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FEMS Microbiology Reviews 22 (1998) 127^150 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, RNA polymerase has to associate with a small protein, known as a c-factor. The c-factor directs RNA polymerase to a specific class of promoter sequences. Most bacterial species synthesize several different c-factors that recognize different consensus sequences. This variety in c-factors provides bacteria with the opportunity to maintain basal gene expression as well as for regulation of gene expression in response to altered environmental or developmental signals. This review focuses on the function, regulation and distribution of the 14 different classes of c-factors that are presently known. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Transcription initiation ; Eubacteria ; Sigma-factor; Promoter consensus sequence Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Two families of bacterial c-proteins . . . . . . . . . . . . . . . . . . . . . . . 3. The c70 -family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Group 1. Primary c-factors . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Group 2. The nonessential primary-like c-factors . . . . . . . . . 3.2.1. Subgroup 2.1. Stationary-phase c-factors . . . . . . . . . . . 3.2.2. Subgroup 2.2. Cyanobacterial c-factors . . . . . . . . . . . . 3.2.3. Subgroup 2.3. The c-factors of high-GC Gram-positive 3.3. Group 3. Alternative c-factors . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Subgroup 3.1. Flagellar c-factors . . . . . . . . . . . . . . . . 3.3.2. Subgroup 3.2. Extracytoplasmic function c-factors . . . . 3.3.3. Subgroup 3.3. Heat shock c-factors . . . . . . . . . . . . . . . 3.3.3.1. Sigma 32 and related c-factors . . . . . . . . . . . . 3.3.3.2. Sigma B and related c-factors . . . . . . . . . . . . . 3.4. Subgroup 3.4. Sigma-factors involved in sporulation . . . . . . . ....... ....... ....... ....... ....... ....... ....... bacteria ....... ....... ....... ....... ....... ....... ....... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 129 130 131 134 134 135 135 135 136 136 137 137 138 138 * Present address: Washington University, School of Medicine, Department of Molecular Microbiology, 660 S. Euclid Ave. Campus Box 8230, St. Louis, MO 63110, USA. Tel.: +1 (314) 362 3691; Fax: +1 (314) 362 1232; E-mail: [email protected] 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 15-10-98 128 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 3.4.1. Sigma H . . . . . . . . . . . . . . . . . . 3.4.2. Sigma F . . . . . . . . . . . . . . . . . . 3.4.3. Sigma E . . . . . . . . . . . . . . . . . . 3.4.4. Sigma G . . . . . . . . . . . . . . . . . . 3.4.5. Sigma K . . . . . . . . . . . . . . . . . . 4. The c54 -family . . . . . . . . . . . . . . . . . . . . . 5. Factors that a¡ect transcription initiation . 6. Anti-sigma-factors . . . . . . . . . . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... 1. Introduction Bacteria encode several thousands of di¡erent proteins, which are necessary for normal cell functions or for adaptation to environmental changes. These proteins are not required at the same time or in the same amount. Regulation of gene expression therefore enables the cell to control the production of proteins needed for its life cycle or for adaptation to extracellular changes. This regulation in turn makes it possible for the bacterium to adequately adapt to rapid changes in the environment. The various steps during transcription and translation are therefore subject to di¡erent regulatory mechanisms. The most prominent step in gene regulation is the initiation of transcription in which the DNA-dependent RNA polymerase (RNAP) is the key enzyme. Depending on the growth rate, 1500 to 11400 RNAP molecules are present in an Escherichia coli cell [1]. As calculated by the synthesis of stable RNA, 24% to 79% of these molecules respectively, are actually engaged in the synthesis of RNA at any particular time [1]. The RNAP or the RNAP core enzyme is the catalytic machinery for the synthesis of RNA from a DNA template [2]. However, RNAP cannot initiate transcription by itself. Initiation of transcription requires an additional polypeptide known as a c-factor. Sigma-factors are a family of relatively small proteins that can associate in a reversible way with the RNAP core enzyme. Together, the c-factor and the RNAP core enzyme form an initiation-speci¢c enzyme, the RNAP holoenzyme [3,4]. Initiation of transcription in eubacteria is a multistep process which begins with the binding of RNAP holoenzyme, containing c70 or a c70 -related c-factor, to a speci¢c DNA sequence recognized by this en- ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ...... ...... ...... ...... ...... ...... ...... ...... ...... ...... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... ..... 139 139 139 139 140 140 141 142 142 143 zyme as a promoter [5^7] (Fig. 1). Recognition of a promoter sequence is directed primarily by the csubunit of the RNAP holoenzyme. The binding of RNAP holoenzyme to the promoter results in a socalled `closed complex' which is converted into an `open complex' by melting of a short region of DNA within the sequence bound by the enzyme. In the resulting ternary complex, the RNAP holoenzyme starts to produce small 2 to 12 bp RNA molecules, but remains located at the promoter. After dissociation of the c-subunit, the RNAP core enzyme moves along the DNA, meanwhile synthesizing the nascent RNA molecule. A locally unwound region of DNA moves with the enzyme. The RNAP core enzyme and RNA are released when a terminator structure or factor is encountered and the DNA is then fully restored to duplex condition [7]. The frequency at which the RNAP holoenzyme initiates transcription, also known as the `strength' of a promoter, is in£uenced by the promoter sequence and the conformation of the DNA in the promoter region. The c-factor recognizes two conserved sequences in the promoter region, known as the promoter consensus sequence [8]. The model proposed in Fig. 1 is supported by footprint analyses in which c-factor or fragments of c-factor have been shown to bind speci¢cally to promoter DNA sequences [9^11] and by speci¢c base pair and amino acid substitutions in the promoter consensus sequences or c-factor [6,12^15]. Most bacterial species synthesize several di¡erent c-factors which direct the RNAP holoenzyme to distinct classes of promoters with a di¡erent consensus sequence. This variety in c-factors provides the bacterium with the opportunity to maintain basal gene expression as well as for regulation of gene expression in response to speci¢c environmental stimuli. FEMSRE 612 15-10-98 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 129 Fig. 1. Schematic representation of several steps required for the initiation of transcription of RNAP containing c70 or a c70 -related cfactor. The RNAP holoenzyme binds to the promoter to form the closed complex. Conformational changes in the closed complex result in an open form in which the 310 promoter region is melting out. In this open form the RNAP holoenzyme starts to produce small RNA molecules. A ternary complex is formed. Finally the RNAP of which the c-factor is dissociated starts to transcribe an mRNA molecule. See text for details. 