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Annu. Rev. Microbiol. 2000. 54:499–518

Copyright
c 2000 by Annual Reviews. All rights reserved

FUNCTIONAL MODULATION OF
ESCHERICHIA COLI RNA POLYMERASE
Akira Ishihama
National Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka
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411-8540, Japan; e-mail: aishiham@lab.nig.ac.jp

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Key Words transcription apparatus, sigma factor, transcription factor,

stationary phase
■ Abstract The promoter recognition specificity of Escherichia coli RNA poly-
merase is modulated by replacement of the σ subunit in the first step and by interaction
with transcription factors in the second step. The overall differentiated state of ∼2000
molecules of the RNA polymerase in a single cell can be estimated after measurement
of both the intracellular concentrations and the RNA polymerase-binding affinities for
all seven species of the σ subunit and 100–150 transcription factors. The anticipated
impact from this line of systematic approach is that the prediction of the expression
hierarchy of ∼4000 genes on the E. coli genome can be estimated.

CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
MODULATION OF RNA POLYMERASE SPECIFICITY BY
REPLACEMENT OF THE σ SUBUNIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
SIGMA REPLACEMENT DURING GROWTH TRANSITION FROM
EXPONENTIAL TO STATIONARY PHASE . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
LEVEL AND ACTIVITY CONTROL OF SIGMA-S . . . . . . . . . . . . . . . . . . . . . . . 503
ACTIVITY CONTROL OF THE SIGMA SUBUNITS BY ANTI-SIGMA FACTORS 507
MODULATION OF RNA POLYMERASE SPECIFICITY BY
TRANSCRIPTION FACTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508
FACTORS AFFECTING SELECTIVE UTILIZATION OF
TRANSCRIPTION APPARATUS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
MULTIPLE PATHWAYS FOR STATIONARY-PHASE ADAPTATION . . . . . . . . . . 510
CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511

INTRODUCTION

The RNA polymerase of Escherichia coli is composed of the core enzyme (sub-
unit composition, α 2ββ’) with the catalytic activity of RNA polymerization, and
one of the seven different species of σ subunit, each responsible for recognition

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500 ISHIHAMA

of a specific set of promoters (28, 29, 31, 40). The total number of core enzyme
molecules in a growing E. coli cell is ∼2000 (38, 43), which is less than the total
number of genes (∼4000) on the E. coli genome (7). Together these findings ac-
centuate the importance of RNA polymerase to choose which genes to transcribe
and how often (39, 40). Because replacement of the σ subunit is the most efficient
way to alter the promoter recognition properties of the transcription apparatus,
the replacement of one σ species of core enzyme-associated σ subunit by another
is believed to be the major mechanism for switching of the transcription pattern.
Along this line, competition between available σ subunits should be a key deter-
minant of which group of genes is transcribed (22, 108; H Maeda, N Fujita, A
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Ishihama, submitted for publication). To test the σ competition model, quanti-

tative and comparative measurements of the intracellular concentrations and the
core enzyme-binding affinities of all seven σ subunits are absolutely required.
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The holoenzyme alone is able to recognize the simple promoters of genes that
are constitutively expressed and to initiate transcription at constant rates. Most of
the genes in bacteria are, however, subject to regulation in response to changes in
environmental conditions. Thus, for transcription of the majority of E. coli genes,
one or more additional accessory factors are required (39, 40). Among the same
group of genes under the control of a single σ species, the order of transcription
level is therefore determined by the promoter strength and the initiation efficiency,
assisted or inhibited by transcription factors. From the genome sequence of E. coli,
the total number of DNA-binding proteins that more or less influence transcription
can be estimated to be ∼240–260 (85; N Fujita, unpublished data). Most of these
DNA-binding proteins, which either activate or repress transcription of specific
genes, interact directly with the RNA polymerase and modulate its specificity
of transcription initiation (and elongation in some cases). The known transcrip-
tion factors can be classified into groups by their structure and mode of function
(40–42). Overall, therefore, the transcription specificity of RNA polymerase core
enzyme is modulated in two steps: by molecular interaction with the σ factors in
the first step and with the transcription factors, usually DNA bound, in the sec-
ond step. The expression hierarchy of the ∼4000 genes on the E. coli genome
must be determined to a great extent by the relative levels of transcription appara-
tus composed of core enzymes combined with different σ subunits and different
transcription factors. This review summarizes our up-to-date knowledge of such
functional differentiation of RNA polymerase in E. coli, focusing on its alteration
during the growth phase transitions of E. coli cultures.

