Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004? 2004542287290Short CommunicationModulation of AraC proteinsG. V.

Plano

Molecular Microbiology (2004) 54(2), 287–290 doi:10.1111/j.1365-2958.2004.04306.x

MicroCommentary

Modulation of AraC family member activity by

protein ligands
Gregory V. Plano* regulation of T3SS genes and then focus on the role that
Department of Microbiology and Immunology, University specific protein interactions play in controlling the activity
of Miami School of Medicine, Miami, FL 33136, USA. of these proteins, including the novel interactions
described by Ha et al. (2004) in this issue and those
described by Dasgupta et al. (2004).
Summary
Members of the AraC family of transcriptional regulators
A number of AraC family transcriptional activators are defined by a 100 amino acid segment of sequence
bind low-molecular-weight ligands that modulate the similarity that corresponds to a DNA binding domain that
activity of these proteins. Recently, it has become contains two helix–turn–helix DNA binding motifs (Martin
clear that the activity of several virulence-related and Rosner, 2001). A few members of the AraC family
AraC family members is regulated through the direct contain only the aforementioned DNA binding domain;
interaction of protein ligands. These interactions, however, most AraC proteins contain both a C-terminal
in general, function to activate or repress the tran- DNA binding domain and an N-terminal dimerization and/
scription of virulence genes in response to specific or ligand binding domain. Several AraC proteins use DNA
extracellular stimuli. The identification and character- looping to repress transcription, a mechanism that was
ization of several protein ligands that modify the first demonstrated for AraC itself. Binding of arabinose to
activity of AraC family members in Pseudomonas AraC dimers converts AraC from a repressor that loops
aeruginosa and Salmonella enterica are discussed DNA between distant AraC binding sites to a transcrip-
herein. tional activator that binds to two adjascent sites. Although
not all AraC family members use DNA looping, the pres-
ence or absence of a bound ligand may still modify the
Virulence-associated type III secretion systems (T3SSs)
function of the regulator. For example, the binding of urea
are found in several genera of Gram-negative bacteria
to UreR, an AraC family protein that controls transcription
that directly interact with animal or plant cells. Bacteria
of the urease operon in Providencia stuartii, significantly
use these complex macromolecular machines to inject
increases the affinity of UreR for its DNA binding site
antihost proteins, termed effectors, directly into eukaryotic
(Gendlina et al., 2002).
cells (see Plano et al., 2001 for a review). Injected effector
Signalling molecules of AraC family members are nor-
proteins, in general, function to disrupt or activate host cell
mally small compounds such as sugars that directly bind
processes in a manner that benefits the pathogen at the
to an AraC family regulator and modulate its activity. How-
host’s expense. Expression of a functional T3SS is an
ever, Darwin and Miller (2001) identified a protein (a type
energy expensive proposition for a bacterial cell; thus,
III secretion chaperone) that directly interacts with an
T3SS genes are tightly regulated in response to specific
AraC transcriptional activator and is required for efficient
environmental signals (Francis et al., 2002). The environ-
transcription of select T3SS genes. This study provides
mental signals that control the expression of T3SS genes
the first evidence that large molecules such as proteins
and the complexity of the regulatory networks that
may function as activators of AraC family members. Con-
respond to these signals vary widely among pathogens
versely, work presented by Ha et al. (2004) and Dasgupta
that employ these systems. Not surprisingly, members of
et al. (2004) demonstrate that proteins can also directly
the AraC family of transcriptional activators play a central
interact with AraC family members in a manner that inhib-
role in many of these regulatory networks. Here I will
its transcription. Together, these findings indicate that pro-
briefly discuss the role of AraC family members in the
tein interactions play an important role in influencing the
activity of a number of virulence-related AraC transcrip-
tional activators.
Accepted 8 July, 2004. *For correspondence. E-mail
gplano@med.miami.edu; Tel. (+1) 305 243 6310; Fax (+1) 305 243 For the purpose of this MicroCommentary I will focus
4623. on T3SSs from two pathogens that employ diverse strat-