2. Two families of bacterial c-proteins Based on sequence similarity, the bacterial c-fac- tors can be grouped into two families, the c70 - and c54 -family, with little if any sequence identity between them [16,17]. The majority of the c-factors FEMSRE 612 15-10-98 130 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 Fig. 2. Structure function maps of c70 - and c54 -family of c-factors. Regions shown as a solid box represent the most conserved parts of the c-factors. A detailed functional assignment for the subregions of the c70 -family proteins is displayed in the expanded view. Region III of the c54 -family contains three structures indicated as X-link, HTH and the RpoN box. They are involved in DNA binding. in eubacteria belong to the c70 -family. This family is named after the 70 kDa primary c-factor from E. coli [18]. All eubacteria contain one or more c-factors belonging to the c70 -family, which is divided into several structurally and functionally related (sub)groups. Many eubacteria also contain a c-factor which belongs to the c54 -family [19], named for the 54 kDa nitrogen regulation c-factor of E. coli. The c54 -family contains only one group of c-factors which 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¡erent names and even di¡erent c-factors having identical names. 3. The c70-family Sequence comparison of c70 -family proteins from di¡erent eubacteria led to the identi¢cation of four highly conserved amino acid regions [20]. As additional sequence data became available these regions FEMSRE 612 15-10-98 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 have been subdivided further [18] (Fig. 2). The Nterminal 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, important for the e¤cient initiation of transcription [21]. Subregion 1.2 is probably required for open complex formation [21]. Deleting region 1.2 prevents RNAP from progressing beyond the closed complex. This subregion 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 be divided in four subregions. Region 2.1 is important for interaction with the subunits of core RNAP, while region 2.3 is involved in DNA melting. Important for DNA binding is subregion 2.4 which has been implicated in the recognition of the 310 promoter element, a region located approximately 10 nucleotides upstream of the start point of transcription (+1) [8]. Mutations in region 2.4 alter the speci¢c interaction with the 310 promoter element [12,22,23]. Recently, region 2.5 was identi¢ed as being involved in contacting nucleotides at positions 314 and 315 in E. coli promoters [24], known in Bacillus subtilis as the 316 promoter consensus sequence [25]. Regions 3 and 4 are divided in two subregions. Subregion 3.1 contains a helix-turn-helix DNA-binding motif and the less conserved region 3.2 may be involved in binding the RNAP core enzyme [18,26]. Region 3.2 is not present in the c-factors K and H. Subregion 4.1 has been proposed to bind certain transcriptional activators during the initiation of transcription [27,28]. Subregion 4.2 also recognizes the promoter sequence as mutations in this region a¡ect the ability of the RNAP holoenzyme to recognize a promoter element approximately 335 nucleotides relative to the start point of transcription [8,12,13,29]. î crystal structure of a fragment of E. coli The 2.6 A 70 c comprising amino acid 114 to 448 was solved recently [30]. This fragment contains a part of region 1.2, the complete regions 2.1, 2.2, 2.3, and all but the C-terminal ¢ve residues of region 2.4. The crystal structure indicates that all the conserved regions are closely associated with one another and that region 1.2 stabilizes the fold of the other regions. Residues involved in core RNAP binding lie on one face of the structure. On the opposite face are residues 131 that interact with the 310 region and residues involved in promoter melting. The c70 -protein family can be divided into three di¡erent functionally and structurally related groups [18] (Fig. 3, Table 1). Group 1 comprises the primary c-factors responsible for the transcription of most genes expressed in exponentially growing cells of which the majority are essential for cell survival. Group 2 comprises the c-factors which are quite similar to the primary c-factors in sequence but are nonessential for cell growth. The third group consists of the so-called alternative c-factors. These c-factors di¡er considerably in amino acid sequence from the primary c-factors and control the transcription of speci¢c regulons. The three groups of the c70 -family and the various subgroups are described below. 3.1. Group 1. Primary c-factors Group 1 is composed of the primary c-factors present in all known eubacteria [31]. They are responsible for the transcription of most genes expressed in exponentially growing cells and are essential for cell survival. Recent data suggest that there is only one primary c-factor present in any given eubacterial species [31]. The primary c-factor in E. coli is encoded by the rpoD gene and is known as c70 [20]. In Mycobacterium spp. this c-factor is known as MysA, in Streptomyces spp. as HrdB and in B. subtilis and other Gram-positive bacteria as SigA or cA [31]. The promoter consensus sequence recognized by the primary c-factor in E. coli and B. subtilis is found to be similar in many other eubacteria [32,33]. This promoter sequence is characterized by two stretches of six nucleotides, TTGACA and TATAAT, centered around positions 335 and 310 respectively, from the transcription start site (+1) (Table 2). A dinucleotide TG at positions 315 and 314 is also conserved in promoters of Gram-positive bacteria but less conserved in E. coli promoters [25,33]. The hexamer sequences around 335 and 310 are most often separated by a stretch of 16 to 18 nonconserved nucleotides called the spacer. Deviations from the consensus sequences usually result in reduced promoter activity [25,34,35]. However, certain prokaryotic promoters can function in the absence of a 335 promoter element if they have an `extended 310' promoter element, TGnTATAAT [36,37]. The FEMSRE 612 15-10-98 132 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 Fig. 3. Phylogenetic relationship of the c-factors of the c70 -family. The tree was generated by the program Fitch of the Phylip package version 3.5c [205] using a PAM matrix-based distance correction from a multiple sequence alignment made be the program Multalin version 4.0 [206]. The scale bar represents 0.2 substitution per site. The sequences used in the alignment were translated from the Genbank entries given in parentheses: Escherichia coli RpoE (P34086), KatF (X16400), RpoH (M20668), FliA (L36677), RpoD (U23083); Salmonella typhimurium RpoE (P34086), RpoS (U05011) ; Streptomyces aureofaciens HrdE (M90412), HrdA (M90410); Haemophilus in£uenzae RpoE (P44790); Photobacterium sp. strain SS9 (L41688); Pseudomonas aeruginosa AlgU (U49151), RpoS (D26134); Mycobacterium tuberculosis 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 enterocolitica FliA (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 coelicolor HrdB (X52983), HrdC (P18184), SigF (L11648); Stigmatella aurantiaca SigB (Z14970); Rhodobacter capsulatus RpoD (Z68306); Proteus mirabilis RpoH (D50830); Agrobacterium tumefaciens RpoD (P33452) ; Streptomyces