MODULATION OF RNA POLYMERASE SPECIFICITY BY

REPLACEMENT OF THE σ SUBUNIT
Seven different species of σ subunits, σ 70, σ N (also called σ 54), σ S (σ 38), σ H (σ 32),
σ F (σ 28), σ E (σ 24), and σ FecI, have been identified in E. coli (28, 29, 31, 40), each
participating in transcription of a specific set of genes (Figure 1, see color insert).
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RNA POLYMERASE FUNCTIONAL MODULATION 501

Most of the housekeeping genes expressed during exponential-phase growth are

transcribed by the holoenzyme containing σ 70 (the rpoD gene product), while the
holoenzyme Eσ S is essential for transcription of some stationary-phase specific
genes (32, 65). Stress response genes are transcribed by RNA polymerase holoen-
zymes containing alternative minor σ subunits. The holoenzyme Eσ N transcribes
those genes, which are activated by a deficiency of nitrogen (70) and some other
stress response genes (90); the holoenzyme Eσ H transcribes the genes for heat
shock proteins (103); Eσ F is needed for expression of the third wave of flagellar
and chemotaxis genes (30); the holoenzyme Eσ E is responsible for transcription
of genes whose products deal with protein defects such as misfolded proteins in
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the periplasm, caused, for example, by heat shock (20, 81, 82, 87); and the fecI
gene product, which was originally identified as a positive regulatory gene for the
ferric citrate transport system (80), is now known to be a member of the extra-
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cytoplasmic function subfamily of σ factors (3, 67) based on its protein sequence
(hereafter referred to as σ FecI) and is involved in transcriptional activation of the
fec operon (19, 25).
The intracellular concentration of RNA polymerase in the steady state of grow-
ing E. coli W3350 cells is maintained at a constant level that is characteristic of
the rate of cell growth (38, 43). In a rich medium, the total number of core en-
zyme molecules is ∼2000 per genome equivalent of DNA, among which about
one third are disengaged from the DNA. Because RNA chain elongation is cat-
alyzed by core enzyme without an associated σ subunit, the combined number
(1200 molecules) of all seven σ subunits (46, 50) is more than the total number
(600–700 molecules) of core enzyme molecules that are not involved in tran-
scription and thus are available for binding σ (Figure 2, see color insert). The
majority of free RNA polymerase molecules in the cytosol should therefore be
in the holoenzyme form, associated with one of the σ subunits. These findings
support the model that competition takes place between the σ subunits for bind-
ing a limited supply of core enzyme. To estimate the relative levels of different
holoenzyme forms, two parameters must be determined: the intracellular concen-
trations of all seven σ subunits and the binding affinity of core enzyme to each σ
subunit.
The concentration of each σ subunit is subject to variation depending on the cell
growth conditions, although the concentration of core enzyme stays constant at a
level that is characteristic of the rate of cell growth (38, 43). The first systematic
determination of the intracellular concentrations of all seven σ subunits has been
performed for the laboratory strain W3110 type-A, which contains the intact forms
of both σ S and σ F (47). The results indicate that the intracellular concentration is
highest for the σ 70 subunit in both exponential and stationary phases and under vari-
ous stress conditions (46, 50, 68). In exponential-phase cells, two of the alternative
σ subunits, σ N and σ F, are present in significant concentrations, but, in addition,
the level of σ S becomes detectable in stationary phase (Figure 1, see color insert).
The core-binding affinities have been determined in vitro by measuring the
amount of core enzyme-bound σ subunit in the presence of various amounts of
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502 ISHIHAMA

each σ subunit and a fixed amount of core enzyme (H Maeda, N Fujita, A Ishi-
hama submitted for publication). Among the seven σ subunits from E. coli, σ 70
was found to have the highest affinity to the core enzyme. The affinities of the other
six σ subunits ranged downwards by 16-fold from σ 70 (0.26 nM) to σ S, which has
the weakest binding activity (4.28 nM) (Figure 2, see color insert). From these
two lines of experimental data, we can now estimate the intracellular concentra-
tion of each holoenzyme. The numbers of each free holoenzyme formed in an
E. coli cell during exponential growth are thus calculated to be 550 molecules
of Eσ 70, 95 molecules of Eσ F, and 55 molecules of Eσ N (Figure 2, see color
insert).
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SIGMA REPLACEMENT DURING GROWTH TRANSITION

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FROM EXPONENTIAL TO STATIONARY PHASE

Bacterial populations in nature often exist in the stationary phase or a state of
partial or complete starvation. The term stationary phase is used to denote a fixed
physiological state regardless of what factors led to growth cessation. The sta-
tionary phase is synonymous with the starvation for only an ideal case in which
the limiting nutrient leading to growth cessation can be specified, but even in
laboratory culture conditions the mechanism of cell growth cessation usually in-
volves multiple factors. Bacteria are capable of sensing the maximum cell density
(or “quorum”) and then can grow under a given condition and communicate this
to other members of the same species by production of extracellular signaling
molecules. This allows the single-celled prokaryotes to function in some respects
as if they are multicellular organisms (55, 86). After entry into stationary phase,
a number of genes, that are not expressed during exponential phase are expressed
in a sequential manner (reviewed in 41, 42). N-Acyl homoserine lactones are one
group of the diffusible extracellular quorum-sensing signaling molecules (bacte-
rial pheromones) that are used for cell-cell communication in some bacteria (88).
The synthesis of N-acyl homoserine lactones also arises as a natural response to
starvation and entry into stationary phase for E. coli (37), but the role of these
molecules has not yet been established for this species. A family of diketopiper-
azines such as cyclo(1Ala-Val) and cyclo(Pro-Tyr) appear to function as signal
molecules in the quorum-sensing systems in certain bacteria (35). Upon entry into
stationary phase, a bacterial culture can divide into two populations, one entering
into dormant phase (sporulation for gram-positive bacteria) and the other into pro-
grammed cell death (or prokaryotic apoptosis) (34, 54). The choice between these
fates seems to be under genetic control (e.g. see 101). Mutations in the rpoS gene
often confer growth advantage for stationary-phase cultures (47, 104). Phages can
express anti-cell death functions to favor their replication in infected cells (18).
The total number of detectably expressed genes among the >4000 putative
genes on the E. coli genome is <1000 in exponentially growing cells, as esti-
mated from the protein patterns on two-dimensional gel electrophoresis (96). The
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RNA POLYMERASE FUNCTIONAL MODULATION 503