© 2004 Blackwell Publishing Ltd

288 G. V. Plano

A B

ExsA PrtA
Cu2+ PrtA Protein X sopE sigD

(+) SipC

ExsA (+) TTSS genes NC …

…
SPI-1
SicA
InvF (+) TTSS genes NC
ExsD
SicA …
ExsA ExsD Protein X
ExsC SicA
SipC
SicA
InvF

ExsD ExsC

anti-activator anti-anti-activator

Fig. 1. T3SS gene regulation by AraC family transcriptional activators ExsA and InvF.
A. Model for the regulation of P. aeruginosa T3SS genes by ExsA. Prior to activation of the secretion process the cytosolic anti-activator ExsD
directly interacts with ExsA and inhibits ExsA-dependent transcription. The cytosolic anti-anti-activator ExsC, a putative secretion chaperone, is
sequestered by Protein X, a hypothetical secreted protein. Contact with an eukaryotic cell triggers secretion of Protein X, freeing ExsC to interact
with ExsD. Sequestration of ExsD allows ExsA-dependent transcription of T3SS genes. In the presence of extracellular Cu2+, the PrtA protein is
expressed and directly interacts with ExsA leading to a complete inhibition of T3SS gene transcription. NC, needle complex.
B. Model for the regulation of S. enterica T3SS genes by InvF. SicA is a type III chaperone that is required for efficient secretion of SipC. Contact
with an eukaryotic cell triggers SipC secretion. Free SicA directly interacts with InvF and functions with InvF to activate transcription of a subset
of SPI-1 T3SS genes and several effector-encoding genes (sopE and sigD) located outside of SPI-1. Active forms of ExsA and InvF are
represented by the shaded circles. Transcriptional activation is represented by the thick solid arrows. Identified protein interactions are indicated
by a thick solid line. Dashed lines indicate events that occur after activation of the secretion process.

egies to regulate T3SS gene transcription. These systems
are: (i) the T3SS of Pseudomonas aeruginosa and (ii) the
Salmonella enterica SPI-1 T3SS. In both of these T3SSs,
the activation of gene transcription appears to occur in two
stages, each in response to distinct environmental signals
(Fig. 1). In the first stage the bacterial cell encounters the
appropriate environmental cues that trigger transcription
of T3SS genes. The end result of this first stage of tran-
scriptional activation is the expression of an assembled,
yet inactive T3SS. The second stage of transcription is
triggered by signals that activate the type III secretion
process. The triggering of secretion results in a concom-
itant increase in T3SS gene transcription. This feedback
control mechanism ensures adequate expression of T3SS
components and substrates for sustained secretory activ-
ity. Although both of the aforementioned T3SSs employ a
similar two-tiered regulatory scheme, the molecular mech-
anism by which each of these organisms regulates tran-
scription are quite unique (Fig. 1).
The P. aeruginosa T3SS is highly homologous to the
plasmid-encoded T3SS of the human pathogenic yersin-
iae (Plano et al., 2001). Likewise, both T3SSs employ a
single central AraC family transcriptional activator to reg-
ulate transcription of T3SS genes (ExsA in P. aeruginosa
and VirF in the enteropathogenic yersiniae). These pro-
teins, which share 56% amino acid sequence identity,
both possess a predicted C-terminal DNA binding domain
(the defining feature of the AraC family) and an N-terminal
domain of unknown function. P. aeruginosa strains defi-
cient in expression of ExsA fail to express T3SS genes.
The environmental signals that induce the initial
transcription of P. aeruginosa T3SS genes are not well-
defined, though the expression of these genes is not
constitutive. The report by Ha et al. (2004) in this issue illus-
trates one mechanism that P. aeruginosa uses to regulate
expression of T3SS genes. This paper describes the iden-
tification and initial characterization of a small 67 residue
protein, PrtA, which directly interacts with ExsA and in
doing so blocks ExsA-dependent transcription. Evidently,
PrtA functions to repress ExsA-dependent transcription of
T3SS genes under conditions that trigger PrtA expression.
The prtA locus was initially identified during a screen for
promoters that were specifically induced in a mouse skin
burn infection model. Subsequently, Ha et al. (2004) dem-
onstrated that expression of PrtA was specifically induced
by extracellular copper, and required the copper respon-
sive CopR-CopS two-component regulatory system. Thus,
PrtA functions, at least in part, to block expression of the
T3SS in the presence of extracellular copper.
The rationale for blocking T3SS gene transcription in
the presence of extracellular copper is not readily appar-
ent. The authors hypothesize that the energy expensive
T3SS is shut down during copper stress, and perhaps
during other cellular stresses, to allow the cell to better
cope with these life-threatening situations. This idea is
further supported by a recent publication by Rietsch et al.
(2004), which shows that the metabolic state of the bac-
terial cell directly influences the transcription of T3SS
genes in P. aeruginosa. Conversely, extracellular copper
released from eukaryotic cells could serve as an indicator

