Nonribosomal Peptide Synthetase Biosynthetic Clusters of ESKAPE Pathogens

Natural Product
Reports
View Article Online
REVIEW View Journal | View Issue
Nonribosomal peptide synthetase biosynthetic

Cite this: Nat. Prod. Rep., 2017, 34, 981
clusters of ESKAPE pathogens
ab
Andrew M. Gulick
Covering: up to 2017.
Published on 23 June 2017. Downloaded on 8/26/2019 2:58:54 PM.
Natural products are important secondary metabolites produced by bacterial and fungal species that play
important roles in cellular growth and signaling, nutrient acquisition, intra- and interspecies
communication, and virulence. A subset of natural products is produced by nonribosomal peptide
synthetases (NRPSs), a family of large, modular enzymes that function in an assembly line fashion.
Because of the pharmaceutical activity of many NRPS products, much effort has gone into the
exploration of their biosynthetic pathways and the diverse products they make. Many interesting NRPS
pathways have been identified and characterized from both terrestrial and marine bacterial sources.
Recently, several NRPS pathways in human commensal bacterial species have been identified that
produce molecules with antibiotic activity, suggesting another source of interesting NRPS pathways may
be the commensal and pathogenic bacteria that live on the human body. The ESKAPE pathogens
(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,
Pseudomonas aeruginosa, and Enterobacter spp.) have been identified as a significant cause of human
bacterial infections that are frequently multidrug resistant. The emerging resistance profile of these
organisms has prompted calls from multiple international agencies to identify novel antibacterial targets
and develop new approaches to treat infections from ESKAPE pathogens. Each of these species contains
several NRPS biosynthetic gene clusters. While some have been well characterized and produce known
Received 5th May 2017
natural products with important biological roles in microbial physiology, others have yet to be
investigated. This review catalogs the NRPS pathways of ESKAPE pathogens. The exploration of novel
DOI: 10.1039/c7np00029d
NRPS products may lead to a better understanding of the chemical communication used by human
rsc.li/npr pathogens and potentially to the discovery of novel therapeutic approaches.
1. The biochemistry and structural biology of the non- 4.1.2. Aureusimine biosynthesis
ribosomal peptide synthetases 4.1.3. Biological role of aureusimine
1.1 NRPS modular architecture 5. Klebsiella pneumoniae
1.2 Structural dynamics of NRPSs 5.1. Enterobactin
2. NRPSs in ESKAPE pathogens 5.1.1. Structure of enterobactin
2.1. ESKAPE pathogens 5.1.2. Biosynthesis of enterobactin
2.2. NRPS biosynthetic gene clusters 5.1.2.1. The chorismate utilizing enzymes
3. Enterococcus faecium 5.1.2.2. The enterobactin NRPSs
3.1. Cluster 1 5.1.3. Biological role of enterobactin
3.2. Cluster 2 5.2. Yersiniabactin
4. Staphylococcus aureus 5.2.1. Structure of yersiniabactin
4.1. Aureusimine 5.2.2. Biosynthesis of yersiniabactin
4.1.1. Structure of aureusimine 5.2.3 Biological role of yersiniabactin and other K. pneu-
moniae siderophores
5.3. Colibactin
a
Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 5.3.1. Colibactin structure
14203, USA. E-mail: gulick@hwi.buffalo.edu
b
5.3.2. Colibactin biosynthesis
Department of Structural Biology, University at Buffalo, 700 Ellicott Street, Buffalo,
5.3.3. Colibactin biology
NY 14203, USA
This journal is © The Royal Society of Chemistry 2017 Nat. Prod. Rep., 2017, 34, 981–1009 | 981
View Article Online
Natural Product Reports Review
6. Acinetobacter baumannii 8. Enterobacter spp

6.1. Acinetobactin 9. Conclusions
6.1.1 Acinetobactin structure 10. Acknowledgement
6.1.2 Acinetobactin biosynthesis 11. Notes and references
6.1.3 Acinetobactin biology
6.2. Fimsbactin
6.2.1 Fimsbactin structure 1. The biochemistry and structural
6.2.2 Fimsbactin biosynthesis biology of the nonribosomal peptide
6.3. An uncharacterized NRPS cluster involved in biolm synthetases
formation and motility
6.3.1. Biological identication of a novel NRPS cluster 1.1 NRPS modular architecture
6.3.2 Proteins within the uncharacterized NRPS BGC The history of peptide natural products from marine and
6.4. Additional uncharacterized NRPSs terrestrial microbial sources offers a view of the parallel
7. Pseudomonas aeruginosa advances in the elds of chemistry, biochemistry, microbiology
7.1. Pyoverdine and genetics. Following the identication of diverse natural
7.1.1. Structure of pyoverdine products with antibiotic and anticancer activity in the 1960s
7.1.2. Pyoverdine biosynthesis and 1970s, the following decades saw the unraveling of the
7.1.3. Biological role of pyoverdine enzymes that were responsible for the ribosome- and RNA-
7.2. Pyochelin independent production of certain peptide products. Early
7.2.1. Structure of pyochelin work from Lipmann and colleagues with the high molecular
7.2.2. Pyochelin biosynthesis weight proteins that catalyzed synthesis of gramicidin demon-
7.2.3. A pyochelin-related cluster for the synthesis of dihy- strated that specic amino acids are activated through an ade-
droaeruginoic acid nylation reaction1 and subsequently covalently bound to a thiol
7.2.4. Biological role of pyochelin on the enzyme.2 The observation that phosphopantethenic acid
7.3. L-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) was present on the gramicidin synthetase3 and that peptide
7.3.1. Structure of AMB intermediates bound this cofactor as a thioester4 led to
7.3.2. AMB biosynthetic cluster a protein thiotemplate model for nonribosomal peptide
7.3.3 Biological role of AMB synthesis in which the amino acid and growing peptide are
7.4. Pyoluteorin delivered by a carrier domain to neighboring catalytic domains.
7.4.1 Pyoluteorin structure The subsequent genomic revolution identied many genes
7.4.2. Biosynthesis of pyoluteorin that are responsible for peptide production by nonribosomal
7.5. Additional uncharacterized NRPS clusters of P. peptide synthetases (NRPSs).5,6 The encoded proteins were
aeruginosa organized into domains7 whose relationship to known proteins
7.5.1. PA1221 enabled the prediction of domain function.8 The conrmation
7.5.2. PA3327 of biochemical activity catalyzed by the core domains9,10 estab-
7.5.3. PA4078 lished the remarkable catalytic strategy used by the multido-
7.5.4. PA7-cluster main NRPSs.11–16
NRPSs are organized into modules; generally, each module
contains the catalytic domains that are responsible for the
Andrew M. Gulick is a Principal incorporation of a single amino acid into the peptide product.
Research Scientist at the Some NRPS pathways, oen within fungal organisms,17,18
Hauptman-Woodward Medical encode all of the modules on a single gene that encodes
Research Institute and also a single, large multimodular protein. More commonly, NRPS
a faculty member in the Depart- pathways contain multiple large NRPS proteins that interact
ment of Structural Biology in the through a series of both intra- and intermolecular transfers in
Jacobs School of Medicine and the synthesis of the peptide product. A standard NRPS module
Biomedical Sciences at the contains a single peptidyl carrier protein (PCP), a small 8 kDa
University at Buffalo. He received domain that is homologous to the acyl carrier proteins used in
a Ph.D. in Experimental fatty acid transport and synthesis.19,20 The PCP domains contain
Oncology at the McArdle Labo- a conserved serine residue that is post-translationally modied
ratory for Cancer Research at the through the activity of a phosphopantetheinyl transferase
University of Wisconsin. He then (PPTase) with a phosphopantetheine cofactor that is derived
joined the Department of Biochemistry at the University of Wis- from coenzyme A.21 This phosphopantetheine serves as a ex-
consin for postdoctoral training in Structural Biology. His lab ible linker that harbors a thiol onto which the amino acid and
explores the structural basis of enzymatic catalysis, with a partic- peptide intermediates are installed as a thioester. A module
ular focus on the complex machinery of natural product generally will also contain an adenylation domain, most
biosynthesis. commonly positioned upstream of the PCP, that catalyzes a two-
982 | Nat. Prod. Rep., 2017, 34, 981–1009 This journal is © The Royal Society of Chemistry 2017
View Article Online
Review Natural Product Reports
step reaction to activate and then load the amino acid onto the chain reversing incorporation of a ureido dipeptide linkage in
pantetheine cofactor. In the initial adenylate-forming partial the production of the nucleoside antibiotic pacidomycin.39 The
reaction, the amino acid substrate and ATP react to form an condensation domain of the nal module of the nocardicin
amino acyl-adenylate and pyrophosphate. In a second step, the NRPS cluster, NocB, catalyzes the formation of the b-lactam
pantetheine thiol attacks the carbonyl carbon of the amino acid moiety within this antibiotic.40 Thioesterase domains also have
substrate to displace the AMP, which serves as a facile leaving been shown to catalyze unusual activities. Several fungal NRPSs
group for this reaction.22,23 The nal catalytic domain within contain thioesterase domains that catalyze the condensation of
standard NRPS modules is a condensation domain. Two PCPs two b-ketoacids that subsequently cyclize to form furanone cores
from upstream and downstream modules meet in the active site of several pigment molecules.41,42 The thioesterase domains have
of the condensation domain to allow the upstream amino acid also been implicated in the Dieckmann cyclization in bacterial
or peptide to be transferred to the amino acid that is loaded tetramate natural products43,44 as well as b-lactam formation in
onto the downstream PCP. The condensation domain is the production of sulfazecin.45 Although not quite as chemically
responsible for the extension of the growing peptide and is diverse as the chemical reactions catalyzed above, adenylation
therefore the true peptide synthetase domain. domains also have been observed to catalyze unexpected reac-
Because the peptide is covalently bound to the PCP domains tions. Amide formation is catalyzed by an unusual adenylation
during synthesis, NRPSs require a domain that catalyzes release domain that by-passes the need for a carrier domain or
of the completed peptide into solution. Most commonly, this a condensation domain in the elongation of the L-b-lysine oli-
activity resides in a C-terminal thioesterase domain that gopeptide of the antibiotic streptothricin.46 Finally, the presence
contains a catalytic serine, or less commonly a cysteine, that of domains that play a more structural role further complicates
forms a stable acyl-enzyme intermediate.24 This intermediate the prediction of product from an NRPS sequence.47 Additional
can then be resolved through hydrolysis to release the free characterization of novel NRPS pathways will continue to inform
linear peptide, or via cyclization with the N-terminal amine.25 the prediction and conrmation of novel activities.
Additional variations on this theme include the cyclization
through a nucleophilic side chain present on the peptide such
as an internal lysine in the siderophore pyoverdine26 and anti- 1.2 Structural dynamics of NRPSs
biotics tyrocidin and bacitracin,27 or the b-hydroxyl moiety of The biochemistry of the NRPS enzymes raises questions about
the N-terminal acyl chain in surfactin.28 The presence of the the structural mechanisms used by these large modular proteins
acyl-enzyme intermediate also allows for the oligomerization of that are responsible for their assembly line mechanism.
the peptide product in certain instances where a peptide that Numerous reviews exist,15,16,22 including several that are quite
has been completed through one cycle of synthesis by the NRPS recent,48–55 that detail what is known about the structures of both
modules, can serve as the nucleophile to attack a second catalytic domains and larger multidomain and modular proteins.
peptide intermediate. The joining of two pentapeptides in Indeed, this is an exciting time for the structural studies of NRPSs
gramicidin29 or of three units in enterobactin,30 for example, as many structures of multidomain proteins were determined in
allow for the production of the nal peptide oligomer. 2016.56–63 This review will briey describe the structures of indi-
Enhancing these core activities of the NRPS domains, and vidual domains and the implications of the recent structures on
further diversifying the nature of the peptide products, are the necessary dynamics to transfer the substrates and interme-
integrated auxiliary domains that catalyze additional chemical diates between different catalytic domains (Fig. 1).
modications.31 Among the most common of these supple- NRPS carrier protein domains are members of the family of
mentary activities are S-adenosylmethionine (SAM)-dependent acyl-carrier protein domains that play many roles in acyl group
methyltransferases,32,33 epimerases,34,35 and domains that cata- transport.19,20 The PCP domains are 70–80 residues in length and
lyze the heterocyclization of serine, threonine, and cysteine are composed of four a-helices and intervening loops. Helices a1,
residues to form oxazoline and thiazoline rings. The latter are a2, and a4 are longer in length and mostly parallel, whereas helix
variant condensation domains that share 25–30% sequence a3 is shorter and oriented roughly perpendicular to the other
identity with peptide bond-forming condensation domains.27,36 three helices. The serine onto which the phosphopanetheine
Additionally, some NRPS proteins terminate not in a thio- cofactor is placed is part of a conserved Gx(D/H)S motif, where
esterase domain but rather in a reductase domain that catalyzes the second position is most commonly a hydrophobic residue
the NADPH dependent formation of a terminal aldehyde that ranging in size from glycine to larger aromatic residues.19
itself may be subject to further spontaneous or enzyme cata- The NRPS condensation domains are 400–450 residues in
lyzed modication.37,38 length and contain two lobes that surround a large central cle
The organization of NRPS domains and modules might containing the active site.55 The lobes have been observed in
suggest that a straightforward bioinformatic approach would multiple conformations (Fig. 1B).64–66 The open cle contains
allow the identication of the nal peptide product from gene the binding sites for the upstream (donor) PCP domain, as well
sequence. However, the facile prediction of peptide product as the downstream (acceptor) PCP. The substrates bound to the
from domain and module organization is further challenged by pantetheine arms then meet for transfer of the upstream amino
the observation that catalytic domains oen perform unexpected acid or peptide to the amino acid that has previously been
chemical transformations. For example, the condensation loaded onto the downstream PCP. Recent structures have been
domain of the didomain protein PacN catalyzes the peptide obtained with pantetheine chains bound to the acceptor
View Article Online