accumulation of extracellular quorum-sensing signals ultimately leads to repres-

sion of most of these growth-related genes and, instead, induces the expression
of a new set of genes that are specific for survival in the stationary phase. Up to
now, ∼100 genes have been identified that are expressed only in stationary phase
(27, 41, 42, 64, 65). The precise selection of genes expressed in stationary phase,
however, differs depending on the factors that led to cessation of cell growth. This
drastic change in gene expression pattern is accompanied by a change in activity
and specificity of both the transcription apparatus and the translation machinery
(reviewed in 40, 41). The most significant changes are preceded by the appearance
of the stationary-phase-specific σ S subunit (for reviews, see 32, 64, 65), which is
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involved in transcription of most, if not all, of the stationary-phase genes (Table 1).
The σ S subunit is maximally expressed at the onset of stationary phase, the
maximum level being ∼30% that of σ 70, or 230 molecules per cell (46, 50; Figure
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2, see color insert). The onset of σ S synthesis is signaled by changes in metabolism

that lead to reductions in growth. Even during the exponential-growth phase,
therefore, the synthesis of σ S can be induced when cells are exposed to conditions
that are unfavorable for growth, so that, at very slow growth rates, E. coli contains
σ S even in exponential phase.

LEVEL AND ACTIVITY CONTROL OF SIGMA-S

The synthesis and accumulation of σ S are controlled at multiple levels, includ-

ing transcription, translation, protein turnover, and activity control (Figure 3, see
color insert). Transcription control of rpoS involves a number of factors, includ-
ing ppGpp and polyphosphate as positive regulators and cyclic AMP (cAMP) and
UDP-glucose as negative regulators (reviewed in 64). Upon increase in the con-
centration of the stringent control signal ppGpp, transcription of rpoS is enhanced
(24, 62). ppGpp binds to the β subunit of RNA polymerase (13) and thereby in-
hibits transcription of stringently controlled genes. However, as for other genes
under the positive control of ppGpp such as those for the amino acid biosynthetic
operons, the molecular mechanism of transcription activation by ppGpp remains
unsolved. The mechanism(s) by which rpoS transcription is stimulated by de-
creases in the concentrations of the catabolite repression signal cAMP (32, 64) or
UDP-glucose (8) also remain unknown.
Translation of rpoS messenger RNA (mRNA) is stimulated under various stress
conditions by several regulatory factors, including the RNA-binding Hfq (HF-1)
protein (10, 11, 73) and the small regulatory DsrA-RNA (63), and is repressed by
the histonelike protein H-NS (52). These factors modulate the secondary structure
of the ribosome-binding region of rpoS mRNA. Hfq was originally identified as a
host factor (HF-1) for phage Qβ replication (23). After gene cloning (51), it was
identified as one of the most abundant E. coli proteins associated with both nucleoid
and ribosomes (92, 93). Several lines of evidence indicate that Hfq binds a set of
mRNAs including rpoS mRNA and regulates the efficiency of their translation by
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TABLE 1 Stationary-phase–specific genes under the control of σ S

Gene Gene function Reference(s)

Genes involved in cell morphology and division

bolA Control of PBP6 synthesis 64, 65
cfa Cyclopropane fatty acid synthase 64, 65
csgBA Curly fimbriae formation 64, 65
csgCDEF Curly fimbriae formation 64, 65
fic Cell division control 64, 65
ftsQAZ Septum formation 65
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gabP γ-Aminoburytic acid permease 88a

hdeAB Periplasmic proteins 65
htrE Pili construction protein 64, 65
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osmB Outer membrane lipoprotein 64, 65

osmC Outer membrane lipoprotein 9a
osmE Outer membrane lipoprotein 14a
osmY Periplasmic protein 64, 65
pqi5 Membrane protein 65
potF (o381) Periplasmic putrescine-binding protein 88a
proU Glycine betaine and proline transport 83
proP Glycine betaine and proline permease 65
vanABCDF D-Ala-D-ala dipeptide transport 63a
vanX D-Ala-D-ala dipeptidase 63a
yhiU Two-membrane drug efflux pump 89a
Genes involved in energy metabolism and anabolism
acnA Aconitase 15a
acs Acetyl-CoA synthetase 89a
aldB Aldehyde dehydrogenase 65
cbdAB Cytochrome bd-II oxidase 65
cyxAB Cytochrome oxidase III 64, 65
frd Fumarate reductase 65
galEKT Galactose use 65
glpD Glyacerol-3-phosphate dehydrogenase 65
hmp Flavohemoglobin Hmp 65
hyaABCDEF Hydrogenase I (hydrogen oxidation) 65
nrz NRZ nitrate reductase 12c
o371 Glucose dehydrogenase B homolog 89a
poxB Pyruvate oxidase (acetate synthesis) 65
tam (o252) trans-Aconitate methyltransferase 12a
Genes involved in protein and nucleic acid breakdown
appY Transcription factor for appABC 65
clpA Clp protease subunit 65
wrbA TrpR repressor binding protein 65
xthA Exonuclease III 64, 65