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 54, 287–290


of host cell lysis or tissue necrosis, essentially signalling
that the T3SS is no longer required for host cell intoxica-
tion. Finally, expression of the T3SS may be shut down in
the presence of extracellular copper simply because cop-
per cations may block the function of the calcium-
regulated T3SS. Regardless of its ultimate function, PrtA
provides the bacterial cell a mechanism to directly down-
regulate the activity of ExsA in response to an extracellu-
lar signal (Fig. 1A).
In contrast to the relatively straightforward regulatory
circuits currently appreciated in P. aeruginosa, the regu-
lation of S. enterica SPI-1 T3SS genes involves a complex
regulatory cascade that includes at least four AraC family
members (RtsA, HilC, HilD and InvF) and one OmpR
family member (HilA) (Lostroh and Lee, 2001). Further-
more, regulation of these T3SS-associated transcription
factors involves a complex web of additional regulatory
circuits that respond to a number of environmental sig-
nals. Environmental conditions that maximize T3SS gene
expression include high osmolarity, low aeration and neu-
tral pH. These conditions induce the bacterial cell to
express and assemble the type III secretion machinery;
however, secretion of effector proteins is not initiated until
a specific extracellular signal, such as contact with an
eukaryotic cell, is encountered. The initiation of secretion
immediately imposes new demands for secretion sub-
strates and accessory proteins. Hence, the SPI-1 T3SS
and most other T3SSs have evolved a feedback mecha-
nism to upregulate transcription of T3SS genes upon acti-
vation of the secretion process. Interestingly, the
molecular mechanisms used to coordinate secretion and
transcription in the various T3SSs are markedly different
(Fig. 1).
Activation of the P. aeruginosa T3SS triggers enhanced
transcription of essentially all ExsA-dependent genes.
Dasgupta et al. (2004) show that P. aeruginosa uses a
unique anti-activator and anti-anti-activator mechanism to
couple ExsA-dependent T3SS gene transcription to the
secretion process (Fig. 1A). ExsD is a cytosolic anti-
activator that, like PrtA, directly interacts with ExsA and
inhibits ExsA-dependent transcription prior to activation
of the secretion process (McCaw et al., 2002). It appears
that PrtA and ExsD represents the first protein ligands
described that directly block the function of an AraC family
transcriptional activator. ExsC, on the other hand, is a
cytosolic anti-anti-activator that blocks ExsD function after
secretion is triggered (Dasgupta et al., 2004). The mech-
anism by which ExsC anti-anti-activator activity is coordi-
nated with secretion has not been established; however,
Dasgupta et al. (2004) point out that ExsC possesses
many of the characteristics of a type III secretion chaper-
one. This suggests that prior to the activation of secretion
ExsC is sequestered by an as yet unidentified T3SS sub-
strate (Protein X). Secretion of Protein X liberates ExsC
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 54, 287–290
Modulation of AraC proteins 289
which then interacts with the anti-activator ExsD. Seques-
tration of ExsD by ExsC allows ExsA to actively transcribe
T3SS genes.
Expression of a subset of S. enterica T3SS genes is
also linked to the type III secretion process through an
AraC family member (InvF) and a type III secretion chap-
erone (SicA) (Darwin and Miller, 2000; 2001) (Fig. 1B).
SicA, the type III chaperone for SipB and SipC, directly
interacts with InvF. The binding of SicA to InvF does not
affect InvF stability; furthermore, SicA does not appear to
directly bind DNA. Thus, SicA appears to function as a
protein coactivator for InvF that is essential for efficient
transcription of genes encoding a number of secreted
effector proteins, including several proteins encoded out-
side of the SPI-1 pathogenicity island. Limited expression
of InvF–SicA-dependent genes occurs even in the
absence of a functional T3SS indicating that secretion per
se is not essential for expression of these genes.
The identification of multiple proteins (SicA, ExsD and
PrtA) that influence T3SS gene transcription through
direct interaction with AraC family proteins suggests that
