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Bacterial nucleoid-associated
proteins, nucleoid structure and
gene expression
Shane C. Dillon and Charles J. Dorman
Abstract | Emerging models of the bacterial nucleoid show that nucleoid-associated
proteins (NAPs) and transcription contribute in combination to the dynamic nature of
nucleoid structure. NAPs and other DNA-binding proteins that display gene-silencing and
anti-silencing activities are emerging as key antagonistic regulators of nucleoid structure.
Furthermore, it is becoming clear that the boundary between NAPs and conventional
transcriptional regulators is quite blurred and that NAPs facilitate the evolution of novel
gene regulatory circuits. Here, NAP biology is considered from the standpoints of both gene
regulation and nucleoid structure.

Stable RNA genes

Genes that code for ribosomal
RNA (rRNA) and tRNA.
Compared with mRNA, rRNA
and tRNA are chemically very
stable.
Department of Microbiology,
School of Genetics and
Microbiology, Moyne
Institute of Preventive
Medicine, Trinity College,
Dublin 2, Ireland.
Correspondence to C.J.D.
e‑mail: cjdorman@tcd.ie
doi:10.1038/nrmicro2261
Published online
8 February 2010
Despite differences in the cellular organization of
Eukarya, Bacteria and Archaea, it is important to remem-
ber that all three domains rely on DNA to store and rep-
licate genetic information. In each domain the genetic
material has to be organized for storage in ways that are
compatible with DNA replication, chromosome segrega-
tion and gene expression. Proteins that alter the shape
of the DNA to make it more compact and that have the
potential to influence transcription have been identified
in all three kingdoms of life. In eukaryotes these pro-
teins are known as histones, and their ability to influence
chromatin structure and transcription is understood in
considerable detail. Initially, this depth of understand-
ing influenced our appreciation of those proteins that
carry out analogous tasks in bacteria, leading them to
be referred to as ‘histone-like’ proteins1,2. However, this
term is becoming less appropriate as the distinct nature
of these bacterial proteins is revealed. They are now
collectively referred to as nucleoid-associated proteins
(NAPs), which more accurately reflects their cellular
location without implying that they have any structural
similarity to histones. Members of the NAP group are
numerous, diverse and unevenly understood, and the
current list is probably not exhaustive (TABLE 1). Archaeal
NAPs are less widely studied, although several have been
investigated in detail (TABLE 1).
Most NAPs possess DNA-binding activity and an abil-
ity to alter the trajectory of the DNA molecule (that is, the
direction taken by the DNA through three-dimensional
space) by bending, wrapping or bridging it. In addition,
many NAPs are able to influence transcription in either
a positive or negative manner. An appreciation of the
higher-order superstructures that link gene expression
and the architecture of the bacterial genome requires
an understanding of the basic organization of the
nucleoid (BOX 1).
Transcription and the nucleoid structure
Transcription and looped domain boundaries.
Experimental data show that there is a good correlation
between transcriptional activity and the number and sta-
bility of looped DNA domains in a particular region3,4,5.
Transcriptional activity varies with the physiological
state of the cell. Fast-growing bacteria have more activity,
especially at genes coding for ribosome components and
other parts of the translation machinery, and have more
looped DNA domains. When bacterial growth slows, as
happens during starvation or at the onset of the station-
ary phase of growth in batch culture, there is less tran-
scriptional activity and a corresponding relaxation in the
looped domain structure of the nucleoid (FIG. 1).
Among the most active promoters in Escherichia coli
are those of the stable RNA genes, in particular genes
coding for ribosomal RNA, which are encoded by seven
rRNA operons6. Expression of rRNA genes is controlled
by the stringent response, which is a mechanism that
stops the production of components of the translational
machinery under restrictive growth conditions. During
the stringent response, the alarmone guanosine tetra-
phosphate (ppGpp) and the polymerase-interacting
DnaK suppressor protein (DksA) facilitate a targeted
weakening of RNA polymerase activity at stringently

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Table 1 | Nucleoid-associated proteins of eukaryotes, archaea and bacteria

