Eubacterial sigma-factors

M.M.S.M. Woësten *Department of Bacteriology, Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Universiteit Utrecht,

P.O. Box 80.165, 3508 TD Utrecht, The Netherlands

Received 20 November 1997; received in revised form 25 June 1998; accepted 30 June 1998

Abstract

The initiation of transcription is the most important step for gene regulation in eubacteria. To initiate transcription, RNApolymerase has to associate with a small protein, known as a c-factor. The c-factor directs RNA polymerase to a specific classof promoter sequences. Most bacterial species synthesize several different c-factors that recognize different consensussequences. This variety in c-factors provides bacteria with the opportunity to maintain basal gene expression as well as forregulation of gene expression in response to altered environmental or developmental signals. This review focuses on thefunction, regulation and distribution of the 14 different classes of c-factors that are presently known. z 1998 Federation ofEuropean Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Transcription initiation; Eubacteria; Sigma-factor; Promoter consensus sequence

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1282. Two families of bacterial c-proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1293. The c70-family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

3.1. Group 1. Primary c-factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1313.2. Group 2. The nonessential primary-like c-factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

3.2.1. Subgroup 2.1. Stationary-phase c-factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1343.2.2. Subgroup 2.2. Cyanobacterial c-factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1353.2.3. Subgroup 2.3. The c-factors of high-GC Gram-positive bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

3.3. Group 3. Alternative c-factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1353.3.1. Subgroup 3.1. Flagellar c-factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1363.3.2. Subgroup 3.2. Extracytoplasmic function c-factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1363.3.3. Subgroup 3.3. Heat shock c-factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

3.3.3.1. Sigma 32 and related c-factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1373.3.3.2. Sigma B and related c-factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

3.4. Subgroup 3.4. Sigma-factors involved in sporulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

0168-6445 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.PII: S 0 1 6 8 - 6 4 4 5 ( 9 8 ) 0 0 0 1 1 - 4

FEMSRE 612 15-10-98

* Present address: Washington University, School of Medicine, Department of Molecular Microbiology, 660 S. Euclid Ave. CampusBox 8230, St. Louis, MO 63110, USA. Tel.: +1 (314) 362 3691; Fax: +1 (314) 362 1232; E-mail: Wosten@borcim.wustl.edu

FEMS Microbiology Reviews 22 (1998) 127^150

3.4.1. Sigma H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393.4.2. Sigma F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393.4.3. Sigma E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393.4.4. Sigma G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393.4.5. Sigma K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

4. The c54-family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1405. Factors that a¡ect transcription initiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1416. Anti-sigma-factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1427. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

1. Introduction

Bacteria encode several thousands of di¡erent pro-teins, which are necessary for normal cell functionsor for adaptation to environmental changes. Theseproteins are not required at the same time or in thesame amount. Regulation of gene expression there-fore enables the cell to control the production ofproteins needed for its life cycle or for adaptationto extracellular changes. This regulation in turnmakes it possible for the bacterium to adequatelyadapt to rapid changes in the environment. The var-ious steps during transcription and translation aretherefore subject to di¡erent regulatory mechanisms.The most prominent step in gene regulation is theinitiation of transcription in which the DNA-depen-dent RNA polymerase (RNAP) is the key en-zyme. Depending on the growth rate, 1500 to11400 RNAP molecules are present in an Escherichiacoli cell [1]. As calculated by the synthesis of stableRNA, 24% to 79% of these molecules respectively,are actually engaged in the synthesis of RNA at anyparticular time [1]. The RNAP or the RNAP coreenzyme is the catalytic machinery for the synthesis ofRNA from a DNA template [2]. However, RNAPcannot initiate transcription by itself. Initiation oftranscription requires an additional polypeptideknown as a c-factor. Sigma-factors are a family ofrelatively small proteins that can associate in a re-versible way with the RNAP core enzyme. Together,the c-factor and the RNAP core enzyme form aninitiation-speci¢c enzyme, the RNAP holoenzyme[3,4].

Initiation of transcription in eubacteria is a multi-step process which begins with the binding of RNAPholoenzyme, containing c70 or a c70-related c-factor,to a speci¢c DNA sequence recognized by this en-

zyme as a promoter [5^7] (Fig. 1). Recognition of apromoter sequence is directed primarily by the c-subunit of the RNAP holoenzyme. The binding ofRNAP holoenzyme to the promoter results in a so-called `closed complex' which is converted into an`open complex' by melting of a short region ofDNA within the sequence bound by the enzyme. Inthe resulting ternary complex, the RNAP holoen-zyme starts to produce small 2 to 12 bp RNA mol-ecules, but remains located at the promoter. Afterdissociation of the c-subunit, the RNAP core en-zyme moves along the DNA, meanwhile synthesizingthe nascent RNA molecule. A locally unwound re-gion of DNA moves with the enzyme. The RNAPcore enzyme and RNA are released when a termina-tor structure or factor is encountered and the DNAis then fully restored to duplex condition [7].

The frequency at which the RNAP holoenzymeinitiates transcription, also known as the `strength'of a promoter, is in£uenced by the promoter se-quence and the conformation of the DNA in thepromoter region. The c-factor recognizes two con-served sequences in the promoter region, known asthe promoter consensus sequence [8]. The model pro-posed in Fig. 1 is supported by footprint analyses inwhich c-factor or fragments of c-factor have beenshown to bind speci¢cally to promoter DNA sequen-ces [9^11] and by speci¢c base pair and amino acidsubstitutions in the promoter consensus sequences orc-factor [6,12^15]. Most bacterial species synthesizeseveral di¡erent c-factors which direct the RNAPholoenzyme to distinct classes of promoters with adi¡erent consensus sequence. This variety in c-fac-tors provides the bacterium with the opportunity tomaintain basal gene expression as well as for regu-lation of gene expression in response to speci¢c en-vironmental stimuli.

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2. Two families of bacterial cc-proteins

Based on sequence similarity, the bacterial c-fac-

tors can be grouped into two families, the c70- andc54-family, with little if any sequence identity be-tween them [16,17]. The majority of the c-factors

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Fig. 1. Schematic representation of several steps required for the initiation of transcription of RNAP containing c70 or a c70-related c-factor. The RNAP holoenzyme binds to the promoter to form the closed complex. Conformational changes in the closed complex resultin an open form in which the 310 promoter region is melting out. In this open form the RNAP holoenzyme starts to produce smallRNA molecules. A ternary complex is formed. Finally the RNAP of which the c-factor is dissociated starts to transcribe an mRNA mol-ecule. See text for details.

M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 129

in eubacteria belong to the c70-family. This family isnamed after the 70 kDa primary c-factor from E.coli [18]. All eubacteria contain one or more c-fac-tors belonging to the c70-family, which is dividedinto several structurally and functionally related(sub)groups. Many eubacteria also contain a c-fac-tor which belongs to the c54-family [19], named forthe 54 kDa nitrogen regulation c-factor of E. coli.The c54-family contains only one group of c-factorswhich are not essential for certain growth conditions.The identi¢cation of c-factors in various bacterial

species has led to similar c-factors having di¡erentnames and even di¡erent c-factors having identicalnames.

3. The cc70-family

Sequence comparison of c70-family proteins fromdi¡erent eubacteria led to the identi¢cation of fourhighly conserved amino acid regions [20]. As addi-tional sequence data became available these regions

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Fig. 2. Structure function maps of c70- and c54-family of c-factors. Regions shown as a solid box represent the most conserved parts ofthe c-factors. A detailed functional assignment for the subregions of the c70-family proteins is displayed in the expanded view. Region IIIof the c54-family contains three structures indicated as X-link, HTH and the RpoN box. They are involved in DNA binding.

