Role of escherichia coli rpos and associated genes in defense against oxidative damage

Free Radical Biology & Medicine,Vol. 21, No~ 7, pp. 975-993, 1996 Copyright © 1996 ElsevierScienceInc. Printed in the USA. All rights reserved 0891-5849/96 $15.00 + .00

PII s0891-5849(96)00154-2

ELSEVIER

Review Article ROLE

OF

E s c h e r i c h i a coli r p o S A N D A S S O C I A T E D GENES DEFENSE AGAINST OXIDATIVE DAMAGE

A . EISENSTARK, *t

IN

M. J. CALCUTT, *t M. BECKER-HAPAK,* and A . IVANOVA*

*Cancer Research Center, and *Departments of Biological Sciences and Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri, USA

(Received 20 December 1995; Revised 28 February 1996; Accepted 7 March 1996)

Abstract--The first phenotype described for mutations in the Escherichia coli rpoS gene was hypersensitivity to near-ultraviolet radiation and to its oxidative photoproduct, hydrogen peroxide. Initially named nur, this gene is now known to code for a sigma factor, and has acquired new names such as katF and rpoS. The role of its protein product (sigma-38) is to regulate a battery of genes as cells enter and rest in stationary phase. Some of the gene products are involved in protection against oxidants (e.g., catalases) and repair of oxidative damage (e.g., exonuclease III). Sigma38 may also modulate transcription of certain growth phase genes, including hydroperoxidase I and glutathione reductase. Sigma-38 activity is regulated at transcriptional, translational, and protein stabilization levels. This review describes the complex mechanisms whereby sigma-38 controls various genes, the interaction of sigma-38 with other regulators, and a possible role of sigma-38 in bacterial virulence. Copyright © 1996 Elsevier Science Inc.

Keywords--Escherichia coli, Oxidative damgage, rpoS

environmental conditions, there is an orchestration of transcription, translation, and protein stabilization of needed gene products. Numerous insights into mechanisms whereby cells protect themselves from damaging ROS, as well as repair of oxidative damage, have come from the study of bacterial mutants that are deficient in these functions. This has been especially true of studies of cells deficient in superoxide dismutases, 1-4 hydroperoxidases, 5,6 and the capacity to repair D N A damage as a result of ROS.7-9 Mutations of rpoS, a ROS-involved regulatory gene, have been particularly useful in unraveling the strategy that Escherichia coli cells use to protect themselves from oxidative damage. Over 20 years ago, Tuveson 1° isolated a mutant (nur; now known as rpoS or katF of E. coli) that is hypersensitive to oxidative near-ultraviolet radiation as well as to hydrogen peroxide. This mutant is deficient in hydroperoxidase II (HPII). It was later shown that nur (rpoS) does not encode a component of the HPII catalase, but is a regulator of the HPII structural gene, katE. 7 It was subsequently found also to regulate synthesis of exonuclease III (xthA), an important enzyme in the repair of oxidatively damaged DNA. 7 Indeed, the

INTRODUCTION

The bacterial cell accomplishes its two missions (to survive and to reproduce) often under harsh and diverse conditions, including damaging bursts of reactive oxygen species (ROS). Its survival may often be at the expense of host cells, thus causing disease. Because not all gene products are needed at all times nor under all Address correspondence to: A. Eisenstark, Cancer Research Center, 3501 Berrywood Drive, Columbia, MO 65201. Abraham Eisenstark is the Byler Distinguished Professor UMC (Emeritus), and Director of the Cancer Research Center, Columbia, MO. Born Warsaw, Poland, he received his Ph.D. (Bacteriology, Univ. Illinois). With Guggenheim, NSF, and NIH Fellowships, his sabbaticals were at Copenhagen, Leicester, Delft, and Paris. He has been Director, Biological Sciences, UMC, and Molecular Biology Section Head, NSF, Washington. He has over 100 research articles on bacterial genetics, most recent of which on oxidative damage by near-UV radiation. Michael Calcun (Ph.D., Biochemistry, Univ. Leicester) is Research Asst. Prof., UMC. His publications deal mainly with molecular genetics of antibiotic production and resistance in Streptomyces. Michelle Becker-Hapak (B.S. degrees in Microbiology and Chemistry, New Mexico State Univ.), CRC Laboratory Manager, is now at Howard Hughes Medical Inst., Washington Univ., St. Louis. Anna Ivanova (Ph.D., Inst. Oncology, Acad. Sci., Kiev) is a Postdoctoral Fellow, CRC. Her publications deal with genes involved in oxidative damage and tumor immunology. 975