Protein or group DNA DNA DNA binding motif Molecular Native protomer refs
of proteins wrapping bridging bending mass
Eukaryotes
Core histones, Yes ND ND A ~10 bp periodic oscillation of AA/TT/TA 11–14 kDa Homodimer 115
H2A, H2B, H3 elements in-phase with each other and
and H4 out-of-phase with ~10 bp periodic GCs
Linker histones, ND Yes ND AT-rich DNA ~21 kDa Homodimer 116
H1 and H5
Smc ND Yes ND AT-rich DNA able to form secondary ~140 kDa Heterodimer (for example, 117
structures SMC1–SMC3)
Hmg ND ND Yes AT-tract sites 11–38 kDa Homodimer or heterodimer 118
(for example, HMG1–HMG2)
Euryarchaeota
Archaeal histones Yes ND ND (A/T)3NN(G/C)3NN ~7.5 kDa Homodimer or heterodimer 119
HMfA and HMfB
Lrp Yes Yes ND ND 15–22 kDa Homodimer 120
Alba ND Yes ND ND ~10 kDa Homotetramer 20
MC1 ND ND Yes AT-rich DNA ~10 kDa Homodimer 121
HU ND ND Yes ND ~10 kDa Homodimer 122
SMC ND Yes ND ND ~135 kDa Homodimer 123
Crenarchaeota
Lrp Yes Yes ND ND ~18 kDa Homodimer 124
Cren7 ND ND Yes ND ~7 kDa Monomer 125
Sul7d ND ND Yes ND ~7 kDa Monomer 126
Alba ND Yes ND ND ~10 kDa Homodimer or 127
homotetramer
SMC ND Yes ND ND ~100 kDa Homodimer 128
CC1 ND ND ND ND ~6 kDa Monomer 129
Gram-negative bacteria
HU Yes ND Yes A DNA structural motif in dsDNA joined ~9 kDa Heterodimer (for example, 42,
to either dsDNA or ssDNA, with a mild HUα–HUβ) 130
preference for AT-rich or curved DNA
Lrp Yes Yes ND (T/C)AG(A/T/C)A(A/T)ATT(A/T)T(A/T/G) ~18 kDa Homodimer 69
CT(A/G)
MukB ND Yes ND ND ~175 kDa Homodimer 57
Fis Yes Yes Yes A6 tracts and AT tracts ~11 kDa Homodimer 77,79
(helically
phased
sites)
H-NS ND Yes ND AT-rich DNA and TCGATAAATT ~15 kDa Homodimer or heterodimer 19
(H-NS–StpA)
IHF ND ND Yes (A/T)ATCAANNNNTT(A/G) ~11 kDa Heterodimer (IHFα– IHFβ) 44,45
Dps ND ND ND ND ~19 kDa Monomer or dodecamer 94
StpA ND Yes ND AT-rich DNA ~15 kDa Homodimer or heterodimer 29
(StpA–H-NS)
CbpA ND ND ND Curved DNA ~33 kDa Homodimer or heterodimer 92
(CbpA–CbpM)
CbpB ND ND ND Curved DNA ~33 kDa Monomer 102
EbfC ND Suggested ND GTNAC ~11 kDa Homodimer 93
MvaT ND Yes ND AT-rich DNA Homodimer 26
Gram-positive bacteria
MukB ND Yes ND Preference for ssDNA ~130 kDa Homodimer 131
Lrp Yes Yes ND ND ~17 kDa Homodimer 72
HU ND ND Yes ND ~10 kDa Homodimer 37
Lsr2 ND Yes ND AT-rich DNA ~12 kDa Homodimer 24
Hlp ND ND ND ND ~21 kDa Monomer 38
MrgA ND ND ND ND ~17 kDa Monomer or dodecamer 132
Alba, acetylation lowers binding affinity; CbpA, curved-DNA-binding protein A; CbpB, curved-DNA-binding protein B (also known as Rob); CbpM, chaperone
modulatory protein; Dps, DNA protection from starvation; dsDNA, double-stranded DNA; Fis, factor for inversion stimulation; Hlp, histone-like protein; Hmg,
high mobility group; H-NS, histone-like nucleoid-structuring; IHF, integration host factor; Lrp, leucine-responsive regulatory protein; MrgA, metalloregulation
DNA-binding stress protein; ND, not determined; Smc, structural maintenance of chromosome; ssDNA, single-stranded DNA.