M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150130

have been subdivided further [18] (Fig. 2). The N-terminal region or region 1, is the least conserved.Subregion 1.1 is found only in the primary c-factors(c70) in which it modulates DNA binding and is,once the closed complex has been formed, importantfor the e¤cient initiation of transcription [21]. Sub-region 1.2 is probably required for open complexformation [21]. Deleting region 1.2 prevents RNAPfrom progressing beyond the closed complex. Thissubregion is found in all c-factors in this family,except for the extra cytoplasmic function c-factors.Region 2 is the most conserved region and can bedivided in four subregions. Region 2.1 is importantfor interaction with the subunits of core RNAP,while region 2.3 is involved in DNA melting. Impor-tant for DNA binding is subregion 2.4 which hasbeen implicated in the recognition of the 310 pro-moter element, a region located approximately 10nucleotides upstream of the start point of transcrip-tion (+1) [8]. Mutations in region 2.4 alter the spe-ci¢c interaction with the 310 promoter element[12,22,23]. Recently, region 2.5 was identi¢ed asbeing involved in contacting nucleotides at positions314 and 315 in E. coli promoters [24], known inBacillus subtilis as the 316 promoter consensus se-quence [25]. Regions 3 and 4 are divided in twosubregions. Subregion 3.1 contains a helix-turn-helixDNA-binding motif and the less conserved region3.2 may be involved in binding the RNAP core en-zyme [18,26]. Region 3.2 is not present in the c-fac-tors K and H. Subregion 4.1 has been proposed tobind certain transcriptional activators during the ini-tiation of transcription [27,28]. Subregion 4.2 alsorecognizes the promoter sequence as mutations inthis region a¡ect the ability of the RNAP holoen-zyme to recognize a promoter element approximately335 nucleotides relative to the start point of tran-scription [8,12,13,29].

The 2.6 Aî crystal structure of a fragment of E. colic70 comprising amino acid 114 to 448 was solvedrecently [30]. This fragment contains a part of region1.2, the complete regions 2.1, 2.2, 2.3, and all but theC-terminal ¢ve residues of region 2.4. The crystalstructure indicates that all the conserved regionsare closely associated with one another and that re-gion 1.2 stabilizes the fold of the other regions. Res-idues involved in core RNAP binding lie on one faceof the structure. On the opposite face are residues

that interact with the 310 region and residues in-volved in promoter melting.

The c70-protein family can be divided into threedi¡erent functionally and structurally related groups[18] (Fig. 3, Table 1). Group 1 comprises the primaryc-factors responsible for the transcription of mostgenes expressed in exponentially growing cells ofwhich the majority are essential for cell survival.Group 2 comprises the c-factors which are quitesimilar to the primary c-factors in sequence but arenonessential for cell growth. The third group consistsof the so-called alternative c-factors. These c-factorsdi¡er considerably in amino acid sequence from theprimary c-factors and control the transcription ofspeci¢c regulons. The three groups of the c70-familyand the various subgroups are described below.

3.1. Group 1. Primary c-factors

Group 1 is composed of the primary c-factorspresent in all known eubacteria [31]. They are re-sponsible for the transcription of most genes ex-pressed in exponentially growing cells and are essen-tial for cell survival. Recent data suggest that there isonly one primary c-factor present in any given eu-bacterial species [31]. The primary c-factor in E. coliis encoded by the rpoD gene and is known as c70

[20]. In Mycobacterium spp. this c-factor is known asMysA, in Streptomyces spp. as HrdB and in B. sub-tilis and other Gram-positive bacteria as SigA or cA

[31]. The promoter consensus sequence recognized bythe primary c-factor in E. coli and B. subtilis isfound to be similar in many other eubacteria[32,33]. This promoter sequence is characterized bytwo stretches of six nucleotides, TTGACA and TA-TAAT, centered around positions 335 and 310 re-spectively, from the transcription start site (+1) (Ta-ble 2). A dinucleotide TG at positions 315 and 314is also conserved in promoters of Gram-positive bac-teria but less conserved in E. coli promoters [25,33].The hexamer sequences around 335 and 310 aremost often separated by a stretch of 16 to 18 non-conserved nucleotides called the spacer. Deviationsfrom the consensus sequences usually result in re-duced promoter activity [25,34,35]. However, certainprokaryotic promoters can function in the absence ofa 335 promoter element if they have an `extended310' promoter element, TGnTATAAT [36,37]. The

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M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 131

FEMSRE 612 15-10-98

Fig. 3. Phylogenetic relationship of the c-factors of the c70-family. The tree was generated by the program Fitch of the Phylip packageversion 3.5c [205] using a PAM matrix-based distance correction from a multiple sequence alignment made be the program Multalin ver-sion 4.0 [206]. The scale bar represents 0.2 substitution per site. The sequences used in the alignment were translated from the Genbankentries given in parentheses: Escherichia coli RpoE (P34086), KatF (X16400), RpoH (M20668), FliA (L36677), RpoD (U23083); Salmo-nella typhimurium RpoE (P34086), RpoS (U05011); Streptomyces aureofaciens HrdE (M90412), HrdA (M90410); Haemophilus in£uenzaeRpoE (P44790); Photobacterium sp. strain SS9 (L41688); Pseudomonas aeruginosa AlgU (U49151), RpoS (D26134); Mycobacterium tuber-culosis RpoE (Z95120), SigE (U87242), SigF (Z92771); Myxococcus xanthus CarQ (X71062), SigB (X55500), RpoD (U20669), SigC(L12992); Synechocystis sp. strain PCC6803 RpoE (D90906), SigF (D90906); Bacillus megaterium SigH (X59070); Yersinia enterocoliticaFliA (L33466), RpoS (U22043); Bacillus subtilis SigH (M29693), SigF (M15744), SigA (M10089), SigB (M34995), SigE (p06222), SigK(M15744), SigG (X57547), RpoD (M20144); Clostridium acetobutylium SigG (U07420), SigE (Z23079), SigK (L23317); Steptomyces coeli-color HrdB (X52983), HrdC (P18184), SigF (L11648); Stigmatella aurantiaca SigB (Z14970); Rhodobacter capsulatus RpoD (Z68306); Pro-teus mirabilis RpoH (D50830); Agrobacterium tumefaciens RpoD (P33452); Streptomyces griseocarneus WhiG (X68709); Vibrio parahae-molyticus LafS (U52957); Mycobacterium smegmatis MysB (U09863), MysA (U09821); Caulobacter crescentus RpoD (U35138), RpoH(U39791); Synechococcus sp. strain PCC7002 SigA (U15574), SigE (U82485); Anabaena sp. strain PCC7120 SigA (M60046), SigC(M95759), SigB (M95760); Corynebacterium glutamicum SigB (Z49824); Erwinia carotovora RpoS (U66542); Legionella pneumophila FliA(X98892); Vibrio cholerae RpoH (U44432).

M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150132

FEMSRE 612 15-10-98

Tab

le1

Fun

ctio

nsan

ddi

stri

buti

onof

the

vari

ous

euba

cter

ialc

-fac

tors

Gro

upO

rgan

ism

,na

mec

-fac

tor

Fun

ctio

nsR

efer

ence

1.P

rim

aryc

-fac

tors

Gra

m-n

egat

ive

bact

eria

,c

70;

Gra

m-p

osit

ive

bact

eria

,c

A;

Myc

obac

teri

a,M

ysA

;S

trep

tom

yces

,H

rdB

Maj

orsi

gma-

fact

orin

expo

nent

ial

grow

ing

cells

regu

late

sho

use

keep

ing

gene

s[3

1]

2.N

ones

sent

ial

prim

ary-

like

c-f

acto

rs2.