Fig. 1 Structures of NRPS enzymes. (A) Surface representation of multidomain NRPS enzymes indicating domain architecture. Complete
modules of SrfA-C (PDB 2VSQ), AB3403 (4ZXI), EntF (5JA1), and LgrA (5ES9). Surfaces are colored to highlight condensation (green), adenylation
N-terminal (pink) and C-terminal (red) subdomains, PCP (blue), thioesterase (yellow) and formyltransferase (cyan) domains. The EntF structure
also illustrates a bound MLP, YbdZ (orange) (B). Ribbon diagrams of condensation domains from CDA synthetase (5DU9) and AB3403, as well as
the homologous epimerization (5ISX) and cyclization (5EJD) domains. (C) Ribbon diagrams of adenylation domains showing GrsA in the ade-
nylate-forming conformation (1AMU), the functional interaction of the EntE adenylation domain with the EntB carrier protein domain in the
thioester-forming conformation (4IZ6), and the interaction between the MLP and N-terminal subdomain of the adenylation domain of SlgN1
(4GR5). (D) Ribbon diagrams of the thioesterase domain from fengycin NRPS FenB (2CB9) and the functional interaction between the PCP and
thioesterase of EntF (3TEJ). Additionally shown is the ribbon diagram from the formyltransferase domain of LgrA.
site.56,58 While no structures exist of a condensation domain smaller subdomains have also been referred to as the Acore and
bound to a donor PCP, structures of TqaA, the homologous Asub domains.50 Members of this enzyme family catalyze two
terminal cyclization domain of a fungal NRPS,62 and an epi- partial reactions, the initial adenylation step followed by
merization domain of the gramicidin synthetase GrsA57 provide a second thioester-forming reaction. In the case of luciferase,
models for the binding of the PCP to the condensation domain the second reaction is an oxidative decarboxylation leading to
donor site. the light-producing intermediate. The active site is positioned
The NRPS adenylation domain is a member of the structur- between the two subdomains and early structures of members
ally homologous ANL superfamily of adenylate-forming of this enzyme family pointed to the possibility that the C-
enzymes. This superfamily contains three subfamilies, the terminal subdomain would adopt two distinct conformations
NRPS adenylation domains, the acyl- and aryl-CoA synthetases, to catalyze the two partial reactions.67–70 This was subsequently
and the beetle luciferase enzymes.22 Members of this enzyme conrmed through biochemical and structural studies of
family are 500–700 residues in length and contain two sub- multiple family members.22 This large conformational change
domains. A larger N-terminal subdomain contains 400–600 was also proposed to play a role in transporting the PCP
residues, while the smaller C-terminal subdomain is less vari- between different catalytic domains of the modular NRPSs.71
able in size at 120–140 residues in length. The larger and
View Article Online
In addition to the carrier protein and the core condensation observations were observed in the recent structures of an
and adenylation domains, structures exist for the additional initiation module from the gramicidin synthetase.60 The rst
NRPS domains, including the chain terminating thioester72,73 module of LgrA contains a formyltransferase domain upstream
and reductase37,38,74,75 domains, as well as the initiating for- of adenylation and PCP domains, forming a tridomain protein
myltransferase domain (Fig. 1D).60 that was structurally characterized in multiple catalytic states.
While structures of individual NRPS domains allow for The LgrA structures also demonstrate the domain alternation of
understanding the reaction mechanisms of a single step in the the adenylation domain C-terminal subdomain upon move-
NRPS biosynthesis, structures of multidomain NRPS proteins ment of the PCP between the adenylate- and thioester-forming
provide more detailed views of the modular architecture. conformations. Further, the C-terminal subdomain adopts an
Didomain structures of the PCP-thioesterase76,77 and the PCP- extended conformation, projecting away from the N-terminal
adenylation domains78–80 illustrate the domain interfaces that subdomain, to allow the substrate bound to the PCP to reach
occur between catalytic domains and the PCP. These structures to the formyltransferase domain.60
show that the PCP helix a2, which starts with the pantetheiny- These recent structural observations58 provide snap shots
lated serine, as well as the loop that joins helices a1 and a2 form that help to piece together the complete structural cycle.
the PCP interface with catalytic domains. Further, in the Because the NRPS module can adopt the conformation to
structures containing the holo-PCP, the cofactor is directed into catalyze simultaneously peptide bond formation in the
the active site of the enzyme. Crystallization of the adenylation– condensation domain while also catalyzing substrate adenyla-
PCP complexes78–80 was achieved with a mechanism based tion within the adenylation domain, the enzyme is able to prime
inhibitor that captured the attack of the pantetheine thiol on the next cycle of synthesis while the PCP is engaged with the
a vinylsulfonamide analog of the adenylate intermediate within condensation domain.51 In comparison to the magnicent rate
the active site of the adenylation domain.81 In these structures, enhancements of many enzymes, the modular NRPS enzymes
the C-terminal subdomain is rotated into the thioester-forming are rather slow, with turnover constants ranging from 1 to 100
conformation, forming the pantetheine tunnel that enables the turnovers per minute.30,83,84 This is true for many secondary
pantetheine to reach the active site. metabolites and perhaps limited evolutionary pressure, or none
The structures of multidomain NRPS proteins provides at all, to evolutionary pressure to accelerate natural product
further understanding of the domain interactions and the biosynthesis. Nonetheless, these new structural results suggest
dynamic nature of the NRPS structural cycle. The determination that the efficiency of the catalytic cycle could be enhanced by the
in 2008 of the structure of SrfA-C, the terminal module from the ability of two NRPS domains within a module to be active at the
surfactin biosynthetic cluster provided the rst view of how same time.
a complete NRPS module was organized (Fig. 1A).82 This land- Also apparent from the structures of the three terminal
mark structure showed a signicant interface was formed modules was the lack of conservation of the positions of the
between the condensation domain and the larger N-terminal downstream thioesterase domain.58,59,82 The lack of interactions
adenylation subdomain. Much more limited interactions were between the thioesterase domain and the remaining catalytic
made between these domains and the C-terminal thioesterase domains suggests that there may be limited multimodular
domain. The structure was consistent with the hypothesis that organization that govern interactions between adjacent
movement of the adenylation C-terminal subdomain could modules. Indeed the Schmeing group has recently successfully
accompany transport of the PCP to the condensation domain crystallized the inter-module structure of a fragment of DhbF,
and adenylation domain active sites.82 a bimodular protein that is used to produce the siderophore
This proposal has been supported by recent structures of two bacillibactin. The NRPS fragment contains an adenylation–
holo-NRPS modules of the same domain architecture as SrfA- PCP–condensation domain, crossing the border between
C.58,59 The structure of the A. baumannii NRPS protein AB3403 modules and extending to the downstream condensation
illustrated the interaction of the PCP with the upstream domain.63 The structure shows that no interactions exist
condensation domain while a structure of the E. coli EntF between the adenylation domain and the condensation domain
protein showed the interaction of the PCP with the adenylation of the subsequent module. Instead the condensation domain
domain (Fig. 1A). Accompanying the movement of the PCP from cradles the “back face” of the carrier protein domain. If one
the condensation domain to the adenylation domain, the ade- considers the downstream thioesterase domain to be part of the
nylation domain C-terminal subdomain rotates through the two “next module”, this structure and the termination modules
catalytic conformations.22 In AB3403, the adenylation domain suggest limited interactions may exist between the core
adopts the adenylate-forming conformation to allow the PCP to condensation–adenylation domains of one module and the
engage the condensation domain. Upon completion of the domains of the downstream modules.
adenylation reaction, the C-terminal subdomain rotates to the
thioester-forming conformation as seen in EntF to deliver the 2. NRPSs in ESKAPE pathogens
PCP to the adenylation domain active site and form the
conformation that is used to catalyze the thioesterication 2.1. ESKAPE pathogens
reaction. A 2008 editorial commentary85 introduced the phrase “ESKAPE
While the above structural studies on modular NRPS bugs” to represent a panel of human pathogens that were
proteins used termination modules, equally exciting increasingly resistant to many of the most common antibiotics.
View Article Online
The frequency of nosocomial infections caused by Enterococcus separated by genes that play no role in pyoverdine biosyn-
faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acineto- thesis.102 We therefore also examined the surrounding genes of
bacter baumannii, Pseudomonas aeruginosa, and Enterobacter any uncharacterized BGCs described below and, if discovered,
spp., particularly in older and immunocompromised patients comment on the proximity of additional genes that may be
in health care settings, illustrated a need to develop novel involved in the biosynthetic pathway.
antimicrobial agents. The use of the term ESKAPE highlighted Herein, the NRPS clusters within the ESKAPE pathogen
the ability of these organisms to “escape” from the antimicro- genomes are described. Where possible, benchmark strains
bial activities of the commonly used antibiotics. A growing that contain the most common NRPSs for a species are used.
consensus of the importance of these species has led to a call to BGCs that are present in less common strains are also
action from the Infectious Diseases Society of America as well as described. Where known, the product is identied and any
the development of public/private partnerships across the associated biology is presented, including the role of the
globe.86,87 Indeed, concern has been raised for the relative lack product in microbial physiology or pathogenesis.
of novel treatments in the pipeline that are needed to reverse
the growing threat posed by multidrug resistant ESKAPE
infections.88,89 3. Enterococcus faecium
Beyond the signicant clinical impact of these pathogens, The Gram-positive streptococci include the genus Enterococcus,
they also serve as “paradigms of pathogenesis, transmission, which includes both E. faecalis and E. faecium. Both strains are
and resistance”.85 Their study can provide details about the very common in the human gastrointestinal tract as commensal
fundamental basis of microbial physiology and interaction with bacteria and have been observed to cause human infections.103
the host. There is value in understanding the unique properties The two species differ in their metabolic prole and, more
that make these organisms particularly suited to survive in the signicantly, in their antibiotic resistance proles. E. faecium is
host and, further, in exploring the features encoded within their much more commonly resistant to both ampicillin and vanco-
genomes that promote their virulence. mycin.104 Vancomycin-resistant enterococci (VREs) are the
As many NRPS products have been identied as virulence second most common cause of antibiotic resistant nosocomial
factors or as novel signaling molecules, a survey of the NRPS infection behind only methicillin resistant S. aureus (MRSA).105
biosynthetic gene clusters that are present in ESKAPE patho- NRPS clusters are rare in the sequenced strains of E. faecium
gens could offer insight into the fundamental microbiology of and there are no characterized NRPS clusters or products.
these organisms and could potentially identify novel targets for Indeed, the rst strain of E. faecium that was sequenced,
antimicrobial discovery. designated DO or TX0016, does not contain any NRPS
sequences, nor does the rst complete sequence of a vancomy-
2.2. NRPS biosynthetic gene clusters cin resistant strain, Aus0004.106 Further, searching a panel107 of
three faecal and four clinical samples fails to identify genes
Several recent reviews have focused on the small molecules encoding NRPS proteins. Interestingly, E. faecium strains lack
produced by the human microbiota.90–94 The combined tools of acyl-CoA synthetase genes as a whole and the only member of
mass spectrometry and the revolution in genomic sequencing the adenylate forming ANL superfamily22 that they contain
revealed much of the biosynthetic potential of the bacteria appears to be a D-alanyl-carrier protein ligase needed for cell
that co-exist upon and within the human body. This infor- wall biosynthesis. No NRPS BGCs have been identied in the
mation has in turn led to the identication of novel biosyn- AntiSMASH database.
thetic pathways for products with important biological The Basic Local Alignment Search Tool (BLAST),108 available
activity.95 at the NCBI, was used to identify several NRPS protein
This review will focus specically on the NRPS products that sequences from alternate E. faecium strains. Four strains con-
are produced by the ESKAPE pathogens. For each species, bio- tained the larger cluster described below, while one strain
informatic approaches were used to identify known and contained an additional NRPS cluster. Two of the identied
uncharacterized biosynthetic gene clusters (BGCs).96 Recent strains were from unpublished clinical isolates while two,
efforts at generating curated and publicly available databases including the strain KAC16100 described in Table 1, were iso-
such as the Minimal Information about a Biosynthetic Gene lated from fermented soybean samples.109
Cluster (MIBiG),97 and the Antibiotics and Secondary Metabolite
Analysis Shell (antiSMASH),98 as well as additional online
tools,99–101 facilitate the identication of and prediction of the 3.1. Cluster 1
products of novel BGCs. The largest NRPS protein identied in E. faecium is a ve
The boundaries of a BGC can be difficult to predict. Multiple module protein that is 5296 residues in length. This gene was
genes of a biosynthetic pathway are oen expressed on a single present in four strains: KAC16100, KAC16106, EnGen0026, and
operon derived from a single mRNA transcript containing Hp_22-12. In the latter two strains, the gene is reported to be
multiple genes that are separated by fewer than 50 nucleotides. divided into three separate genes. Whether this represents
However, sometimes the genes of a BGC are separated into sequencing errors or the introduction of true stop codons is
multiple operons. The pyoverdine BGC of P. aeruginosa, for unknown. However, the proteins encoded by the smaller genes
example, contains 15 genes on multiple operons that are are identical to the full length gene of the two KAC strains and
View Article Online
Table 1 NRPS clusters within Enterococcus faeciuma
Locus ID (accession) Length (AA) Predicted function
Strain KAC16100 (Genbank Accession NZ_LDNJ01000053)

WP_047937824 (NpsA) 5296 Five module NRPS
WP_002343040 131 Aspartate decarboxylase
WP_053002117 303 HAD hydrolase
WP_047929904 (NpsB) 808 A–T–Te
WP_053002116 211 PPTase
Strain KAC16100 (Genbank Accession NZ_LDNJ01000090)

ACH93_RS12695 (WP_002318470) 135 Hydrolase
ACH93_RS12690 213 Hypothetical
ACH93_RS12685 (WP_047937945) 222 Hypothetical
ACH93_RS12680 (WP_047937944) 239 Dehydrogenase
ACH93_RS12675 (WP_047937943) 987 A–T–C

a
Abbreviations used in all tables to represent NRPS domains: A, adenylation; C, condensation; Ep, epimerization; FT, formyltransferase; M,
methyltransfase; Re, reductase; T, thiolation or PCP; U, domains of unknown function.
the protein boundaries are both positioned within a condensa- elements and raising the possibility that the rst two genes in
tion domain leading to the suspicion that the truncated genes this cluster are not involved in the BGC.
are more likely a result of sequencing errors.
This BGC contains two NRPS proteins, designated NpsA and
NpsB in strain KAC16100. The smaller NpsB contains a single
4. Staphylococcus aureus
module that contains an adenylation, PCP, and thioesterase S. aureus is a Gram-positive bacterium and a common source of
domain. The larger NpsA appears to be the initiating protein, as nosocomial infections, occuring in a wide variety of care facil-
the rst module of NpsA contains only an adenylation and ities. Roughly 60% of humans carry S. aureus intermittently and
carrier protein. The remaining four complete modules harbor another 20% carry S. aureus strains at all times.89 S. aureus is the
condensation, adenylation, and carrier domains. The fourth primary cause of epidermal and so-tissue infections and can
module contains an additional epimerization domain. Aer the also cause a number of invasive infections, including abscesses,
h module of NpsA is a condensation domain that may hand pneumoniae, and sepsis.110 The prevalence of antibiotic resis-
the peptide off to the PCP of the shorter protein (we note with tant S. aureus, particularly methicillin resistant (MRSA) strains
caution here that the generic “Nps” nomenclature has been that can represent as much as 50% of S. aureus isolates in some
used for proteins in multiple species to designate uncharac- regions,89 has risen over the past decade. This startling trend
terized NRPS proteins). Also present in the operon are two only raises the importance of efforts to develop new treatment
additional biosynthetic enzymes showing homology to aspar- options.111
tate decarboxylase and HAD family hydrolases. Finally, the
cluster also contains a PPTase to convert the apo to holo PCP
4.1. Aureusimine
domains.
4.1.1. Structure of aureusimine. The S. aureus genome
encodes a single NRPS BGC that has been assigned to the
3.2. Cluster 2 natural product aureusimine, a cyclic dipeptide formed through
The second cluster from E. faecium appears only in a single the condensation of either tyrosine or phenylalanine with valine
strain of E. faecium. The operon contains seven genes, to form pyrazinones (1) and (2). The cluster is conserved in S.
including a gene encoding a single NRPS module. The cluster aureus and also S. epidermidis, S. lugdunensis, and S. capitus.112
also encodes four hypothetical proteins of unknown function The molecule is thought to be produced through the reductive
as well as a hydrolase and an NAD(P)H-dependent dehydro- release of a dipeptide aldehyde, which spontaneously cyclizes.
genase. A search of the NCBI protein sequence database with The aureusimine biosynthetic gene cluster contains two genes,
BLAST nds no homologs above 40% sequence identity for named ausA and ausB.113 A search of other S. aureus strains
any of the nal ve proteins of this cluster demonstrating that identies NRPS genes of shorter lengths that are 99% iden-
this is a unique, orphan BGC. The 135 residue protein tical to AusA, suggesting either sequencing errors or bonade
matches other sequences, as does the initial 110 residues of mutations that have separated the NRPS gene into two multi-
protein ACH93_RS12690, which shows 100% identity with domain proteins. Aureusimine was identied nearly simulta-
other E. faceium strains. In strain EnGen0038, accession neously by the Fischbach112 and Magarvey113 groups. It was also
AHXW01000024, the neighboring genes are transposon and noted that the phenylalanine isoform had been identied
integrase genes suggesting the presence of mobile genetic previously and called phevalin.114
View Article Online
the regulation of a variety of genes encoding proteins involved

with electron transfer properties and redox signaling.
Further investigation of the role of the aureusimines in the
biology of S. aureus and human keratinocytes demonstrated
that the aureusimines were more highly expressed under bio-
lm conditions compared to planktonic growth, and that the
phenylalanine isoform (2) was more prevalent than the tyrosine
isoform.118 This same study concluded that the addition of
exogenous aureusimine had no effects on S. aureus growth and
no dramatic effects on the extracellular metabolome. Further,
the presence of aureusimine had modest effects on the gene
expression prole of 24 genes in human keratinocytes.
S. aureus is an intracellular pathogen that is able to with-
stand the environment of the phagolysosome by disruption of
Fig. 2 Aureusimine biosynthesis. The production of aureusimine has lysosomal membranes through the use of the so-called phenol
been demonstrated in biochemical experiments with holo-AusA.
soluble modulins, which are necessary but not sufficient for cell
Cyclization of the dipeptide aldehyde is believed to occur spontane-
lysis and escape from the phagosome.119 A search for additional
ously. Multidomain NRPS proteins are represented here and in
subsequent figures as circles and labeled as described in Table 1. genes that play a role in intracellular survival and phagosomal
escape identied ausA and ausB.120 While the authors concluded
that the aureusimines are not “major virulence factors”, they
4.1.2. Aureusimine biosynthesis. Aureusimine has been raised the possibility that they may contribute to S. aureus
shown to be produced through the activity of a single, dimod- virulence in strain- and tissue-specic manners.120
ular NRPS protein called AusA (Fig. 2).38,115 The aus operon also Finally, a recent report suggests the dipeptide aldehyde, and
contains ausB, encoding a PPTase to convert the AusA protein to not the cyclic pyrazinone of aureusimine, could be the bioactive
the holo form. The rst module of AusA encodes an adenylation compound.121 Examination of NRPS BGCs from microbiome
and PCP domain, while the second module contains adenyla- DNA identied clusters that are present in >90% of stool
tion, PCP, and reductase domains. Although the adenylation samples from the Human Microbiome Project and almost
domains have not been biochemically analyzed in isolation, the exclusively within gut bacteria. These NRPSs produce multiple
sequence of the adenylation domain pockets suggest that the dipeptides, including Val–Phe- and Phe–Phe-aldehyde. These
rst module activates valine while the second module activates linear aldehydes of aureusimine A and B were shown to be
the aromatic substrates. The full length protein has been pro- potent inhibitors of cathepsins, a family of lysosomal cysteine
bed for adenylation activity both in the presence and absence of proteases that play a role in antigen presenting within macro-
phenylalanine- and valine-specic inhibitors, supporting the phages and dendritic cells. As the cyclization is an aerobic step
promiscuous activity of the adenylation domain of module 1.115 in the biosynthesis of the pyrazinones, the aldehydes may have
The NRPS protein AusA is relatively straight-forward with two a sufficiently long half-life in the anaerobic environment of the
modules that contribute to the production of the dipeptides gut in which they are produced to play a biological role.121
(Fig. 2), along with a minor production of pyrazinone derived
from a Val–Leu dipeptide. The C-terminal reductase domain
catalyzes the release of the dipeptide as an aldehyde, which is 5. Klebsiella pneumoniae
believed to cyclize spontaneously in the energetically favorable
reaction that benets from the aromatic stabilization of the Klebsiella pneumoniae is a common human pathogen that has
pyrazinone product.38,115 The reductase domain has been been identied as the cause of many health-care associated
structurally characterized38 demonstrating that the domain infections of the lung, urinary tract, abdominal cavity, and
adopts a standard Rossmann fold shared by other short chain eye.122,123 Several reports note the presence and rapid dissemi-
dehydrogenase enzymes. nation of carbapenem-resistant K. pneumoniae in long-term care
4.1.3. Biological role of aureusimine. As some cyclized facilities.124,125 Perhaps even more concerning, hypervirulent
dipeptides display antibacterial properties, aureusimine was strains of K. pneumoniae (designated hvKP)126,127 that were
assayed for growth inhibition against E. coli, B. subtilis, and originally identied in Asia appear to be spreading across the
a panel of actinomycetes from the human skin microbiome. No globe. The hypervirulent strains exhibit the ability to infect
effect on the tested bacteria was identied.112 Although origi- a younger, healthier population as well as the ability to spread
nally identied as being related to the virulence of S. aureus,113 metastatically from the initial sites of infection. To date, most of
subsequent analysis demonstrated that the mutant strain used the strains appear to be sensitive to antibiotics; however, the
in the initial studies contained a second mutation in the saeS potential for the acquisition of a drug-resistance phenotype by
gene, a regulator of known virulence factors.116,117 Performing these hvKP strains is high,128,129 raising the possibility of
the genetic array analysis with an ausA mutant strain showed a pathogen that is both highly virulent and highly antimicrobial
resistant. K. pneumoniae contains several NRPS BGCs that are
common to many Gram-negative enteric bacteria.
View Article Online
5.1. Enterobactin utilizing enzymes converts chorismate to the aryl moieties