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Box 1 | Organization of the bacterial nucleoid
Escherichia coli and its close relatives continue to provide the paradigm for our
understanding of chromosome storage in the nucleoid. The chromosome exists
in a space that is subject to molecular crowding, where entropic forces can influence
the path taken by the DNA. The DNA polymer adopts a self-avoiding random walk
characterized by a thermal persistence length (the distance traversed by the molecule
before it randomly takes a turn) of about 150 bp, equivalent to a distance of 50 nm.
This produces a cloud of randomly coiled DNA with a diameter of around 10 μm.
Subdivision of the chromosome into around 400 independent loops of ~10 kb provides
further compaction, and negative supercoiling of the loops further reduces the
diameter of the folded chromosome110.
Negative supercoiling of DNA is mainly a product of the activity of topoisomerases,
but it is also influenced at a local level by the activities of DNA polymerase and RNA
polymerase. Nucleoid-associated proteins and other DNA-binding proteins can
constrain supercoils, altering the level of effective DNA supercoiling in the
chromosome111. This decreases the superhelical density of the DNA in E. coli from a
value of –0.05 for naked DNA to –0.025 for the protein-bound molecule. This is of
considerable importance, because in many cases transcriptional promoter activity is
sensitive to the local effective level of supercoiling and the activity of promoters can
be increased or decreased in response to superhelical variation112.
controlled promoters 7,8. Experiments using RNA
polymerase tagged with GFP show the presence of so-
called ‘transcription factories’ (or ‘transcription foci’) in
bacteria undergoing rapid growth (FIG. 1). These struc-
tures become dispersed in bacteria in which the stringent
response is active but not in mutants deficient in RelA9,
a key regulator of this response7. Each cell possesses a
small number of these factories (on average, three)9,10.
However, given the observed correlation between high
transcription levels and domain maintenance, most of
the genes along the chromosome are expressed too infre-
quently or at too low a level to influence the domain
structure of the nucleoid. Therefore, one must consider
other elements with the potential to have an impact on
nucleoid architecture.
NAPs and looped domains. some NAPs have proper-
ties that could allow them to act as boundary elements
(that is, the structures that mark the beginning and
end of chromosomal domain loops), and there is evi-
dence from genetics and bioinformatics studies that
Superhelical density some NAPs do indeed serve this function11,12. NAPs
The average number of with DNA-bridging activity are especially attractive
superhelical turns per helical
candidates. chromatin immunoprecipitation-on-
turn of the DNA, usually
abbreviated as σ. chip (chIP-on-chip) studies have shown that the NAP
histone-like nucleoid-structuring protein (H-Ns)
Transcription factories binds AT-rich DNA throughout the genomes of
Foci in the nucleoid where E. coli 13,14 and Salmonella enterica15,16. The distribution
highly transcribed promoters,
such as those of the ribosomal
of these binding sites is consistent with the likely loca-
RNA genes, coalesce during tions of domain loop boundaries12. Factor for inversion
periods of high transcriptional stimulation (Fis), another NAP, also binds throughout
activity. the genome and, like H-Ns, is especially abundant at
intergenic sites 13. It has also been reported to form
DNA bridging
Pertaining to a protein that DNA–protein–DNA bridges17. The expression pattern
binds to two distinct DNA of Fis is in keeping with the dynamic nature of looped
molecules or different domain boundaries, in that Fis is most abundant in the
portions of the same DNA early exponential phase of growth, when the looped
molecule simultaneously
through two DNA-binding
domains are most plentiful. Both Fis and H-Ns target
domains, producing a promoters of the rRNA genes, perhaps indicating that
DNA–protein–DNA bridge. their bridging activities support superstructures such as
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transcription factories (FIG. 1). However, bacteria depleted
of NAPs, including H-Ns and Fis, can maintain their
nucleoid structure18, which contradicts the proposed
structural role for NAPs. Although this discrepancy may
reflect the limitations in the tools available for nucleoid
analysis at the required scale or a divergence between the
experimental and physiological conditions, it may also
suggest that additional factors could be important for
the maintenance of nucleoid structure, perhaps acting in
concert with NAPs to form domain boundaries.
Bridge builders. When considering the potential for
NAPs to engage in both nucleoid structuring and gene
regulation, it is useful to examine their physical impact
on the DNA to which they bind. The importance of the
DNA-bridging activity that is displayed by proteins such
as H-Ns in enteric bacteria and acetylation lowers bind-
ing affinity protein (Alba) in archaea has been reported
in several studies19,20,21. In these studies, the authors used
single-molecule techniques such as optical tweezers
to study DNA–protein–DNA bridges, allowing these
bridges to be assessed for their potential to influence
processes such as transcription initiation and nucleoid
architecture19. In vitro, bridge formation by H-Ns is sen-
sitive to Mg 2+, such that the protein oligomerizes along
linear duplex DNA in the absence of Mg 2+ and forms
DNA–protein–DNA bridges on the same DNA molecules
in the presence of 10 mM Mgcl2 (REF. 133). Bridge forma-
tion lends itself to the control of transcription, because
the bridges have the potential to trap RNA polymerase at
or exclude it from promoters, thereby silencing the genes
concerned. Furthermore, evidence from a range of stud-
ies has shown that H-Ns and RNA polymerase colocalize
at bacterial promoters, consistent with a trapping mecha-
nism13,22. It is thought that DNA–protein–DNA bridges
are repressive for transcription and must be disrupted
for gene expression to commence. It is becoming clear
that bridging is opposed by a wide range of mechanisms,
each of which is based on a type of interaction with DNA
that renders the bridges untenable21,23. Examples of such
anti-bridging activity include DNA bending and DNA
wrapping, which are activities that are associated with
most DNA-binding proteins (FIG. 2).
Dual-purpose NAPs
The number of bacterial proteins in the NAP class is
gradually increasing, and their classification is discussed
below (TABLE 1). NAPs contribute to both nucleoid struc-
ture and gene regulation, and they may perform both
roles simultaneously.
H-NS: genome guardian and universal repressor. Proteins
with an amino acid sequence related to that of H-Ns
are widely found in bacteria and have been studied in
pathogens such as Mycobacterium tuberculosis 24, Vibrio
cholerae25 and Pseudomonas aeruginosa26. A protein capa-
ble of complementing an H-Ns deficiency has also been
isolated in mice27. Perhaps it is conservation of function
rather than amino acid sequence that is crucial, and an
ability to form DNA–protein–DNA bridges may emerge
as the common feature of these different proteins.
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a Exponential phase of growth b Stationary phase of growth

RNA polymerase Transcription

H–NS Fis
at RNA promoters factories

Figure 1 | Nucleoid-associated proteins and DNA supercoiling influence nucleoid structure. a | The folded
chromosome is organized into looped domains that are negatively supercoiled during the exponential phase of growth.
In this phase, the abundant nucleoid-associated proteins histone-like nucleoid-structuring protein (H-NS) and factor for
Nature Reviews | Microbiology
inversion stimulation (Fis) bind throughout the nucleoid and are associated with the seven ribosomal RNA operons. As
shown here in two cases, these are organized into superstructures called transcription factories. b | In stationary phase the
rRNA operons are quiescent and Fis is almost undetectable. The chromosome has fewer looped domains, and those that
are visible consist of relaxed DNA.