1.St

atio

nary

phas

ec

-fac

tor

Ent

erob

acte

ria,

Pse

udom

onas

,R

poS

Maj

orsi

gma-

fact

ordu

ring

entr

yin

tost

atio

nary

phas

e.G

loba

lre

gula

tor

ofge

neex

pres

sion

ince

llsex

pose

dto

hype

rosm

otic

orac

idst

ress

[43]

2.2.

Cya

noba

cter

ialc

-fac

tors

Syn

echo

cocc

us,

Syn

echo

cyst

is,

Ana

baen

a,Si

gB-E

Con

trol

ling

gene

expr

essi

ondu

ring

circ

adia

nre

spon

ses,

carb

onan

dni

trog

enav

aila

bilit

yor

post

-exp

onen

tial

-gro

wth

phas

e

[31,

65]

2.3.

The

c-f

acto

rsof

high

-GC

Gra

m-p

osit

ive

bact

eria

Myc

obac

teri

a,M

ysB

;C

oryn

ebac

teri

a,Si

gB;

Str

epto

myc

es,

Hrd

A,

Hrd

C-E

Unk

now

n[3

1]

3.A

lter

nati

vec

-fac

tors

3.1.

Fla

gella

c-f

acto

rE

nter

obac

teri

a,c

28;

Str

epto

myc

es,

Whi

G;

Bac

illus

subt

ilis,

cD

Exp

resi

onof

chem

otax

is,

late

£age

llar

orea

rly

spor

ulat

ion

gene

s[7

4,87

]

3.2.

EC

Fc

-fac

tor

Pse

udom

onas

aeru

gino

sa,

Alg

U;

Esc

heri

chia

coli,

Fec

I;P

seud

omon

as£u

ores

cens

,P

brA

;M

yxoc

occu

sxa

nthu

s,C

arQ

;S

.co

elic

olor

,E

.co

li,M

ycob

acte

ria,

cE

;P

seud

omon

asS

yrin

gae,

Hpr

L;

Alc

alig

enes

eutr

ophu

s,C

rnH

;B

.su

btili

s,Si

gV-Z

Exp

ress

ion

ofge

nes

invo

lved

inal

gina

tebi

osyn

thes

is,

iron

upta

ke,

caro

teno

idbi

o-sy

nthe

sis,

anti

biot

icpr

oduc

tion

,in

duct

ion

ofvi

rule

nce

fact

ors,

resi

stan

tsto

coba

ltan

dni

ckel

,ou

ter

mem

bran

epr

otei

nsor

surv

ival

ofhi

ghte

mpe

ratu

re

[75]

3.3.

Hea

tsh

ockc

-fac

tors

3.3.

1.Si

gma

32an

dre

late

dc

-fac

tors

Gra

m-n

egat

ive

bact

eria

,c

32;

M.

xant

hus,

SigB

;S

.au

rant

iaca

,Si

gCE

xpre

ssio

nof

gene

sdu

ring

stre

ss,

frui

ting

body

form

atio

nor

mat

urat

ion

ofm

yxos

pore

s[7

6,10

8,14

]

3.3.

2.Si

gma

Ban

dre

late

dc

-fac

tors

B.

subt

ilis,

Sta

pylo

cocc

us,c

B;

Myc

obac

teri

a,S

trep

tom

yces

,Si

gFE

xpre

ssio

nof

gene

sdu

ring

stre

ssor

late

spor

ulat

ion

[78,

120,

121]

3.4.

Spor

ulat

ionc

-fac

tors

3.4.

1.Si

gma

HB

acill

us,

Clo

stri

dium

,c

HT

rans

crip

tion

ofea

rly

spor

ulat

ion

and

post

-ex

pone

ntia

l-gr

owth

-pha

sege

nes

[79,

129]

3.4.

2.Si

gma

FB

acill

us,

Clo

stri

dium

,c

FT

rans

crip

tion

ofea

rly

fore

spor

ege

nes

[79,

129]

3.4.

3.Si

gma

EB

acill

us,

Clo

stri

dium

,c

ET

rans

crip

tion

ofea

rly

mot

her-

cell

gene

s[7

9,12

9]3.

4.4.

Sigm

aG

Bac

illus

,C

lost

ridi

um,c

GT

rans

crip

tion

ofla

tefo

resp

ore

gene

s[7

9,12

9]3.

4.5.

Sigm

aK

Bac

illus

,C

lost

ridi

um,c

KT

rans

crip

tion

ofla

tem

othe

r-ce

llge

nes

[79,

129]

4.T

hec

54-f

amily

Rhi

zobi

um,

Rho

doba

cter

,P

seud

omon

as,

Bac

illus

,C

aulo

bact

er,

Ent

erob

acte

riac

eae

Exp

ress

ion

ofge

nes

invo

lved

in:

nitr

ogen

¢xat

ion,

nitr

ate

utili

zati

on,

glut

amin

esy

nthe

tase

,di

carb

oxyl

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M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 133

so-called `extended 310' promoter provides a verystrong recognition signal for the c70-subunit whichcan overcome both the lack of a 335 promoter ele-ment as well as less conserved 310 region [38].

Recognition of the 310 and 335 region of theprimary c promoter has been studied extensivelyby site-speci¢c mutagenesis. Amino acid substitutionstudies within region 2.4 (Fig. 2) of c70 of E. colishowed that two amino acids at positions 437 and440 interact with the 310 region of c70 promoters.These amino acids, glutamine at position 437 andthreonine at position 440, are in contact with theT/A base pair at position 312 of the 310 promoterelement [12,22,39]. Mutagenesis studies within region4.2 have shown that the arginine residues at posi-tions 588 and 584 of c70, respectively, interact withthe G and C residues of the 335 region TTGACA[12].

3.2. Group 2. The nonessential primary-like c-factors

The c-factors in this group are nonessential forexponential cell growth [18]. They are highly similarto the primary c-factors in the amino acid sequenceof their DNA-binding regions, which suggests thatboth groups of c-factors recognize similar promotersequences. The enterobacteria, the high-GC contentGram-positive bacteria and the cyanobacteria con-

tain c-factors which belong to group 2 [31]. TheEnterobacteriaceae and Pseudomonas contain agroup 2 c-factor, known as the stationary-phase-spe-ci¢c c-factor, cS or RpoS [40]. All cyanobacterialgroup 2 c-factors are closely related based on aminoacid sequences, but di¡er from the primary c-factorsof the corresponding organisms (Fig. 3). The group 2c-factors from Gram-positive bacteria with a high-GC content do not form such a tight grouping. Thehigh-GC content Gram-positive bacteria, cyanobac-teria and Chloro£exus aurantiacus are the only or-ganisms in which multiple group 2 c-factors havebeen found [31]. The subgroups of group 2 are de-scribed below.

3.2.1. Subgroup 2.1. Stationary-phase c-factorsIn their natural environment, bacteria frequently

experience nutrient limitation and a variety of phys-ical and chemical stresses. Consequently, they endureand survive long periods without growth [41]. Tosurvive environmental stress some bacteria producehighly resistant spores while others enter into thestationary-growth phase [41,42]. Entry into the sta-tionary phase is characterized by the synthesis ofstress proteins [41]. In E. coli and other enteric bac-teria the general stress response is mediated by cS

(RpoS, c38) encoded by the rpoS gene [43]. In lessclosely related bacteria only the Pseudomonas spp.