incorporated into the products.133,144 This section will focus on
5.1.1. Structure of enterobactin. Enterobactin (8) was
these pathways (Fig. 3) and set the stage for their use in not only
discovered almost 50 years ago, where it was identied from
enterobactin, but also yersiniabactin, acinetobactin, msbac-
both S. typhimurium130 and E. coli.131 Enterobactin is a trilactone
tin, and pyochelin biosynthesis in the sections to follow.
composed of three molecules of serine that are cyclized through
The DHB moiety of enterobactin is produced from cho-
ester linkages between their carboxylates and the side chain
rismate (3) by the sequential activities of EntC, EntB, and EntA.
hydroxyls. Each of the three amines of this trilactone is joined
via an amide linkage to a molecule of 2,3-dihydroxybenzoic acid EntC is a Mg2+-dependent isochorismate synthase that converts
(7, DHB).132–134 The six catechol oxygens form the ligands for the chorismate to isochorismate (4).141,145 The isochorismate is then
hydrolyzed by the isochorismatase domain of the bifunctional
Fe3+ ion, providing an extremely high affinity binding motif
EntB to produce 2,3-dihydro-2,3-dihydroxybenzoate (6) and
with a proton-independent formation constant reported as 1052
pyruvate,146 which is then converted to DHB by the activity of the
for the iron–siderophore complex.135
short-chain dehydrogenase/reductase protein EntA.136,147
5.1.2. Biosynthesis of enterobactin. Enterobactin is
EntC is a member of the MST family of enzymes that initiate
produced through the activities of ve proteins encoded in the
these chorismate conversion steps involved in the production of
ent operon.136–139 EntA, EntB, and EntC are responsible for the
production of the DHB molecule, while the NRPS proteins are menaquinone, siderophores, or tryptophan.148,149 Additional
housed on EntE, a freestanding adenylation domain, and EntF, members of this family include PchA of pyochelin biosynthesis,
BasJ of acinetobactin biosynthesis, and Irp9 of yersiniabactin
which contains the module for the incorporation of serine. As
biosynthesis. Interestingly while the initial step in the reac-
one of the oldest characterized products of an NRPS, as well as
tion—the isomerization to isochorismate—is shared, different
a relatively simple NRPS product from E. coli, enterobactin
members of the family form different products. Thus, the
biosynthesis served as a model system (within the lab of
common isochorismate intermediate can be converted to
Professor Christopher Walsh) and was utilized for much of the
salicylate (5) through a second activity within the MST protein,
foundational biochemical studies to understand the function of
NRPS enzymes. Further, the ve biosynthetic proteins from E. as in the case of Irp9 of yersiniabactin biosynthesis, or a sepa-
coli have all been crystallized and their structures determined, rate isochorismate lyase protein as with PchB. Alternatively,
some BGCs use a separate enzyme to retain the hydroxyl at the
including the three enzymes required for DHB synthesis140–142
meta position to produce (6). Such enzymes include EntB and
and the NRPS proteins in varying functional complexes,58,59,79,80
BasF, which both contain the isochorismatase domain as part
providing further insight into the structural mechanisms.
of a fusion with an NRPS acyl-carrier protein domain.140,146 In
Although limited structural and biochemical data exist on the
enterobactin biosynthesis, the nal step in the production of
enterobactin enzymes from K. pneumoniae, the enzymes are 70
DHB (7) is catalyzed by EntA, which catalyzes the aromatization
to 90% identical to the E. coli proteins. The biosynthesis will
therefore focus on data obtained with E. coli enzymes. of the product of the EntB reaction.136,142,150
5.1.2.1. The chorismate utilizing enzymes. Many side- 5.1.2.2. The enterobactin NRPSs. The enterobactin NRPS
system encompasses three proteins, EntE, EntB, and EntF, that
rophores utilize DHB (7) or salicylate (5) capping groups that
combine three molecules of DHB and three molecules of serine
play critical roles in iron interaction.143 A series of chorismate-
into the nal enterobactin trilactone (Fig. 4).30,146,151 Additionally
included in the full biosynthetic cluster is EntD, the PPTase that
converts the apo to holo carrier protein domains.146 The free-
standing adenylation domain EntE rst loads a molecule of
DHB onto the aryl carrier protein (ArCP) domain of the
bifunctional protein EntB. As described above, EntB also
contains the isochorismatase domain that is responsible for
DHB synthesis. EntF is a four domain NRPS protein with
a condensation–adenylation–PCP–thioesterase architecture.
The EntF adenylation domain loads a molecule of serine onto
the downstream PCP. The loaded PCP then migrates to the
condensation domain where it awaits delivery of the DHB from
a loaded EntB ArCP domain. The condensation domain cata-
lyzes amide bond formation via nucleophilic attack of the
loaded serine amino group on the carbonyl of the DHB–ArCP
thioester of EntB. The EntF PCP then delivers the resulting N-
Fig. 3 Chorismate utilizing enzymes in NRPS siderophore pathways. (2,3-dihydroxybenzyl)serine to the EntF thioesterase domain.
Members of the MST family of chorismate-utilizing enzymes convert The catalytic serine (Ser1138) of the EntF thioesterase
chorismate to isochorismate. MbtI and Irp9 are able to perform
domain attacks the thioester linkage to form a covalent
a second reaction to convert isochorismate 4 to salicylate 5, while
PchB is a monofunctional salicylate synthase. EntB, BasF, and FbsC are enzyme–peptide intermediate at the thioesterase domain active
isochorismatase enzymes. EntA and FbsD are dehydrogenases that site. A second cycle then provides a second N-(2,3-dihydrox-
produce 2,3-dihydroxybenzoate 7. ybenzyl)serine to the thioesterase domain. In the second cycle,
View Article Online
chain (Fig. 5). The nal molecule is assembled from building

blocks of salicylate, three cysteine residues, and a malonic acid
group that is contributed by an integrated polyketide synthase
(PKS) module.83 The three cysteine residues are all cyclized at
varying oxidation states and present as either thiazolidine or
thiazoline rings. Additionally, methyl groups derived from three
molecules of S-adenosylmethionine (SAM) are utilized by inte-
grated methyltransferase domains within the PKS and terminal
NRPS module to produce the nal product.
5.2.2. Biosynthesis of yersiniabactin. As with enterobactin,
to our knowledge, there are no biochemical studies on the
synthesis of yersiniabactin using the proteins from K. pneumo-
niae. Most of the studies, again elegantly performed by the
Walsh lab, were done with the homologous proteins from Yer-
sinia pestis. As the sequence identities between K. pneumoniae
and Y. pestis are greater than 95%, we again describe the

biosynthetic pathway using the Y. pestis model system.
The yersiniabactin BGC encodes ve proteins (Fig. 5). YbtS is
a salicylate synthase of the MST superfamily discussed above
that carries out both the conversion of chorismate to iso-
chorismate and the salicylate synthase reaction.157 The NRPS
Fig. 4 Enterobactin biosynthesis. Enterobactin biosynthesis has been
proteins include YbtE, a freestanding adenylation domain that
demonstrated biochemically for the homologous enzymes from E.
coli. EntE and EntB form a single module for incorporation of DHB, activates the initial salicylate molecule, and two large multido-
while EntF forms a single module for serine. EntB contains the thio- main NRPSs named high molecular weight protein 1 and 2
lation domain as well as the isochorismatase domain (IC) used in DHB (HMWP1 and HMWP2), as well as an auxiliary reductase protein
biosynthesis. The N-(2,3-dihydroxybenoyl)serine is bound to a cata- called YbtU. The Ybt protein and gene names are used in the Y.
lytic serine within the thioesterase domain of EntF for two subsequent
pestis literature; in Y. enterocolitica, the two large NRPS proteins
cycles of synthesis that iteratively complete the trilactone.
are named Irp1 and Irp2, while YbtE, YbtS, and YbtU are called
Irp5, Irp9, and Irp3 respectively.
HMWP2 contains two NRPS modules and six domains
the serine side chain hydroxyl from the rst synthetic cycle, not
organized as an ArCP-cyclization/condensation–adenylation–
the Ser1138 side chain, is used as the nucleophile in the
PCP–cyclization/condensation–PCP architecture. HMWP1
transacylation reaction. A nal iteration results in the covalently
contains a PKS module containing ve domains necessary to
bound linear trimer of N-(2,3-dihydroxybenzyl)serine moieties,
incorporate and dimethylate a malonyl moiety, followed by
joined through ester linkages between the serine side chain and
a terminal NRPS module containing cyclization/condensation–
carboxylate, that is loaded on the catalytic serine of the thio-
methyltransferase–PCP–thioesterase domains.83 The NRPS
esterase domain. Closure of the ring within the thioesterase
pathway for yersiniabactin synthesis begins with the activation
domain releases enterobactin (8) and frees the catalytic serine
of salicyate by YbtE and the loading of the aryl substrate onto
to begin a new cycle of enterobactin biosynthesis.30
the N-terminal ArCP domain of HMWP2. The single adenylation
5.1.3. Biological role of enterobactin. The association of
domain of HMWP2 loads a cysteine residue onto the two
enterobactin production with K. pneumoniae growth in low iron
downstream PCP domains within this protein in cis, as well as
conditions has been known for almost 40 years.152,153 The
the PCP domain of the HMWP1 protein in trans.157 The
redundancy of siderophore production by pathogens reects
bifunctional cyclization/condensation domains sequentially
not only the signicance of iron acquisition but also may be
catalyze the amide bond formation and cyclization of the
related to the ability of specic siderophores to be involved in
cysteine residues to form the thiazolinyl rings. The freestanding
bacterial growth in particular microenvironments. Because
reductase YbtU then catalyzes the conversion of the second
many studies of K. pneumoniae siderophores consider this
thiazoline ring, but not the others, to a thiazolidine moiety,
functional interplay between the different siderophore systems,
while the immature siderophore is still bound to the nal PCP
the biological role of enterobactin is described along with yer-
domain.83,158 Hydrolysis of the mature peptide results in the
siniabactin in more detail below.
release of yersiniabactin (9).
5.2.3 Biological role of yersiniabactin and other K. pneu-
5.2. Yersiniabactin moniae siderophores. The yersiniabactin and enterobactin
5.2.1. Structure of yersiniabactin. Yersiniabactin (9) was pathways are present on the chromosome in most strains of K.
puried in 1993 (ref. 154) and structurally dened several years pneumoniae. In contrast, hypervirulent strains of K. pneumoniae
later155,156 as a siderophore in Yersinia species and later in (hvKP) contain a large 220 kilobase plasmid that contains genes
additional bacterial species. Like enterobactin, it contains an related to the virulent phenotype.159 Included on the plasmid
aryl cap composed of a salicylate moiety connected to a peptide are the iuc genes responsible for the biosynthesis and uptake of
View Article Online

Fig. 5 Yersiniabactin biosynthesis. Yersiniabactin biosynthesis has been reconstituted for the homologous enzymes from Y. pestis. HMWP2
contains condensation/cyclization domains (Cy) that catalyze peptide bond formation and thiazoline formation. HMWP1 contains a PKS module
containing ketosynthase (KS), acyltransferase (AT), methyltransferase (M), ketoreductase (KR) and acyl carrier protein (ACP) domains in addition to
other domains described earlier.
the NRPS-independent siderophore aerobactin, as well as the division. Transposon mutagenesis allowed the mapping of this
iroB gene that produces glucosylated variants of enterobactin cluster to a large, 54-kilobase region encoding a hybrid PKS/
called salmochelin.160 The reason for this redundancy of side- NRPS BGC that is common to strains that share this cytotoxic
rophore systems is unclear; however, this may reect the phenotype.167 The colibactin pathway has additionally been
differential utility and regulation of preferred siderophores identied in both classical and hypervirulent K.
under certain conditions. Specically, the pH dependence of pneumoniae.168–170
iron affinity and the desire to evade eukaryotic lipocalin 2,161,162 The precise identity of the bioactive product of this complex
a siderophore binding protein that is present in serum, may biosynthetic pathway has not yet been elucidated. However,
dictate which siderophores become most effective in certain shared features have been identied that may be responsible for
pathogenic environments.163 the mechanism of action.171 Because of the highly reactive
Examination of the roles of different K. pneumoniae side- intermediates and the potential for multiple cyclization steps,
rophores can be done with genetic mutations that are unable to different intermediates have been identied in studies that
produce one or more of the compounds. In a murine intranasal used mutant strains to isolate biosynthetic intermediates.
infection model with a classical K. pneumoniae strain, a yersi- Consensus exists that the PKS/NRPS product is produced
niabactin mutant was less virulent than the wild-type or through a “prodrug” mechanism that requires proteolysis to
a enterobactin mutant. However, a double mutant defective for form the nal active product.172,173 The peptidase ClbP is
both siderophores, the only two present in this strain, was responsible for cleavage of an acyl-asparagine moiety from
completely avirulent, demonstrating the importance of iron a precursor during product maturation. The nal colibactin
acquisition.164 molecule is expected to contain an electrophilic warhead that
A more recent study examined the relationship of the four
siderophores, enterobactin, yersiniabactin, aerobactin, and
salmochelin, that are present in an hvKP strain. The aerobactin
BGC is encoded on the hvKP virulence plasmid and is highly
over-expressed165 in the hypervirulent strains. In murine pneu-
monia and subcutaneous infection models, mutants that are
decient for the production of aerobactin were signicantly less
virulent than wild-type cells or a mutant strain that was defec-
tive for the production of the three other siderophores.166
5.3. Colibactin
5.3.1. Colibactin structure. Some pathogenic E. coli strains
Fig. 6 Precolibactin molecules. The bioactive product of the col-
have been observed to induce megalocytosis in eukaryotic host ibactin pathway has not yet been identified. Several precolibactin
cells upon infection. This process is characterized by enlarge- metabolites, including 10 containing the acyl-Asn peptide leader and
ment of the nucleus and the cell without associated cell 11 derived after cleavage of the leader, have been identified.
View Article Online
can cross-link DNA as well as heterocyclic thiazoline rings that vivo167 likely plays a role in this effect. The ability of a 0.2 m
could facilitate interaction with DNA (Fig. 6).174–176 On-going membrane between the bacteria and eukaryotic cells to block
biochemical analysis of the biosynthetic enzymes is providing cytotoxicity suggests a requirement for cell–cell contact between
insight into the exact structure of the colibactin molecule. A the pathogen and the mammalian cell.167 Despite the tremen-
recent proposal suggests that the timing of the hydrolysis of the dous recent progress, the exact nature of the biosynthesis and
acyl-asparagine leader may in fact lead to different precolibactin mode of action of colibactin remains to be fully determined.
molecules, including pyridone forms such as (10) that do not
react with DNA, as well as lactam (11) that is able to cross-link
DNA.174,177 6. Acinetobacter baumannii
5.3.2. Colibactin biosynthesis. The clb BGC encodes 3 A. baumannii is a Gram-negative bacillus that belongs to the
PKSs, 3 NRPSs, and two hybrid PKS/NRPS proteins. Addition- family Moraxellaceae. Infections caused by A. baumannii
ally, seven other enzymes are required for the genotoxicity.167 include both community- and hospital-acquired pneumonia,
Also encoded within the cluster is ClbM, a transporter that is bacteremia, urinary tract infections, and skin, so tissue, and
responsible for export of precolibactin across the cytoplasmic burn infections182,183 and has been observed in outbreaks in
membrane.178 several military hospitals during the wars in Afghanistan and
While the complete biosynthetic steps are unknown, some Iraq.184,185 Increasing the seriousness of nosocomial infections
features have been biochemically explored. The acyl-Asn “pro- and illustrating the challenge of stopping an outbreak, A. bau-
tecting group” that is removed as part of the pro-drug strategy is mannii can live on abiotic surfaces for months and has the
installed by the activity of ClbN, a four domain NRPS that ability to form biolms on both glass and plastic.186 Interest-
activates and epimerizes the asparagine residue.172 The N- ingly, despite a genus name that translates as “non-motile”, A.
terminal condensation domain adds the acyl chain, which is baumannii exhibits a so agar motility phenotype that is
most commonly a myristate fatty acid, however mono- dependent on Type IV pili and other uncharacterized
unsaturated myristate or palmitate moieties have also been factors.187–189
observed.179 Elegant bioinformatic analysis allowed the predic- Also challenging the treatment of A. baumannii infections,
tion of the next module in the pathway to involve the NRPS many strains are highly drug resistant to common antibiotics,
portion of the hybrid ClbB protein, which incorporates an because of the presence of genomic resistance islands that are
alanine residue, affording the production of the acyl-Asn-Ala acquired through horizontal gene transfer from unrelated
peptide.172 In the periplasmic space, the capping acyl-Asn species.182,190 The prevalence of drug resistance has led to the
moiety is cleaved by the ClbP protease.180 The PKS module of informatic191 and experimental192 search for previously
ClbB as well as the PKS proteins ClbC and ClbI incorporate uncharacterized essential genes within A. baumannii that may
a malonyl moiety into the growing natural product. be targeted through novel antibiotic development campaigns.
ClbH is a four domain NRPS protein with an architecture of While nearly all A. baumannii strains contain an NRPS cluster
adenylation–condensation–adenylation–PCP domains. The rst that encodes the production of an aryl-capped peptide side-
adenylation domain activates a serine residue that is trans- rophore, acinetobactin, a second peptide siderophore, ms-
ferred in trans to the freestanding carrier protein ClbE.181 The bactin, has been identied in only a few strains. An additional
loaded serine residue is then sequentially acted on by two NRPS cluster that is responsible for an uncharacterized peptide
dehydrogenases, ClbD and ClbF, to produce aminomalonyl product appears to play a role in motility and biolm formation.
loaded carrier protein. The genotoxic phenotype of a colibactin-
expressing E. coli strain was blocked in mutants for either clbE
alone or the entire clbDEF operon, demonstrating that this 6.1. Acinetobactin
aminomalonyl group is essential for production of the active 6.1.1 Acinetobactin structure. A. baumannii produces
colibactin molecule.181 The second adenylation domain of ClbH a peptide siderophore called acinetobactin (15) containing DHB
activates a methionine residue that is believed to be the source (7) and N-hydroxyhistamine (13) moieties joined by a heterocy-
of the aminocyclopropylcarboxylate moiety that forms the clic moiety (Fig. 7). Originally identied as a oxazoline, acine-
smaller ring of the warhead. Notably, genetic disruption of clbD, tobactin is now recognized as being produced in a pre-
clbE, clbF, clbG, clbJ, clbK, or clbO had no effect on the formation acinetobactin form (14) that is released from the NRPS, which
of the warhead, placing the function of these gene products subsequently isomerizes to mature acinetobactin (15),
further downstream in colibactin biosynthesis.176 harboring an isoxazolidinone ring.193 The interconversion of
The two module NRPS protein ClbJ then incorporates pre-acinetobactin and acinetobactin, both of which can form
a glycine and a cysteine, the latter of which is cyclized into complexes with ferric iron, is pH-dependent, with the pre-
a thiazoline moiety. Finally, the NRPS module of the two acinetobactin species being more stable at pH < 6.194 Both
module PKS/NRPS protein ClbK incorporates a second cysteine forms use the same cellular receptor to allow entry of the ferric
residue that is also cyclized. complexes into the cell, illustrating that a single siderophore
5.3.3. Colibactin biology. Colibactin was originally identi- may be optimized for different pathogenic environments,
ed through its ability to induce megalocytosis on several allowing A. baumannii to thrive in different sites of infection.
mammalian cells upon infection.167 Colibactin's ability to 6.1.2 Acinetobactin biosynthesis. The BGC for acineto-
alkylate DNA in vitro174 and cause DNA double strand breaks in bactin biosynthesis was identied in 2004 containing a multiple
View Article Online
a salicylate rather than DHB aryl cap, has been described by the
Walsh group.197 BasE is a freestanding adenylation domain that
activates DHB and loads it onto the PCP of the isochorismatase–
PCP didomain protein BasF. In this system, BasE and BasF are
homologs of EntE and EntB of the enterobactin system.198 BasA
is a freestanding adenylation domain that loads a molecule of
threonine onto the PCP of BasB, a two domain protein con-
taining a PCP–condensation domain architecture.193
The didomain NRPS protein BasD, in analogy to the PmsD
protein of pseudomonine biosynthesis, which also contains
tandem cyclization domains,197 transfers the DHB moiety from
BasF to the loaded threonine of BasB and cyclizes the threonine
to form a oxazolinyl moiety on BasB. This loaded 2,3-dihy-
droxyphenyloxazolinyl group on BasB is then released through
the nucleophilic attack with N-hydroxyhistamine, catalyzed by
the condensation domain of BasB.197 The combined activity of