griseocarneus WhiG (X68709) ; Vibrio parahaemolyticus 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). FEMSRE 612 15-10-98 FEMSRE 612 15-10-98 3.4.2. Sigma F 3.4.3. Sigma E 3.4.4. Sigma G 3.4.5. Sigma K 4. The c54 -family 3.3. Heat shock c-factors 3.3.1. Sigma 32 and related c-factors 3.3.2. Sigma B and related c-factors 3.4. Sporulation c-factors 3.4.1. Sigma H 3.2. ECF c-factor Mycobacteria, MysB ; Corynebacteria, SigB; Streptomyces, HrdA, HrdC-E 2.3. The c-factors of high-GC Gram-positive bacteria 3. Alternative c-factors 3.1. Flagella c-factor Transcription of early sporulation and postexponential-growth-phase genes Transcription of early forespore genes Transcription of early mother-cell genes Transcription of late forespore genes Transcription of late mother-cell genes Expression of genes involved in: nitrogen ¢xation, nitrate utilization, glutamine synthetase, dicarboxylate transport, xylene degradation, ¢mbriae and £agellar synthesis or levanase production Bacillus, Clostridium, cH Bacillus, Clostridium, cF Bacillus, Clostridium, cE Bacillus, Clostridium, cG Bacillus, Clostridium, cK Rhizobium, Rhodobacter, Pseudomonas, Bacillus, Caulobacter, Enterobacteriaceae Expression of genes during stress, fruiting body formation or maturation of myxospores Expression of genes during stress or late sporulation Expresion of chemotaxis, late £agellar or early sporulation genes Expression of genes involved in alginate biosynthesis, iron uptake, carotenoid biosynthesis, antibiotic production, induction of virulence factors, resistants to cobalt and nickel, outer membrane proteins or survival of high temperature Major sigma-factor during entry into stationary phase. Global regulator of gene expression in cells exposed to hyperosmotic or acid stress Controlling gene expression during circadian responses, carbon and nitrogen availability or post-exponential-growth phase Unknown Major sigma-factor in exponential growing cells regulates house keeping genes Functions Gram-negative bacteria, c32 ; M. xanthus, SigB; S. aurantiaca, SigC B. subtilis, Stapylococcus, cB ; Mycobacteria, Streptomyces, SigF Enterobacteria, c28 ; Streptomyces, WhiG; Bacillus subtilis, cD Pseudomonas aeruginosa, AlgU; Escherichia coli, FecI; Pseudomonas £uorescens, PbrA; Myxococcus xanthus, CarQ ; S. coelicolor, E. coli, Mycobacteria, cE ; Pseudomonas Syringae, HprL ; Alcaligenes eutrophus, CrnH ; B. subtilis, SigV-Z Synechococcus, Synechocystis, Anabaena, SigB-E Enterobacteria, Pseudomonas, RpoS 2.2. Cyanobacterial c-factors 2. Nonessential primary-like c-factors 2.1. Stationary phase c-factor Gram-negative bacteria, c ; Gram-positive bacteria, cA ; Mycobacteria, MysA; Streptomyces, HrdB 1. Primary c-factors 70 Organism, name c-factor Group Table 1 Functions and distribution of the various eubacterial c-factors [79,129] [79,129] [79,129] [79,129] [19] [79,129] [78,120,121] [76,108,14] [75] [74,87] [31] [31,65] [43] [31] Reference M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 133 134 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 so-called `extended 310' promoter provides a very strong recognition signal for the c70 -subunit which can overcome both the lack of a 335 promoter element as well as less conserved 310 region [38]. Recognition of the 310 and 335 region of the primary c promoter has been studied extensively by site-speci¢c mutagenesis. Amino acid substitution studies within region 2.4 (Fig. 2) of c70 of E. coli showed that two amino acids at positions 437 and 440 interact with the 310 region of c70 promoters. These amino acids, glutamine at position 437 and threonine at position 440, are in contact with the T/A base pair at position 312 of the 310 promoter element [12,22,39]. Mutagenesis studies within region 4.2 have shown that the arginine residues at positions 588 and 584 of c70 , respectively, interact with the G and C residues of the 335 region TTGACA [12]. tain c-factors which belong to group 2 [31]. The Enterobacteriaceae and Pseudomonas contain a group 2 c-factor, known as the stationary-phase-speci¢c c-factor, cS or RpoS [40]. All cyanobacterial group 2 c-factors are closely related based on amino acid sequences, but di¡er from the primary c-factors of the corresponding organisms (Fig. 3). The group 2 c-factors from Gram-positive bacteria with a highGC content do not form such a tight grouping. The high-GC content Gram-positive bacteria, cyanobacteria and Chloro£exus aurantiacus are the only organisms in which multiple group 2 c-factors have been found [31]. The subgroups of group 2 are described below. 3.2.1. Subgroup 2.1. Stationary-phase c-factors In their natural environment, bacteria frequently experience nutrient limitation and a variety of physical and chemical stresses. Consequently, they endure and survive long periods without growth [41]. To survive environmental stress some bacteria produce highly resistant spores while others enter into the stationary-growth phase [41,42]. Entry into the stationary phase is characterized by the synthesis of stress proteins [41]. In E. coli and other enteric bacteria the general stress response is mediated by cS (RpoS, c38 ) encoded by the rpoS gene [43]. In less closely related bacteria only the Pseudomonas spp. 3.2. Group 2. The nonessential primary-like c-factors The c-factors in this group are nonessential for exponential cell growth [18]. They are highly similar to the primary c-factors in the amino acid sequence of their DNA-binding regions, which suggests that both groups of c-factors recognize similar promoter sequences. The enterobacteria, the high-GC content Gram-positive bacteria and the cyanobacteria conTable 2 Consensus sequences recognized by various eubacterial c-factors c70 -family Namea Primary c-factors Nonessential primary-like c-factors 2.1. Stationary-phase c-factor Alternative c-factors 3.1. Flagella c-factors 3.2. ECF c-factors 3.3. Heat shock c-factors c70 , RpoD, SigA 3.4. Sporulation c-factors c54 -family a b Consensus sequenceb Reference 335 Spacer 310 TTGACA 16^18 TATAAT [8,33] CTATACT [58] c38 , RpoS c28 , FliA, SigD cE , SigE c32 , RpoH cB , SigB cH , SpoOH cF , SpoIIAC cE , SpoIIGB cG , SpoIIIG cK , SpoIIIC TAAA GAACTT CTTGAAA GTTTAA AGGAWWT WGCATA GKCATATT TGAATA AC 324 15 16^17 11^16 12^14 12^14 14^15 13^15 17^18 16^17 GCCGATAA TCTRA CCCATnT GGGTAT RGAAT GGnRAYAMTW CATACAMT CATACTA CATAnAnTA 312 [74] [75,89,104,105] [107] [78] [139] [129,144] [154,155] [159] [167] cN , RpoN, SigL TGGCAC 5 TTGCW [19] Only the c-factor names for which a consensus recognition sequence has been derived are indicated. Ambiguous codes: N, any base; R, A or G; W, A or T ; Y, C or T; M, A or C; K, G or T. FEMSRE 612 15-10-98 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 135 have been shown to contain a rpoS-like gene [31,43]. While cS is best known for its crucial role in gene regulation during entry into stationary phase [41], it also serves as a global regulator of gene expression in cells exposed to hyperosmotic [44] or acid stress [45]. Genes regulated by cS encode a variety of di¡erent functions being involved in the prevention and repair of DNA damage, cell morphology, modulation of virulence genes, osmoprotection, thermotolerance, glycogen synthesis, membrane and cell envelope functions [43]. Increased expression of stationaryphase