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Table 2Consensus sequences recognized by various eubacterial c-factors

c70-family Namea Consensus sequenceb Reference

335 Spacer 310

Primary c-factors c70, RpoD, SigA TTGACA 16^18 TATAAT [8,33]Nonessential primary-like c-factors2.1. Stationary-phase c-factor c38, RpoS CTATACT [58]Alternative c-factors3.1. Flagella c-factors c28, FliA, SigD TAAA 15 GCCGATAA [74]3.2. ECF c-factors cE, SigE GAACTT 16^17 TCTRA [75,89,104,105]3.3. Heat shock c-factors c32, RpoH CTTGAAA 11^16 CCCATnT [107]

cB, SigB GTTTAA 12^14 GGGTAT [78]3.4. Sporulation c-factors cH, SpoOH AGGAWWT 12^14 RGAAT [139]

cF, SpoIIAC WGCATA 14^15 GGnRAYAMTW [129,144]cE, SpoIIGB GKCATATT 13^15 CATACAMT [154,155]cG, SpoIIIG TGAATA 17^18 CATACTA [159]cK, SpoIIIC AC 16^17 CATAnAnTA [167]

c54-family 324 312

cN, RpoN, SigL TGGCAC 5 TTGCW [19]

aOnly the c-factor names for which a consensus recognition sequence has been derived are indicated.bAmbiguous codes: N, any base; R, A or G; W, A or T; Y, C or T; M, A or C; K, G or T.

M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150134

have been shown to contain a rpoS-like gene [31,43].While cS is best known for its crucial role in generegulation during entry into stationary phase [41], italso serves as a global regulator of gene expression incells exposed to hyperosmotic [44] or acid stress [45].Genes regulated by cS encode a variety of di¡erentfunctions being involved in the prevention and repairof DNA damage, cell morphology, modulation ofvirulence genes, osmoprotection, thermotolerance,glycogen synthesis, membrane and cell envelopefunctions [43]. Increased expression of stationary-phase RpoS-regulated genes requires an increase inthe abundance of cS. The cellular concentration ofcS in E. coli is controlled at the levels of transcrip-tion, translation and protein stability. In E. coli cS iscontrolled by four di¡erent c70 promoters of whichthe second promoter (P2) in vitro is also recognizedby c38 [46]. The RNA-binding protein HF-1 is essen-tial for the translational regulation of rpoS [47]. Oth-er factors such as DNA-binding protein H-NS [48],cyclic AMP [49] and UDP-glucose [50] negativelyin£uence production of cS, while pppGpp [51], ho-moserine lactone [52] and DnaK [53] raise the levelof cS. In exponentially growing cells, cS is very un-stable. Involved in the degradation of cS are thenegative regulator RssB and ClpXP protease [54];mutations in these genes give rise to stable cS. Thedecreased proteolytic turnover of cS during acidshock in S. typhimurium is mediated by MviA [55].

Some promoters that are recognized by cS are alsounder the control of c70 [56,57]. Comparison of 33cS-regulated promoters revealed that the cS pro-moter consensus sequence lacks a 335 region, butcontains a conserved 310 promoter element, CTA-TACT [58] (Table 2). The lack of a 335 region ispossibly compensated by a region upstream of the310 region with an intrinsic DNA curvature [58].However, the presence of DNA-bending regions isoften found in front of strong promoters [59], andthey are not essential for recognition by cS in vitro[60]. So far, no DNA-binding protein that acts spe-ci¢cally as an activator of cS-dependent genes hasbeen identi¢ed [61].

3.2.2. Subgroup 2.2. Cyanobacterial c-factorsThe Gram-negative cyanobacteria are capable of

photosynthetic growth. They are the simplest unicel-lular organisms known to exhibit circadian (daily)

rhythms [62]. The cyanobacterial group 2 c-factorsform a tight cluster of related sequences [31] (Fig. 3).Members of this cluster are the SigB and SigC c-factors of Anabaena sp., and Synechocystis sp. andthe SigB-E c-factors of Synechococcus sp. [31]. Re-cent investigations have shown that SigB (RpoD2) ofSynechococcus sp. controls gene expression duringcircadian responses [31,63]. Mutagenesis of therpoD2 gene resulted in a rhythmic mutant with alow amplitude phenotype. The sigB and sigC genesof Anabaena sp. are expressed under nitrogen-limit-ing conditions but neither of these genes is requiredfor nitrogen ¢xation or heterocyst di¡erentiation[64]. The homologues of SigB and SigC in Synecho-coccus sp. play a role in carbon and nitrogen avail-ability [65]. The sigB increases signi¢cantly whencells are starved for nitrogen and carbon. Mutationof sigC increases transcription of sigB indicating thattheir functions are related to one another. SigE ofSynechococcus sp. may play a role in the transcrip-tion of genes during post-exponential growth in thisorganism and therefore, SigE may be similar in func-tion although its sequence places it outside the RpoSfamily of c-factors [66].

3.2.3. Subgroup 2.3. The c-factors of high-GCGram-positive bacteria

The c-factors belonging to this group are thehrdA, hrdC, hrdD and hrdE genes of Streptomycesspp., mysB of Mycobacterium spp. and sigB of Cor-ynebacterium glutamicum. The c-factors of thesephylogenetically related bacteria are more diversethan those of the cS and cyanobacterial group 2 c-family [31] (Fig. 3). The Streptomyces coelicolor hrdDgene is expressed in exponentially growing cellsand chrdD recognizes the promoters in front ofredD and actII-orf4, two regulatory genes requiredfor the synthesis of antibiotics [67,68]. Although theother genes are expressed and can be disrupted, thefunctions of these nonessential c-factors remainunclear [69^73].

3.3. Group 3. Alternative c-factors

The third group of the c70-family comprises alter-native c-factors that control the transcription of spe-ci¢c regulons during special physiological or devel-opmental conditions [6,20]. In alignment studies,

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subgroups of the alternative c-factors were foundto cluster together and subsequently shown to haverelated functions (Fig. 3, Table 1). This group in-cludes the £agellar [74], extracytoplasmic functional[75], heat shock [76^78] and the sporulation [79] c-factors.

3.3.1. Subgroup 3.1. Flagellar c-factorsMany motile bacteria possess an external organ-

elle, the £agellum, that propels the cell in directionsdictated by diverse sensory signals. More than 50genes, which are divided into three classes, are in-volved in the formation of a functional £agellum inE. coli and S. typhimurium [80]. At the top of the£agellar gene hierarchy are the class 1 genes £hD and£hC of which the products positively control thetranscription of the class 2 genes. Class 2 genes en-code components needed in the early stage of basal-body construction, FlgM and c-factor FliA. FliA orc28 is needed for the expression of class 3 £agellarproteins involved in the ¢lament assembly and genesinvolved in chemotaxis gene hierarchy in various en-terobacteria [74,80,81]. FliA is negatively regulatedby FlgM, which binds to c28 until the basal-bodyhook formation is completed [82]. The cD of B. sub-tilis [83] and WhiG of Streptomyces spp. [84,85] arealso members of the £agellar c-factors. The functionand regulation of cD of B. subtilis is similar to thatof FliA of the enterobacteria [86]. WhiG of theGram-positive mycelial S. coelicolor is similar instructure but has a totally di¡erent function com-pared to FliA. These bacteria can form spores inthe aerial hyphae, a process in which WhiG playsan essential role [87]. In the absence of WhiG theaerial hyphae fail to develop sporulation septa, whilean excess of WhiG causes hyper-sporulation [85].Although WhiG has a totally di¡erent function, theS. coelicolor whiG gene can complement a £iA nullmutant of S. typhimurium to restore £agellar biosyn-thesis and motility [87]. Genetic experiments haveshown that WhiG is also regulated post-translation-ally by FlgM when introduced into S. typhimurium[87]. It is therefore likely that the WhiG recognizesthe same promoter sequence as FliA of S. typhimu-rium. Analysis of c28- and cD-dependent promotersin E. coli and B. subtilis revealed that RNAP con-taining c28 or cD recognizes the 335 promoter ele-ment, TAAA, and the 310 sequence, GCCGATAA,

separated by a spacer of 15 nucleotides [74] (Table2).