the ve NRPS proteins results in the formation of pre-
acinetobactin, which can spontaneously form acinetobactin at
neutral or basic pH.
6.1.3 Acinetobactin biology. As discussed above, side-
rophore biosynthesis represents an increasingly appreciated
virulence target. The low iron environment of the host creates
a competitive environment and the ability to produce the
siderophores that facilitate iron acquisition is an appealing
target.199,200 Acinetobactin production has been examined in
a series of ex vivo and in vivo assays, suggesting that this
phenotype is required in multiple infection models.201 Speci-
cally, while a basD mutant strain was able to physically interact
Fig. 7 Acinetobactin biosynthesis. N-hydroxyhistamine is produced with human epithelial cells in culture, the mutant showed
through the activites of BasG and BasC. The intermolecular loading of a reduced ability to persist or induce apoptosis in the eukaryotic
DHB on the carrier domain of BasF has been demonstrated bio- cells. Finally, the mutant strain showed reduced virulence in the
chemically while the subsequent reactions are assumed based on the
infection of the larvae of the greater wax moth Galleria mello-
homologous reactions of pseudomonine biosynthesis. The tandem
cyclization domains of BasD perform the peptide bond formation and nella and in a mouse sepsis model.201 A more recent study
cyclization to form the oxazoline ring. The condensation domain of suggests a critical role for acinetobactin production in the
BasB then transfers the DHB-oxazoline to 13 to result in the formation establishment of a traumatic wound infections.202
of pre-acinetobactin 14.
6.2. Fimsbactin
transcriptional operons that express proteins involved in the 6.2.1 Fimsbactin structure. While acinetobactin appears to
regulation, uptake, and synthesis of acinetobactin.195,196 As with be the primary siderophore of nearly all A. baumannii strains,
enterobactin described above, three proteins are responsible for a second BGC that encodes another catecholate siderophore
the conversion of chorismate to DHB: BasJ is an isochorismate called msbactin (17) appears in strain ATCC 17978 (Fig. 8).203
synthase, BasF is an isochorismatase that releases pyruvate to Additional A. baumannii strains, as well as the nonpathogenic
form 2,3-dihydro-2,3-dihydroxybenzoate, and EntA is the species A. baylyi, also contain the identical cluster. Six ms-
reductase that catalyzes the reductive aromatization to form bactin isoforms were identied that share a DHB moiety as well
DHB (Fig. 3). Interestingly, the entA gene is not part of the aci- as an oxazoline ring derived from a serine residue. Attached to
netobactin BGC but is located on a different region of the this core are two additional residues, a serine residue that has
chromosome.150 been modied at the side chain through the addition of
The other unusual substrate for acinetobactin is N-hydrox- a second DHB moiety and a hydroxamate containing N-acetyl-N-
yhistamine (13), which is derived from histidine (12) through hydroxyputrescine (16) moiety. Variants contained threonine
the activities of a decarboxylase (BasG) and a hydroxylase substitutions for either serine moiety or a 1,3-diaminopropane
(BasC).196 Along with threonine, these building blocks serve as in place of the putrescine (1,4-diaminobutane).
substrates for the acinetobactin NRPS enzymes – BasA, BasB, 6.2.2 Fimsbactin biosynthesis. The msbactin BGC was
BasD, BasE, and BasF (Fig. 7). The biochemical reconstitution of characterized for the A. baylyi cluster by the Bode group,203 who
acinetobactin biosynthesis has not been reported; however, the provided annotations and a prediction of the biosynthetic
biosynthesis of pseudomonine, a Pseudomonas entomophila pathway. As the proteins from A. baylyi and A. baumannii all
siderophore that differs from acinetobactin by only the use of share 60–90% sequence identity and are organized in the same
View Article Online
NRPS operate with a series of both intra- and intermolecular

interactions and reactions.
FbsE contains an N-terminal ArCP domain, followed by
a condensation domain, a truncated adenylation domain, and
a second PCP. FbsF contains a condensation–adenylation–PCP
domain architecture. And nally, the three domain FbsG
harbors condensation, PCP, and condensation domains.
Several steps can be condently predicted on the basis of the
shared architecture with other NRPS systems, including others
discussed herein. The ArCP of FbsE is proposed to be loaded
with DHB by the activity of FbsH. The adenylation domain of
FbsF could load both the FbsH PCP intramolecularly as well as
the PCP of FbsE intermolecularly with serine. The FbsE
condensation/cyclization domain then transfers the DHB to the
rst serine residue on the FbsE PCP and also cyclizes the serine
to form the oxazoline ring. The module in FbsF would the be

expected to extend the peptide by the addition of the serine
residue, which remains uncyclized.
The subsequent steps have not been biochemically shown
and are less clear, given the non-linear nature of the reactions.
FbsG contains two condensation domains surrounding a PCP.
Two additional reactions are required for completion of the
msbactin molecule, both of which could be carried out by
a condensation domain. In one reaction, a DHB moiety is
installed on the serine side chain, while the second reaction
involves the nucleophilic release of the peptide with the N-
acetyl-N-hydroxyputrescine. The source of the DHB in the initial
Fig. 8 Fimsbactin biosynthesis. The formation of the initial DHB– reaction is unclear and could involve a DHB loaded onto the
oxazoline–serine tripeptide on FbsF is catalyzed using standard NRPS
carrier protein of either FbsE or FbsG. The latter possibility
biochemistry. The final steps catalyzed by FbsG are not yet charac-
terized. The truncated adenylation domain of FbsE is depicted with the would require that the adenylation domain FbsH is able to
cross-hairs through the domain. The biosynthesis of fimsbactin has recognize and load two carrier domains intermolecularly. The
not been demonstrated biochemically but the early steps can be unraveling of the unusual NRPS biochemistry of the msbactin
confidently assigned on the basis of homologous enzymes. system will be an interesting addition to the literature on
alternate NRPS systems. Similarly, the role of msbactin and
the redundancy of these siderophore systems in A. baumannii
genetic fashion, there is high condence in the parallel function remains to be explored.
of the clusters from both members of the acinetobacter
genus.203 The DHB (7) building block is built once again
through the activities of FbsB, FbsC, and FbsD, which serve as 6.3. An uncharacterized NRPS cluster involved in biolm
the homologs of EntC, EntB, and EntA, respectively (Fig. 3). The formation and motility
DHB moiety is activated by a freestanding adenylation domain, 6.3.1. Biological identication of a novel NRPS cluster.
FbsH, which loads the DHB onto a N-terminal ArCP of FbsE Many of the uncharacterized NRPS clusters discussed here and
(Fig. 8). in other studies were identied solely on the basis of bio-
The hydroxamate building block is formed through the informatic cluster mining. However, one NRPS cluster from A.
activities of FbsJ, an ornithine decarboxylase, FbsI, a hydroxy- baumannii has been identied repeatedly in a number of genetic
lase, and FbsK, an acetyltransferase. Together, these three screens for important virulence related phenotypes such as
proteins likely convert ornithine into putrescine, which is then motility, biolm formation, and quorum signaling. These
is hydroxylated and acetylated to the nal NRPS substrate. On studies suggest an interesting new signaling molecule will be
the basis of homologous proteins from pyochelin or aerobactin identied in further biochemical and genetic studies.
biosynthesis, the hydroxylation step likely occurs before the As noted above, bacteria of the acinetobacter genus were
acetylation.204 believed to be amotile. However, many strains do exhibit
With the building blocks in place, the function of three a phenotype known as twitching motility as demonstrated by an
multidomain NRPSs are required along with the freestanding ability to form halos around colonies innoculated on so agar.
adenylation domain FbsH to build the msbactin molecule. Twitching motility is mediated by the cyclical extension and
The three NRPS modules are contained upon FbsE, FbsF, and retraction of Type IV pili, laments of proteinacous projections
FbsG. The domain organization suggests that the msbactin from the poles of the cell. Adherence of the tip of the pilus to the
substratum followed by retraction results in the force required
View Article Online
for motility.205,206 Type IV pili genes are required for A. bau- family to make starter units that may be incorporated into the
mannii motility.187,189 product in the absence of a PCP thioester.
A search for additional factors that are responsible for Very recently, a proposed structure of the product of this
twitching motility in A. baumannii via transposon mutagenesis NRPS BGC has been reported, illustrating the presence of a 3-
identied an NRPS BGC that is present in most strains.187 This hydroxy long chain fatty acid. Interestingly, the authors note the
cluster, containing eight genes, is located immediately down- presence of a Cys–Gly moiety within the structure that may
stream of a gene encoding an acyl homoserine lactone synthase derive from a glutathione adduct of a true product that is
and a luxR type regulator, which are involved in quorum further metabolized.210
signaling regulation. This A. baumannii NRPS cluster was
subsequently identied as being among the most highly upre- 6.4. Additional uncharacterized NRPSs
gulated operons in cells grown in biolms relative to cells from
A. baumannii strain ATCC 17978 contains an additional multi-
planktonic (liquid culture) growth.207 Finally, mutants within
domain NRPS protein that is present in some, but not all, of the
this cluster are defective in forming pellicles, a biolm structure
A. baumannii strains. This protein is encoded at locus A1S_2651
that forms at the liquid/air interface of cultures.208
and is 1332 residues in length; in the AB307-0294 strain,211 the
6.3.2 Proteins within the uncharacterized NRPS BGC.
gene is located at ABBFA_00856, with only ve amino acid
Given the unknown identity of the product of this NRPS cluster,

substitutions over the 1332 residues.
the proteins have simply been identied by their annotation
The protein contains an N-terminal adenylation domain that
names in several strains. Much of the biological characteriza-
spans the rst 520 residues followed by a carrier protein domain
tion described above was performed with A. baumannii strain
from 530 through 596. The C-terminal half of the protein is
ATCC17978. The protein annotation names of this cluster in
a domain of unknown function. Sequence comparison fails to
ATCC17978, running from A1S_0112 through A1S_0119 are
identify any characterized homologs that allow for prediction of
described in Table 2. Our lab has performed initial structural
function. The gene encoding this NRPS does not belong to
characterization of the enzymes using strain A. baumannii
a larger operon. The neighboring genes, while conserved in
AB307-0294, where the identical proteins are referred to as
multiple NRPS strains, do not appear to encode biosynthetic
ABBFA_003406 through ABBFA_003399.
enzymes that are common to other NRPS clusters.
The eight proteins encoded by this cluster include three
bona de NRPS proteins, a freestanding adenylation domain
(A1S_0112), a freestanding carrier protein (A1S_0114), and 7. Pseudomonas aeruginosa
a four domain termination module containing a condensation,
The Gram-negative bacteria P. aeruginosa is an opportunistic
adenylation, PCP, and thioesterase domain architecture
human pathogen. P. aeruginosa is a common cause of hospital-
(A1S_0115). These latter two proteins have been structurally
acquired pneumonia, particularly with immunocompromised,
characterized from strain AB307-0294, where they are named
cancer, and cystic brosis patients.212 The metabolic diversity
ABBFA_003404 and ABBFA_003403, respectively.58,209 Addition-
and the ability to produce a variety of virulence factors
ally, the cluster contains a PPTase and a transmembrane
contribute to the ability of P. aeruginosa to colonize a variety of
protein likely involved in product export. The three remaining
environments.213 Additionally, P. aeruginosa has been a model
proteins show homology to an acyl-CoA dehydrogenase
organism for the understanding of biolm formation and
(A1S_0113), an NADH dependent dehydratase (A1S_0118), and
quorum signaling, and exhibits a high propensity for antimi-
a protein of unknown function (A1S_0117). The protein enco-
crobial resistance.214,215
ded upstream at the A1S_0104 (ABBFA_003414) locus is an acyl-
The genome of P. aeruginosa strain PA01, a standard labo-
CoA synthetase, a family of proteins that produces acyl-CoA
ratory strain, contains three well-characterized NRPS clusters
thioesters. NRPS clusters oen contain members of this
and three additional BGCs encoding NRPS proteins that have
not yet been characterized. These three BGCs appear to be
Table 2 Uncharacterized NRPS clusters of A. baumannii induced in quorum signaling conditions and likely produce
novel signaling molecules. Additionally, several NRPS clusters
Locus ID (accession) Length (AA) Predicted function will be mentioned that appear in alternate strains of P. aerugi-
nosa, along with the more common biosynthetic clusters shared
Strain ATCC17978 (Genbank Accession CP00521)
by most strains.
A1S_0112 (ABO10599) 629 A
A1S_0113 (ABO10600) 597 Acyl-CoA dehydrogenase
A1S_0114 (ABO10601) 86 T 7.1. Pyoverdine
A1S_0115 (ABO10602) 1319 C–A–T–Te
A1S_0116 (ABO10603) 1216 RND exporter 7.1.1. Structure of pyoverdine. Under iron limiting condi-
A1S_0117 (ABO10604) 424 Hypothetical tions, P. aeruginosa produces two peptide siderophores that are
A1S_0118 (ABO10605) 621 NAD-dependent dehydratase both produced by NRPSs. Pyoverdine (19) is a larger molecule
A1S_0119 (ABO10606) 254 PPTase that is built from an initial acyl-peptide composed of 11 amino
acids (Fig. 9), while pyochelin (20, discussed in the next section)
Strain ATCC17978 (Genbank Accession CP00521)
A1S_2651 (ABO13068) 1332 A–T–unknown is a smaller molecule derived from three building blocks. While
both pyochelin and pyoverdine play important roles in different
View Article Online

Fig. 9 Pyoverdine biosynthesis. Four NRPS proteins are involved in the production of the initial pyoverdine precursor (18). Final steps in the
biosynthesis convert precursor (18) to mature pyoverdine (19). The complete biosynthesis of pyoverdine has not been reconstituted in vitro with
the NRPS proteins. Several of the steps in chromophore maturation have been demonstrated with precursor or analog substrates.
environments, pyoverdine is produced at higher levels and elaborated the steps involved in the nal maturation of the
appears to be the primary siderophore under many pathogenic pyoverdine molecule.
conditions. Multiple reviews describe the siderophores of The pyoverdine molecule can be divided into three compo-
Pseudomonas;199,216–218 however, several recent papers have nents, a variable N-terminal sidearm that can include succinate
View Article Online
or succinamide groups. Additionally, a heterocyclic chromo- a fatty acid in the mature pyoverdine molecule was reconciled
phore contains catechol oxygens that contribute to iron with the presence of PvdQ, a protein that showed homology
binding. The third component is a peptide backbone that varies with fatty acid hydrolase enzymes involved in cephalosporin
between different strains of P. aeruginosa.218 In many cases, the biosynthesis.230 The initial adenylation domain of PvdL was
peptide tail is cyclized, oen through an amide linkage with expressed recombinantly and showed specicity for fatty acids
a lysine side chain and the terminal carboxylate. Pyoverdine of 12 or 14 carbons in length. A mutant pvdQ strain produced an
oen contains the non-proteinogenic amino acids 2,4-dia- alternate pyoverdine precursor (18) that contains the extra fatty
minobutyrate (DAB) and N5-formyl-N5-hydroxyornithine acid component that can be hydrolyzed by PvdQ.231,232 Thus, the
(fOHOrn) within the peptide. initial module of PvdL incorporates the myristate (or myr-
The sidearm is derived from g-glutamate, while the chro- istoleic233) fatty acid, which PvdQ then removes during pyo-
mophore is produced via the oxidation and cyclization of verdine maturation. As PvdQ contains a periplasmic signal and
a dipeptide that begins as Tyr3–DAB4. In strain PA01, the pyo- is present in the periplasm,231 this suggests that the acylation
verdine peptide chain contains the sequence Ser5–Arg6–Ser7– step might be involved in trafficking the pyoverdine molecule to
fOHOrn8–Lys9–fOHOrn10–Thr11–Thr12, with the cyclization the periplasm for the nal maturation steps. PvdQ is a member
occuring between Lys9 and Thr12. The iron chelating groups in of the family of NTN hydrolase enzymes that are formed
through the autoproteolysis of a larger protein into an a/b het-
pyoverdine are the catechol oxygens of the chromophore and

the two oxygens from each of the fOHOrn groups. This octa- erodimer, which reveals the catalytic nucleophile at the N-
hedral arrangement allows pyoverdine to coordinate Fe(III) with terminal residue of the b chain. Recently, the structure of
affinity constants as high as 1030 M 1.218 PvdQ bound to the acyl pyoverdine precursor was determined
7.1.2. Pyoverdine biosynthesis. The pyoverdine biosyn- through the use of a circularly permuted enzyme that enabled
thetic genes in strain PA01 are clustered in one region of the the production of a processed but catalytically inactive form of
chromosome however the genes are not expressed as a single the enzyme.234
operon. Instead, the genes are present on both strands of DNA Also containing periplasmic signaling sequences are PvdN,
separated by regulatory genes as well as other genes that encode PvdO, and PvdP,232 suggesting additional steps in maturation
proteins of unrelated function. The biosynthesis of the pyo- occur in the periplasm aer release of the acyl chain by PvdQ.
verdine peptide utilizes four NRPS enzymes and the products of PvdP is homologous to phenoloxidases and tyrosinases sug-
at least nine additional genes (Fig. 9).102 The nonproteinogenic gesting a role in the installation of the second hydroxyl group on
amino acids are provided by PvdH, an aminotransferase that the Tyr3 position. PvdP has recently been shown to be a copper-
produces DAB from aspartate b-semialdehyde,219 and PvdA220,221 dependent tyrosinase that is able to hydroxylate ferribactin and
and PvdF222 which convert ornithine to fOHOrn in sequential initiate the proposed oxidative cascade235,236 to enable the
hydroxylation and acyltransferase steps. The NRPS enzymes formation of the mature chromophore.237 PvdN has now been
that produce the immature pyoverdine peptide are PvdL and shown to catalyze changes to the N-terminal sidearm converting
PvdI, which each contain four modules, and PvdJ and PvdD, the g-glutamate into the succinamide moiety that is commonly
which each contain two modules. observed in pyoverdine isoforms.238 The mechanistic details
The pyoverdine gene cluster also encodes the protein PA2412 provided for this enzyme, supported by the recent crystal
(ref. 223), an MbtH-like protein (MLP).224 These small auxiliary structure of PvdN bound to PLP,239 suggest an oxidative decar-
proteins interact with NRPS adenylation domains to enhance boxylation that results in the production of the alternate
adenylation activity.225,226 Crystal structures of MLPs bound to sidearm. The roles of the remaining proteins, PvdM and PvdO,
an adenylation domain227 or to multidomain NRPS proteins59,63 in pyoverdine biosynthesis have not yet been determined. A
illustrate how the MLP interacts with the N-terminal sub- crystal structure of PvdM (PDB code 3B40) shows structural and
domain over 15 Å away from the active site. Structures of NRPS sequence homology with dipeptidase enzymes. A PvdO crystal
proteins in the presence and absence of the partner MLP have structure shows structural similarity to a formyl-glycine gener-
not yet identied the basis for activation. ating enzyme however PvdO lacks key catalytic residues sug-
Finally, production of pyoverdine requires the activities of gesting an alternate function.240
ve other proteins that are involved in chromophore matura- 7.1.3. Biological role of pyoverdine. The exact role of pyo-
tion. The proteins encoded by pvdM, pvdN, pvdO, pvdP, and verdine production in virulence has been mixed, with studies
pvdQ were originally identied as being necessary for produc- that both support an important role in establishment and
tion of wild-type levels of pyoverdine.102 Studies using maintenance of an infection as well as others that suggest that
biochemical analysis and metabolite proling of mutant strains pyoverdine production is dispensable.241,242 A recent meta
have enabled the discovery of the nal steps in pyoverdine analysis examined the effect of pyoverdine on both plant, insect,
synthesis. and vertebrate infection models, supporting a limited but
Although the pyoverdine peptide contains 11 amino acids, consistent contribution to virulence.243 Furthermore, chemical
the four NRPS proteins harbor a total of 12 modules. Bio- precedent exists for the intervention in the signaling pathways
informatic prediction of the amino acid specicity of the NRPS that induce pyoverdine production. The antifungal 5-uo-
adenylation domains,228,229 allowed module assignments of all rocytosine has been shown to inhibit the transcriptional regu-
four proteins, except for the rst module of PvdL. This module lation of the pyoverdine operon as mediated by the regulator
was predicted to encode preferences for fatty acids. The lack of PvdS.244 While treatment reduced virulence of the P. aeruginosa
View Article Online
in a murine lung infection model, it did not reduce the pyochelin molecule (Fig. 10). Finally, pchC encodes a proof-
pulmonary bacterial load, illustrating a specic impact on reading thioesterase that increases the efficiency via the release
virulence caused by pyoverdine production. of mischarged molecules from the PCP domains of PchE and
PchF.253
Salicylic acid is produced through the activities of PchA and
7.2. Pyochelin
PchB. PchA is an isochorismate synthase254 while PchB catalyzes
7.2.1. Structure of pyochelin. A second siderophore the conversion of isochorismate to salicylate.255 The three NRPS
produced by P. aeruginosa is pyochelin (20), a smaller molecule enzymes then function to combine the starter units.84,256 PchD is
derived from three building blocks and consisting of catechol a freestanding adenylation domain that loads the salicylate
and thiazole moieties (Fig. 10).132,245 While the redundancy of onto the N-terminal carrier protein domain of PchE. Following
iron uptake systems may simply reect the importance of iron transfer of the salicylate to the rst PCP of PchE, the bifunc-
acquisition and the need for multiple “backup” systems, alter- tional cyclization/condensation domain forms the initial
nate functions for secondary siderophores such as pyochelin peptide linkage between the cysteine and salicylate, followed by
have been proposed, including induction of further signaling cyclization to generate the hydroxyphenylthiazolinyl–enzyme
cascades and modulation of host defense mechanisms.246 Pyo- intermediate. The same process is repeated at the third pyo-
chelin consists of a salicylate moiety condensed with two thia-
chelin module located on the PchF, to result in the formation of