RpoS-regulated genes requires an increase in the abundance of cS . The cellular concentration of cS in E. coli is controlled at the levels of transcription, translation and protein stability. In E. coli cS is controlled by four di¡erent c70 promoters of which the second promoter (P2) in vitro is also recognized by c38 [46]. The RNA-binding protein HF-1 is essential for the translational regulation of rpoS [47]. Other factors such as DNA-binding protein H-NS [48], cyclic AMP [49] and UDP-glucose [50] negatively in£uence production of cS , while pppGpp [51], homoserine lactone [52] and DnaK [53] raise the level of cS . In exponentially growing cells, cS is very unstable. Involved in the degradation of cS are the negative regulator RssB and ClpXP protease [54]; mutations in these genes give rise to stable cS . The decreased proteolytic turnover of cS during acid shock in S. typhimurium is mediated by MviA [55]. Some promoters that are recognized by cS are also under the control of c70 [56,57]. Comparison of 33 cS -regulated promoters revealed that the cS promoter consensus sequence lacks a 335 region, but contains a conserved 310 promoter element, CTATACT [58] (Table 2). The lack of a 335 region is possibly compensated by a region upstream of the 310 region with an intrinsic DNA curvature [58]. However, the presence of DNA-bending regions is often found in front of strong promoters [59], and they are not essential for recognition by cS in vitro [60]. So far, no DNA-binding protein that acts speci¢cally as an activator of cS -dependent genes has been identi¢ed [61]. rhythms [62]. The cyanobacterial group 2 c-factors form a tight cluster of related sequences [31] (Fig. 3). Members of this cluster are the SigB and SigC cfactors of Anabaena sp., and Synechocystis sp. and the SigB-E c-factors of Synechococcus sp. [31]. Recent investigations have shown that SigB (RpoD2) of Synechococcus sp. controls gene expression during circadian responses [31,63]. Mutagenesis of the rpoD2 gene resulted in a rhythmic mutant with a low amplitude phenotype. The sigB and sigC genes of Anabaena sp. are expressed under nitrogen-limiting conditions but neither of these genes is required for nitrogen ¢xation or heterocyst di¡erentiation [64]. The homologues of SigB and SigC in Synechococcus sp. play a role in carbon and nitrogen availability [65]. The sigB increases signi¢cantly when cells are starved for nitrogen and carbon. Mutation of sigC increases transcription of sigB indicating that their functions are related to one another. SigE of Synechococcus sp. may play a role in the transcription of genes during post-exponential growth in this organism and therefore, SigE may be similar in function although its sequence places it outside the RpoS family of c-factors [66]. 3.2.2. Subgroup 2.2. Cyanobacterial c-factors The Gram-negative cyanobacteria are capable of photosynthetic growth. They are the simplest unicellular organisms known to exhibit circadian (daily) The third group of the c70 -family comprises alternative c-factors that control the transcription of speci¢c regulons during special physiological or developmental conditions [6,20]. In alignment studies, 3.2.3. Subgroup 2.3. The c-factors of high-GC Gram-positive bacteria The c-factors belonging to this group are the hrdA, hrdC, hrdD and hrdE genes of Streptomyces spp., mysB of Mycobacterium spp. and sigB of Corynebacterium glutamicum. The c-factors of these phylogenetically related bacteria are more diverse than those of the cS and cyanobacterial group 2 cfamily [31] (Fig. 3). The Streptomyces coelicolor hrdD gene is expressed in exponentially growing cells and chrdD recognizes the promoters in front of redD and actII-orf4, two regulatory genes required for the synthesis of antibiotics [67,68]. Although the other genes are expressed and can be disrupted, the functions of these nonessential c-factors remain unclear [69^73]. 3.3. Group 3. Alternative c-factors FEMSRE 612 15-10-98 136 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 subgroups of the alternative c-factors were found to cluster together and subsequently shown to have related functions (Fig. 3, Table 1). This group includes the £agellar [74], extracytoplasmic functional [75], heat shock [76^78] and the sporulation [79] cfactors. 3.3.1. Subgroup 3.1. Flagellar c-factors Many motile bacteria possess an external organelle, the £agellum, that propels the cell in directions dictated by diverse sensory signals. More than 50 genes, which are divided into three classes, are involved in the formation of a functional £agellum in E. 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 the transcription of the class 2 genes. Class 2 genes encode components needed in the early stage of basalbody construction, FlgM and c-factor FliA. FliA or c28 is needed for the expression of class 3 £agellar proteins involved in the ¢lament assembly and genes involved in chemotaxis gene hierarchy in various enterobacteria [74,80,81]. FliA is negatively regulated by FlgM, which binds to c28 until the basal-body hook formation is completed [82]. The cD of B. subtilis [83] and WhiG of Streptomyces spp. [84,85] are also members of the £agellar c-factors. The function and regulation of cD of B. subtilis is similar to that of FliA of the enterobacteria [86]. WhiG of the Gram-positive mycelial S. coelicolor is similar in structure but has a totally di¡erent function compared to FliA. These bacteria can form spores in the aerial hyphae, a process in which WhiG plays an essential role [87]. In the absence of WhiG the aerial hyphae fail to develop sporulation septa, while an excess of WhiG causes hyper-sporulation [85]. Although WhiG has a totally di¡erent function, the S. coelicolor whiG gene can complement a £iA null mutant of S. typhimurium to restore £agellar biosynthesis and motility [87]. Genetic experiments have shown that WhiG is also regulated post-translationally by FlgM when introduced into S. typhimurium [87]. It is therefore likely that the WhiG recognizes the same promoter sequence as FliA of S. typhimurium. Analysis of c28 - and cD -dependent promoters in E. coli and B. subtilis revealed that RNAP containing c28 or cD recognizes the 335 promoter element, TAAA, and the 310 sequence, GCCGATAA, separated by a spacer of 15 nucleotides [74] (Table 2). 