3.3.2. Subgroup 3.2. Extracytoplasmic functionc-factors

The extracytoplasmic function (ECF) c-subgroupof the c70-family is a class of environmentally re-sponsive transcriptional regulators [75]. The aminoacid sequences of these c-factors were so di¡erentfrom the c70-factors that they were initially not rec-ognized as c-factors (Fig. 3). They share similaritywith the primary c-factors in three of the four con-served regions but lack most of region 1 (Fig. 2) [75].Several bacterial species contain members of theECF family in which they control a variety of func-tions in response to speci¢c extracellular environ-mental signals. ECF factors include AlgU, whichregulates alginate biosynthesis in Pseudomonas aeru-ginosa [88,89]. Alginate is a viscous exopolysacchar-ide believed to protect the bacterium from the ad-verse environment of the cystic ¢brosis lung [90].FecI of E. coli [91] and PbrA of Pseudomonas £uo-rescens [92] are both involved in iron uptake. Theycoordinate gene response in low-iron conditions andare both negatively regulated by the Fur repressor[92]. The light inducible biosynthesis of carotenoidin Myxococcus xanthus is controlled by CarQ [93].Carotenoids are often found in non-photosyntheticbacteria as light-protective agents [93]. The S. coeli-color cE promotes transcription of a gene encodingan extracellular agar-degrading enzyme, agarase [75],while cE of Streptomyces antibioticus is required forthe production of the antibiotic actinomycin [94].HprL of Pseudomonas syringae controls transcriptionand secretion of a plant virulence factor and is in-ducible by plant extracts [95]. CnrH plays a role inresistance to nickel and cobalt e¥ux in Alcaligeneseutrophus [96]. The Mycobacterium smegmatis ECFc-factor plays a role in the ability to withstand avariety of stresses [97]. The expression of several out-er membrane proteins in the deep-sea bacteriumPhotobacterium sp. strain SS9 is regulated by cE inresponse to growth under hydrostatic pressure [98].B. subtilis contains ¢ve ECF c-factors SigV, SigW,SigX, SigY and SigZ [99,100]. SigX is required forsurvival at high temperatures [101] but the functionof the other ECF c-factors is unknown. The secondECF c-factor of E. coli, cE, is induced not only by

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heat or ethanol but also by disruption of proteinfolding in the periplasm [102]. Like FliA of S. typhi-murium, the P. aeruginosa AlgU, M. xanthus CarQand E. coli cE are negatively regulated by proteinsthat bind to the c-factor until the speci¢c activity ofthe ECF c-factor is needed [90,102,103].

In spite of the fact that the members of the ECFsubfamily are divergent in amino acid sequence, E.coli, P. aeruginosa, S. coelicolor and M. xanthus ECFc-factors recognize the same promoter sequence[75,89,104,105]. In this promoter sequence, separatedby a spacer of 16 or 17 base pairs, a 335 region,GAACTT and a 310 region, TCTRA, can be iden-ti¢ed (Table 2). The degree of similarity is higher inthe 335 region than in the 310 region. The B. sub-tilis sigX gene can complement an E. coli fecI mutantindicating that the cX recognizes the same promotersequence as E. coli FecI [106].

3.3.3. Subgroup 3.3. Heat shock c-factorsWhen bacteria are exposed to high temperatures

or other environmental stresses, such as ethanol,heavy metals or hydrogen peroxide, they respondrapidly with the synthesis of heat shock proteins[107]. These proteins appear to operate in the pre-vention and repair of protein damage caused by de-naturation and aggregation [77]. In E. coli, the heatshock response is mediated by the positive regulatorprotein c32 [76]. In B. subtilis three classes of heatshock inducible proteins are known [78] of which themajority belong to class II which are regulated by cB

[78]. Class II proteins are not only induced by heatbut also by other stresses. Therefore, cB is known asthe general stress-response c-factor in B. subtilis.

3.3.3.1. Sigma 32 and related c-factors. The heatshock gene response in E. coli and several otherGram-negative bacteria is mediated by c32

[76,108,109] which is encoded by the rpoH gene. InE. coli and other Q-proteobacteria, transcription ofthe rpoH gene is mediated by c70 and a secondheat shock c-factor, cE [108]. At temperatures above50³C, the rpoH of E. coli is regulated by cE only[110]. In Caulobacter crecentus, a member of the K-proteobacteria, the rpoH gene is transcribed from ac70 promoter and is positively autoregulated by a c32

promoter [111]. In the best characterized heat shockgene regulatory mechanism, that of E. coli, the cel-lular concentrations of c32 increase during temper-

ature upshift by increasing c32 synthesis and stabil-ity. Normally the concentration of c32 is low becauseof its half-life of less than 1 min. This instability isdue to the interaction of DnaK with c32. The result-ing complex is degraded by FtsH, a membranebound metalloprotease [112]. After temperature up-shift DnaK is released from c32 because DnaK pref-erentially binds to denatured proteins accumulatingduring stress [113]. The levels of c32 increase 20-fold,resulting in increased initiation of transcription fromc32-dependent promoters. Some of the heat shockproteins (e.g. GroEL and DnaK) function as molec-ular chaperones and support proper folding of cellu-lar proteins, while others have protease activity anddegrade incorrectly folded proteins [107]. After theheat shock proteins have refolded or degraded thedamaged proteins the amount of c32 drops rapidlybecause DnaK binds again to c32 [113].

Most bacteria contain only one rpoH gene, exceptBradyrhizobium japonicum which possesses three dif-ferent rpoH genes [109] of which rpoH2 is essentialfor the synthesis of cellular proteins and is not in-duced by heat shock [109]. The Gram-negative glid-ing bacteria M. xanthus and Stigmatella aurantiacapossess two c-factors, SigB and SigC, which areclosely related to the c32-proteins [114^116](Fig. 3). These bacteria migrate to aggregation cen-ters under conditions of nutrient limitation wherethey form multicellular structures called fruitingbodies [117]. During development a small percentageof the cells in the fruiting bodies di¡erentiates intospherical myxospores. These spores withstandprolonged periods of nutrient deprivation. SigCmay play a role in expression of genes involvedin the negative regulation of the initiation of fruit-ing body formation [116]. SigB is required for theexpression of certain late-stage developmentalgenes and for proper maturation of myxospores[114,115].

In E. coli, 18 c32-depending promoters have beenidenti¢ed. The nucleotide sequences of these pro-moters were aligned to identify a c32 promoter con-sensus sequence [107]. This c32 heat shock promoterconsensus sequence di¡ers from that of c70-depen-dent promoters, with respect to the 335 consensussequence (CTTGAAA), the 310 consensus sequence(CCCATnT) and the length of the spacer, which is11 to 16 nucleotides (Table 2). In the K subdivisions

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of the proteobacteria the c32 promoter consensussequence seems to be less conserved [118]. Basedon nine promoter sequences, a c32 consensus se-quence for these bacteria was derived which showsa 335 region, CTTG, and a 310 region, CY-TATnTnnG, separated by 17 or 18 bp.