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A. EISENSTARKet al.

fact that xthA mutants are also hypersensitive to nearUV and H202 was an important clue that rpoS was a regulatory and not a structural gene. 7 Identification of additional genes that are under rpoS regulation soon followed. It is now known that rpoS encodes a 342 amino acid, 38 kDa protein that functions as an alternate sigma factor (sigma-38) for RNA polymerase. Examination of the deduced sigma-38 sequence revealed that evolutionarily, the RpoS gene product is a member of the primary sigma factor family that includes the principal sigma subunit of RNA polymerase, sigma-70. 5'6 A recent review by Loewen and Hengge-Aronis detailed the role of sigma-38 in protection against oxidative stress and in global gene regulation. 5 Since that article was published, a plethora of diverse genes has been identified that involve sigma-38 in the regulatory process. In addition, significant progress has been made towards unravelling the complex network of gene regulators that interact with sigma-38 or modulate its activity. The emphases of this review are on recent developments as well as the emerging theme of a role for rpoS in bacterial virulence. For further details of pathways that lead to protection against oxidants and repair of oxidatively damaged DNA several reviews are available.3.11-~5 As the contribution of RpoS and its target genes to the growth and survival of E. coli is investigated further, it has become clear that the sigma-38 isoform of RNA polymerase (RNAP) has a multilayered integral role in the latter stages of bacterial development/differentiation. As a simplified starting point, sigma-38 is considered important for transcription of genes whose products are needed while cells enter and rest in dormancy. Simultaneously, it may reduce or even inhibit the expression of genes whose products in excess might be either harmful or not needed (thus, biologically uneconomical) in stationary phase cells. 16,17This inhibition may be carried out either directly or via an indirect mechanism. In either case, there are probably graded responses to changes in nutrient availability, cell density as well as other environmental stimuli. Not only modulation of sigma-38/RNAP polymerase activity but regulation of sigma70/RNAP function occurs during these changes.

lated, depending on metabolic state (e.g., katG, gor, dps, aid). By examination of protein profiles of wildtype and rpoS mutant cells by 2-D PAGE (polyacrylamide gel electrophoresis), about 30 proteins are absent in rpoS mutant cells as compared with wild-type cells. A recent study using lacZ transcriptional fusions indicates that the activities of more than 100 promoters are modulated by sigma-38.18 This number is likely to increase further as new genes are characterized and as the determinants of promoter selectivity by sigma-70 and sigma-38 are unravelled. It should be noted that although RpoS is generally considered to be a positive factor in transcription initiation, a small number of promoters are less active in wild-type cells than in rpoS mutants. It is not yet known how such negative regulation is achieved. Possibly, RpoS acts on another gene, which in turn, negatively effects the final product. Because the cellular content of sigma-38 increases greatly as cells enter stationary phase, it follows that the gene products that sigma-38 regulates are needed for preparation for dormancy, viability, and stress survival during this period and possibly for reemergence into growth phase. 16 Oxidative as well as osmotic changes, heat, and nutritional deprivation are stresses that the dormant cell may encounter. 17 At present, the interrelationship of the different stress protection mechanisms and the signals that trigger these responses are unclear. It is quite possible that multiple signal transduction pathways operate to prepare against various stresses. For example, although RpoS synthesis may be initiated in response to oxidation, the increase in RpoS that follows a stimulus of osmotic shock may occur at the posttranscriptional level. Although it may seem logical that sigma-38 would positively regulate gene products needed to survive in dormancy, it should be pointed out that mutants of some of these show no defects when observed under normal laboratory growth conditions. A typical example is the gene encoding pyruvate oxidase (poxB) which is sigma-38 regulated. ~9"2° Although both rpoS and poxB mutant cells fail to synthesize pyruvate oxidase, the latter cells grow and survive early stationary phase normally. When certain other mutants have been kept in dormancy for many days, the survival rate is very low, 2~ and perhaps death after a longer period of dormancy could be the case for cells that lack poxB.