Plectonemically
supercoiled DNA
DNA with a braided or
interwound appearance
caused by the addition or
removal of helical turns in
topologically closed
double-stranded DNA.
Both H-Ns and its paralogue stpA can constrain
supercoils in DNA28,29, and data from atomic-force micro-
scopy indicate that H-Ns reinforces duplex interwinding
in plectonemically supercoiled DNA30. Negative supercoiling
of the DNA is likely to facilitate the formation of DNA–
H-Ns–DNA bridges, accounting for the coincidence of
DNA-supercoiling sensitivity and H-Ns binding at many
bacterial promoters31. Bridging allows H-Ns to influence
both nucleoid structure and gene expression simultane-
ously. stpA has DNA-binding activity but is best charac-
terized as an RNA-binding protein and RNA chaperone32.
Because these proteins have similar oligomerization
domains (FIG. 3), stpA can interact with H-Ns to form
heteromers, as can other paralogues, such as the plasmid-
encoded sfh33. It has been suggested that the homomers
and heteromers exert distinct effects on transcription,
although supporting data are limited. However, data
from transcriptomics studies show that changes in the
populations of these multimeric forms of NAPs result in
new gene expression patterns34. The activity of H-Ns is
modulated through its interaction with members of the
haemolysin expression-modulating (Hha)/YdgT family
of proteins. These molecules lack DNA-binding activ-
ity of their own and are thought to bind to the oligomeri-
zation region in the amino-terminal domain of H-Ns,
influencing the genes that H-Ns regulates by concentrat-
ing its effects on horizontally acquired genes, including
pathogenicity genes with AT-rich DNA35 (FIG. 3). How
the Hha/YdgT proteins influence H-Ns interactions with
different promoters is currently unknown.
HU: regulator of DNA flexibility. The Hu protein has
two subunits, Huα and Huβ, that are 70% identical in
amino acid sequence. In E. coli, Hu exists in homodimer
or heterodimer forms, depending on the stage of growth
of the bacterium36. The equivalent protein in species
such as Geobacillus stearothermophilus exists only as a
homodimer 37. Perhaps this reflects the differences in the
ecological circumstances of the bacteria, as E. coli and its
close relatives need to adapt in a wider environmental
range. The equivalent protein in Mycobacterium spp.,
histone-like protein (Hlp), consists of two domains, with
the amino-terminal portion of the protein resembling
Hu from E. coli and the carboxy-terminal portion being
similar to the eukaryotic histone H1 (REF. 38). This is a
rare example of a bacterial NAP that is truly histone-like
in its amino acid sequence.
Hu–DNA interactions seem to be nonspecific, but the
protein has a preference for binding to distorted regions
of the DNA, such as bends or four-way junctions39. This
is consistent with the roles of Hu in recombination
and DNA topology management 40. Hu influences the
expression of a wide range of genes in E. coli, including
those involved in central metabolism and respiration41.
It interacts with topoisomerase I, leading to alterations
in the superhelicity of DNA40, which has implications
for both nucleoid structure and gene expression. X-ray
crystallography of Hu has revealed that the protein
can form multimers consisting of octameric units. In
one arrangement, these have the potential to form spi-
ral filaments onto which a negatively supercoiled DNA