3.3.3.2. Sigma B and related c-factors. The sigBgene product is the general c-factor involved in thestress response in B. subtilis [78]. Homologous cB-factors have been found in Staphylococcus aureus[119], Mycobacterium spp. [120], and Streptomycesspp. [121], in the latter two this c-factor is calledSigF (Fig. 3). The function of SigB is similar tothat of RpoS, the general stress c-factor of E. coli[78,122]. Expression of sigB is activated by heat, al-cohol or osmotic stress, but is also activated by theentry of B. subtilis into the stationary phase in richmedium or by depletion of glucose, phosphate oroxygen [78]. Under starvation conditions, SigB isnegatively regulated by the anti-sigma-factorRsbW. In cells containing a high level of ATP,RsbW is bound to SigB, preventing transcriptionof SigB-dependent promoters. SigB is released fromRsbW when the ATP level drops under starvationconditions [123]. RsbX is the negative regulator ofSigB under stress induced conditions, in which RsbUis involved which releases RsbW from SigB [78]. ThesigB gene is transcribed from a cA promoter. How-ever, it is also autoregulated in response to stress orstarvation by a cB promoter [124]. More than 40general stress proteins are regulated by cB, whichtogether provide adaptation to stress and starvation[125]. The SigF of Mycobacterium tuberculosis is sim-ilar in function to the SigB of B. subtilis. This sta-tionary-phase or stress-response c-factor is inducedduring stationary phase, nitrogen depletion, oxida-tive stress, alcohol or cold shock [120]. The aminoacid sequence of S. coelicolor SigF is similar to thatof B. subtilis cB gene product (Fig. 3), however, theirfunctions di¡er. The S. coelicolor sigF encodes a late-stage, sporulation-speci¢c c-factor. Knockout mu-tants of S. coelicolor sigF are unable to sporulatee¡ectively and produce deformed thin-walled spores[121]. The S. coelicolor SigF recognizes the B. subtilisctc cB promoter in vitro, indicating that cB and SigFrecognize the same promoter sequences [126]. Anal-ysis of eight cB-dependent promoters of B. subtilisrevealed a highly conserved 335 region, GTTTAA,

and a 310 region, GGGTAT, separated by a spacerof 12 to 14 nucleotides [78] (Table 2).

3.4. Subgroup 3.4. Sigma-factors involved insporulation

In response to adverse conditions most bacterialspecies undergo physiological changes during whichthe whole cell is transformed into a stress-resistantstate with highly reduced metabolic activity. Somebacteria however, such as the Gram-positive generaBacillus, Clostridium and mycelial Streptomyces dif-ferentiate into highly resistant spores. This e¡ectivelyprotects their genome from an otherwise inevitablefatal end, when living conditions become intolerable.

The sporulation process in Bacillus spp. and Clos-tridium spp. take place within a sporangium, a struc-ture consisting of two unequal cellular compartmentscalled the forespore and the mother cell [127]. Spor-ulation in Bacillus spp. is induced by high cell densityand nutrient limitation [42,128], whereas in Clostri-dium spp. it is induced by cessation of growth due toexcess carbon and nitrogen source or to exposure tooxygen [129].

Spore formation in Bacillus spp. is controlled by acomplex regulatory network which involves ¢ve c-factors: cH, the mother-cell-speci¢c cE- and cK-fac-tors and the forespore-speci¢c factors cF and cG.After the initial commitment to sporulation, in whichcH is involved, the other c-factors are sequentiallyactivated and e¡ect the sequential expression of avariety of proteins necessary for the complex taskof spore assembly [79,130]. The Gram-positive my-celial bacteria Streptomycetes are distant relatives ofBacillus spp. that undergo a quite di¡erent sporula-tion process [131]. When nutrients become limiting,an aerial mycelium is formed of which the polyploidtip compartments become subdivided into haploidprespore compartments. The prespore compartmentsundergo further maturation to give rise to chains of50 or more spores. The c-factors WhiG and SigF arerequired for sporulation in the aerial hyphae of S.coelicolor [87]. WhiG plays a crucial role in trigger-ing the initiation of sporulation. It activates the earlysporulation whi genes of which the translated pro-teins together with WhiG itself are necessary forthe transcription of sigF. SigF controls the last stageof Streptomyces di¡erentiation the spore maturation.

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Based on amino acid sequence, WhiG belongs to the£agellar c-factors and SigF shows the highest simi-larity with cB of B. subtilis.

3.4.1. Sigma HSigma-factor H is encoded by sigH (or spoOH)

present in B. subtilis and Clostridium spp. [129]. Sig-ma H is essential for sporulation in B. subtilis and isthe earliest acting developmental c-factor in thesporulation process [132]. The early sporulationgenes spoOA, spoOF and kinA [133] and the lateracting spoIIA and spoVG genes [134] are transcribeddue to the presence of cH. Sigma H is not only in-volved in sporulation. It also controls the increase inpost-exponential-growth-phase expression of cA

[135], citG which encodes fumarase, and the ftsAZand minCD cell-division operons [136,137]. Sigma His present in vegetative cells but its level is increasedwhen B. subtilis enters the mid-logarithmic-growthstage [132]. Transcription initiation of sigH is undernegative control of regulator AbrB [132]. The abrBgene expression is repressed by phosphorylatedSpoOA, which is activated by signals that promotesporulation [138].

Using eight known cH-regulated promoter sequen-ces for a sequence alignment, a promoter consensussequence was derived which contains a 335 region,AGGAWWT, and a 310 region RGAAT, separatedby a spacer of 12 to 14 bp [139] (Table 2). Studieswith mutants have shown that amino acids at posi-tion 96 and 100 of cH interact with the nucleotide Gat position 312 of the nucleotide stretch RGAAT[23,140].

3.4.2. Sigma FSigma-factor F is encoded by sigF or spoIIAC in

B. subtilis [141] and is probably also present in Clos-tridium spp. [129]. The B. subtilis sigF gene product(cF) is essential for gene expression in the earlyspore. It is not transcribed until shortly after thestart of sporulation [142] and its protein product isspeci¢cally activated within the developing foresporeafter septation [143]. The spoIIAC gene, togetherwith spoIIAA and spoIIAB genes is located in thespoA operon, which is controlled by cH [134]. ThecF activity depends on the phosphorylation state ofSpoIIAA, which is determined by the ATP/ADP ra-tio in the cell [82]. Phosphorylated SpoIIAA binds to

SpoIIAB, preventing SpoIIAB to bind to cF andconsequently inhibit transcription of cF-dependentpromoters [82]. The cF-factor is required for the ap-pearance of two later acting sporulation-speci¢c c-factors, cE and cG, as well as for morphologicaldevelopment of the forespore itself.

Based on the six known cF-dependent promotersand analysis of several cF promoter point muta-tions a consensus sequence [129,144] containinga 335 region, WGCATA, and a 310 region,GGnRAYAMTW, separated by a spacer of 14 or15 bp (Table 2) was derived.