zolidine rings derived from cysteine residues.247 The iron is the hydroxyphenylthiazyl–thiazolinyl–enzyme intermediate on
coordinated by the phenolic oxygen of the salicylate moiety, the PchF. Two steps remain for pyochelin production. First, PchG
two nitrogens of the thiazolidine rings, and an oxygen from the catalyzes the NADPH-dependent reduction in trans to convert
carboxylate.248,249 the thiazolinyl moiety to a thiazolidinyl group.252 In this reduced
7.2.2. Pyochelin biosynthesis. The biosynthetic gene form, the nitrogen is then methylated in a SAM-dependent
cluster for the synthesis of pyochelin consists of seven genes. reaction catalyzed by the embedded methyltransferase of
Two genes, pchA and pchB encode chorismate utilizing enzymes PchF. Release of pyochelin by the terminal thioesterase domain
that are involved in the production of salicylate.250 Three NRPS of PchF completes the process.
genes, pchD, pchE, and pchF,251 along with a thiazolinyl reduc- 7.2.3. A pyochelin-related cluster for the synthesis of
tase encoded by pchG158,252 catalyze the formation of the dihydroaeruginoic acid. Although not present in the standard
laboratory strain PA01, a four gene cluster appears on a mobile
genomic element in the virulent PA14 strain257 of P. aeruginosa.
The BGC contains four NRPS genes that synthesize dihy-
droaeruginoic acid (DHA), a truncated form of pyochelin con-
taining a salicylate cap joined with a single thiazoline ring of
opposite stereochemistry to that observed in pyochelin.258
Present in this BGC is a gene encoding PA14_54940, a bi-
domain protein that is an unusual fusion between an adenyla-
tion domain, which shows predicted specicity for salicylate
over DHB,70 and a salicylate synthase domain that may directly
convert chorismate to salicylate, similar to MbtI of M. tubercu-
losis.259 The cluster then contains two proteins, PA14_54930 and
PA14_54920, that each encode one complete module. The two
proteins share 36% sequence identity over the rst 1000
residues and differ by the presence of a reductase domain at the
C-terminus of PA14_54920. Finally, the BGC also contains
a proofreading thioesterase domain. The cluster is followed by
several other genes, including one encoding a serine O-acetyl-
transferase that may contribute to sufficient cysteine levels for
DHA production.258
The two adenylation domains of PA14_54930 and
PA14_54920 show specicity predictions for cysteine using the
prediction tools of the antiSMASH database, while the two
condensation domains classify as cyclization domains. The
cluster therefore appears very similar to the pyochelin NRPS
with several notable differences.
Fig. 10 Pyochelin biosynthesis. Pyochelin biosynthesis has been First, there are no internal epimerization or methylation
demonstrated biochemically using the three NRPS enzymes PchD,
domains in either NRPS module as is seen in PchE and PchF.
PchE, PchF, as well as the redutase PchG, along with building blocks
DHB and cysteine, along with necessary substrates ATP, NADPH, and While the epimerization and methylation domains are not
SAM. PchE contains an additional epimerization domain (E), while PchF necessary for the production of DHA, the cluster also appears to
contains an N-methyltransferase. be missing a carrier protein for the salicylate module. While the
View Article Online
adenylation domain of PA14_54940 will adenylate the substrate,

there does not appear to be an aryl carrier protein that can serve
to shuttle the salicylate to the downstream condensation
domain. Furthermore, it appears that one of the NRPS modules
encoded by PA14_54930 or PA14_54920 is not needed in the
production of the truncated pyochelin variant. The PA14_54930
protein was able to complement a pchE mutant in P. putida in
the production of enantiopyochelin as it lacks the epimerization
domain.258 The ability of PA14_54920 to complement either
a pchE or pchF mutant was not tested.
Expression of the four gene operon in both the presence or
absence of the downstream operon containing the serine O-
acetyltransferase gene resulted in the production of the same
product, which was shown by mass spectrometry and thin layer
chromatography to be the truncated pyochelin variant, DHA.258
The exact role of the PA14_54920 and the identity of the carrier
protein for the salicylate adenylating domain remain to be
determined.
7.2.4. Biological role of pyochelin. Although present in all
isolates of P. aeruginosa,245 pyochelin appears to be somewhat
less signicant than pyoverdine in a number of clinically rele-
vant isolates. Indeed, it has been classied among a variety of
siderophores with lower affinity for iron that have been labeled
“secondary siderophores”.246 For example, while the gene
encoding the pyochelin synthetase PchF was expressed in the
majority of sputum samples from cystic brosis patients, the
level of transcripts was much lower than several pyoverdine
biosynthetic genes. Further, pyochelin was detected in only 6 of
45 samples tested.242
However, there is also evidence that different siderophores
may be exploited by P. aeruginosa under certain environments.
As a lower complexity molecule, pyochelin may be more useful
Fig. 11 AMB biosynthesis. The AMB residue is produced in two steps
under low iron stress conditions. P. aeruginosa would then
from glutamate through the activities of AmbC and AmbD. The
switch to the more metabolically demanding pyoverdine under formation of the Ala–Glu–Ala tripeptide on AmbE has been shown
extreme iron depletion.260 It has been proposed that in the cystic biochemically to precede conversion to the AMB containing tripeptide,
brosis lung, pyoverdine may be required to establish an initial which occurs while the tripeptide is loaded on AmbE if AmbC and
infection. At later stages in the infection, the accompanying AmbD are present.
damage to the pulmonary tissue may result in inammation
and disruption of the host cells, releasing iron that may make
pyochelin the more relevant siderophore.245 standard E. coli strains. The cluster responsible for AMB
production was therefore isolated by screening a mutant library
of P. aeruginosa for loss of this growth inhibition phenotype.268
7.3. L-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) Four mutants were identied that all mapped to the cluster
7.3.1. Structure of AMB. Another NRPS cluster from P. encoded by genes PA2302 through PA2305 in strain PA01. The
aeruginosa encodes the synthesis of the antimetabolite AMB (22) ve gene cluster was named ambABCDE. The ambA gene
(Fig. 11). AMB is a substituted vinylglycine that along with other encodes a LysE type transmembrane transporter, which is
similar molecules have been shown to inhibit growth of bacte- proposed to be responsible for the export of, and potentially
rial and eukaryotic cells,261–263 possibly through the inhibition of resistance to, the antimetabolite.267 AmbB and AmbE encode
PLP-dependent enzymes.264,265 The literature on AMB biosyn- NRPS proteins, while AmbC and AmbD are members of the
thesis in P. aeruginosa includes an alternate pathway that was family269 of Fe(II)/a-ketoglutarate dependent oxygenases. The
proposed to be responsible for a novel quorum signaling puzzling NRPS architecture, necessary to produce AMB from
molecule based on analysis of the whole cell metabolic what might be expected to be a single amino acid, contains two
prole.266 However, recent biochemical evidence convincingly adenylation, two condensation, and three PCP domains.
demonstrates the ability of the AmbBCDE proteins to catalyze The biosynthetic pathway of AMB has recently been
production of AMB.267 proposed267 as follows (Fig. 11). The adenylation domain of
7.3.2. AMB biosynthetic cluster. Production of AMB by P. AmbB performs two loading steps, attaching an alanine residue
aeruginosa can be assayed through growth inhibition of intramolecularly to the downstream PCP domain and catalyzing
View Article Online
a second intermolecular reaction to load the second PCP fold down-regulation of ambC and ambD expression, respec-
domain of AmbE with a second alanine molecule. The adeny- tively.273 While no precise phenotype has been ascribed to AMB
lation domain of AmbE loads a molecule of glutamate onto the production, these signs suggest a link to quorum signaling
rst PCP domain of AmbE. The condensation domain of AmbB pathways.
transfers the alanine residue to the loaded glutamate, forming
the Ala–Glu dipeptide on the rst AmbB PCP domain. Subse- 7.4. Pyoluteorin
quently the condensation domain of AmbE transfers this
7.4.1 Pyoluteorin structure. Several P. aeruginosa strains
dipeptide to the loaded alanine residue on the second PCP
have been annotated to contain a hybrid NRPS/PKS cluster that
domain of AmbE, resulting in the formation of an Ala–Glu–Ala
encodes proteins for the synthesis of the antibiotic pyoluteorin
tripeptide. This intermediate serves as the substrate for the
(23), a compound more commonly associated with other pseu-
Fe(II)/a-ketoglutarate dependent oxygenases, AmbC and AmbD
domonads.274 The two sequenced P. aeruginosa strains with
as well as the methyltransferase domain of AmbE, which
pyoluteorin clusters are the Liverpool epidemic strains LESB58
together convert the glutamate residue to a molecule of AMB.
and LES431, two virulent strains from cystic brosis
These steps in the synthesis are believed to occur while the
patients.275,276 Pyoluteorin contains a resorcinol moiety fused to
tripeptide is bound to AmbE, and not aer peptide release, as
a dihalogenated pyrrole derived from proline.
the mature Ala–AMB–Ala tripeptide (21) was identied cova-

7.4.2. Biosynthesis of pyoluteorin. The pyoluteorin BGC
lently bound to the PCP.267 While the presence of the AMB-
contains eight biosynthetic genes, two genes involved in regu-
containing tripeptide on the AmbE protein suggests the
lation, and six genes277 that support metabolite transport.278 The
tailoring reactions occur on the PCP-bound peptide, the authors
eight biosynthetic genes encode PltA through PltG, and PltL
note that the exact timing of the steps in the synthesis have not
(Fig. 12). PltF is a freestanding adenylation domain that loads
yet been conclusively demonstrated. Similarly, neither the role
the freestanding carrier protein PltL with proline.279 The proline
of the alanine residues in the Ala–AMB–Ala tripeptide nor the
then is oxidized by the FAD-dependent enzyme PltE and chlo-
source of peptidase activity needed to release the AMB have
rinated by the halogenase PltA.280 NMR studies of PltL loaded
been identied. Inhibition of PLP-dependent enzymes would
with proline demonstrate the interaction of the ligand with the
require a free amine on the AMB residue to form an initial
core of the PCP domain.281 PltF is specic for L-proline and
external aldimine with the cofactor.264 Potentially, the alanine
shows no activity with pyrrolyl-2-carboxylate,279 demonstrating
residues serve as a protective group until the AMB has been
that the subsequent oxidation of the proline derivative occurs
exported from the cell.
while bound to the carrier protein. The dichloropyrrolyl group is
7.3.3 Biological role of AMB. The lack of an obvious one-
then offered to the PKS enzymes PltB, PltC, and PltG for the nal
to-one correspondence between the modules and the
steps.
product may have been partly responsible for the misiden-
tication of the product of the amb operon as a new quorum
7.5. Additional uncharacterized NRPS clusters of P.
signaling molecule.266 An ambB mutant also showed reduced
aeruginosa
production of several quorum signaling molecules, including
2-(2-hydroxyphenyl)thiazole-4-carbaldehyde, a molecule that P. aeruginosa also contains four NRPS clusters that have not yet
the authors named IQS. The subsequent biochemical been characterized. The rst three of these clusters are common
reconstitution of AMB biosynthesis conrmed the product to most sequenced P. aeruginosa strains, while some appear
shown above and suggests that AMB is part of a signaling only in a few strains (Table 3). They are as yet unnamed and are
cascade that includes the production of multiple quorum described below using their protein locus in the P. aeruginosa
signals, iron-acquisition molecules, and the potential PA01 strain. Interestingly, the PA1221, PA3327, and PA4078 BGC
metabolite IQS. are present in other Gram-negative bacteria. The genes are
Beyond its role as an antimetabolite with the ability to organized in the same order and share 50% sequence identity,
inhibit PLP-dependent enzymes, the precise role of AMB in the sometimes as high as 70%. These do not appear to simply be
microbial physiology of P. aeruginosa is unknown. Several
reports correlate the expression of this operon with quorum
signaling and biolm formation. The amb operon is regulated
by LasR and RhlR, two transcription factors that sense and
activate genes in response to the acylhomoserine lactone
quorum signals. Transcriptome analysis showed that the
ambBCDE genes are expressed 30- to 130-times more highly in
wild-type cells compared to lasRrhlR mutants.270 Furthermore,
the amb operon is induced by 10-fold by the addition of the
these acylHSL signals.271 The amb operon has been more
Fig. 12 Pyoluteorin biosynthesis. The early steps in pyoluteorin
recently included in the core set of genes that are controlled by
biosynthesis have been demonstrated with enzymes from related
quorum signals across multiple P. aeruginosa strains.272 Finally, pseuodomonads. The 4,5-dichloropyrrole has been shown to be
expression of BpiB09, a, NADP-dependent reductase that cata- produced while tethered to the carrier protein PltL. Subsequent PKS
lyzes the inactivation of acyl-HSL signals, results in a 30- and 79- enzymes produce the resorcinol moiety.
View Article Online
Table 3 Uncharacterized NRPS clusters of P. aeruginosa
Strain PA01 (Genbank Accession AE004091.2)

PA1221 (NP_249912) 618 A–T
PA1220 (NP_249911) 420 C
PA1219 (NP_249910) 204 Te
PA1218 (NP_249909) 292 Fe/aKG-dependent dioxygenase
PA1217 (NP_249908) 455 Isopropylmalate synthase
PA1216 (NP_249907) 248 Methyltransferase
PA1215 (NP_249906) 492 A
PA1214 (NP_249905) 532 Amidotransferase
PA1213 (NP_249904) 319 Fe/aKG-dependent dioxygenase
PA1212 (NP_249903) 409 MFS transporter
PA1211 (NP_249902) 211 a/b hydrolase (Te)

PA3327 (NP_252017) 2352 C–A–T–C–A–T–Te

PA3328 (NP_252018) 388 FAD-dep monooxygenase
PA3329 (NP_252019) 442 C
PA3330 (NP_252020) 304 NAD-dependent dehydrogenase
PA3331 (NP_252021) 418 Cytochrome P450
PA3332 (NP_252022) 141 Hypothetical protein
PA3333 (NP_252023) 330 Ketoacyl-ACP synthase
PA3334 (NP_252024) 79 ACP
PA3335 (NP_252025) 250 Hypothetical protein
PA3336 (NP_252026) 388 MFS transporter

PA4078 (NP_252767) 991 A–T–Re
PA4079 (NP_252768) 229 NAD-dependent dehydrogenase
Strain PA7 (Genbank Accession CP000744)