Genes regulated by SIGMA-38 Once it was established that rpoS encoded a positive regulator of gene expression, various approaches were adopted to determine the number and identity of the RpoS-regulated genes in E. coli. Table 1 indicates genes that are directly regulated by sigma-38 (e.g., katE, xthA, mccC, boIA), genes that are dually regu-

rpoS homologues in other organisms Homologues of rpoS that have been identified include three Salmonella sp. (S. dublin, S. enterica, and S. typhimurium),5'22-24 Erwinia carotovora,25 Shigella flexneri, 5 Yersinia enterocolitica, 26 and Pseudomonas aeruginosa. 27 Comparisons of the deduced protein se-

977

Role of rpoS in ROS defense Table 1. rpoS-Associated Genes Gene

Gene Product/Phenotype

Map Position

ada aidB b ahp appA a appY~ bolA a cfa ~ clpPX crl csgA a dnaA dps, (pexB) a ficA a fis fur glgS~ got b hdeAB himA, hid himD, hip hipA hipB hns htrE hyaA-hyaP

O6 methylguanine-DNAmethyltransferase Induced by alkylating agents alkyl hydroperoxidase pH 2.5 acid phosphatase: exopolyphosphatase Regulatory protein affecting appA and other genes Morphogene Cyclopropane fatty acid synthase caseinolytic protease Activation of cryptic genes Curlin subunit DNA biosynthesis Global regulator, starvation conditions, sigma 38 dependent expression Filamentation in presence of cyclic AMP in mutant; cell division control site specific DNA inversion ferric uptake regulator Glycogen biosynthesis, rpoS dependent Glutathione oxidoreductase unknown functions; needed for curlin synthesis Integration host factor IHF, alpha subunit; site-specificrecombination Integration host factor IHF, beta subunit Frequency of persistence following inhibition of murein synthesis; DNA-synthesis inhibitor Affecting lethality due to the inhibition of peptidohlycan or DNA histone-like DNA binding protein Periplasmatic protein Hydrogenase-1 small subunit Catalase hydroperoxidase HPII bifunctional enzyme Catalase-peroxidase hydroperoxidase HPI(I) leucine response protein lipoprotein Osmotically inducible lipoprotein HyperosmoticaUy inducible periplasmic protein Trehalose phosphate synthase production Activator, hydrogen peroxide-inducible genes oxygen regulation carboxyl methyltransferase regulatory gene for poxB pyruvate oxidase Gamma-glutamyl phosphate reductase Low-affinity transport system for glycine betaine and proline; proline permease II glycine betaine-binding protein Repetitive sequence responsible for duplications within chromosome-rhsF Repetitive sequence responsible for duplications within chromosome ATP: GTP3'-pyrophosphotransferase Beta-D-galactosidase Repetitive sequence responsible for duplications within chromosome Repetitive sequence responsible for duplications within chromosome RNA polyrnerase, sigma 70 subunit RNA polymerase, sigma subunit virulence regulatory gene in Salmonella typhimurium Trp repressor binding protein Exonuclease III S. typhimurium plasmid virulence genes under spvR regulation

50 95 13.7 22.5 12.6 9.8 37.6 9.8 5.7 23 0.3 18.3 75.4 73.1 15.5 68.7 77.2 78.7 38.6 20.8 34.2 34.3 6.0 10.2 22.3 39 88.6 20.1 61.7 28.9 99.3 42.6 89.7 89.5 61.8 95 19 5.7 93.3 60.3 81.1 78.1 63 7.8 15.9 11.3 69.1 61.7

katE~ katGb lrp nlpD osmB osmY~ otsAB a oxyR oxyS pcm poxA poxB a proA proP proV rhsA, rhsF rhsB relA lacZ rhsC rhsD rpoD rpoS, katF spvR a wrbA ~ xthA a spvABCD

21.9 39.4

Genes involved in RpoS regulation. Unless otherwise sepcified, these are E. coli gene designationsand phenotypes, as tabulatedby Bachmannt9 and periodically updated, using WWW: http:/lwww.ai.sri.com/ecocyc/install.html.Further descriptions of the genes and gene functions may be found in the text, or in designated references. Superscripts: Genes known to be directly regulated by RpoS, without any known OxyR involvement. b Genes found to be regulated by both OxyR (mainly in growth phase) and RpoS (mainly in stationary phase).