this type of regulation may be common in T3SSs. Indeed,
recent evidence demonstrates that both IpgC, a type III
chaperone, and MxiE, an AraC family transcriptional acti-
vator, are required for efficient transcription of specific
T3SS genes in Shigella flexneri (Mavris et al., 2002). Like-
wise, the Yersinia enterocolitica chromosomal Ysa T3SS
contains a chaperone (SycB) homologous to IpgC and
SicA and an AraC regulator (YsaE) homologous to InvF
and MxiE. Not surprisingly, recent studies confirm that
both YsaE and SycB are required for efficient transcription
of select Ysa T3SS genes (Walker and Miller, 2004). In
the human pathogenic yersiniae, two secreted proteins,
YopD and YscM and their respective chaperones, SycD
and SycH, are required to prevent VirF-dependent tran-
scription prior to the triggering of secretion. In addition to
type III chaperones, other proteins that directly interact
with AraC family proteins, such as PrtA, may be present
in the chromosome of pathogens that employ T3SSs.
Indeed, direct protein–protein interactions may be a com-
mon means for bacteria to regulate virulence-related AraC
family proteins and perhaps other AraC family proteins.
The molecular mechanisms by which the identified pro-
tein interactions regulate AraC family member activities
are unknown. Darwin and Miller (2001) have identified an
InvF consensus binding sequence and located potential
InvF binding sites upstream of three InvF–SicA regulated
genes. They also show that InvF binds DNA in the pres-
ence or absence of SicA in vitro. In contrast, SicA does
not bind DNA and is not required for InvF stability. The
authors hypothesize that binding of SicA to InvF may alter
the conformation of InvF in a manner that promotes a
productive interaction with RNA polymerase. Alternatively,
the InvF–SicA complex may have an increased affinity for
290 G. V. Plano
InvF DNA binding sites. Another possibility is that SicA,
which functions as a homodimer, may directly facilitate
InvF homodimer formation. The stoichiometry of the InvF–
SicA complex is not known: however, many AraC proteins
are active as homodimers. In contrast to SicA, the inter-
action of either PrtA or ExsD with ExsA blocks ExsA-
dependent transcription. Binding of these proteins to ExsA
may alter the conformation of the regulator in a manner
that decreases its affinity for DNA binding sites or blocks
productive contacts with RNA polymerase. Alternatively,
interaction of these proteins with ExsA may prevent ExsA
dimerization.
In conclusion, the studies by Darwin and Miller (2001),
Ha et al. (2004) and Dasgupta et al. (2004) demonstrate
that transcriptional activation by AraC family members
can be regulated through the binding of protein ligands.
These interactions can lead either to transcriptional acti-
vation or to an inhibition of transcription. Further studies
are required to answer key questions that remain and,
without doubt, are ongoing. These studies will include
determining the molecular mechanism by which these
protein ligands either promote or inhibit transcription. In
particular, DNA footprinting studies will be required to
map Protein–DNA contacts in the presence and absence
of the protein ligands. Further analysis of these protein
interactions will include mapping the domain and amino
acid residues involved in binding the protein ligands and
identifying any conformational changes induced by these
interactions. These types of studies will be of particular
interest for PrtA and ExsD that both directly interact with
ExsA. Do these unrelated proteins bind at the same site
on ExsA? Do they inhibit transcription by the same mech-
anism? The study of these proteins, their interactions and
their effect on transcription of T3SS genes will likely lead
to the discovery of novel means of genetic regulation and
further our knowledge of the AraC family of transcriptional
regulators.
Acknowledgements
I thank Ken Fields and Lisa Plano for their excellent com-
ments on the manuscript. I also acknowledge helpful discus-
sions with George Munson on AraC family proteins.
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 54, 287–290
Research in my laboratory is supported by the National Insti-
tutes of Health.
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