PSPA7_2112 (ABR82062) 258 Hypothetical protein
PSPA7_2111 (ABR85185) 115 Cupin domain
PSPA7_2110 (ABR85498) 309 Amine oxygenase
PSPA7_2109 (ABR83084) 1145 A–T–Re
PSPA7_2108 (ABR84094) 155 Hypothetical protein
misclassied genomic samples because of the low overall an MFS transporter suggests the production of a novel product
sequence identities, and likely represent horizontal transfer of that is excreted from the cell.
the entire BGCs between different species. 7.5.2. PA3327. A ten-gene cluster beginning at PA3327
Although none of the products of the following BGCs has encodes several NRPS proteins, along with other enzymes and
been identied, these three clusters from strain PA01 frequently proteins likely to be involved in export of the natural product.
appear in transcriptomic studies that aim to understand the The NRPS protein PA3327 is a dimodular NRPS, containing two
genes that are regulated in association with biolm and viru- extending modules and a C-terminal thioesterase domain.
lence related phenotypes, including the production of acyl- Additionally, the cluster contains a freestanding condensation
homoserine lactones, the Pseudomonas Quinolone Signal domain at PA3329 and a freestanding carrier protein at PA3334.
(PQS) and phenazines.270–272,282,283 Additional proteins within the cluster include a mono-
7.5.1. PA1221. The PA1221 cluster encodes 11 proteins. oxygenase, an NAD-dependent dehydrogenase, a cytochrome
Several have homology to known proteins while several are P450, a ketoacyl-ACP synthase, and two hypothetical proteins.
uncharacterized. There are three NRPS proteins, a didomain 7.5.3. PA4078. In contrast to the other clusters from strain
adenylation and PCP, and freestanding condensation, thio- PA01, the NRPS gene PA4078 appears in a much smaller operon,
esterase, and adenylation domains. The didomain protein with only one additional gene appearing to be co-transcribed.
PA1221 was used for structural studies to identify the functional The PA4078 gene encodes a three-domain adenylation–PCP–
interactions of the NRPS adenylation and PCP domains. PA1221 reductase gene, while the downstream PA4079 gene encodes an
showed adenylating activity with valine, among the proteino- NAD-dependent dehydrogenase. While there are additional
genic amino acids tested.78 The cluster contains two Fe/a-keto- enzymes encoded on neighboring genes, located on both
glutarate dependent oxygenases; PA1218 shows reasonable strands, none can be condently assigned to belong to any
homology with phytanoyl-CoA dioxygenases, suggesting it may NRPS associated families such as carrier proteins, acyl-CoA
add a hydroxyl group to an acyl-CoA thioester. The presence of synthetases, or PKS related proteins.
View Article Online
Table 4 Uncharacterized NRPS cluster of E. cloacae microorganisms and the complicated role that these natural
products play both in the environment and in the pathogenic
setting of the host. These natural products, by denition, have
Strain s611 (Genbank Accession NZ_AXOM01000000) overcome many of the challenges that face medicinal chemists
WP_023480884 66 MLP in the development of novel therapeutics. Specically, they are
WP_023480734 4466 C–A–T–C–A–T–C–A–T–Te bioactive molecules that are able to enter the cell or are able to
WP_023480967 237 PPTase exert their effect from the environment through the activity of
WP_023480995 67 MLP
specic receptors. Further, they are soluble in the aqueous
WP_080263008 3632 C–A–T–C–A–T–C–A–T–Epim
WP_051372113 2366 C–A–T–C–A–T–Te biological environment and are stable, at least at the time scale
with which they need to operate. It is not surprising then that
many natural products have been exploited in the development
of pharmaceuticals.
7.5.4. PA7-cluster. The clinical isolate strain P. aeruginosa The natural products produced by all bacteria, and by human
PA7 (ref. 257) contains a ve-gene operon containing two pathogens in particular, can provide insight into bacterial
hypothetical proteins, a cupin domain, an amine oxygenase, metabolism and how cells adapt to diverse environments.
and a three-domain NRPS. Upstream is an additional operon Identifying the biosynthetic potential of ESKAPE pathogens will
containing genes encoding an editing thioesterase and a 528- be valuable to understand the role these molecules play. The
residue adenylation domain at PSPA7_2121. This upstream demonstration of the specic conditions under which BGCs are
operon also encodes a predicted isopropylmalate synthase and induced is an equally valuable goal that should accompany
an acyl-CoA dehydrogenase, reminiscent of the A1S_0112 discovery of the specic products. Understanding the regulatory
operon of A. baumannii. networks that involve novel signaling molecules will facilitate
the establishment of the roles these molecules play.
This is an exciting and important time in the history of
8. Enterobacter spp
antimicrobial discovery. The prevalence of antibiotic resistance
The nal group of ESKAPE pathogens belong to genus Enter- and the lack of novel compounds in the pipeline raises concern
obacter. Enterobacter species are facultative anaerobic Gram- about the rise of highly pathogenic organisms that are able to
negative bacilli that are a common cause of nosocomial infec- overcome the treatments currently in use. This possibility
tions, including blood stream and urinary tract infections, and emphasizes the continued need to explore the fundamental
menengitis.89,284 Enterobacter are increasingly resistant to biochemistry of natural product biosynthesis.
common antibacterials, a phenotype that is mediated by resis-
tance factors encoded on a transmissible plasmid.
Enterobacter strains are oen not classied into individual 10. Acknowledgement
species because of the difficulty in phenotypic classication and
I would like to thank all of the past and present members of my
recent genotypic classications dene an Enterobacter cloacae
lab, whose hard work has allowed us to contribute to the
complex composed of multiple heterogeneous subspecies.285
understanding of the structural biology of NRPS proteins. I
Among the most common causes of human infections are E.
would particularly like to thank Daniel C. Bailey and Lisa S.
hormaechei and E. cloacae.284–286 We therefore focused our search
Mydy for helpful comments on this manuscript. Work in my lab
for the NRPS clusters identied within these two species. The
is supported by the National Institute of General Medical
genome of E. cloacae strain ATCC 13047, reported as the rst
Sciences (GM-116957) and the National Institute of Allergy and
complete genome sequence, contains only a single NRPS
Infectious Diseases (AI-116998) of the National Institutes of
operon encoding enterobactin.287 The same was true of E. hor-
Health.
maechei strain ATCC 49162.288 Examination of the databases for
additional NRPS clusters identies colibactin and an additional
uncharacterized BGCs as noted below. 11. Notes and references
E. cloacae strain s611 contains a BGC containing six genes
(Table 4). Interestingly, it begins with an MLP, followed by 1 W. Gevers, H. Kleinkauf and F. Lipmann, Proc. Natl. Acad.
a large NRPS gene. Following this three module NRPS protein is Sci. U. S. A., 1968, 60, 269–276.
a PPTase, a second MLP, and two additional NRPS proteins. 2 W. Gevers, H. Kleinkauf and F. Lipmann, Proc. Natl. Acad.
This unusual architecture containing two MLPs that share 79% Sci. U. S. A., 1969, 63, 1335–1342.
sequence identity, raises the possibility that the adenylation 3 C. C. Gilhuus-Moe, T. Kristensen, J. E. Bredesen, T. Zimmer
domains could be differentially regulated by these auxiliary and S. G. Laland, FEBS Lett., 1970, 7, 287–290.
proteins. 4 H. Kleinkauf, R. Roskoski Jr and F. Lipmann, Proc. Natl.
Acad. Sci. U. S. A., 1971, 68, 2069–2072.
9. Conclusions 5 G. Mittenhuber, R. Weckermann and M. A. Marahiel, J.
Bacteriol., 1989, 171, 4881–4887.
The past few decades have seen tremendous advances in our 6 R. Weckermann, R. Furbass and M. A. Marahiel, Nucleic
understanding of the biosynthetic capabilities of Acids Res., 1988, 16, 11841.
View Article Online
7 K. Turgay, M. Krause and M. A. Marahiel, Mol. Microbiol., 36 C. G. Marshall, M. D. Burkart, T. A. Keating and C. T. Walsh,
1992, 6, 529–546. Biochemistry, 2001, 40, 10655–10663.
8 M. A. Marahiel, FEBS Lett., 1992, 307, 40–43. 37 P. Kinatukara, K. D. Patel, A. S. Haque, R. Singh,
9 M. Gocht and M. A. Marahiel, J. Bacteriol., 1994, 176, 2654– R. S. Gokhale and R. Sankaranarayananan, J. Struct. Biol.,
2662. 2016, 194, 368–374.
10 T. Stachelhaus, H. D. Mootz, V. Bergendahl and 38 M. A. Wyatt, M. C. Mok, M. Junop and N. A. Magarvey,
M. A. Marahiel, J. Biol. Chem., 1998, 273, 22773–22781. ChemBioChem, 2012, 13, 2408–2415.
11 D. E. Cane, C. T. Walsh and C. Khosla, Science, 1998, 282, 39 W. Zhang, B. Ostash and C. T. Walsh, Proc. Natl. Acad. Sci.
63–68. U. S. A., 2010, 107, 16828–16833.
12 M. A. Marahiel, Chem. Biol., 1997, 4, 561–567. 40 N. M. Gaudelli, D. H. Long and C. A. Townsend, Nature,
13 C. T. Walsh, Nat. Chem. Biol., 2015, 11, 620–624. 2015, 520, 383–387.
14 C. T. Walsh, Nat. Prod. Rep., 2016, 33, 127–135. 41 J. Braesel, S. Gotze, F. Shah, D. Heine, J. Tauber,
15 M. A. Marahiel and L. O. Essen, Methods Enzymol., 2009, C. Hertweck, A. Tunlid, P. Stallforth and D. Hoffmeister,
458, 337–351. Chem. Biol., 2015, 22, 1325–1334.
16 M. Strieker, A. Tanovic and M. A. Marahiel, Curr. Opin. 42 E. Geib, M. Gressler, I. Viediernikova, F. Hillmann,
Struct. Biol., 2010, 20, 234–240. I. D. Jacobsen, S. Nietzsche, C. Hertweck and M. Brock,
17 G. Weber, K. Schorgendorfer, E. Schneider-Scherzer and Cell Chem. Biol., 2016, 23, 587–597.
E. Leitner, Curr. Genet., 1994, 26, 120–125. 43 J. A. V. Blodgett, D. C. Oh, S. Cao, C. R. Currie, R. Kolter and
18 A. Wiest, D. Grzegorski, B. W. Xu, C. Goulard, S. Rebuffat, J. Clardy, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 11692–
D. J. Ebbole, B. Bodo and C. Kenerley, J. Biol. Chem., 2002, 11697.
277, 20862–20868. 44 L. Lou, G. Qian, Y. Xie, J. Hang, H. Chen, K. Zaleta-Rivera,
19 J. Crosby and M. P. Crump, Nat. Prod. Rep., 2012, 29, 1111– Y. Li, Y. Shen, P. H. Dussault, F. Liu and L. Du, J. Am.
1137. Chem. Soc., 2011, 133, 643–645.
20 A. C. Mercer and M. D. Burkart, Nat. Prod. Rep., 2007, 24, 45 R. Li, R. A. Oliver and C. A. Townsend, Cell Chem. Biol.,
750–773. 2016, 24, 24–34.
21 R. H. Lambalot, A. M. Gehring, R. S. Flugel, P. Zuber, 46 C. Maruyama, J. Toyoda, Y. Kato, M. Izumikawa, M. Takagi,
M. LaCelle, M. A. Marahiel, R. Reid, C. Khosla and K. Shin-ya, H. Katano, T. Utagawa and Y. Hamano, Nat.
C. T. Walsh, Chem. Biol., 1996, 3, 923–936. Chem. Biol., 2012, 8, 791–797.
22 A. M. Gulick, ACS Chem. Biol., 2009, 4, 811–827. 47 T. Huang, L. Li, N. L. Brock, Z. Deng and S. Lin,
23 U. Linne, A. Schafer, M. T. Stubbs and M. A. Marahiel, FEBS ChemBioChem, 2016, 17, 1421–1425.
Lett., 2007, 581, 905–910. 48 T. Kittila, A. Mollo, L. K. Charkoudian and M. J. Cryle,
24 T. A. Keating, D. E. Ehmann, R. M. Kohli, C. G. Marshall, Angew. Chem., Int. Ed. Engl., 2016, 55, 9834–9840.
J. W. Trauger and C. T. Walsh, ChemBioChem, 2001, 2, 99– 49 C. C. Ladner and G. J. Williams, J. Ind. Microbiol.
107. Biotechnol., 2016, 43, 371–387.
25 J. W. Trauger, R. M. Kohli, H. D. Mootz, M. A. Marahiel and 50 T. M. Schmeing, Clin. Invest. Med., 2016, 39, E220–E226.
C. T. Walsh, Nature, 2000, 407, 215–218. 51 A. M. Gulick, Curr. Opin. Chem. Biol., 2016, 35, 89–96.
26 J. Ravel and P. Cornelis, Trends Microbiol., 2003, 11, 195– 52 B. R. Miller and A. M. Gulick, Methods Mol. Biol., 2016, 1401,
200. 3–29.
27 D. Konz, A. Klens, K. Schorgendorfer and M. A. Marahiel, 53 K. J. Weissman, Nat. Chem. Biol., 2015, 11, 660–670.
Chem. Biol., 1997, 4, 927–937. 54 R. D. Sussmuth and A. Mainz, Angew. Chem., Int. Ed., 2017,
28 R. M. Kohli, J. W. Trauger, D. Schwarzer, M. A. Marahiel and 56, 3770–3821.
C. T. Walsh, Biochemistry, 2001, 40, 7099–7108. 55 K. Bloudoff and T. M. Schmeing, Biochim. Biophys. Acta,
29 M. Krause and M. A. Marahiel, J. Bacteriol., 1988, 170, 4669– 2017, DOI: 10.1016/j.bbapap.2017.05.010.
4674. 56 K. Bloudoff, D. A. Alonzo and T. M. Schmeing, Cell Chem.
30 A. M. Gehring, I. Mori and C. T. Walsh, Biochemistry, 1998, Biol., 2016, 23, 331–339.
37, 2648–2659. 57 W. H. Chen, K. Li, N. S. Guntaka and S. D. Bruner, ACS
31 C. T. Walsh, H. Chen, T. A. Keating, B. K. Hubbard, Chem. Biol., 2016, 11, 2293–2303.
H. C. Losey, L. Luo, C. G. Marshall, D. A. Miller and 58 E. J. Drake, B. R. Miller, C. Shi, J. T. Tarrasch, J. A. Sundlov,
H. M. Patel, Curr. Opin. Chem. Biol., 2001, 5, 525–534. C. L. Allen, G. Skiniotis, C. C. Aldrich and A. M. Gulick,
32 J. Burmester, A. Haese and R. Zocher, Biochem. Mol. Biol. Nature, 2016, 529, 235–238.
Int., 1995, 37, 201–207. 59 B. R. Miller, E. J. Drake, C. Shi, C. C. Aldrich and
33 C. Hacker, M. Glinski, T. Hornbogen, A. Doller and A. M. Gulick, J. Biol. Chem., 2016, 291, 22559–22571.
R. Zocher, J. Biol. Chem., 2000, 275, 30826–30832. 60 J. M. Reimer, M. N. Aloise, P. M. Harrison and
34 U. Linne, S. Doekel and M. A. Marahiel, Biochemistry, 2001, T. M. Schmeing, Nature, 2016, 529, 239–242.
40, 15824–15834. 61 D. P. Dowling, Y. Kung, A. K. Cro, K. Taghizadeh,
35 T. Stachelhaus and C. T. Walsh, Biochemistry, 2000, 39, W. L. Kelly, C. T. Walsh and C. L. Drennan, Proc. Natl.
5775–5787. Acad. Sci. U. S. A., 2016, 113, 12432–12437.
View Article Online
62 J. Zhang, N. Liu, R. A. Cacho, Z. Gong, Z. Liu, W. Qin, 86 H. W. Boucher, G. H. Talbot, D. K. Benjamin Jr, J. Bradley,
C. Tang, Y. Tang and J. Zhou, Nat. Chem. Biol., 2016, 12, R. J. Guidos, R. N. Jones, B. E. Murray, R. A. Bonomo and
1001–1003. D. Gilbert, Clin. Infect. Dis., 2013, 56, 1685–1694.
63 M. J. Tarry, A. S. Haque, K. H. Bui and T. M. Schmeing, 87 R. A. Stavenger and M. Winterhalter, Sci. Transl. Med., 2014,
Structure, 2017, 25, 783–793. 6, 228ed7.
64 K. Bloudoff, D. Rodionov and T. M. Schmeing, J. Mol. Biol., 88 H. W. Boucher, G. H. Talbot, J. S. Bradley, J. E. Edwards,
2013, 425, 3137–3150. D. Gilbert, L. B. Rice, M. Scheld, B. Spellberg and
65 T. A. Keating, C. G. Marshall, C. T. Walsh and A. E. Keating, J. Bartlett, Clin. Infect. Dis., 2009, 48, 1–12.
Nat. Struct. Biol., 2002, 9, 522–526. 89 J. N. Pendleton, S. P. Gorman and B. F. Gilmore, Expert Rev.
66 S. A. Samel, G. Schoenanger, T. A. Knappe, M. A. Marahiel Anti-Infect. Ther., 2013, 11, 297–308.
and L. O. Essen, Structure, 2007, 15, 781–792. 90 M. S. Donia and M. A. Fischbach, Science, 2015, 349,
67 E. Conti, N. P. Franks and P. Brick, Structure, 1996, 4, 287– 1254766.
298. 91 N. Garg, T. Luzzatto-Knaan, A. V. Melnik, A. M. Caraballo-
68 E. Conti, T. Stachelhaus, M. A. Marahiel and P. Brick, EMBO Rodriguez, D. J. Floros, D. Petras, R. Gregor,
J., 1997, 16, 4174–4183. P. C. Dorrestein and V. V. Phelan, Nat. Prod. Rep., 2016,
69 A. M. Gulick, V. J. Starai, A. R. Horswill, K. M. Homick and 34, 194–216.