3.4.3. Sigma ESigma-factor E is encoded by sigE (spoIIGB)

present in B. subtilis [145] and Clostridium acetobu-tylicum [129]. Sigma E is involved in the transcrip-tion of early sporulation genes in the mother-cellcompartment in B. subtilis. Transcription of sigE re-quires cA and phosphorylated SpoOA [146,147]. Theprimary translation product of sigE, pro-cE, is inac-tive as a c-factor and appears just after the onset ofsporulation [148]. After formation of the septumpro-cE is converted into active cE by removal of29 N-terminal amino acids by a sporulation-speci¢cprotease SpoIIGA which is coexpressed with pro-cE

[148,149]. Processing of pro-cE only occurs after aspeci¢c signal protein SpoIIR, controlled by cF trig-gers the reaction [150]. Sigma E blocks formation ofa second polar septum in the mother cell by activat-ing the spoIID [151] and spoIIID [152] genes and anumber of other genes [153]. These genes have incommon a promoter consensus sequence consistingof a 335 region, GKCATATT and a 310 region,CATACAMT, separated by a spacer of 13 to 15 bp[154,155] (Table 2).

Results from amino acid substitution experimentsshowed that nucleotide C in the 335 region is rec-ognized by a histidine residue at position 219 of cE

[154]. A substitution of the glutamine residue at po-sition 217 in cE of B. subtilis to arginine resulted in ac-factor that could direct transcription from cK-de-pendent promoters [154], but no longer from cE-de-pendent promoters.

3.4.4. Sigma GSigma G is encoded by sigG (spoIIIG) and is like

cE present in B. subtilis [156] and in C. acetobutyli-

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cum [157]. SigG causes RNAP to transcribe the latesporulation genes in the forespore. The B. subtilis cG

transcription is regulated by cF as well as being au-toregulated [144,156,158]. Transcription of cG is in-duced shortly after septation. Like cF, cG activity isinhibited by SpoAB and is presumably counteractedby SpoIIAA [82] The cG-dependent genes encode afamily of small, acid-soluble spore proteins [158]which are activated after the forespore is pinchedo¡ as a free protoplast within the mother cell. SigmaG recognizes the consensus promoter elements 335,TGAATA, and 310, CATACTA, separated by aspacer of 17 or 18 bp [159] (Table 2).

3.4.5. Sigma KSigma-factor K is involved in the transcription of

late sporulation genes in the mother-cell compart-ment in Bacillus spp. and probably Clostridium spp.as well [79,129]. In B. subtilis, cK transcription de-pends on a sporulation-site-speci¢c rearrangement ofthe chromosome which joins the spoIVCB andspoIIIC genes to a single cistron sigK [160]. The re-arrangement is due to the site-speci¢c recombinaseSpoIVCA. Transcription of the spoIVCA gene is ac-tivated by SpoIIID [161]. The rearrangement doesnot occur in Bacillus thuringiensis, Bacillus megate-rium and C. acetobutylicum in which sigK is a con-tiguous gene [129,157]. Transcription of cK occursby cE RNAP and SpoIIID [162]. The product ofsigK in B. subtilis and B. thuringiensis is an inactivepro-protein called pro-cK which is converted to ac-tive cK after proteolytic removal of 20 amino acids[163]. Active cK is produced in the mother-cell com-partment late in sporulation where it regulates theformation of spore coat genes [164], promotes thesynthesis of dipicolinic acid synthetase [165] and ini-tiates a negative feedback loop controlling the tran-scription of sigE [166].

Alignment of six cK-dependent promoters revealeda 335 consensus promoter element of only two nu-cleotides, AC, and a 310 region, CATAnAnTA, sep-arated by 16 or 17 nucleotides [167] (Table 2).

4. The cc54-family

The c54-family is structurally and functionally dis-tinct from the c70-family. These proteins have no

detectable sequence similarity with the primary c-factors. Based on the homology between c54-pro-teins, three distinct regions have been recognized(Fig. 2) [16]. Region I is glutamine rich and com-prises 25 to 50 amino acids. This region is followedby a more variable region II, which usually containsbetween 60 and 110 amino acids with a signi¢cantexcess of acidic residues. The carboxyterminal regionIII is some 400 amino acids long and contains the X-link region [168], which has been shown to cross-linkto DNA and two well-conserved motifs : a potentialhelix-turn-helix (HTH) region and the RpoN box.Both motifs are involved in recognition of the pro-moter region of c54-dependent promoters [169]. TheRpoN box is characterized by a stretch of ten con-served amino acids, ARRTVAKYRE. As assayed byband shift analysis, single point mutations near ami-no acids 363 and 383 of E. coli RpoN destroyed theability of c54 to bind DNA [15].

The initiation of transcription of RNAP holoen-zyme containing c54 di¡ers from that of the c70-fam-ily [19]. Whereas the RNAP holoenzyme containinga member of the c70-family often initiates transcrip-tion in the absence of transcriptional activators,transcription from all known c54-dependent pro-moters requires the presence of an activator protein.By contrast, the c54 holoenzyme can form a closedpromoter complex but is incapable of proceeding toopen complex formation in the absence of an activa-tor protein [170]. The activator protein binds to anenhancer which is located 100 bp or more upstreamof the transcriptional start site of a c54 promoter andpossesses a nucleoside triphosphatase activity bywhich it catalyzes the open complex formation byinteraction with RNAP holoenzyme. The activator/polymerase interaction often requires bending of theDNA. Promoters without an intrinsic single bend inthe DNA between enhancer and promoter are de-pendent on the integration factor (IHF) which canintroduce a bend in a stretch of DNA [171]. Theopen complex can be maintained without the pres-ence of an activator. Several of the RpoN activatorshave been characterized in di¡erent bacterial species[172,173]. Examples are NtrC in Rhodobacter spp.,Klebsiella pneumoniae and NifA in Rhizobium spp.both involved in nitrogen ¢xation, DctD, involvedin dicarboxylate transport in Rhizobium spp., FhlAactivator of the formate regulon in E. coli [174] and

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XylR involved in the degradation of aromatic com-pounds in Pseudomonas putida [175].

Sigma 54 encoded by rpoN was ¢rst identi¢ed inE. coli and S. typhimurium as the c-factor requiredfor transcription of nitrogen-regulated genes e.g. glu-tamine synthetase [176,177]. Today, a wide variety ofc54-dependent genes of Gram-negative and Gram-positive bacteria is known. These genes have in com-mon that they are not essential for growth [19]. Oneexception has to be made for some genes in M. xan-thus because the rpoN product in this bacterium isprobably essential [178]. Examples of RpoN-depend-ent genes include those coding for proteins involvedin the formate dehydrogenase in E. coli, dicarboxy-late transport in P. putida, Aztobacter vinelandii andRhizobium meliloti, ¢mbriae synthesis in P. aerugino-sa, Moraxella bovis and Neisseria gonorrhoea, £agel-lar synthesis in C. crescentus and P. aeruginosa, le-vanase production in B. subtilis [173], nitrogen¢xation in K. pneumoniae, Rhizobium sp. and Rhodo-bacter sp. and xylene degradation in P. putida. Mostc54 containing eubacterial species contain one singlec54 gene, but B. japonicum, and Rhodobacter sphaer-oides contain two di¡erent rpoN genes [179,180]. In-activation of one of these genes in B. japonicum andR. sphaeroides has no in£uence on the phenotype ofthese bacteria. Expression of rpoN is usually constit-utive but in some cases, such as in Rhodobacter cap-sulatus, it is regulated by oxygen and nitrogen levels[181]. In C. crescentus it is regulated temporally dur-ing the cell cycle [182]. In B. japonicum rpoN1 isregulated in response to oxygen while rpoN2 is neg-atively autoregulated [180]. Negatively autoregulatedrpoN genes are also found in P. putida and Rhi-zobium etli [183].