(Continued )
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RNA POLYMERASE FUNCTIONAL MODULATION 505

TABLE 1 (Continued )
Gene Gene function Reference (s)

Genes involved in gene regulation, DNA replication, and nucleoid configuration

aidB Methylation damage repair of DNA 64, 65
cbpA Curved DNA-binding protein 65
dnaN DNA polymerase III β-subunit 96a
dps (pexB) DNA binding protein 64, 65
hip (himD) IHF subunit 65
hns (osmZ) H-NS 65
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rob (cbpB) Curved DNA-binding protein 51a

rpoS RNA polymerase σ S 91a
topA DNA topoisomerase I 65
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Genes involved in production of storage products

glgCAF Glycogen synthesis 65
glgS Glycogen synthesis 64, 65
otsA (pexA) Trehalose synthesis 64, 65
otsB (perX) Trehalose-6-phosphate phosphatase 64, 65
Genes for stress resistance
ahpCF Alkyl hydroperoxide reductase 70a
ecp-thtrE Thermotolerance/osmotolerance 88a
cpxRA Disaggregation of misfolded proteins 16a
gadAB Glutamic acid decarboxylase 12b, 15b
gor Glutathione oxidoreductase 65
katE Catalase HPII 64, 65
katG Catalase-peroxidase HPI 64, 65
ldc Lysine decarboxylase 51b
oxyR Transcription factor for oxy regulon 70a
pcm L-Isoaspartyl protein methyltransferase 96b
pspABCE Phage shock proteins 65
sodC Periplasmic superoxide dismutase 25a
sprA Virulence to animals 29a
treA (osmA) Periplasmic trehalase 64, 65
treF Cytoplasmic trehalase 35a
usp Universal stress proteins 21a
Unknown functions
csiDEF Carbon starvation-induced proteins 64, 65
f186 89a
f 253a 89a
o215 89a
pexCDEF Carbon starvation-induced proteins 65
yciG 65
yohF 65
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modulating the secondary structure of as-yet-unidentified sites near the translation

initiation codon (10, 11, 74). The regulatory RNA encoded by oxyS is known to
compete for Hfq and, as a result, inhibits translation of the rpoS mRNA (106).
Structural modification of rpoS mRNA can also be mediated through direct
RNA-RNA interaction with DsrA RNA (63, 91). DsrA, an 87-nucleotide untrans-
lated RNA, acts as a positive riboregulator of RpoS synthesis (91), by enhancing
translation of rpoS mRNA through RNA:RNA interactions with rpoS mRNA (63).
DsrA is required for the optimal translation of rpoS mRNA with the help of Hfq
(11, 73, 91), and, accordingly, dsrA mutants fail to accumulate σ S in stationary
phase. The product of dksA (dnaK suppressor) is also required for the optimal
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translation of rpoS mRNA, but the region of rpoS mRNA required to see the
effects of dsrA mutations was identified on rpoS between codons 8 and 73 (98),
suggesting that the contact sequence of DksA is located downstream of the trans-
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lation initiation site on rpoS mRNA. The histone-like protein H-NS plays a dual
role by interfering with both transcription and translation of rpoS (52, 102). The
target of H-NS binding on the rpoS mRNA is located upstream from the initia-
tion codon. Thus, DsrA RNA antagonizes the H-NS–mediated inhibition of rpoS
mRNA translation. LeuO, however, represses dsrA expression and thereby reduces
rpoS translation at low temperature (52).
The σ S protein is subject to rapid turnover in exponential-phase E. coli cells.
The increase in σ S level in stationary-phase E. coli results, at least in part, from a
large increase in the stability of σ S protein (105). The clpP gene product is one pro-
tease that is responsible for degradation of σ S (89, 98). The target site for the ClpP
protease action apparently resides in a 20-amino-acid stretch near the middle of
the σ S protein (89). The σ S factor also becomes stable in rssB mutants because the
activity of ClpP protease is enhanced by RssB [or SprE (MviA for Salmonella ty-
phimurium)], a response regulator (73). Phosphorylation of S. typhimurium MviA
(RssB/SprE for E. coli) is required for this regulation, but no evidence has been
obtained to support the model that acetyl phosphate contributes to the phosphoryla-
tion of MviA, although the σ S level increases about fivefold when acetate is used as
a carbon source (15). Rss/SprE contains a unique C-terminal output domain and is
the first known response regulator involved in the control of protein turnover (73).
On the other hand, the ClpP protease is activated by the ClpP-specific chaperone
ClpX (26, 97, 99) and inhibited by the general chaperone DnaK (65). LrhA, a LysR
homolog, promotes degradation of σ S, indirectly activating the ClpP protease by
modulating the activity of RssB/SprE (25).
The promoter recognition specificity of Eσ S is not entirely understood because
the promoters of the stationary-specific genes so far analyzed do not show any
distinctive consensus sequence and are mostly recognized by both Eσ 70 and Eσ S
holoenzymes in vitro (53, 94, 95). Several lines of evidence indicate that some
additional factors are involved in the control of activity and specificity of these
two different forms of RNA polymerase holoenzyme. Under stress conditions,
there are marked increases in the intracellular concentrations of compatible solutes,
including stress protectants such as potassium glutamate, trehalose, and glycine
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RNA POLYMERASE FUNCTIONAL MODULATION 507