J. C. Escalante-Semerena, Biochemistry, 2003, 42, 2866– 92 J. A. Gilbert, R. A. Quinn, J. Debelius, Z. Z. Xu, J. Morton,
2873. N. Garg, J. K. Jansson, P. C. Dorrestein and R. Knight,
70 J. J. May, N. Kessler, M. A. Marahiel and M. T. Stubbs, Proc. Nature, 2016, 535, 94–103.
Natl. Acad. Sci. U. S. A., 2002, 99, 12120–12125. 93 N. Koppel and E. P. Balskus, Cell Chem. Biol., 2016, 23, 18–
71 A. M. Gulick, A. R. Horswill, J. B. Thoden, J. C. Escalante- 30.
Semerena and I. Rayment, Acta Crystallogr., Sect. D: Biol. 94 E. P. Trautman and J. M. Crawford, Curr. Top. Med. Chem.,
Crystallogr., 2002, 58, 306–309. 2016, 16, 1705–1716.
72 S. D. Bruner, T. Weber, R. M. Kohli, D. Schwarzer, 95 A. Zipperer, M. C. Konnerth, C. Laux, A. Berscheid, D. Janek,
M. A. Marahiel, C. T. Walsh and M. T. Stubbs, Structure, C. Weidenmaier, M. Burian, N. A. Schilling, C. Slavetinsky,
2002, 10, 301–310. M. Marschal, M. Willmann, H. Kalbacher, B. Schittek,
73 S. A. Samel, B. Wagner, M. A. Marahiel and L. O. Essen, J. H. Brotz-Oesterhelt, S. Grond, A. Peschel and B. Krismer,
Mol. Biol., 2006, 359, 876–889. Nature, 2016, 535, 511–516.
74 J. F. Barajas, R. M. Phelan, A. J. Schaub, J. T. Kliewer, 96 M. H. Medema and M. A. Fischbach, Nat. Chem. Biol., 2015,
P. J. Kelly, D. R. Jackson, R. Luo, J. D. Keasling and 11, 639–648.
S. C. Tsai, Chem. Biol., 2015, 22, 1018–1029. 97 M. H. Medema, R. Kottmann, P. Yilmaz, M. Cummings,
75 A. Chhabra, A. S. Haque, R. K. Pal, A. Goyal, R. Rai, S. Joshi, J. B. Biggins, K. Blin, I. de Bruijn, Y. H. Chooi, J. Claesen,
S. Panjikar, S. Pasha, R. Sankaranarayanan and R. C. Coates, P. Cruz-Morales, S. Duddela, S. Dusterhus,
R. S. Gokhale, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, D. J. Edwards, D. P. Fewer, N. Garg, C. Geiger,
5681–5686. J. P. Gomez-Escribano, A. Greule, M. Hadjithomas,
76 D. P. Frueh, H. Arthanari, A. Koglin, D. A. Vosburg, A. S. Haines, E. J. Helfrich, M. L. Hillwig, K. Ishida,
A. E. Bennett, C. T. Walsh and G. Wagner, Nature, 2008, A. C. Jones, C. S. Jones, K. Jungmann, C. Kegler,
454, 903–906. H. U. Kim, P. Kotter, D. Krug, J. Masschelein,
77 Y. Liu, T. Zheng and S. D. Bruner, Chem. Biol., 2011, 18, A. V. Melnik, S. M. Mantovani, E. A. Monroe, M. Moore,
1482–1488. N. Moss, H. W. Nutzmann, G. Pan, A. Pati, D. Petras,
78 C. A. Mitchell, C. Shi, C. C. Aldrich and A. M. Gulick, F. J. Reen, F. Rosconi, Z. Rui, Z. Tian, N. J. Tobias,
Biochemistry, 2012, 51, 3252–3263. Y. Tsunematsu, P. Wiemann, E. Wyckoff, X. Yan, G. Yim,
79 J. A. Sundlov and A. M. Gulick, Acta Crystallogr., Sect. D: Biol. F. Yu, Y. Xie, B. Aigle, A. K. Apel, C. J. Balibar,
Crystallogr., 2013, 69, 1482–1492. E. P. Balskus, F. Barona-Gomez, A. Bechthold, H. B. Bode,
80 J. A. Sundlov, C. Shi, D. J. Wilson, C. C. Aldrich and R. Borriss, S. F. Brady, A. A. Brakhage, P. Caffrey,
A. M. Gulick, Chem. Biol., 2012, 19, 188–198. Y. Q. Cheng, J. Clardy, R. J. Cox, R. De Mot, S. Donadio,
81 C. Qiao, D. J. Wilson, E. M. Bennett and C. C. Aldrich, J. Am. M. S. Donia, W. A. van der Donk, P. C. Dorrestein,
Chem. Soc., 2007, 129, 6350–6351. S. Doyle, A. J. Driessen, M. Ehling-Schulz, K. D. Entian,
82 A. Tanovic, S. A. Samel, L. O. Essen and M. A. Marahiel, M. A. Fischbach, L. Gerwick, W. H. Gerwick, H. Gross,
Science, 2008, 321, 659–663. B. Gust, C. Hertweck, M. Hoe, S. E. Jensen, J. Ju, L. Katz,
83 D. A. Miller, L. Luo, N. Hillson, T. A. Keating and L. Kaysser, J. L. Klassen, N. P. Keller, J. Kormanec,
C. T. Walsh, Chem. Biol., 2002, 9, 333–344. O. P. Kuipers, T. Kuzuyama, N. C. Kyrpides, H. J. Kwon,
84 H. M. Patel and C. T. Walsh, Biochemistry, 2001, 40, 9023– S. Lautru, R. Lavigne, C. Y. Lee, B. Linquan, X. Liu,
9031. W. Liu, A. Luzhetskyy, T. Mahmud, Y. Mast, C. Mendez,
85 L. B. Rice, J. Infect. Dis., 2008, 197, 1079–1081. M. Metsa-Ketela, J. Mickleeld, D. A. Mitchell,
B. S. Moore, L. M. Moreira, R. Muller, B. A. Neilan,
M. Nett, J. Nielsen, F. O'Gara, H. Oikawa, A. Osbourn,
View Article Online
M. S. Osburne, B. Ostash, S. M. Payne, J. L. Pernodet, 104 C. J. Kristich, L. B. Rice and C. A. Arias, in Enterococci: From
M. Petricek, J. Piel, O. Ploux, J. M. Raaijmakers, Commensals to Leading Causes of Drug Resistant Infection,
J. A. Salas, E. K. Schmitt, B. Scott, R. F. Seipke, B. Shen, ed. M. S. Gilmore, D. B. Clewell, Y. Ike and N. Shankar,
D. H. Sherman, K. Sivonen, M. J. Smanski, M. Sosio, Boston, 2014.
E. Stegmann, R. D. Sussmuth, K. Tahlan, C. M. Thomas, 105 H. Y. Chiang, E. N. Perencevich, R. Nair, R. E. Nelson,
Y. Tang, A. W. Truman, M. Viaud, J. D. Walton, M. Samore, K. Khader, M. L. Chorazy, L. A. Herwaldt,
C. T. Walsh, T. Weber, G. P. van Wezel, B. Wilkinson, A. Blevins, M. A. Ward and M. L. Schweizer, Infect Control
J. M. Willey, W. Wohlleben, G. D. Wright, N. Ziemert, Hosp. Epidemiol., 2017, 38, 203–215.
C. Zhang, S. B. Zotchev, R. Breitling, E. Takano and 106 M. M. Lam, T. Seemann, D. M. Bulach, S. L. Gladman,
F. O. Glockner, Nat. Chem. Biol., 2015, 11, 625–631. H. Chen, V. Haring, R. J. Moore, S. Ballard,
98 K. Blin, M. H. Medema, R. Kottmann, S. Y. Lee and M. L. Grayson, P. D. Johnson, B. P. Howden and
T. Weber, Nucleic Acids Res., 2017, 45, D555–D559. T. P. Stinear, J. Bacteriol., 2012, 194, 2334–2341.
99 C. A. Dejong, G. M. Chen, H. Li, C. W. Johnston, 107 W. van Schaik, J. Top, D. R. Riley, J. Boekhorst,
M. R. Edwards, P. N. Rees, M. A. Skinnider, A. L. Webster J. E. Vrijenhoek, C. M. Schapendonk, A. P. Hendrickx,
and N. A. Magarvey, Nat. Chem. Biol., 2016, 12, 1007–1014. I. J. Nijman, M. J. Bonten, H. Tettelin and R. J. Willems,
100 M. A. Skinnider, C. A. Dejong, P. N. Rees, C. W. Johnston, BMC Genomics, 2010, 11, 239.
H. Li, A. L. Webster, M. A. Wyatt and N. A. Magarvey, 108 S. F. Altschul, W. Gish, W. Miller, E. W. Myers and
Nucleic Acids Res., 2015, 43, 9645–9662. D. J. Lipman, J. Mol. Biol., 1990, 215, 403–410.
101 M. Wang, J. J. Carver, V. V. Phelan, L. M. Sanchez, N. Garg, 109 E. B. Kim, G. D. Jin, J. Y. Lee and Y. J. Choi, PLoS One, 2016,
Y. Peng, D. D. Nguyen, J. Watrous, C. A. Kapono, 11, e0153279.
T. Luzzatto-Knaan, C. Porto, A. Bouslimani, A. V. Melnik, 110 L. S. Miller and J. S. Cho, Nat. Rev. Immunol., 2011, 11, 505–
M. J. Meehan, W. T. Liu, M. Crusemann, P. D. Boudreau, 518.
E. Esquenazi, M. Sandoval-Calderon, R. D. Kersten, 111 E. J. Choo and H. F. Chambers, Infect. Chemother., 2016, 48,
L. A. Pace, R. A. Quinn, K. R. Duncan, C. C. Hsu, 267–273.
D. J. Floros, R. G. Gavilan, K. Kleigrewe, T. Northen, 112 M. Zimmermann and M. A. Fischbach, Chem. Biol., 2010,
R. J. Dutton, D. Parrot, E. E. Carlson, B. Aigle, 17, 925–930.
C. F. Michelsen, L. Jelsbak, C. Sohlenkamp, P. Pevzner, 113 M. A. Wyatt, W. Wang, C. M. Roux, F. C. Beasley,
A. Edlund, J. McLean, J. Piel, B. T. Murphy, L. Gerwick, D. E. Heinrichs, P. M. Dunman and N. A. Magarvey,
C. C. Liaw, Y. L. Yang, H. U. Humpf, M. Maansson, Science, 2010, 329, 294–296.
R. A. Keyzers, A. C. Sims, A. R. Johnson, 114 M. E. Alvarez, C. B. White, J. Gregory, G. C. Kydd, A. Harris,
A. M. Sidebottom, B. E. Sedio, A. Klitgaard, C. B. Larson, H. H. Sun, A. M. Gillum and R. Cooper, J. Antibiot., 1995, 48,
P. C. Boya, D. Torres-Mendoza, D. J. Gonzalez, D. B. Silva, 1165–1167.
L. M. Marques, D. P. Demarque, E. Pociute, E. C. O'Neill, 115 D. J. Wilson, C. Shi, A. M. Teitelbaum, A. M. Gulick and
E. Briand, E. J. Helfrich, E. A. Granatosky, E. Glukhov, C. C. Aldrich, Biochemistry, 2013, 52, 926–937.
F. Ryffel, H. Houson, H. Mohimani, J. J. Kharbush, 116 F. Sun, H. Cho, D. W. Jeong, C. Li, C. He and T. Bae, PLoS
Y. Zeng, J. A. Vorholt, K. L. Kurita, P. Charusanti, One, 2010, 5, e15703.
K. L. McPhail, K. F. Nielsen, L. Vuong, M. Elfeki, 117 M. A. Wyatt, W. Wang, C. M. Roux, F. C. Beasley,
M. F. Traxler, N. Engene, N. Koyama, O. B. Vining, D. E. Heinrichs, P. M. Dunman and N. A. Magarvey,
R. Baric, R. R. Silva, S. J. Mascuch, S. Tomasi, S. Jenkins, Science, 2011, 333, 1381.
V. Macherla, T. Hoffman, V. Agarwal, P. G. Williams, 118 P. R. Secor, L. K. Jennings, G. A. James, K. R. Kirker,
J. Dai, R. Neupane, J. Gurr, A. M. Rodriguez, A. Lamsa, E. D. Pulcini, K. McInnerney, R. Gerlach, T. Livinghouse,
C. Zhang, K. Dorrestein, B. M. Duggan, J. Almaliti, J. K. Hilmer, B. Bothner, P. Fleckman, J. E. Olerud and
P. M. Allard, P. Phapale, L. F. Nothias, T. Alexandrov, P. S. Stewart, PLoS One, 2012, 7, e40973.
M. Litaudon, J. L. Wolfender, J. E. Kyle, T. O. Metz, 119 M. Grosz, J. Kolter, K. Paprotka, A. C. Winkler, D. Schafer,
T. Peryea, D. T. Nguyen, D. VanLeer, P. Shinn, A. Jadhav, S. S. Chatterjee, T. Geiger, C. Wolz, K. Ohlsen, M. Otto,
R. Muller, K. M. Waters, W. Shi, X. Liu, L. Zhang, T. Rudel, B. Sinha and M. Fraunholz, Cell. Microbiol.,
R. Knight, P. R. Jensen, B. O. Palsson, K. Pogliano, 2014, 16, 451–465.
R. G. Linington, M. Gutierrez, N. P. Lopes, W. H. Gerwick, 120 S. Blattner, S. Das, K. Paprotka, U. Eilers, M. Krischke,
B. S. Moore, P. C. Dorrestein and N. Bandeira, Nat. D. Kretschmer, C. W. Remmele, M. Dittrich, T. Muller,
Biotechnol., 2016, 34, 828–837. C. Schuelein-Voelk, T. Hertlein, M. J. Mueller, B. Huettel,
102 U. A. Ochsner, P. J. Wilderman, A. I. Vasil and M. L. Vasil, R. Reinhardt, K. Ohlsen, T. Rudel and M. J. Fraunholz,
Mol. Microbiol., 2002, 45, 1277–1287. PLoS Pathog., 2016, 12, e1005857.
103 F. Lebreton, R. J. L. Willems and M. S. Gilmore, in 121 C. J. Guo, F. Y. Chang, T. P. Wyche, K. M. Backus,
Enterococci: From Commensals to Leading Causes of Drug T. M. Acker, M. Funabashi, M. Taketani, M. S. Donia,
Resistant Infection, ed. M. S. Gilmore, D. B. Clewell, Y. Ike S. Nayfach, K. S. Pollard, C. S. Craik, B. F. Cravatt,
and N. Shankar, Boston, 2014. J. Clardy, C. A. Voigt and M. A. Fischbach, Cell, 2017, 168,
517–526.
View Article Online
122 Y. Keynan and E. Rubinstein, Int. J. Antimicrob. Agents, 150 W. F. Penwell, B. A. Arivett and L. A. Actis, PLoS One, 2012,
2007, 30, 385–389. 7, e36493.
123 B. M. Kuehn, J. Am. Med. Assoc., 2013, 309, 1573–1574. 151 D. E. Ehmann, C. A. Shaw-Reid, H. C. Losey and
124 G. C. Lee and D. S. Burgess, Ann. Clin. Microbiol. C. T. Walsh, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 2509–
Antimicrob., 2012, 11, 32. 2514.
125 K. Prabaker and R. A. Weinstein, Curr. Opin. Crit. Care, 152 J. M. Lodge, P. Williams and M. R. Brown, J. Bacteriol., 1986,
2011, 17, 472–479. 165, 353–356.
126 A. S. Shon, R. P. Bajwa and T. A. Russo, Virulence, 2013, 4, 153 R. D. Perry and C. L. San Clemente, J. Bacteriol., 1979, 140,
107–118. 1129–1132.
127 A. S. Shon and T. A. Russo, Future Microbiol., 2012, 7, 669– 154 H. Haag, K. Hantke, H. Drechsel, I. Stojiljkovic, G. Jung and
671. H. Zahner, J. Gen. Microbiol., 1993, 139, 2159–2165.
128 Y. Liu, X. Y. Li, L. G. Wan, W. Y. Jiang, J. H. Yang and 155 C. E. Chambers, D. D. McIntyre, M. Mouck and P. A. Sokol,
F. Q. Li, Microb. Drug Resist., 2014, 20, 150–155. BioMetals, 1996, 9, 157–167.
129 L. K. Siu, D. B. Huang and T. Chiang, BMC Infect. Dis., 2014, 156 H. Drechsel, H. Stephan, R. Lotz, H. Haag, H. Zahner,
14, 176. K. Hantke and G. Jung, Liebigs Ann., 1995, 1727–1733.
130 J. R. Pollack and J. B. Neilands, Biochem. Biophys. Res. 157 A. M. Gehring, E. DeMoll, J. D. Fetherston, I. Mori,
Commun., 1970, 38, 989–992. G. F. Mayhew, F. R. Blattner, C. T. Walsh and R. D. Perry,
131 I. G. O'Brien, G. B. Cox and F. Gibson, Biochim. Biophys. Chem. Biol., 1998, 5, 573–586.
Acta, 1970, 201, 453–460. 158 K. M. Meneely, T. A. Ronnebaum, A. P. Riley,
132 J. H. Crosa and C. T. Walsh, Microbiol. Mol. Biol. Rev., 2002, T. E. Prisinzano and A. L. Lamb, Biochemistry, 2016, 55,
66, 223–249. 5423–5433.
133 C. T. Walsh, J. Liu, F. Rusnak and M. Sakaitani, Chem. Rev., 159 Y. T. Chen, H. Y. Chang, Y. C. Lai, C. C. Pan, S. F. Tsai and
1990, 90, 1105–1129. H. L. Peng, Gene, 2004, 337, 189–198.
134 K. N. Raymond, E. A. Dertz and S. S. Kim, Proc. Natl. Acad. 160 S. I. Müller, M. Valdebenito and K. Hantke, BioMetals, 2009,
Sci. U. S. A., 2003, 100, 3584–3588. 22, 691–695.
135 W. R. Harris, C. J. Carrano, S. R. Cooper, S. R. Sofen, 161 M. A. Bachman, S. Lenio, L. Schmidt, J. E. Oyler and
A. E. Avdeef, J. V. McArdle and K. N. Raymond, J. Am. J. N. Weiser, mBio, 2012, 3, e00224-11.
Chem. Soc., 1979, 101, 6097–6104. 162 M. A. Bachman, J. E. Oyler, S. H. Burns, M. Caza, F. Lepine,
136 J. Liu, K. Duncan and C. T. Walsh, J. Bacteriol., 1989, 171, C. M. Dozois and J. N. Weiser, Infect. Immun., 2011, 79,
791–798. 3309–3316.
137 M. S. Nahlik, T. J. Brickman, B. A. Ozenberger and 163 M. Valdebenito, A. L. Crumbliss, G. Winkelmann and
M. A. McIntosh, J. Bacteriol., 1989, 171, 784–790. K. Hantke, Int. J. Med. Microbiol., 2006, 296, 513–520.
138 F. Rusnak, W. S. Faraci and C. T. Walsh, Biochemistry, 1989, 164 M. S. Lawlor, C. O'Connor and V. L. Miller, Infect. Immun.,
28, 6827–6835. 2007, 75, 1463–1472.
139 F. Rusnak, M. Sakaitani, D. Drueckhammer, J. Reichert and 165 T. A. Russo, R. Olson, U. Macdonald, D. Metzger,
C. T. Walsh, Biochemistry, 1991, 30, 2916–2927. L. M. Maltese, E. J. Drake and A. M. Gulick, Infect.
140 E. J. Drake, D. A. Nicolai and A. M. Gulick, Chem. Biol., 2006, Immun., 2014, 82, 2356–2367.
13, 409–419. 166 T. A. Russo, R. Olson, U. MacDonald, J. Beanan and
141 S. Sridharan, N. Howard, O. Kerbarh, M. Blaszczyk, C. Abell B. A. Davidson, Infect. Immun., 2014, 82, 3325–3333.
and T. L. Blundell, J. Mol. Biol., 2010, 397, 290–300. 167 J. P. Nougayrede, S. Homburg, F. Taieb, M. Boury,
142 J. A. Sundlov, J. A. Garringer, J. M. Carney, A. S. Reger, E. Brzuszkiewicz, G. Gottschalk, C. Buchrieser, J. Hacker,
E. J. Drake, W. L. Duax and A. M. Gulick, Acta Crystallogr., U. Dobrindt and E. Oswald, Science, 2006, 313, 848–851.
Sect. D: Biol. Crystallogr., 2006, 62, 734–740. 168 S. Conlan, C. Deming, Y. C. Tsai, A. F. Lau, J. P. Dekker,
143 L. E. Quadri, Mol. Microbiol., 2000, 37, 1–12. J. Korlach and J. A. Segre, Genome Announc., 2014, 2,
144 O. Kerbarh, E. M. Bulloch, R. J. Payne, T. Sahr, F. Rebeille e01332-14.
and C. Abell, Biochem. Soc. Trans., 2005, 33, 763–766. 169 C. Struve, C. C. Roe, M. Stegger, S. G. Stahlhut,
145 J. Liu, N. Quinn, G. A. Berchtold and C. T. Walsh, D. S. Hansen, D. M. Engelthaler, P. S. Andersen,
Biochemistry, 1990, 29, 1417–1425. E. M. Driebe, P. Keim and K. A. Krogfelt, mBio, 2015, 6,
146 A. M. Gehring, K. A. Bradley and C. T. Walsh, Biochemistry, e00630.
1997, 36, 8495–8503. 170 J. Putze, C. Hennequin, J. P. Nougayrede, W. Zhang,
147 M. Sakaitani, F. Rusnak, N. R. Quinn, C. Tu, T. B. Frigo, S. Homburg, H. Karch, M. A. Bringer, C. Fayolle,
G. A. Berchtold and C. T. Walsh, Biochemistry, 1990, 29, E. Carniel, W. Rabsch, T. A. Oelschlaeger, E. Oswald,
6789–6798. C. Forestier, J. Hacker and U. Dobrindt, Infect. Immun.,
148 K. M. Meneely, J. A. Sundlov, A. M. Gulick, G. R. Moran and 2009, 77, 4696–4703.
A. L. Lamb, J. Am. Chem. Soc., 2016, 138, 9277–9293. 171 E. P. Balskus, Nat. Prod. Rep., 2015, 32, 1534–1540.
149 K. M. Meneely, Q. Luo and A. L. Lamb, Arch. Biochem. 172 C. A. Brotherton and E. P. Balskus, J. Am. Chem. Soc., 2013,
Biophys., 2013, 539, 70–80. 135, 3359–3362.
View Article Online
173 X. Bian, J. Fu, A. Plaza, J. Herrmann, D. Pistorius, 197 W. M. Wuest, E. S. Sattely and C. T. Walsh, J. Am. Chem.
A. F. Stewart, Y. Zhang and R. Muller, ChemBioChem, Soc., 2009, 131, 5056–5057.
2013, 14, 1194–1197. 198 E. J. Drake, B. P. Duckworth, J. Neres, C. C. Aldrich and
174 M. I. Vizcaino and J. M. Crawford, Nat. Chem., 2015, 7, 411– A. M. Gulick, Biochemistry, 2010, 49, 9292–9305.
417. 199 A. L. Lamb, Biochim. Biophys. Acta, 2015, 1854, 1054–1070.
175 C. A. Brotherton, M. Wilson, G. Byrd and E. P. Balskus, Org. 200 M. Miethke and M. A. Marahiel, Microbiol. Mol. Biol. Rev.,
Lett., 2015, 17, 1545–1548. 2007, 71, 413–451.
176 X. Y. Bian, A. Plaza, Y. M. Zhang and R. Muller, Chem. Sci., 201 J. A. Gaddy, B. A. Arivett, M. J. McConnell, R. Lopez-Rojas,
2015, 6, 3154–3160. J. Pachon and L. A. Actis, Infect. Immun., 2012, 80, 1015–
177 E. P. Trautman, A. R. Healy, E. E. Shine, S. B. Herzon and 1024.
J. M. Crawford, J. Am. Chem. Soc., 2017, 139, 4195–4201. 202 I. D. Fleming, M. A. Krezalek, N. Belogortseva, A. Zaborin,
178 J. J. Mousa, R. C. Newsome, Y. Yang, C. Jobin and J. Defazio, L. Chandrasekar, L. A. Actis, O. Zaborina and
S. D. Bruner, Biochem. Biophys. Res. Commun., 2017, 482, J. C. Alverdy, J. Trauma Acute Care Surg., 2017, 82, 557–565.
1233–1239. 203 A. Proschak, P. Lubuta, P. Grun, F. Lohr, G. Wilharm, V. De
179 M. I. Vizcaino, P. Engel, E. Trautman and J. M. Crawford, J. Berardinis and H. B. Bode, ChemBioChem, 2013, 14, 633–
Am. Chem. Soc., 2014, 136, 9244–9247. 638.