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Binding site for
protein antagonist
H-NS
DNA bridging DNA coating
DNA wrapping DNA bending
H-NS displaced H-NS displaced
Figure 2 | Antagonistic DNA-binding modes of nucleoid-associated
Nature Reviewsproteins.
| Microbiology
Histone-like nucleoid-structuring protein (H-NS) binds to DNA and influences its
structure. This binding can result in DNA coating (top right), in which the protein binds
to two sites that are close together on the same strand, or DNA bridging (top left), in
which it binds to two sites that are further apart and causes the DNA between them
to form a loop. The integrity of the resulting bridge is compromised by the intervention
of a DNA-wrapping protein (green; bottom left) or by a DNA-bending protein (turquoise;
bottom right). The alternative interactions that result from DNA-binding of these
proteins make the DNA–protein–DNA bridge untenable, and H-NS becomes displaced.
can be wrapped in modelling experiments42, suggesting
a plausible mechanism by which Hu can influence the
organization of the nucleoid by directly guiding the path
of the DNA. However, evidence that this form of Hu is
the functional unit in vivo is lacking.
In addition to its influence on DNA superhelicity, Hu
contributes to DNA flexibility by bending the duplex.
Hu reduces the effective stiffness of DNA over short
distances at low protein concentrations, but it stiffens
DNA at high concentrations21. Hu-induced flexibility
facilitates DNA loop formation, which is important for
both gene regulation and the architecture of the chromo-
some43. Interestingly, H-Ns has the opposite effect, and
DNA loops form much more easily in bacteria that lack
this NAP43.
IHF and DNA U-turns. Integration host factor (IHF)
was described originally as a cofactor in the site-specific
recombination of bacteriophage-λ44. IHF is related to Hu
at the level of amino acid sequence (FIG. 3) but has a distinct
mode of interaction with DNA. Although Hu engages
in DNA wrapping in a sequence-independent manner,
IHF binds to a well-conserved nucleotide sequence and
DNA writhe
This approximates to our introduces a u-turn into the DNA, centred at the bind-
intuitive sense of DNA ing site45. In E. coli and related bacteria, IHF is composed
supercoiling. When helical of an α-subunit and β-subunit. The αβ heterodimer is
turns are added to or the predominant active form, although transcriptomics
subtracted from relaxed DNA,
the DNA adopts a minimum
data from Salmonella enterica subsp. enterica serovar.
energy state by winding the Typhimurium show that the αα and ββ forms are also
helical axis about itself. biologically active46. The IHF protein influences global
NATuRE REvIEWs | Microbiology
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REVIEWS
transcription in both E. coli 47 and S. Typhimurium46. IHF
has some of the characteristics of a conventional trans-
cription factor, being capable of recruiting σ54-containing
RNA polymerase to promoters48. Its DNA-bending activ-
ity can influence transcription by facilitating contact
between regulatory proteins and RNA polymerase49.
However, other mechanisms have also been described.
For example, the ilvPG promoter of the ilvGMEDA operon
of E. coli is activated by IHF through the release of free
energy that is normally stored in the IHF-binding site,
making this energy available to assist with the formation
of the open transcription complex at the promoter 50. IHF
quenches the tendency of the DNA at the IHF-binding
site to become single-stranded, transferring this tendency
to the next-nearest labile region, which in this case is the
target promoter. This is thought to occur through dis-
placement, by protein-mediated DNA bending, of the
torsional energy that arises from DNA twist in negatively
supercoiled DNA. When this displaced torsion is applied
to a neighbouring double-stranded DNA sequence that
is prone to base-pairing disruption, a single-stranded
‘bubble’ may form51. Any DNA segment that is prone
to the formation of single-stranded DNA may be vul-
nerable to supercoiling-induced duplex destabilization
(sIDD)51. The ability to displace sIDD is not restricted to
IHF; Fis performs a similar function at a stable RNA gene
promoter 52. However, the promoter concerned, that of
the leuV tRNA operon, is unusual in that it possesses
one binding site for Fis rather than three sites arranged
in-phase along the double helix52. other well-characterized
stable RNA gene promoters, such as that of the tyrT
tRNA gene, use a torsional transmission mechanism in
which DNA writhe is stabilized by Fis, bound at three
consecutive sites, and converted into DNA untwisting
at the promoter 30,53. Bioinformatic analysis has iden-
tified sIDD-prone regions at the promoters of E. coli
genes that respond to environmental stress, although
evidence that this is exploited as a global regulatory
mechanism is lacking.
IHF also affects chromosome replication initiation
at the chromosomal origin, oriC, as do Fis and Hu54,
and influences the timing of replication initiation at the
datA locus55. IHF is a component of several site-specific
recombination systems, and it affects transposition56.
IHF therefore has roles in nucleoid structuring, chro-
mosome replication and DNA rearrangements, in addi-
tion to its role in transcription. In all of these cases, the
primary role of IHF is in remodelling DNA at a local
level through the introduction of u-turns.
MukB and chromosome structural maintenance.
E. coli depends on the chromosome partition protein
MukB for efficient segregation of its daughter chro-
mosomes at cell division57. MukB-deficient mutants
exhibit chromosomal decondensation and fail to seg-
regate daughter chromosomes at cell division; sup-
pressor mutations correcting these phenotypes map
to topA, which encodes topoisomerase I, and result in
increased negative supercoiling 58. This implies that a
functional link exists between DNA supercoiling and
MukB activity. consistent with this, binding of MukB
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1 10 20 30 40 50 60 70 80 90 100 110 120 130

H-NS
Protein interaction Linker DNA binding
1 10 20 30 40 50 60 70 80 90 100 110 120 130
StpA
Protein interaction Linker DNA binding

1 10 20 30 40 50 60 70
Hha
Loop
α-helix Secondary
1 10 20 30 40 50 60 70
structure
YdgT β-sheet
Histone_HNS
1 10 20 30 40 50 60 70 80 90
HTH_8
HUα
Hha domain Pfam
domains
1 10 20 30 40 50 60 70 80 90 Bac_DNA binding
IHFα
AsnC_trans_reg
1 10 20 30 40 50 60 70 80 90
Fis

1 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

Lrp
Protein interaction Protein interaction

Figure 3 | Nucleoid-associated protein secondary structure and domain organization. The predicted secondary
structures113 and Pfam domain organizations114 of the best characterized nucleoid-associated proteins (NAPs) of Salmonella
enterica subsp. enterica serovar Typhimurium are shown. The secondary structures of the α-subunits of integration host
factor (IHF) and HU are identical to those of the β-subunits (not shown). The locations of α-helices, β-sheets and loop
regions show that structural modularity is a feature of related NAPs (for example, histone-like nucleoid-structuring
protein (H-NS) and StpA). Those proteins that have the same Pfam domain belong to a family of related proteins as
determined by Hidden Markov Model analysis of protein sequences114. Fis, factor for inversion stimulation; Hha,
haemolysin expression-modulating protein; Lrp, leucine-responsive regulatory protein.