betaine, and of some storage products such as glycogen and polyphosphate (e.g. see
78). These compounds influence the activity and specificity of RNA polymerase to
various degrees. The activity of σ S holoenzyme is modulated by glutamate (17, 83)
and trehalose (59) at the steps of Eσ S holoenzyme formation and Eσ S holoenzyme
binding to certain promoters. Transcription by Eσ S of some osmoregulated genes
such as osmB and osmY takes place in the presence of ≥0.5 M concentrations of
potassium glutamate, which completely inhibit the activity of Eσ 70 in vitro (17).
The optimal concentrations of trehalose for maximal transcription by Eσ 70 and
Eσ S are ∼0.5 and 1.0 M, respectively (59).
RNA polymerase from stationary-phase cells of E. coli is associated with an
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acidic compound(s) and exhibits an altered promoter selectivity (77). The RNA
polymerase-associated acidic compounds were found to be inorganic polyphos-
phate (poly P) (60), which is known to accumulate in stationary-phase cells (55).
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The ubiquitous occurrence of poly P is suggestive of some important physiological

role(s) for this polymer, and a number of hypotheses have been proposed for its
function (56, 84). Because mutants that are defective in the ppk gene encoding
polyphosphate kinase (PPK) are also defective in survival in the stationary phase
(60), poly P is now believed to play a role in bacterial adaptation to the stationary
phase. At low salt concentrations, poly P inhibits transcription in vitro by both
Eσ 70 and Eσ S holoenzymes. Upon increasing the concentration of potassium glu-
tamate, however, poly P inhibition is relieved for the Eσ S holoenzyme, but not for
Eσ 70, suggesting that poly P may play a role in the promoter selectivity control
of RNA polymerase in E. coli growing under high-osmolarity conditions and in
stationary phase (60).
The crl gene stimulates transcription of csgBA, the locus encoding the major
subunits of curli (the surface structures in stationary phase). crl-null alleles influ-
ence the expression of σ S-regulated genes in a fashion similar to an rpoS-null allele.
Crl stimulates the activity of the rpoS regulon by stimulating σ S activity during sta-
tionary phase (79). The mechanism of σ S activity control by Crl is not yet known.

ACTIVITY CONTROL OF THE SIGMA SUBUNITS BY

ANTI-SIGMA FACTORS
A new frame of transcription regulation has been discovered, in which the activity
of RNA polymerase σ subunit is controlled by so-called anti-σ factors (for a
review, see 36). An anti-σ factor is defined by the ability to form a complex
with its cognate σ subunit and thereby inhibits the σ function. This definition
excludes other σ subunits that compete for available core enzyme. The control of
σ activities by anti-σ factors has been well established in Bacillus subtilis (12, 57).
Among seven E. coli σ subunits, anti-σ factors have been identified for σ 70, σ F,
and σ E (cited in 48).
The fliA gene, one of the class II genes within the transcription hierarchy of
the flagellar biosynthetic pathway, encodes the flagella-specific RNA polymerase
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508 ISHIHAMA

σ subunit σ F, which is responsible for transcription of class III genes (30). A

negative regulatory gene, flgM, for class III genes encodes anti-σ F factor, which
inhibits σ F activity (61). FlgM exists as a binary complex with σ F in the early
phase of flagella formation, but it is secreted out of the cell after completion of
the flagella hook-basal body, resulting in reactivation of the σ F subunit. The
contact site for FlgM on the σ F subunit is located within region 4 (45), as for
class II transcription factors (28, 40). Under the steady state of E. coli growth, a
large fraction of the σ F subunit stays as a complex with FlgM (48), and thus the
actual concentration of Eσ F holoenzyme must be considerably lower than the σ F
level.
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The first member of the extracytoplasmic function subfamily of σ subunit iden-

tified in E. coli is σ E, which was originally identified as a factor required for
survival on exposure to extremely high temperature (19). Increasing the amounts
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of outer membrane proteins increases σ E-dependent gene expression, whereas re-