180 D. Dubois, O. Baron, A. Cougnoux, J. Delmas, N. Pradel, 204 K. M. Meneely and A. L. Lamb, Biochemistry, 2007, 46,
M. Boury, B. Bouchon, M. A. Bringer, J. P. Nougayrede, 11930–11937.
E. Oswald and R. Bonnet, J. Biol. Chem., 2011, 286, 205 L. L. Burrows, Annu. Rev. Microbiol., 2012, 66, 493–520.
35562–35570. 206 J. S. Mattick, Annu. Rev. Microbiol., 2002, 56, 289–314.
181 A. O. Brachmann, C. Garcie, V. Wu, P. Martin, R. Ueoka, 207 S. Rumbo-Feal, M. J. Gomez, C. Gayoso, L. Alvarez-Fraga,
E. Oswald and J. Piel, Chem. Commun., 2015, 51, 13138– M. P. Cabral, A. M. Aransay, N. Rodriguez-Ezpeleta,
13141. A. Fullaondo, J. Valle, M. Tomas, G. Bou and M. Poza,
182 M. J. McConnell, L. Actis and J. Pachon, FEMS Microbiol. PLoS One, 2013, 8, e72968.
Rev., 2013, 37, 130–155. 208 S. K. Giles, U. H. Stroeher, B. A. Eijkelkamp and
183 A. Y. Peleg, H. Seifert and D. L. Paterson, Clin. Microbiol. M. H. Brown, BMC Microbiol., 2015, 15, 116.
Rev., 2008, 21, 538–582. 209 C. L. Allen and A. M. Gulick, Acta Crystallogr., Sect. D: Biol.
184 J. H. Calhoun, C. K. Murray and M. M. Manring, Clin. Crystallogr., 2014, 70, 1718–1725.
Orthop. Relat. Res., 2008, 466, 1356–1362. 210 S. Rumbo-Feal, A. Perez, T. A. Ramelot, L. Alvarez-Fraga,
185 E. F. Keen 3rd, B. J. Robinson, D. R. Hospenthal, J. A. Vallejo, A. Beceiro, E. J. Ohneck, B. A. Arivett,
W. K. Aldous, S. E. Wolf, K. K. Chung and C. K. Murray, M. Merino, S. E. Fiester, M. A. Kennedy, L. A. Actis,
Burns, 2010, 36, 819–825. G. Bou and M. Poza, Front. Cell. Infect. Microbiol., 2017, 7,
186 D. E. Karageorgopoulos and M. E. Falagas, Lancet Infect. 108.
Dis., 2008, 8, 751–762. 211 M. D. Adams, K. Goglin, N. Molyneaux, K. M. Hujer,
187 K. M. Clemmer, R. A. Bonomo and P. N. Rather, H. Lavender, J. J. Jamison, I. J. MacDonald, K. M. Martin,
Microbiology, 2011, 157, 2534–2544. T. Russo, A. A. Campagnari, A. M. Hujer, R. A. Bonomo
188 B. A. Eijkelkamp, U. H. Stroeher, K. A. Hassan, and S. R. Gill, J. Bacteriol., 2008, 190, 8053–8064.
M. S. Papadimitrious, I. T. Paulsen and M. H. Brown, 212 J. B. Lyczak, C. L. Cannon and G. B. Pier, Microbes Infect.,
FEMS Microbiol. Lett., 2011, 323, 44–51. 2000, 2, 1051–1060.
189 C. M. Harding, E. N. Tracy, M. D. Carruthers, P. N. Rather, 213 C. Winstanley, S. O'Brien and M. A. Brockhurst, Trends
L. A. Actis and R. S. Munson Jr, mBio, 2013, 4, e00360-13. Microbiol., 2016, 24, 327–337.
190 L. Poirel, R. A. Bonnin and P. Nordmann, IUBMB Life, 2011, 214 K. Poole, Front. Microbiol., 2011, 2, 65.
63, 1061–1067. 215 M. A. Welsh and H. E. Blackwell, FEMS Microbiol. Rev.,
191 H. U. Kim, T. Y. Kim and S. Y. Lee, Mol. BioSyst., 2010, 6, 2016, 40, 774–794.
339–348. 216 I. J. Schalk and L. Guillon, Environ. Microbiol., 2013, 15,
192 T. C. Umland, L. W. Schultz, U. Macdonald, J. M. Beanan, 1661–1673.
R. Olson and T. A. Russo, mBio, 2012, 3, e00113-12. 217 P. Visca, F. Imperi and I. L. Lamont, Trends Microbiol., 2007,
193 E. S. Sattely and C. T. Walsh, J. Am. Chem. Soc., 2008, 130, 15, 22–30.
12282–12284. 218 C. Cezard, N. Farvacques and P. Sonnet, Curr. Med. Chem.,
194 J. A. Shapiro and T. A. Wencewicz, ACS Infect. Dis., 2016, 2, 2015, 22, 165–186.
157–168. 219 C. S. Vandenende, M. Vlasschaert and S. Y. Seah, J.
195 C. W. Dorsey, A. P. Tomaras, P. L. Connerly, Bacteriol., 2004, 186, 5596–5602.
M. E. Tolmasky, J. H. Crosa and L. A. Actis, Microbiology, 220 P. Visca, A. Ciervo and N. Orsi, J. Bacteriol., 1994, 176, 1128–
2004, 150, 3657–3667. 1140.
196 K. Mihara, T. Tanabe, Y. Yamakawa, T. Funahashi, 221 L. Ge and S. Y. Seah, J. Bacteriol., 2006, 188, 7205–7210.
H. Nakao, S. Narimatsu and S. Yamamoto, Microbiology, 222 B. J. McMorran, H. M. Kumara, K. Sullivan and
2004, 150, 2587–2597. I. L. Lamont, Microbiology, 2001, 147, 1517–1524.
View Article Online
223 E. J. Drake, J. Cao, J. Qu, M. B. Shah, R. M. Straubinger and 248 D. Cobessi, H. Celia and F. Pattus, J. Mol. Biol., 2005, 352,
A. M. Gulick, J. Biol. Chem., 2007, 282, 20425–20434. 893–904.
224 R. H. Baltz, J. Ind. Microbiol. Biotechnol., 2011, 38, 1747– 249 K. Schlegel, J. Lex, K. Taraz and H. Budzikiewicz, Z.
1760. Naturforsch., C: J. Biosci., 2006, 61, 263–266.
225 E. A. Felnagle, J. J. Barkei, H. Park, A. M. Podevels, 250 L. Serino, C. Reimmann, P. Visca, M. Beyeler, V. D. Chiesa
M. D. McMahon, D. W. Drott and M. G. Thomas, and D. Haas, J. Bacteriol., 1997, 179, 248–257.
Biochemistry, 2010, 49, 8815–8817. 251 C. Reimmann, L. Serino, M. Beyeler and D. Haas,
226 W. Zhang, J. R. Heemstra Jr, C. T. Walsh and H. J. Imker, Microbiology, 1998, 144(11), 3135–3148.
Biochemistry, 2010, 49, 9946–9947. 252 C. Reimmann, H. M. Patel, L. Serino, M. Barone,
227 D. A. Herbst, B. Boll, G. Zocher, T. Stehle and L. Heide, J. C. T. Walsh and D. Haas, J. Bacteriol., 2001, 183, 813–820.
Biol. Chem., 2013, 288, 1991–2003. 253 C. Reimmann, H. M. Patel, C. T. Walsh and D. Haas, J.
228 G. L. Challis, J. Ravel and C. A. Townsend, Chem. Biol., 2000, Bacteriol., 2004, 186, 6367–6373.
7, 211–224. 254 C. Gaille, C. Reimmann and D. Haas, J. Biol. Chem., 2003,
229 T. Stachelhaus, H. D. Mootz and M. A. Marahiel, Chem. 278, 16893–16898.
Biol., 1999, 6, 493–505. 255 C. Gaille, P. Kast and D. Haas, J. Biol. Chem., 2002, 277,
230 Y. Kim and W. G. Hol, Chem. Biol., 2001, 8, 1253–1264. 21768–21775.

231 E. J. Drake and A. M. Gulick, ACS Chem. Biol., 2011, 6, 1277– 256 L. E. Quadri, T. A. Keating, H. M. Patel and C. T. Walsh,
1286. Biochemistry, 1999, 38, 14941–14954.
232 E. Yeterian, L. W. Martin, L. Guillon, L. Journet, 257 D. G. Lee, J. M. Urbach, G. Wu, N. T. Liberati,
I. L. Lamont and I. J. Schalk, Amino Acids, 2010, 38, 1447– R. L. Feinbaum, S. Miyata, L. T. Diggins, J. He,
1459. M. Saucier, E. Deziel, L. Friedman, L. Li, G. Grills,
233 M. Hannauer, M. Schafer, F. Hoegy, P. Gizzi, P. Wehrung, K. Montgomery, R. Kucherlapati, L. G. Rahme and
G. L. Mislin, H. Budzikiewicz and I. J. Schalk, FEBS Lett., F. M. Ausubel, Genome Biol., 2006, 7, R90.
2012, 586, 96–101. 258 A. Maspoli, N. Wenner, G. L. Mislin and C. Reimmann,
234 K. D. Clevenger, R. Mascarenhas, D. Catlin, R. Wu, BioMetals, 2014, 27, 559–573.
N. L. Kelleher, E. J. Drake, A. M. Gulick, D. Liu and 259 A. J. Harrison, M. Yu, T. Gardenborg, M. Middleditch,
W. Fast, ACS Chem. Biol., 2017, 12, 643–647. R. J. Ramsay, E. N. Baker and J. S. Lott, J. Bacteriol., 2006,
235 P. Dorrestein and T. P. Begley, Bioorg. Chem., 2005, 33, 136– 188, 6081–6091.
148. 260 Z. Dumas, A. Ross-Gillespie and R. Kummerli, Proc. Biol.
236 P. C. Dorrestein, K. Poole and T. P. Begley, Org. Lett., 2003, Sci., 2013, 280, 20131055.
5, 2215–2217. 261 D. L. Pruess, J. P. Scannell, M. Kellett, H. A. Ax, J. Janecek,
237 P. Nadal-Jimenez, G. Koch, C. R. Reis, R. Muntendam, T. H. Williams, A. Stempel and J. Berger, J. Antibiot., 1974,
H. Raj, C. M. Jeronimus-Stratingh, R. H. Cool and 27, 229–233.
W. J. Quax, J. Bacteriol., 2014, 196, 2681–2690. 262 J. P. Scannel, D. L. Pruess, T. C. Demny, L. H. Sello and
238 M. T. Ringel, G. Drager and T. Bruser, J. Biol. Chem., 2016, T. Williams, J. Antibiot., 1972, 25, 122–127.
291, 23929–23938. 263 X. Lee, M. D. Azevedo, D. J. Armstrong, G. M. Banowetz and
239 E. J. Drake and A. M. Gulick, Acta Crystallogr., Sect. F: Struct. C. Reimmann, Environ. Microbiol. Rep., 2013, 5, 83–89.
Biol. Commun., 2016, 72, 403–408. 264 H. Gehring, R. R. Rando and P. Christen, Biochemistry,
240 Z. Yuan, F. Gao, G. Bai, H. Xia, L. Gu and S. Xu, Biochem. 1977, 16, 4832–4836.
Biophys. Res. Commun., 2017, 484, 195–201. 265 R. R. Rando, Nature, 1974, 250, 586–587.
241 D. W. Reid, G. J. Anderson and I. L. Lamont, Am. J. Physiol.: 266 J. Lee, J. Wu, Y. Deng, J. Wang, C. Wang, J. Wang, C. Chang,
Lung Cell. Mol. Physiol., 2009, 297, L795–L802. Y. Dong, P. Williams and L. H. Zhang, Nat. Chem. Biol.,
242 A. F. Konings, L. W. Martin, K. J. Sharples, L. F. Roddam, 2013, 9, 339–343.
R. Latham, D. W. Reid and I. L. Lamont, Infect. Immun., 267 N. Rojas Murcia, X. Lee, P. Waridel, A. Maspoli, H. J. Imker,
2013, 81, 2697–2704. T. Chai, C. T. Walsh and C. Reimmann, Front. Microbiol.,
243 E. T. Granato, F. Harrison, R. Kummerli and A. Ross- 2015, 6, 170.
Gillespie, Front. Microbiol., 2016, 7, 1952. 268 X. Lee, A. Fox, J. Sufrin, H. Henry, P. Majcherczyk, D. Haas
244 F. Imperi, F. Massai, M. Facchini, E. Frangipani, and C. Reimmann, J. Bacteriol., 2010, 192, 4251–4255.
D. Visaggio, L. Leoni, A. Bragonzi and P. Visca, Proc. Natl. 269 R. P. Hausinger, Crit. Rev. Biochem. Mol. Biol., 2004, 39, 21–68.
Acad. Sci. U. S. A., 2013, 110, 7458–7463. 270 M. Schuster, C. P. Lostroh, T. Ogi and E. P. Greenberg, J.
245 P. Cornelis and J. Dingemans, Front. Cell. Infect. Microbiol., Bacteriol., 2003, 185, 2066–2079.
2013, 3, 75. 271 V. E. Wagner, D. Bushnell, L. Passador, A. I. Brooks and
246 P. Cornelis, Appl. Microbiol. Biotechnol., 2010, 86, 1637– B. H. Iglewski, J. Bacteriol., 2003, 185, 2080–2095.
1645. 272 S. Chugani, B. S. Kim, S. Phattarasukol, M. J. Brittnacher,
247 C. D. Cox, K. L. Rinehart Jr, M. L. Moore and J. C. Cook Jr, S. H. Choi, C. S. Harwood and E. P. Greenberg, Proc. Natl.
Proc. Natl. Acad. Sci. U. S. A., 1981, 78, 4256–4260. Acad. Sci. U. S. A., 2012, 109, E2823–E2831.
View Article Online
273 P. Bijtenhoorn, H. Mayerhofer, J. Muller-Dieckmann, 281 M. J. Jaremko, D. J. Lee, S. J. Opella and M. D. Burkart, J. Am.
C. Utpatel, C. Schipper, C. Hornung, M. Szesny, S. Grond, Chem. Soc., 2015, 137, 11546–11549.
A. Thurmer, E. Brzuszkiewicz, R. Daniel, K. Dierking, 282 S. de Bentzmann, C. Giraud, C. S. Bernard, V. Calderon,
H. Schulenburg and W. R. Streit, PLoS One, 2011, 6, e26278. F. Ewald, P. Plesiat, C. Nguyen, D. Grunwald, I. Attree,
274 Q. Yan, B. Philmus, J. H. Chang and J. E. Loper, eLife, 2017, K. Jeannot, M. O. Fauvarque and C. Bordi, PLoS Pathog.,
6, e22835. 2012, 8, e1003052.
275 P. Salunkhe, C. H. Smart, J. A. Morgan, S. Panagea, 283 A. Zaborin, S. Gerdes, C. Holbrook, D. C. Liu, O. Y. Zaborina
M. J. Walshaw, C. A. Hart, R. Geffers, B. Tummler and and J. C. Alverdy, PLoS One, 2012, 7, e34883.
C. Winstanley, J. Bacteriol., 2005, 187, 4908–4920. 284 W. Y. Liu, C. F. Wong, K. M. Chung, J. W. Jiang and
276 I. Kukavica-Ibrulj, A. Bragonzi, M. Paroni, C. Winstanley, F. C. Leung, PLoS One, 2013, 8, e74487.
F. Sanschagrin, G. A. O'Toole and R. C. Levesque, J. 285 A. Paauw, M. P. Caspers, F. H. Schuren, M. A. Leverstein-
Bacteriol., 2008, 190, 2804–2813. van Hall, A. Deletoile, R. C. Montijn, J. Verhoef and
277 X. Huang, A. Yan, X. Zhang and Y. Xu, Gene, 2006, 376, 68– A. C. Fluit, PLoS One, 2008, 3, e3018.
78. 286 S. Ohad, C. Block, V. Kravitz, A. Farber, S. Pilo, R. Breuer
278 B. Nowak-Thompson, N. Chaney, J. S. Wing, S. J. Gould and and E. Rorman, J. Appl. Microbiol., 2014, 116, 1315–1321.
J. E. Loper, J. Bacteriol., 1999, 181, 2166–2174. 287 Y. Ren, Y. Ren, Z. Zhou, X. Guo, Y. Li, L. Feng and L. Wang,
279 M. G. Thomas, M. D. Burkart and C. T. Walsh, Chem. Biol., J. Bacteriol., 2010, 192, 2463–2464.
2002, 9, 171–184. 288 H. Hoffmann, S. Stindl, W. Ludwig, A. Stumpf, A. Mehlen,
280 P. C. Dorrestein, E. Yeh, S. Garneau-Tsodikova, D. Monget, D. Pierard, S. Ziesing, J. Heesemann,
N. L. Kelleher and C. T. Walsh, Proc. Natl. Acad. Sci. U. S. A. Roggenkamp and K. H. Schleifer, J. Clin. Microbiol.,
A., 2005, 102, 13843–13848. 2005, 43, 3297–3303.

Nonribosomal Peptide Synthetase Biosynthetic Clusters of ESKAPE Pathogens

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Nonribosomal Peptide Synthetase Biosynthetic Clusters of ESKAPE Pathogens

Uploaded by

Copyright:

Available Formats

Natural Product

Nonribosomal peptide synthetase biosynthetic

Natural Product Reports Review

6. Acinetobacter baumannii 8. Enterobacter spp

Review Natural Product Reports

Natural Product Reports Review

Review Natural Product Reports

Natural Product Reports Review

Review Natural Product Reports

Table 1 NRPS clusters within Enterococcus faeciuma

Locus ID (accession) Length (AA) Predicted function

Strain KAC16100 (Genbank Accession NZ_LDNJ01000053)

Strain KAC16100 (Genbank Accession NZ_LDNJ01000090)

ACH93_RS12665 (WP_047937941) 346 Hypothetical

Natural Product Reports Review

the regulation of a variety of genes encoding proteins involved

Review Natural Product Reports

5.1. Enterobactin utilizing enzymes converts chorismate to the aryl moieties

Natural Product Reports Review

chain (Fig. 5). The nal molecule is assembled from building

and Y. pestis are greater than 95%, we again describe the

Review Natural Product Reports

Natural Product Reports Review

Review Natural Product Reports

the condensation domain of BasB.197 The combined activity of

Natural Product Reports Review

NRPS operate with a series of both intra- and intermolecular

to form the oxazoline ring. The module in FbsF would the be

Review Natural Product Reports

Given the unknown identity of the product of this NRPS cluster,

Natural Product Reports Review

Review Natural Product Reports

pyoverdine are the catechol oxygens of the chromophore and

Natural Product Reports Review

chelin module located on the PchF, to result in the formation of

Review Natural Product Reports

adenylation domain of PA14_54940 will adenylate the substrate,

Natural Product Reports Review

the mature Ala–AMB–Ala tripeptide (21) was identied cova-

Review Natural Product Reports

Table 3 Uncharacterized NRPS clusters of P. aeruginosa

Locus ID (accession) Length (AA) Predicted function

Strain PA01 (Genbank Accession AE004091.2)

Strain PA01 (Genbank Accession AE004091.2)

PA3327 (NP_252017) 2352 C–A–T–C–A–T–Te

Strain PA01 (Genbank Accession AE004091.2)

Strain PA7 (Genbank Accession CP000744)

Natural Product Reports Review

Review Natural Product Reports

Natural Product Reports Review

69 A. M. Gulick, V. J. Starai, A. R. Horswill, K. M. Homick and 34, 194–216.

Review Natural Product Reports

Natural Product Reports Review

Review Natural Product Reports

Am. Chem. Soc., 2014, 136, 9244–9247. 638.

Natural Product Reports Review

230 Y. Kim and W. G. Hol, Chem. Biol., 2001, 8, 1253–1264. 21768–21775.

Review Natural Product Reports

You might also like