3.3.2. Subgroup 3.2. Extracytoplasmic function c-factors The extracytoplasmic function (ECF) c-subgroup of the c70 -family is a class of environmentally responsive transcriptional regulators [75]. The amino acid sequences of these c-factors were so di¡erent from the c70 -factors that they were initially not recognized as c-factors (Fig. 3). They share similarity with the primary c-factors in three of the four conserved regions but lack most of region 1 (Fig. 2) [75]. Several bacterial species contain members of the ECF family in which they control a variety of functions in response to speci¢c extracellular environmental signals. ECF factors include AlgU, which regulates alginate biosynthesis in Pseudomonas aeruginosa [88,89]. Alginate is a viscous exopolysaccharide believed to protect the bacterium from the adverse environment of the cystic ¢brosis lung [90]. FecI of E. coli [91] and PbrA of Pseudomonas £uorescens [92] are both involved in iron uptake. They coordinate gene response in low-iron conditions and are both negatively regulated by the Fur repressor [92]. The light inducible biosynthesis of carotenoid in Myxococcus xanthus is controlled by CarQ [93]. Carotenoids are often found in non-photosynthetic bacteria as light-protective agents [93]. The S. coelicolor cE promotes transcription of a gene encoding an extracellular agar-degrading enzyme, agarase [75], while cE of Streptomyces antibioticus is required for the production of the antibiotic actinomycin [94]. HprL of Pseudomonas syringae controls transcription and secretion of a plant virulence factor and is inducible by plant extracts [95]. CnrH plays a role in resistance to nickel and cobalt e¥ux in Alcaligenes eutrophus [96]. The Mycobacterium smegmatis ECF c-factor plays a role in the ability to withstand a variety of stresses [97]. The expression of several outer membrane proteins in the deep-sea bacterium Photobacterium sp. strain SS9 is regulated by cE in response to growth under hydrostatic pressure [98]. B. subtilis contains ¢ve ECF c-factors SigV, SigW, SigX, SigY and SigZ [99,100]. SigX is required for survival at high temperatures [101] but the function of the other ECF c-factors is unknown. The second ECF c-factor of E. coli, cE , is induced not only by FEMSRE 612 15-10-98 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 heat or ethanol but also by disruption of protein folding in the periplasm [102]. Like FliA of S. typhimurium, the P. aeruginosa AlgU, M. xanthus CarQ and E. coli cE are negatively regulated by proteins that bind to the c-factor until the speci¢c activity of the ECF c-factor is needed [90,102,103]. In spite of the fact that the members of the ECF subfamily are divergent in amino acid sequence, E. coli, P. aeruginosa, S. coelicolor and M. xanthus ECF c-factors recognize the same promoter sequence [75,89,104,105]. In this promoter sequence, separated by a spacer of 16 or 17 base pairs, a 335 region, GAACTT and a 310 region, TCTRA, can be identi¢ed (Table 2). The degree of similarity is higher in the 335 region than in the 310 region. The B. subtilis sigX gene can complement an E. coli fecI mutant indicating that the cX recognizes the same promoter sequence as E. coli FecI [106]. 3.3.3. Subgroup 3.3. Heat shock c-factors When bacteria are exposed to high temperatures or other environmental stresses, such as ethanol, heavy metals or hydrogen peroxide, they respond rapidly with the synthesis of heat shock proteins [107]. These proteins appear to operate in the prevention and repair of protein damage caused by denaturation and aggregation [77]. In E. coli, the heat shock response is mediated by the positive regulator protein c32 [76]. In B. subtilis three classes of heat shock inducible proteins are known [78] of which the majority belong to class II which are regulated by cB [78]. Class II proteins are not only induced by heat but also by other stresses. Therefore, cB is known as the general stress-response c-factor in B. subtilis. 3.3.3.1. Sigma 32 and related c-factors. The heat shock gene response in E. coli and several other Gram-negative bacteria is mediated by c32 [76,108,109] which is encoded by the rpoH gene. In E. coli and other Q-proteobacteria, transcription of the rpoH gene is mediated by c70 and a second heat shock c-factor, cE [108]. At temperatures above 50³C, the rpoH of E. coli is regulated by cE only [110]. In Caulobacter crecentus, a member of the Kproteobacteria, the rpoH gene is transcribed from a c70 promoter and is positively autoregulated by a c32 promoter [111]. In the best characterized heat shock gene regulatory mechanism, that of E. coli, the cellular concentrations of c32 increase during temper- 137 ature upshift by increasing c32 synthesis and stability. Normally the concentration of c32 is low because of its half-life of less than 1 min. This instability is due to the interaction of DnaK with c32 . The resulting complex is degraded by FtsH, a membrane bound metalloprotease [112]. After temperature upshift DnaK is released from c32 because DnaK preferentially binds to denatured proteins accumulating during stress [113]. The levels of c32 increase 20-fold, resulting in increased initiation of transcription from c32 -dependent promoters. Some of the heat shock proteins (e.g. GroEL and DnaK) function as molecular chaperones and support proper folding of cellular proteins, while others have protease activity and degrade incorrectly folded proteins [107]. After the heat shock proteins have refolded or degraded the damaged proteins the amount of c32 drops rapidly because DnaK binds again to c32 [113]. Most bacteria contain only one rpoH gene, except Bradyrhizobium japonicum which possesses three different rpoH genes [109] of which rpoH2 is essential for the synthesis of cellular proteins and is not induced by heat shock [109]. The Gram-negative gliding bacteria M. xanthus and Stigmatella aurantiaca possess two c-factors, SigB and SigC, which are closely related to the c32 -proteins [114^116] (Fig. 3). These bacteria migrate to aggregation centers under conditions of nutrient limitation where they form multicellular structures called fruiting bodies [117]. During development a small percentage of the cells in the fruiting bodies di¡erentiates into spherical myxospores. These spores withstand prolonged periods of nutrient deprivation. SigC may play a role in expression of genes involved in the negative regulation of the initiation of fruiting body formation [116]. SigB is required for the expression of certain late-stage developmental genes and for proper maturation of myxospores [114,115]. In E. coli, 18 c32 -depending promoters have been identi¢ed. The nucleotide sequences of these promoters were aligned to identify a c32 promoter consensus sequence [107]. This c32 heat shock promoter consensus sequence di¡ers from that of c70 -dependent promoters, with respect to the 335 consensus sequence (CTTGAAA), the 310 consensus sequence (CCCATnT) and the length of the spacer, which is 11 to 16 nucleotides (Table 2). In the K subdivisions FEMSRE 612 15-10-98 138 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 of the proteobacteria the c32 promoter consensus sequence seems to be less conserved [118]. Based on nine promoter sequences, a c32 consensus sequence for these bacteria was derived which shows a 335 region, CTTG, and a 310 region, CYTATnTnnG, separated by 17 or 18 bp. 3.3.3.2. Sigma B and related c-factors. The sigB gene product is the general c-factor involved in the stress response in B. subtilis [78]. Homologous cB factors have been found in Staphylococcus aureus [119], Mycobacterium spp. [120], and Streptomyces spp. [121], in the latter two this c-factor is called SigF (Fig. 3). The function of SigB is similar to that of RpoS, the general stress c-factor of E. coli [78,122]. Expression of sigB is activated by heat, alcohol or osmotic stress, but is also activated by the entry of B. subtilis into the stationary phase in rich medium or by depletion of glucose, phosphate or oxygen [78]. Under starvation conditions, SigB is negatively regulated by the anti-sigma-factor RsbW. In cells containing a high level of ATP, RsbW is bound to SigB, preventing transcription of SigB-dependent promoters. SigB is released from RsbW when the ATP level drops under starvation conditions [123]. RsbX