ducing outer membrane proteins results in a decrease in σ E-dependent transcription
(69). Overexpression of the dsb genes, coding for proteins involved in disulfide
bond formation, and the surA and fkpA genes, coding for distinct peptidyl-prolyl
isomerases, can compensate for and/or complement some of σ E mutations (71),
indicating that σ E is required for expression of the repair systems of denatured
proteins under certain stress conditions. The product of rseA, located downstream
of the rpoE gene within the same operon negatively regulates σ E (16, 72). The N
terminus of RseA interacts directly with σ E.
After screening for proteins that are associated with the σ 70 subunit, Jishage
& Ishihama (48) isolated Rsd (regulator of sigma D). Isolated Rsd forms a binary
complex with purified σ 70 and thereby inhibits its function. It was therefore
proposed that Rsd is an anti-σ 70 factor. Rsd is not present in exponentially growing
E. coli cells (48, 49), and in these conditions most of the free RNA polymerase
(not engaged in RNA synthesis) must be in the Eσ 70 holoenzyme form, which can
be immediately used for transcription of the growth-related genes. On entry into
stationary phase, Rsd starts to be synthesized and reaches its maximum level in
the early stationary phase (49). As in the case of FglM-σ F interaction, the contact
target for Rsd on σ 70 is located within region 4. Together, these observations
indicate that, in the stationary-phase E. coli, some of the unused σ 70 subunits are
stored in inactive form as a complex with Rsd, the anti-σ 70 factor.

MODULATION OF RNA POLYMERASE SPECIFICITY BY

TRANSCRIPTION FACTORS
Each holoenzyme recognizes and transcribes a different set of genes, but tran-
scription of some genes requires, in addition, accessory proteins or nucleotide
factors. The functional specificity of RNA polymerase holoenzyme is modulated
through interaction with 1 or 2 (in some cases) of ∼100–150 different transcrip-
tion factors. These factors can be classified based on their contact subunit(s)
P1: FQP/LVF P2: FQP
August 4, 2000 14:36 Annual Reviews AR110-15

RNA POLYMERASE FUNCTIONAL MODULATION 509

on RNA polymerase (40; Figure 4, see color insert). Regulatory proteins were
originally classified as activators, or, if they were known to inhibit transcription, as
repressors. More recent studies, however, indicate that both activators and repres-
sors can have dual functions, activating or inhibiting transcription from different
promoters depending on where they bind to the DNA (e.g. 1, 14, 44). More-
over, both activators and repressors seem to make direct contact with the RNA
polymerase to function [both are hereafter referred to simply as transcription fac-
tors (40)].
Many stationary-phase genes are transcribed by two different systems, de-
pending on cell growth conditions; one is σ S dependent but transcription factor
Annu. Rev. Microbiol. 2000.54:499-518. Downloaded from arjournals.annualreviews.org

independent, and the other is σ 70 dependent but transcription factor dependent.

Such genes usually carry multiple promoters, to be recognized by each form of
the transcription apparatus. It is surprising, however, that the two systems ini-
by INSERM-multi-site account on 03/04/09. For personal use only.

tiate transcription at the same site in some cases. For instance, some carbon
catabolite genes such as dps or pexB, encoding stationary-specific DNA-binding
protein Dps with regulatory and protective roles, are transcribed by Eσ 70 in cAMP-
receptor protein (CRP)–dependent fashion, but can also be transcribed by Eσ S in
the absence of cAMP-CRP; thus, the expression of these genes is cAMP-CRP
dependent in growing phase, but becomes independent of cAMP-CRP in station-
ary phase (66). Furthermore, the dps promoter is activated by OxyR when it is
transcribed by Eσ 70 during growth, but in the stationary phase it is transcribed
by Eσ S without the support of OxyR [integration host factor (IHF) is required
instead (2)]. Thus, the same transcriptional start is achieved both by a combina-
tion of Eσ 70 with stress-responsive transcription factors and by Eσ S holoenzyme.
Other recent evidence substantiates the suggestion that σ S overlaps functionally
with σ 70 (53, 94, 95). The RNA polymerase holoenzymes carrying other minor
σ subunits such as Eσ N, Eσ H, Eσ F, and Eσ FecI recognize and transcribe genes
involved in adaptation to imbalance in nitrogen and other nutrients, in acquisition
of heat-shock resistance, in generation of flagella, and in an iron citrate uptake
system, respectively. In sharp contrast to the σ S-dependent system, the promoters
under the control of these minor σ subunits cannot be recognized by the Eσ 70
holoenzyme, and the holoenzymes containing these minor σ subunits are unable
to transcribe σ 70-dependent genes.

FACTORS AFFECTING SELECTIVE UTILIZATION OF

TRANSCRIPTION APPARATUS
More than 100 promoters have been identified, which can be recognized either in
vivo or in vitro by RNA polymerase holoenzyme containing σ S, but no significant
consensus sequences have been identified. Instead, a number of σ 70-dependent
promoters are transcribed in vitro by the Eσ S holoenzyme (33, 53, 94, 95). One
possibility is that the Eσ S holoenzyme recognizes a specific DNA conformation
such as bent DNA regions (21). The action of DNA gyrase and the association
P1: FQP/LVF P2: FQP
August 4, 2000 14:36 Annual Reviews AR110-15