to DNA results in the introduction of protein-stabilized
negative supercoils59. Furthermore, mutations in the
mukB gene alter DNA supercoiling in plasmids as well
as in the chromosome, which is consistent with a direct
role for this protein in modulating DNA topology 60.
MukB has also been implicated in the formation of inde-
pendent topological domains in the E. coli chromosome,
probably in association with DNA gyrase61.
Lrp: a physiological barometer. leucine-responsive
regulatory protein (lrp) influences the transcription of
~10% of the genes in E. coli and, depending on the target
gene, the activity of lrp can be potentiated, inhibited or
unaffected by leucine62. The lrp regulon includes genes
involved in nutrient uptake and amino acid metabolism.
lrp also has a role in microbial virulence, regulating
genes involved in the phase-variable expression of pili.
It binds to DNA at a degenerate consensus sequence63
and has a profound influence on the trajectory of the
bound DNA64.
lrp exists in variable oligomeric states: it can be
detected in dimeric, octameric and hexadecameric
forms, with leucine binding favouring the dissocia-
tion of the hexadecamer to the octamer 65. The DNA-
binding domain of lrp is located in the amino-terminal
portion of the protein, and in the multimers these
domains are on the surface66–69. lrp binding patterns
at genes that have been examined in detail reveal either
a series of approximately equally spaced lrp-binding
sites or a continuous region of around 100 bp that is
altered by lrp in its sensitivity to DNase I digestion64.
These data are consistent with a model in which lrp
octamers (or hexadecamers) bind and wrap the DNA.
The observation of regularly spaced sites of hyper-
sensitivity to DNase I is certainly consistent with DNA
wrapping by lrp70. In addition to bending and wrap-
ping the DNA, lrpc from Bacillus subtilis has been
reported to bridge DNA71,72. All of these features have
implications for the ability of lrp to influence nucleoid
structure and to regulate transcription.
lrp influences stable RNA expression in E. coli by
cooperating with H-Ns to repress rRNA operon tran-
scription73. The lrp gene is positively controlled by
ppGpp, probably in an indirect manner 74. This means
that lrp levels are highest at the end of the exponen-
tial phase of growth, when the demand for stable RNA
and other components of the translational machinery
is declining. This would correlate with the negative
action of lrp on rRNA transcription.
The versatile Fis protein. Fis has been studied
intensively from the perspective of both genome
organization and gene regulation. Fis activity has
a major impact on the transcriptomes of E. coli and