is the negative regulator of SigB under stress induced conditions, in which RsbU is involved which releases RsbW from SigB [78]. The sigB gene is transcribed from a cA promoter. However, it is also autoregulated in response to stress or starvation by a cB promoter [124]. More than 40 general stress proteins are regulated by cB , which together provide adaptation to stress and starvation [125]. The SigF of Mycobacterium tuberculosis is similar in function to the SigB of B. subtilis. This stationary-phase or stress-response c-factor is induced during stationary phase, nitrogen depletion, oxidative stress, alcohol or cold shock [120]. The amino acid sequence of S. coelicolor SigF is similar to that of B. subtilis cB gene product (Fig. 3), however, their functions di¡er. The S. coelicolor sigF encodes a latestage, sporulation-speci¢c c-factor. Knockout mutants of S. coelicolor sigF are unable to sporulate e¡ectively and produce deformed thin-walled spores [121]. The S. coelicolor SigF recognizes the B. subtilis ctc cB promoter in vitro, indicating that cB and SigF recognize the same promoter sequences [126]. Analysis of eight cB -dependent promoters of B. subtilis revealed a highly conserved 335 region, GTTTAA, and a 310 region, GGGTAT, separated by a spacer of 12 to 14 nucleotides [78] (Table 2). 3.4. Subgroup 3.4. Sigma-factors involved in sporulation In response to adverse conditions most bacterial species undergo physiological changes during which the whole cell is transformed into a stress-resistant state with highly reduced metabolic activity. Some bacteria however, such as the Gram-positive genera Bacillus, Clostridium and mycelial Streptomyces differentiate into highly resistant spores. This e¡ectively protects their genome from an otherwise inevitable fatal end, when living conditions become intolerable. The sporulation process in Bacillus spp. and Clostridium spp. take place within a sporangium, a structure consisting of two unequal cellular compartments called the forespore and the mother cell [127]. Sporulation in Bacillus spp. is induced by high cell density and nutrient limitation [42,128], whereas in Clostridium spp. it is induced by cessation of growth due to excess carbon and nitrogen source or to exposure to oxygen [129]. Spore formation in Bacillus spp. is controlled by a complex regulatory network which involves ¢ve cfactors: cH , the mother-cell-speci¢c cE - and cK -factors and the forespore-speci¢c factors cF and cG . After the initial commitment to sporulation, in which cH is involved, the other c-factors are sequentially activated and e¡ect the sequential expression of a variety of proteins necessary for the complex task of spore assembly [79,130]. The Gram-positive mycelial bacteria Streptomycetes are distant relatives of Bacillus spp. that undergo a quite di¡erent sporulation process [131]. When nutrients become limiting, an aerial mycelium is formed of which the polyploid tip compartments become subdivided into haploid prespore compartments. The prespore compartments undergo further maturation to give rise to chains of 50 or more spores. The c-factors WhiG and SigF are required for sporulation in the aerial hyphae of S. coelicolor [87]. WhiG plays a crucial role in triggering the initiation of sporulation. It activates the early sporulation whi genes of which the translated proteins together with WhiG itself are necessary for the transcription of sigF. SigF controls the last stage of Streptomyces di¡erentiation the spore maturation. FEMSRE 612 15-10-98 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 Based on amino acid sequence, WhiG belongs to the £agellar c-factors and SigF shows the highest similarity with cB of B. subtilis. 3.4.1. Sigma H Sigma-factor H is encoded by sigH (or spoOH) present in B. subtilis and Clostridium spp. [129]. Sigma H is essential for sporulation in B. subtilis and is the earliest acting developmental c-factor in the sporulation process [132]. The early sporulation genes spoOA, spoOF and kinA [133] and the later acting spoIIA and spoVG genes [134] are transcribed due to the presence of cH . Sigma H is not only involved in sporulation. It also controls the increase in post-exponential-growth-phase expression of cA [135], citG which encodes fumarase, and the ftsAZ and minCD cell-division operons [136,137]. Sigma H is present in vegetative cells but its level is increased when B. subtilis enters the mid-logarithmic-growth stage [132]. Transcription initiation of sigH is under negative control of regulator AbrB [132]. The abrB gene expression is repressed by phosphorylated SpoOA, which is activated by signals that promote sporulation [138]. Using eight known cH -regulated promoter sequences for a sequence alignment, a promoter consensus sequence was derived which contains a 335 region, AGGAWWT, and a 310 region RGAAT, separated by a spacer of 12 to 14 bp [139] (Table 2). Studies with mutants have shown that amino acids at position 96 and 100 of cH interact with the nucleotide G at position 312 of the nucleotide stretch RGAAT [23,140]. 3.4.2. Sigma F Sigma-factor F is encoded by sigF or spoIIAC in B. subtilis [141] and is probably also present in Clostridium spp. [129]. The B. subtilis sigF gene product (cF ) is essential for gene expression in the early spore. It is not transcribed until shortly after the start of sporulation [142] and its protein product is speci¢cally activated within the developing forespore after septation [143]. The spoIIAC gene, together with spoIIAA and spoIIAB genes is located in the spoA operon, which is controlled by cH [134]. The cF activity depends on the phosphorylation state of SpoIIAA, which is determined by the ATP/ADP ratio in the cell [82]. Phosphorylated SpoIIAA binds to 139 SpoIIAB, preventing SpoIIAB to bind to cF and consequently inhibit transcription of cF -dependent promoters [82]. The cF -factor is required for the appearance of two later acting sporulation-speci¢c cfactors, cE and cG , as well as for morphological development of the forespore itself. Based on the six known cF -dependent promoters and analysis of several cF promoter point mutations a consensus sequence [129,144] containing a 335 region, WGCATA, and a 310 region, GGnRAYAMTW, separated by a spacer of 14 or 15 bp (Table 2) was derived. 3.4.3. Sigma E Sigma-factor E is encoded by sigE (spoIIGB) present in B. subtilis [145] and Clostridium acetobutylicum [129]. Sigma E is involved in the transcription of early sporulation genes in the mother-cell compartment in B. subtilis. Transcription of sigE requires cA and phosphorylated SpoOA [146,147]. The primary translation product of sigE, pro-cE , is inactive as a c-factor and appears just after the onset of sporulation [148]. After formation of the septum pro-cE is converted into active cE by removal of 29 N-terminal amino acids by a sporulation-speci¢c protease SpoIIGA which is coexpressed with pro-cE [148,149]. Processing of pro-cE only occurs after a speci¢c signal protein SpoIIR, controlled by cF triggers the reaction [150]. Sigma E blocks formation of a second polar septum in the mother cell by activating the spoIID [151] and spoIIID [152] genes and a number of other genes [153]. These genes have in common a promoter consensus sequence consisting of 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 experiments showed that nucleotide C in the 335 region is recognized by a histidine residue at position 219 of cE [154]. A substitution of the glutamine residue at position 217 in cE of B. subtilis to arginine resulted in a c-factor that could direct transcription from cK -dependent promoters [154], but no longer from cE -dependent promoters. 