510 ISHIHAMA

of DNA-binding proteins combine to cause growth-dependent changes in the con-

figuration of the E. coli chromosome, often called the nucleoid, which affect the
pattern of gene transcription as well as protecting the genome DNA from stress-
induced damage. In agreement with the in vivo findings, transcription in vitro by
Eσ S is much higher when directed by templates with low superhelical density (58).
The enhancing effect of decreased superhelicity of template DNA on transcrip-
tion by Eσ S is additive with that of trehalose and potassium glutamate, indicating
that the changes in the cytoplasmic composition and the nucleoid conformation
independently produce growth-dependent changes in gene transcription.
A nucleoid-associated histone-like protein, H-NS, functions as a general si-
Annu. Rev. Microbiol. 2000.54:499-518. Downloaded from arjournals.annualreviews.org

lencer for gene transcription, repressing the expression of many and diverse genes
(4, 83). Mutations in H-NS can lead to overexpression of stress-induced genes in
the absence of stresses. Because the binding of H-NS to target DNA sequences is
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sensitive to both intracellular potassium glutamate levels (increased after osmotic

shock) and the level of DNA superhelicity (decreased during stationary phase), the
enhancing effect of high potassium glutamate concentration and low DNA superhe-
licity on transcription of σ S-dependent genes may be caused not only by activation
of Eσ S holoenzyme but also by release from the inhibition by H-NS (17, 83).
The protein composition of E. coli nucleoid changes markedly depending on
cell growth phase (93). Dps or PexB is a DNA-binding protein that appears in
E. coli only in stationary phase and is involved, together with H-NS and IHF, in
the condensation of the nucleoid into a more compact configuration. The dps or
pexB gene is recognized by Eσ S holoenzyme only when its promoter structure is
altered by the binding of another histone-like protein, IHF (2, 66), whose level
also increases in stationary phase (5, 93). Both Dps and IHF are required for the
efficient expression of σ S-dependent genes required for starvation survival (2, 76).
On the other hand, the DNA-binding protein Fis, most abundant in exponential
phase (93), generally activates transcription of genes that are highly expressed
in exponentially growing cells (9, 75, 107) and represses stationary-specific gene
transcription (76, 100). Accordingly, the level of Fis protein changes dramatically
upon nutrient upshift, from <100 molecules per cell in stationary phase to >50,000
in exponential phase (6, 93).

MULTIPLE PATHWAYS FOR STATIONARY-PHASE

ADAPTATION
The highly significant difference in survival levels between rpoS+ and rpoS strains
strongly suggests a major role for rpoS (and/or the genes under its control) in
stationary-phase survival. E. coli W3110 is an attenuated laboratory strain that has
been widely used as a standard for E. coli genetics and as a model strain in the
genome program. However, there are at least five independent lineages of strain
W3110 that differ in their content of two σ subunits, σ S and σ F (47). Because
mutations in the rpoS gene render the wild-type rpoS+ strain nonviable in stationary
phase, natural variants lacking the intact σ S must presumably have acquired yet
P1: FQP/LVF P2: FQP
August 4, 2000 14:36 Annual Reviews AR110-15

RNA POLYMERASE FUNCTIONAL MODULATION 511

unidentified suppressor mutations. Thus, it should be noted that different genetic

systems and mechanisms must be present in E. coli for adaptation to stationary
phase. In this respect, care should be taken not to draw conclusions from results
obtained by using different bacterial strains with different genetic backgrounds.

CONCLUSION
The stationary-phase–associated changes in the pattern of global gene expression
in E. coli are mainly mediated by modulation of the specificity of RNA polymerase
Annu. Rev. Microbiol. 2000.54:499-518. Downloaded from arjournals.annualreviews.org

by replacement of the promoter recognition subunit σ 70 with σ S. Accordingly, the

intracellular concentration of σ S increases when cultures stop growing. In addition,
the activities of Eσ 70 and Eσ S are regulated in different ways by changes in both
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the cytoplasmic composition and the conformation of the nucleoid. The overall
findings raise the possibility that each stationary-specific promoter carries a specific
sequence that is recognized by Eσ S under a specific condition or in the presence of a
specific factor. If so, the promoter sequence recognized by Eσ S must vary between
groups that require different conditions or factors for function. However, because
some E. coli strains lacking σ S can successfully enter stationary phase, alternative
pathways must exist, which allow bacterial adaptation to the stationary phase.

ACKNOWLEDGMENTS
Work at the National Institute of Genetics (NIG) was supported by grants-in-aid
from the Ministry of Education, Science, Sports and Culture of Japan, and the
CREST fund from the Japanese Science and Technology Corporation. The author
thanks his colleagues in NIG and collaborators from 70 laboratories in 20 countries.
The author thanks Richard S Hayward for critical reading of the manuscript and
helpful comments.

Visit the Annual Reviews home page at www.AnnualReviews.org

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by INSERM-multi-site account on 03/04/09. For personal use only.

Figure 1 Sigma subunits of Escherichia coli. E. coli contains seven species of σ subunit.
The RNA polymerase holoenzyme containing each σ subunit recognizes and transcribes a
specific set of genes. The intracellular concentrations of all seven σ subunits were deter-
mined for both exponential and stationary phases of E. coli W3110 (46, 50, 68).

Figure 2 Intracellular concentration of each holoenzyme form. The intracellular concen-

trations of all seven σ subunits were determined by quantitative Western blot analysis (46,
50, 68), while the dissociation constant between the σ subunit and the core enzyme was
determined by FPLC gel chromatography (Maeda H et al, submitted for publication). The
concentrations of all seven holoenzyme forms were calculated from these two values.
P1: FDS
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Figure 3 Regulation of the level and activity of σ s subunit. The synthesis of σ s subunit is
regulated at both transcription and translation steps by a number of regulatory factors. The
metabolic stability of σ s subunit is also controlled. The activity of holoenzyme containing
σ s subunit is specifically activated or repressed by a number of stationary-phase specific
factors. Transcription of some σ s-dependent genes is modulated by AppY, BolA, CRP, Fis,
IHF, Lrp (for a review see 65).