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Duplex slithering
In the absence of constraints
(for example, due to protein
binding), the DNA duplex is
free to migrate in the
plectonemically interwound,
supercoiled structure by
adopting a slithering motion.
NATuRE REvIEWs | Microbiology
S. Typhimurium75,76. It binds as a homodimer to a con-
sensus sequence that is usually 17 bp in length and
AT rich, except at positions 2 and 16, where G or c
residues are commonly found77. Dimeric Fis bends the
DNA at its binding site78, allowing DNA to bind to
the helix–turn–helix motif in each monomer 79. like
some of the other NAPs reviewed here, Fis contrib-
utes to many DNA-based cellular activities, including
DNA transcription, replication and recombination80.
Bioinformatic analysis has detected potential bind-
ing sites for Fis throughout the E. coli genome, and
chromatin immunoprecipitation data show that it does
indeed bind to many locations, especially those outside
the coding regions of genes13. This is consistent with
information from in silico work showing that Fis pre-
fers to bind to sequences that are commonly located at
transcription promoters81.
Fis can repress or activate promoters, depending
on the position of its binding site relative to that of
RNA polymerase. Fis represses transcription initia-
tion either by imposing a blockade at the target pro-
moter and excluding RNA polymerase or by a more
subtle mechanism, in which it modulates the RNA
polymerase-mediated isomerization of the closed
transcription complex to an open complex. This raises
the activation barrier to values that make transcription
initiation very unlikely 82,83.
Fis can also be a conventional transcription activa-
tor, making physical contact with RNA polymerase84. In
addition, it acts from binding sites located at a distance
from the promoter, through several distinct mecha-
nisms that involve local DNA topology. one mechanism
involves the displacement of DNA twisting from an
upstream site to the target promoter by Fis-mediated
DNA bending 52. This allows exploitation of sIDD at
the binding site through a mechanism similar to that
described above for IHF40. Another mechanism concerns
the preservation of DNA writhe through Fis binding at a
negatively supercoiled region of the duplex 85. This main-
tains a micro-domain of negatively supercoiled DNA at
the target promoter, sustaining transcription when DNA
elsewhere in the genome is becoming relaxed. This same
type of interaction may serve to impart structure to the
genome by impeding duplex slithering, an activity that is
also within the scope of IHF and H-Ns81.
There is an interesting correlation between Fis and
genes belonging to the stringent response regulon, of
which fis itself is a member 86. Fis activity is not restricted
to genes of the stringent response, however, and it can
affect transcription across the genome77,78. stringently
regulated promoters contain a Gc-rich discriminator
sequence downstream of the –10 position, and they
are sensitive to changes in DNA supercoiling. Many
of the stringently controlled promoters encode stable
RNA, and the relationships between the different fac-
tors that affect these promoters are complex and, in
some cases, controversial87. The involvement of Fis is
not straightforward, because the protein is implicated at
two levels, at the least. The first is direct, with Fis bind-
ing to sites upstream of the stable-RNA gene promoter.
The other is indirect and arises because Fis represses
© 2010 Macmillan Publishers Limited. All rights reserved
REVIEWS
the promoters of the genes encoding the A and B subu-
nits of DNA gyrase, which produces negative supercoil-
ing in DNA. Depending on growth conditions, Fis is
also capable of activating or repressing the transcription
of topA88. Regulating the expression of the two major
topoisomerases allows Fis to influence DNA supercoil-
ing globally, which has the potential to affect sensitive
promoters at the stable RNA genes. As discussed above,
it has also been suggested that Fis can preserve a nega-
tively supercoiled DNA micro-domain at the promoters
by binding to its sites there89.
An appreciation of the expression pattern of Fis is key
to understanding its physiological relevance as a tran-
scriptional regulator. The protein is maximally expressed
at entry to the exponential phase of growth, correlating
with the rise in demand for components of the transla-
tion apparatus to support rapid growth. Fis is therefore
well placed to stimulate and sustain the activity of the
promoters of the relevant genes. Fis negatively regulates
its own gene, and the concentration of the protein drops
to almost undetectable levels by the time the bacterium
enters the stationary phase. one of the consequences
of this drop in Fis levels is the removal of its inhibitory
control of the gene encoding RNA polymerase σ-factor
Rpos, the σ-factor that reprogrammes RNA polymerase
to transcribe genes required for adaptation to the cessa-
tion of growth90. Rpos also exerts a negative influence,
presumably indirectly, on the transcription of fis 91. In
this way, Fis, like other NAPs, influences the global tran-
scription pattern in response to changes in growth phase
and physiological state80.
CbpA. curved-DNA-binding protein (cbpA) is related
to the chaperone protein DnaJ but, unlike DnaJ, has
DNA-binding activity and has been grouped with the
NAPs. It is found in E. coli and many other bacteria, in
which it contributes to growth at low and high tem-
peratures and is required for normal cell division. It
forms a complex with chaperone modulatory protein
(cbpM), which inhibits its activity. cbpM is subject to
proteolysis by lon and clpAP, but only when it is not
in a complex with cbpA. The transcription of cbpA is
under the control of lrp and is modulated by leucine;
it is also controlled by Rpos92.
EbfC: a new type of NAP. Borrelia burgdorferi, the cause
of lyme disease, expresses Ebfc, which is a DNA bind-
ing protein that binds as a homodimer to a specific DNA
sequence and bends the DNA. The crystal structures of
Ebfc from B. burgdorferi and of the corresponding protein
from Haemophilus influenzae exhibit an unusual tweezer-
like motif that is required for DNA binding. Although
Ebfc is involved in gene regulation, the widespread distri-
bution of the consensus binding site in the DNA suggests
that it contributes to genome structuring 93.
Dps: an unusual NAP. Although DNA protection during
starvation protein (Dps), a ferritin-like protein, is usu-
ally grouped with the NAPs, evidence that it is directly
involved in transcriptional regulation is largely lack-
ing. The purified protein from E. coli has DNA-binding
voluME 8 | MARcH 2010 | 191
REVIEWS
activity 94 and is usually expressed in the stationary
phase, when the protein is thought to provide the DNA
with protection from damage. Transcription of dps in
the stationary phase requires Rpos and IHF94 and is
influenced directly by at least two other NAPs, Fis and
a the promoters of the genes encoding its paralogues, stpA
Lag phase–log phase Log phase–stationary
transition phase transition
Log cell number
H-NS
IHF
Fis
Time
b
Log phase
RpoS
H-NS
RpoD
–35 –10
Fis
Stationary phase
H-NS IHF
RpoS
–35 –10
Figure 4 | complex regulation of transcription by
multiple nucleoid-associated proteins. a | The expression
Nature Reviews | Microbiology
patterns of the four nucleoid-associated proteins (NAPs),
DNA protection from starvation protein (Dps), factor for
inversion stimulation (Fis), histone-like nucleoid-structur-
ing protein (H-NS) and integration host factor (IHF). A
typical bacterial growth curve is shown, with the lag
phase–log phase and log phase–stationary phase
transitions indicated. The expression curves of the
four NAPs102 are representational summaries. b | The
regulatory inputs at the promoter of dps. During the early
log phase of growth, Fis is abundant and binds to the dps
promoter, creating a repression complex that traps RNA
polymerase containing the RNA polymerase σ-factor
RpoD 82. H-NS has a negative influence at all stages of
growth. RNA polymerase containing the σ-factor RpoS
fails to gain access to the dps promoter as long as the
Fis–RpoD–promoter repression complex is intact. This
complex becomes less tenable as Fis concentrations fall
throughout the log phase and reach almost undetectable
levels with the onset of stationary phase. RNA polymerase
containing RpoS can then overcome the repressive
influence of H-NS; IHF then makes a positive contribution
to dps promoter function. Levels of Dps therefore increase
at the end of the exponential phase of growth.
192 | MARcH 2010 | voluME 8
© 2010 Macmillan Publishers Limited. All rights reserved
H-Ns (FIG. 4). It is also subject to indirect control through
Rpos, the expression of which is influenced by Fis; the
mRNA encoding Rpos also has a sensitivity at the post-
transcriptional level, along with hns mRNA, to the small
regulatory RNA dsrA and the RNA chaperone Hfq95.
The cross-regulation by IHF, Fis and H-Ns at the dps
promoter is not unique. For example, H-Ns represses
and sfh96, and hns is itself under the control of Fis, such
that Fis offsets the autorepressive action of H-Ns97. lrp
Dps
negatively autoregulates its own gene98 and is an activator
Protein concentration
of stpA, the transcription of which is strongly enhanced
in bacteria grown in minimal medium99.
Crp: the NAP–transcription factor interface. cyclic
AMP regulatory protein (crp; also known as catabolite
gene-activator protein (cAP)) has been traditionally
Growth
curve regarded as a conventional transcription factor 100. It is
active as a dimer and binds to a well-conserved DNA
sequence, the location of which at target promoters
allows the protein to act as a repressor or activator of
transcription. It requires a cofactor, cAMP, for binding
to DNA, and this provides a link between crp activity
and the physiology of the cell. crp was one of the first
gene-regulatory proteins to be studied in detail, and it
has been considered in the context of some paradig-
matic examples of transcription control, including the
lac operon of E. coli. To some extent, this may account
for the view that crp is a standard regulator of promoter
dps
activity. Nevertheless, it has been appreciated for many
years that crp controls a very large regulon of genes, and
recent chIP-on-chip studies have confirmed this view
and extended it by showing that crp binds to hundreds
of sites on the E. coli chromosome101. This has led to
the suggestion that crp has many of the characteristics
associated with NAPs101. like NAPs, crp is effective at
remodelling DNA owing to the introduction of a bend
dps
of approximately 90˚ at its binding site. The behav-
iour of crp and its wide distribution across the genome,
combined with its very precise interactions with RNA
polymerase when it acts as a transcriptional activator,
point to the existence of a spectrum of regulatory pro-
teins rather than independent categories with clearly
distinct properties.
Miscellaneous NAPs and NAP-like proteins. some
chromosome replication proteins have been described
as NAPs102, including DnaA, seqA and chromosome
initiation inhibitor (IciA). seqA binds to the GATc
sequences in DNA that are methylated or hemimethy-
layted in association with the DNA replication cycle,
and it contributes negatively to the control of chromo-
some replication initiation103. Mutants deficient in seqA
exhibit altered expression of genes with GATc motifs
in their promoter regions104. IciA and DnaA act with
mutual antagonism at oriC, with IciA seeking to inhibit
the initiation process105. IciA is a member of the lysR
family of transcription factors, a group that is not usu-
ally considered to include NAPs. on the other hand, the
lysR-like DNA-binding protein leuo has widespread
effects on the transcriptome of Gram-negative bacteria
www.nature.com/reviews/micro
 REVIEWS