3.4.4. Sigma G Sigma G is encoded by sigG (spoIIIG) and is like cE present in B. subtilis [156] and in C. acetobutyli- FEMSRE 612 15-10-98 140 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 cum [157]. SigG causes RNAP to transcribe the late sporulation genes in the forespore. The B. subtilis cG transcription is regulated by cF as well as being autoregulated [144,156,158]. Transcription of cG is induced shortly after septation. Like cF , cG activity is inhibited by SpoAB and is presumably counteracted by SpoIIAA [82] The cG -dependent genes encode a family of small, acid-soluble spore proteins [158] which are activated after the forespore is pinched o¡ as a free protoplast within the mother cell. Sigma G recognizes the consensus promoter elements 335, TGAATA, and 310, CATACTA, separated by a spacer of 17 or 18 bp [159] (Table 2). 3.4.5. Sigma K Sigma-factor K is involved in the transcription of late sporulation genes in the mother-cell compartment in Bacillus spp. and probably Clostridium spp. as well [79,129]. In B. subtilis, cK transcription depends on a sporulation-site-speci¢c rearrangement of the chromosome which joins the spoIVCB and spoIIIC genes to a single cistron sigK [160]. The rearrangement is due to the site-speci¢c recombinase SpoIVCA. Transcription of the spoIVCA gene is activated by SpoIIID [161]. The rearrangement does not occur in Bacillus thuringiensis, Bacillus megaterium and C. acetobutylicum in which sigK is a contiguous gene [129,157]. Transcription of cK occurs by cE RNAP and SpoIIID [162]. The product of sigK in B. subtilis and B. thuringiensis is an inactive pro-protein called pro-cK which is converted to active cK after proteolytic removal of 20 amino acids [163]. Active cK is produced in the mother-cell compartment late in sporulation where it regulates the formation of spore coat genes [164], promotes the synthesis of dipicolinic acid synthetase [165] and initiates a negative feedback loop controlling the transcription of sigE [166]. Alignment of six cK -dependent promoters revealed a 335 consensus promoter element of only two nucleotides, AC, and a 310 region, CATAnAnTA, separated by 16 or 17 nucleotides [167] (Table 2). 4. The c54-family The c54 -family is structurally and functionally distinct from the c70 -family. These proteins have no detectable sequence similarity with the primary cfactors. Based on the homology between c54 -proteins, three distinct regions have been recognized (Fig. 2) [16]. Region I is glutamine rich and comprises 25 to 50 amino acids. This region is followed by a more variable region II, which usually contains between 60 and 110 amino acids with a signi¢cant excess of acidic residues. The carboxyterminal region III is some 400 amino acids long and contains the Xlink region [168], which has been shown to cross-link to DNA and two well-conserved motifs: a potential helix-turn-helix (HTH) region and the RpoN box. Both motifs are involved in recognition of the promoter region of c54 -dependent promoters [169]. The RpoN box is characterized by a stretch of ten conserved amino acids, ARRTVAKYRE. As assayed by band shift analysis, single point mutations near amino acids 363 and 383 of E. coli RpoN destroyed the ability of c54 to bind DNA [15]. The initiation of transcription of RNAP holoenzyme containing c54 di¡ers from that of the c70 -family [19]. Whereas the RNAP holoenzyme containing a member of the c70 -family often initiates transcription in the absence of transcriptional activators, transcription from all known c54 -dependent promoters requires the presence of an activator protein. By contrast, the c54 holoenzyme can form a closed promoter complex but is incapable of proceeding to open complex formation in the absence of an activator protein [170]. The activator protein binds to an enhancer which is located 100 bp or more upstream of the transcriptional start site of a c54 promoter and possesses a nucleoside triphosphatase activity by which it catalyzes the open complex formation by interaction with RNAP holoenzyme. The activator/ polymerase interaction often requires bending of the DNA. Promoters without an intrinsic single bend in the DNA between enhancer and promoter are dependent on the integration factor (IHF) which can introduce a bend in a stretch of DNA [171]. The open complex can be maintained without the presence of an activator. Several of the RpoN activators have 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, involved in dicarboxylate transport in Rhizobium spp., FhlA activator of the formate regulon in E. coli [174] and FEMSRE 612 15-10-98 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 XylR involved in the degradation of aromatic compounds in Pseudomonas putida [175]. Sigma 54 encoded by rpoN was ¢rst identi¢ed in E. coli and S. typhimurium as the c-factor required for transcription of nitrogen-regulated genes e.g. glutamine synthetase [176,177]. Today, a wide variety of c54 -dependent genes of Gram-negative and Grampositive bacteria is known. These genes have in common that they are not essential for growth [19]. One exception has to be made for some genes in M. xanthus because the rpoN product in this bacterium is probably essential [178]. Examples of RpoN-dependent genes include those coding for proteins involved in the formate dehydrogenase in E. coli, dicarboxylate transport in P. putida, Aztobacter vinelandii and Rhizobium meliloti, ¢mbriae synthesis in P. aeruginosa, Moraxella bovis and Neisseria gonorrhoea, £agellar synthesis in C. crescentus and P. aeruginosa, levanase production in B. subtilis [173], nitrogen ¢xation in K. pneumoniae, Rhizobium sp. and Rhodobacter sp. and xylene degradation in P. putida. Most c54 containing eubacterial species contain one single c54 gene, but B. japonicum, and Rhodobacter sphaeroides contain two di¡erent rpoN genes [179,180]. Inactivation of one of these genes in B. japonicum and R. sphaeroides has no in£uence on the phenotype of these bacteria. Expression of rpoN is usually constitutive but in some cases, such as in Rhodobacter capsulatus, it is regulated by oxygen and nitrogen levels [181]. In C. crescentus it is regulated temporally during the cell cycle [182]. In B. japonicum rpoN1 is regulated in response to oxygen while rpoN2 is negatively autoregulated [180]. Negatively autoregulated rpoN genes are also found in P. putida and Rhizobium etli [183]. Together with at least one activator, c54 enables RNAP to transcribe c54 -dependent promoter sequences (TGGCAC-N5-TTGC) located between positions 326 and 311 ( þ 1) of the transcription start site [19] (Table 2). Changes of only 1 bp in the spacing between the GG and GC motifs inactivate the promoter [19]. 