Figure 4 Classification of E. coli transcription factors. Transcription factors have been

classified on the basis of the contact subunit of RNA polymerase (40, 42).
 Annual Review of Microbiology
Volume 54, 2000

CONTENTS
THE LIFE AND TIMES OF A CLINICAL MICROBIOLOGIST, Albert
Balows 1
ROLE OF CYTOTOXIC T LYMPHOCYTES IN EPSTEIN-BARR
VIRUS-ASSOCIATED DISEASES, Rajiv Khanna, Scott R. Burrows 19
BIOFILM FORMATION AS MICROBIAL DEVELOPMENT, George
O'Toole, Heidi B. Kaplan, Roberto Kolter 49
MICROBIOLOGICAL SAFETY OF DRINKING WATER, U. Szewzyk,
R. Szewzyk, W. Manz, K.-H. Schleifer 81
THE ADAPTATIVE MECHANISMS OF TRYPANOSOMA BRUCEI
FOR STEROL HOMEOSTASIS IN ITS DIFFERENT LIFE-CYCLE
ENVIRONMENTS, I. Coppens, P. J. Courtoy 129
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THE DEVELOPMENT OF GENETIC TOOLS FOR DISSECTING THE

BIOLOGY OF MALARIA PARASITES, Tania F. de Koning-Ward,
Chris J. Janse, Andrew P. Waters 157
by INSERM-multi-site account on 03/04/09. For personal use only.

NUCLEIC ACID TRANSPORT IN PLANT-MICROBE

INTERACTIONS: The Molecules That Walk Through the Walls, Tzvi
Tzfira, Yoon Rhee, Min-Huei Chen, Talya Kunik, Vitaly Citovsky
187
PHYTOPLASMA: Phytopathogenic Mollicutes, Ing-Ming Lee, Robert E.
Davis, Dawn E. Gundersen-Rindal 221
ROOT NODULATION AND INFECTION FACTORS PRODUCED BY
RHIZOBIAL BACTERIA, Herman P. Spaink 257
ALGINATE LYASE: Review of Major Sources and Enzyme
Characteristics, Structure-Function Analysis, Biological Roles, and
Applications, Thiang Yian Wong, Lori A. Preston, Neal L. Schiller 289
INTERIM REPORT ON GENOMICS OF ESCHERICHIA COLI, M.
Riley, M. H. Serres 341
ORAL MICROBIAL COMMUNITIES: Biofilms, Interactions, and
Genetic Systems, Paul E. Kolenbrander 413
ROLES OF THE GLUTATHIONE- AND THIOREDOXIN-
DEPENDENT REDUCTION SYSTEMS IN THE ESCHERICHIA COLI
AND SACCHAROMYCES CEREVISIAE RESPONSES TO OXIDATIVE
STRESS, Orna Carmel-Harel, Gisela Storz 439
RECENT DEVELOPMENTS IN MOLECULAR GENETICS OF
CANDIDA ALBICANS, Marianne D. De Backer, Paul T. Magee, Jesus
Pla 463
FUNCTIONAL MODULATION OF ESCHERICHIA COLI RNA
POLYMERASE, Akira Ishihama 499
BACTERIAL VIRULENCE GENE REGULATION: An Evolutionary
Perspective, Peggy A. Cotter, Victor J. DiRita 519
LEGIONELLA PNEUMOPHILA PATHOGENESIS: A Fateful Journey
from Amoebae to Macrophages, M. S. Swanson, B. K. Hammer
567
THE DISEASE SPECTRUM OF HELICOBACTER PYLORI : The
Immunopathogenesis of Gastroduodenal Ulcer and Gastric Cancer, Peter
B. Ernst, Benjamin D. Gold 615
PATHOGENICITY ISLANDS AND THE EVOLUTION OF
MICROBES, Jörg Hacker, James B. Kaper 641
DNA SEGREGATION IN BACTERIA, Gideon Scott Gordon, Andrew
Wright 681
 POLYPHOSPHATE AND PHOSPHATE PUMP, I. Kulaev, T.
Kulakovskaya 709
ASSEMBLY AND FUNCTION OF TYPE III SECRETORY SYSTEMS,
Guy R. Cornelis, Frédérique Van Gijsegem 735
PROTEINS SHARED BY THE TRANSCRIPTION AND
TRANSLATION MACHINES, Catherine L. Squires, Dmitry Zaporojets
775
HOLINS: The Protein Clocks of Bacteriophage Infections, Ing-Nang
Wang, David L. Smith, Ry Young 799
OXYGEN RESPIRATION BY DESULFOVIBRIO SPECIES, Heribert
Cypionka 827
REGULATION OF CARBON CATABOLISM IN BACILLUS
SPECIES, J. Stülke, W. Hillen 849
IRON METABOLISM IN PATHOGENIC BACTERIA, Colin Ratledge,
Lynn G Dover 881
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