Together with at least one activator, c54 enablesRNAP to transcribe c54-dependent promoter se-quences (TGGCAC-N5-TTGC) located between po-sitions 326 and 311 ( þ 1) of the transcription startsite [19] (Table 2). Changes of only 1 bp in the spac-ing between the GG and GC motifs inactivate thepromoter [19].

5. Factors that a¡ect transcription initiation

The sequence of promoter elements is just one ofmany factors that determine the overall e¤ciency of

a particular promoter [184]. The binding of theRNAP holoenzyme to the promoter and the transi-tion from closed to open complex are a¡ected by theDNA conformation in the promoter region and byaccessory proteins.

Gene expression depends on the supercoiling ofthe genome, which is normally negative [185]. Localdi¡erences in the level of supercoiling exist and canbe modulated by environmental signals such as tem-perature, anaerobiosis and osmolarity. The densityof DNA supercoils a¡ects the £exibility of the pro-moter spacer region in both closed and open com-plex formation and thus a¡ects initiation of tran-scription by the RNAP holoenzyme in a positive ornegative way [186].

Many sequence-speci¢c DNA-binding proteins areknown that can in£uence promoter activity. For ex-ample there are 40 proteins which can activate orprevent transcription of c70-regulated promoters inE. coli [187]. These proteins allow the bacterium toregulate the expression of subsets of genes in re-sponse to external and internal stimuli. Genes thatare responding to a common stimulus often use thesame regulatory elements and are therefore referredto as being part of a regulon. Activators of thesepromoters usually bind to the DNA between posi-tions 330 and 380 of the transcription start site[187]. The 335 sequences of these promoters oftenshow a poor homology to the standard c70 335 pro-moter sequence [188]. Repressors bind primarily tolocations that overlap with the polymerase-bindingsite [187]. Promoters recognized by the c54 holoen-zyme are, however, always subject to positive con-trol. Activators of c54-dependent promoters typicallybind around 100 bp upstream of the promoter, butin some cases this is more than 1 kb [19].

Histone-like proteins, such as H-NS in E. coli,may inhibit transcription initiation. H-NS binds any-where on DNA, but it shows a preference for curvedDNA [189]. The transcription of many stationary-phase-responsive genes is inhibited by H-NS [41].

Other, non-proteinaceous factors, that can alsoa¡ect promoter activity are DNA methylation andthe regulatory nucleotide ppGpp. DNA methylationmay a¡ect the binding of RNAP holoenzyme ortranscriptional regulators. Several ¢mbrial operonsin E. coli are regulated by this mechanism [190].

ppGpp is synthesized by RelA, an enzyme that is

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activated in response to amino acid starvation in theso-called stringent response [191]. High concentra-tions of ppGpp result in the cessation of RNA syn-thesis and the down regulation of energy-consumingprocesses. Some promoters are activated by ppGpp,like the RpoS promoter of the general stress c-factorS [192]. Promoters which are negatively regulated byppGpp all contain a G+C rich nucleotide elementbetween the 310 promoter element and the tran-scriptional start site [191,193]. This element is calledthe `stringent box' or `discriminator'. In E. coli, ithas the consensus sequence GCGCCnCC [193].

6. Anti-sigma-factors

Proteins that negatively regulate transcription byinteraction with a speci¢c c-factor are known asanti-c-factors [82]. The binding of an anti-c-factorto its cognate c-factor is probably reversible. It en-ables the cell to respond adequately to environmen-tal signals when the alternative c-factor is alreadyavailable and does not need to be synthesized. Theanti-c-factor binds the c-factor until it is needed fortranscription. In S. typhimurium, the activity of c28 isregulated by FlgM [194]. FlgM functions as an anti-sigma-factor and regulates its own expression. Itbinds to c28 and blocks its activity until the hook-basal body is assembled into the membrane, at whichtime expression of £agellin and other £agellar genesis derepressed. After completion of the basal-bodyhook formation, FlgM is exported through thebasal-body hook liberating c28. Once the £agellum¢lament has been capped, FlgM can no longer beexported. FlgM then binds c28 and the synthesis ofthe £agellin and expression of chemotaxis genesceases.

Anti-sigma-factors of sporulation, stress and ECFrelated c-factors are also known. In B. subtilis threec-factors are negatively regulated by an anti-c-fac-tor. The spore formation is mediated by the anti-c-factor SpoIIAB which interacts with the early spor-ulation cF and the late sporulation cG [195,196].Anti-c-factor RsbW is bound to the stress relatedcB in exponentially growing cells [82]. The regulationof SpoIIAB and RsbW is similar. In conditions ofATP excess these anti-c-factors are bound to theircognate c-factor. In conditions of ADP excess

SpoIIAB and RsbW are bound to the dephosphory-lated anti-anti-c-factor SpoIIAA and RsbV respec-tively, releasing the active c-factor. The ECF c-fac-tor CarQ is negatively regulated by anti-c-factorCarR [103]. In the dark CarR is bound to CarQ.Light inactivates CarR by degradation or light-inhib-ition translation of CarR [103]. AlgU, an ECF c-factor of P. aeruginosa, regulates the alginate biosyn-thesis and is negatively regulated by anti-c-factorMucA [197]. The cE of E. coli is negatively regulatedby RseB and anti-c-factor RseA [198,199]. RseA isan inner membrane protein of which the cytoplasmicdomain binds to cE and inhibits cE directed tran-scription. RseB is a periplasmic protein and speci¢-cally interacts with the C-terminal periplasmic do-main of RseA.

7. Conclusions

Bacterial genome projects are proving importantinformation about the presence of genes for variousc-factors in di¡erent species. When a c-factor isidenti¢ed in a bacterial genome, it is reasonable toassume that genes regulated by this c-factor in otherbacteria are similarly regulated in this organism. Forexample when a c28 homolog is identi¢ed, it is highlyprobable that some of the £agellar genes of this bac-terium are regulated by c28. The genome of Haemo-philus in£uenzae codes for four c-factors: c70, theheat shock related c32 and two c-factors belongingto the ECF family [200]. Synechocystis contains eightdi¡erent c-factors: one primary c-factor RpoD1,four c-factors belonging to the nonessential pri-mary-like c-factors, two c-factors belonging to theECF family and the sporulation related c-factor cF

[201]. The small genomes of Helicobacter pylori andBorrelia burgdorferi contain only three c-factors[202,203]. H. pylori contains c70, the £agella relatedc28 and c54. Instead of c28 B. burgdorferi harbors thestationary-phase c-factor c38. The seven c-factors ofE. coli are c70, c38, c 32, c28, c54 and the ECF c-factors cE and cFecI [204]. Analysis of the genome ofB. subtilis revealed four new potential ECF c-factorsmaking it a total of 14 di¡erent c-factors: primaryc-factor cA, heat shock c-factor cB, £agella relatedcD, ¢ve sporulation related c-factors, ¢ve ECF c-factors and cL belonging to the c54-family [100].

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To date, B. subtilis is the species with the largestnumber of c-factors, even though it does not containthe largest genome size of the bacteria described.There is however, a relationship between the numberof c-factors, the bacterial genome size and the moresevere conditions a bacteria can survive.

Consensus sequences derived from experimentallydetermined promoter sequences are valuable for theidenti¢cation of promoter sequences in genomicDNA sequences. The number of genes from whicha hypothetical protein can be translated is increasingrapidly. Identi¢cation of promoters in front of thesegenes by the use of promoter consensus sequencescan help to classify these genes and may even helpto predict a possible function for the encoded pro-teins.

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