5. Factors that a¡ect transcription initiation The sequence of promoter elements is just one of many factors that determine the overall e¤ciency of 141 a particular promoter [184]. The binding of the RNAP holoenzyme to the promoter and the transition from closed to open complex are a¡ected by the DNA conformation in the promoter region and by accessory proteins. Gene expression depends on the supercoiling of the genome, which is normally negative [185]. Local di¡erences in the level of supercoiling exist and can be modulated by environmental signals such as temperature, anaerobiosis and osmolarity. The density of DNA supercoils a¡ects the £exibility of the promoter spacer region in both closed and open complex formation and thus a¡ects initiation of transcription by the RNAP holoenzyme in a positive or negative way [186]. Many sequence-speci¢c DNA-binding proteins are known that can in£uence promoter activity. For example there are 40 proteins which can activate or prevent transcription of c70 -regulated promoters in E. coli [187]. These proteins allow the bacterium to regulate the expression of subsets of genes in response to external and internal stimuli. Genes that are responding to a common stimulus often use the same regulatory elements and are therefore referred to as being part of a regulon. Activators of these promoters usually bind to the DNA between positions 330 and 380 of the transcription start site [187]. The 335 sequences of these promoters often show a poor homology to the standard c70 335 promoter sequence [188]. Repressors bind primarily to locations that overlap with the polymerase-binding site [187]. Promoters recognized by the c54 holoenzyme are, however, always subject to positive control. Activators of c54 -dependent promoters typically bind around 100 bp upstream of the promoter, but in 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 anywhere on DNA, but it shows a preference for curved DNA [189]. The transcription of many stationaryphase-responsive genes is inhibited by H-NS [41]. Other, non-proteinaceous factors, that can also a¡ect promoter activity are DNA methylation and the regulatory nucleotide ppGpp. DNA methylation may a¡ect the binding of RNAP holoenzyme or transcriptional regulators. Several ¢mbrial operons in E. coli are regulated by this mechanism [190]. ppGpp is synthesized by RelA, an enzyme that is FEMSRE 612 15-10-98 142 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 activated in response to amino acid starvation in the so-called stringent response [191]. High concentrations of ppGpp result in the cessation of RNA synthesis and the down regulation of energy-consuming processes. Some promoters are activated by ppGpp, like the RpoS promoter of the general stress c-factor S [192]. Promoters which are negatively regulated by ppGpp all contain a G+C rich nucleotide element between the 310 promoter element and the transcriptional start site [191,193]. This element is called the `stringent box' or `discriminator'. In E. coli, it has the consensus sequence GCGCCnCC [193]. 6. Anti-sigma-factors Proteins that negatively regulate transcription by interaction with a speci¢c c-factor are known as anti-c-factors [82]. The binding of an anti-c-factor to its cognate c-factor is probably reversible. It enables the cell to respond adequately to environmental signals when the alternative c-factor is already available and does not need to be synthesized. The anti-c-factor binds the c-factor until it is needed for transcription. In S. typhimurium, the activity of c28 is regulated by FlgM [194]. FlgM functions as an antisigma-factor and regulates its own expression. It binds to c28 and blocks its activity until the hookbasal body is assembled into the membrane, at which time expression of £agellin and other £agellar genes is derepressed. After completion of the basal-body hook formation, FlgM is exported through the basal-body hook liberating c28 . Once the £agellum ¢lament has been capped, FlgM can no longer be exported. FlgM then binds c28 and the synthesis of the £agellin and expression of chemotaxis genes ceases. Anti-sigma-factors of sporulation, stress and ECF related c-factors are also known. In B. subtilis three c-factors are negatively regulated by an anti-c-factor. The spore formation is mediated by the anti-cfactor SpoIIAB which interacts with the early sporulation cF and the late sporulation cG [195,196]. Anti-c-factor RsbW is bound to the stress related cB in exponentially growing cells [82]. The regulation of SpoIIAB and RsbW is similar. In conditions of ATP excess these anti-c-factors are bound to their cognate c-factor. In conditions of ADP excess SpoIIAB and RsbW are bound to the dephosphorylated anti-anti-c-factor SpoIIAA and RsbV respectively, releasing the active c-factor. The ECF c-factor CarQ is negatively regulated by anti-c-factor CarR [103]. In the dark CarR is bound to CarQ. Light inactivates CarR by degradation or light-inhibition translation of CarR [103]. AlgU, an ECF cfactor of P. aeruginosa, regulates the alginate biosynthesis and is negatively regulated by anti-c-factor MucA [197]. The cE of E. coli is negatively regulated by RseB and anti-c-factor RseA [198,199]. RseA is an inner membrane protein of which the cytoplasmic domain binds to cE and inhibits cE directed transcription. RseB is a periplasmic protein and speci¢cally interacts with the C-terminal periplasmic domain of RseA. 7. Conclusions Bacterial genome projects are proving important information about the presence of genes for various c-factors in di¡erent species. When a c-factor is identi¢ed in a bacterial genome, it is reasonable to assume that genes regulated by this c-factor in other bacteria are similarly regulated in this organism. For example when a c28 homolog is identi¢ed, it is highly probable that some of the £agellar genes of this bacterium are regulated by c28 . The genome of Haemophilus in£uenzae codes for four c-factors: c70 , the heat shock related c32 and two c-factors belonging to the ECF family [200]. Synechocystis contains eight di¡erent c-factors: one primary c-factor RpoD1, four c-factors belonging to the nonessential primary-like c-factors, two c-factors belonging to the ECF family and the sporulation related c-factor cF [201]. The small genomes of Helicobacter pylori and Borrelia burgdorferi contain only three c-factors [202,203]. H. pylori contains c70 , the £agella related c28 and c54 . Instead of c28 B. burgdorferi harbors the stationary-phase c-factor c38 . The seven c-factors of E. coli are c70 , c38 , c 32 , c28 , c54 and the ECF cfactors cE and cFecI [204]. Analysis of the genome of B. subtilis revealed four new potential ECF c-factors making it a total of 14 di¡erent c-factors: primary c-factor cA , heat shock c-factor cB , £agella related cD , ¢ve sporulation related c-factors, ¢ve ECF cfactors and cL belonging to the c54 -family [100]. FEMSRE 612 15-10-98 M.M.S.M. Woësten / FEMS Microbiology Reviews 22 (1998) 127^150 To date, B. subtilis is the species with the largest number of c-factors, even though it does not contain the largest genome size of the bacteria described. There is however, a relationship between the number of c-factors, the bacterial genome size and the more severe conditions a bacteria can survive. Consensus sequences derived from experimentally determined promoter sequences are valuable for the identi¢cation of promoter sequences in genomic DNA sequences. The number of genes from which a hypothetical protein can be translated is increasing rapidly. 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