and is an effective antagonist of H-Ns23. These proteins,
which have a preference for binding to AT-rich DNA, lie
along the spectrum between clear-cut examples of NAPs
and conventional transcriptional regulators. This spec-
trum may provide many opportunities for the evolution
and refinement of gene regulatory switches.
Concluding remarks
one of the features of NAPs that makes them excel-
lent regulators of gene expression at the global level is
their promiscuity in their interactions with DNA. NAP-
binding sites, discussed above, tend to be AT-rich, which
is a feature associated with promoters. Thus, NAPs are
DNA-binding proteins with requirements for binding
that tend to direct them to promoters. once they have
bound to a target promoter, their subsequent interac-
tions with that DNA have the potential to determine the
expression status of the gene concerned.
Bridging of DNA may be incompatible with DNA
wrapping or DNA bending, but all three activities are
associated with proteins in this class (FIG. 2). This provides
the basis for a simple genetic switch, whereby mutually
antagonistic activities between NAPs set the expression
state of the target gene. The variables in this relationship
include the availability of the antagonists. H-Ns is present
at an almost constant level per chromosome copy 106,
whereas its paralogues, stpA and sfh, show variation in
their levels in the cell during growth107. Fis is available
at high concentrations for only a portion of the growth
cycle; IHF also has a growth phase component in its
expression pattern, as does Hu, with the added variable
of its subunit composition. The expression pattern of Dps
is the reciprocal of that of Fis. Even without the added
sophistication of ligand binding or covalent modification
in response to environmental signals, this growth phase
dependency provides a basis for a crude but wide-ranging
global regulatory system. control of crp activity by cAMP
availability possibly represents a more advanced form of
regulation. This second messenger is a useful reporter
of cellular physiology, because its concentration varies as
the reciprocal of glucose concentration108. The cAMP–crp
system regulates virulence-gene expression in a number
of pathogens, highlighting the integrated nature of the
infectious and housekeeping functions of these cells and
the pivotal part that is played by cAMP–crp in both109.
In addition, it is important to note that in many cases
cAMP–crp acts in concert with or antagonistically to
other NAPs and transcription factors100. Furthermore,
the simple requirements of NAPs for DNA binding and
their mutually antagonistic activities may allow these pro-
teins to contribute to the process by which horizontally
acquired genes become integrated into the pre-existing
regulatory networks of the cell.
In the immediate future there is scope for more
work using imaging techniques to investigate the role
of NAPs as agents of nucleoid structure in the context of
DNA replication and transcription, with the intention
of obtaining a real-time picture of the nucleoid as it under-
goes its major molecular transactions. current knowl-
edge of NAP biology allows hypotheses to be formed
about the ways in which these proteins can contribute to
the evolution of gene regulation; these hypotheses need
to be tested in ways that take into account the nucleoid-
structuring roles of NAPs. By deepening our understand-
ing of these fundamental processes, we will enhance our
ability to manipulate bacteria to our benefit.

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Acknowledgements
We thank N. Ní Bhriain for critical comments on the manu-
script. Work in the authors’ laboratory is supported by grants
from Science Foundation Ireland and the Wellcome Trust.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
Entrez Genome Project: http://www.ncbi.nlm.nih.gov/
genomeprj
Bacillus subtilis | Borrelia burgdorferi | Escherichia coli |
Geobacillus stearothermophilus | Haemophilus influenzae |
Mycobacterium tuberculosis | Pseudomonas aeruginosa |
Salmonella enterica | Vibrio cholerae
UniProtKB: http://www.uniprot.org
CbpA | Crp | DksA | DnaA | Dps | EbfC | Fis | Hlp | H-NS | HUα |
HUβ | IciA | LeuO | Lrp | MukB | RelA | RpoS | SeqA | StpA
FURTHER INFORMATION
Author’s homepage:
http://www.tcd.ie/Microbiology/research/cj_dorman.php
Pfam: http://pfam.sanger.ac.uk
All liNks Are Active iN the oNliNe PDf
voluME 8 | MARcH 2010 | 195