quences reveal that the sigma-38 proteins from the are very similar, s h a r i n g 8 9 - 9 1 % identity, w h e r e a s 75% o f r e s i d u e s in the P s e u d o m o n a s a e r u g i n o s a p r o t e i n are identical. P r o t e i n s of s i m i l a r R p o S activity h a v e also b e e n reported in

Enterobacteria

monas campestris, Pseudomonas putida, and Rhizob i u m m e l i l o t i , 28 b u t the s e q u e n c e s o f these genes have

Klebsiella, 5

n o t yet b e e n reported. T o date, n o g e n e s that are funct i o n a l l y h o m o l o g o u s to r p o S have b e e n reported in gram-positive organisms. The complete nucleotide sequence of the H a e m o -

Acetobacter

methanolicus,

Xantho-

978

A. EISENSTARK et aL

philus influenzae Rd chromosome was recently published. 29 Although the deduced complement of gene products includes several sigma factors, no rpoS sequence is present. Whether this bacterium uses a completely different system/mechanism to protect against oxidative damage and other perils in stationary phase or whether the Rd strain is atypical compared to some of the more pathogenic variants remains to be established. Interestingly, H. influenzae Rd has a catalase gene homologous to the katE of E. coli; 3° however, because this organism does not have rpoS, it remains an open question as to how it is regulated. RpoS regulation of heterologous genes in E. coli There are now several reports of genes that are not native to common laboratory strains of E. coli but are regulated by RpoS when introduced into such strains. The microcin genes of coliform bacteria are a good example. Some microcin genes encode small peptides, which, when introduced into cells on a plasmid, seem to act as antibiotics by inhibiting protein synthesis during stationary phase. 5 Another example is the rhsA gene, which is not universally distributed among natural E. coli isolates. 31 Multicopy plasmids bearing a small internal portion of the rhsA gene impart a viability block in cultures grown to stationary phase. This toxic effect is enhanced in rpoS mutants. 31 How RpoS potentiates this toxicity has yet to be determined. Another example of sigma-38 regulation of heterologous genes in E. coli is the carotenoid biosynthesis genes from Erwinia herbicola. 25'32When a DNA segment containing six of these genes is introduced into E. coli on a plasmid, the resulting transformants synthesize carotenoids with a typical beta-carotene like spectra. However, when this plasmid is introduced into a katF::TnlO (rpoS) strain, only a very small amount of carotenoid is produced. 25'32 When the plasmid is transformed into wild-type strains, the carotenoids act primarily as scavengers of singlet oxygen rather than as free radical absorbers. 25'32

ORGANIZATION AND EXPRESSION OF THE

rpoS GENE

Genetic organization of the rpoS region of the E. coli chromosome (fig. 1) Although rpoS was initially regarded as an independent transcriptional unit, it is now clear that rpoS is part of a dicistronic operon within a cluster of genes that are involved in stationary phase survival. 33-4° These genes, surE, pcm, nlpD, and rpoS, are all transcribed in the direction surE to rpoS. 35 Transcriptional analysis indicates that surE-peru and nlpD-rpoS constitute two

operons separated by 141 nucleotides. Although related to rpoS in function, the upstream surE-pcm operon does not appear to effect rpoS expression, nor is there any evidence for the involvement of sigma-38 in surE-pcm synthesis. The nlpD gene encodes a lipoprotein whose function is incompletely known. 34 Sequence homology to a cell wall lysis enzyme and data from the effect of NlpD overexpression suggest that NlpD may play a role in cell wall formation. Although nlpD mutants do show a reduced viability after prolonged incubation in stationary phase, the phenotype is not as dramatic as that seen for surE, pcm, or rpoS mutants. The function of surE is unknown, but disruption of this gene results in reduced viability in stationary phase and an impaired ability to withstand elevated temperatures and osmotic s t r e s s . 35 The surE promoter appears to be responsible for transcription of both surE and pcm, because these open reading frames overlap by four base pairs. The pcm gene encodes a protein methyl transferase that transfers methyl groups to L-isoaspartyl residues that result from the spontaneous chemical breakdown of ageing proteins. Thus, the pcm gene product is involved in the repair of damaged proteins. Although pcm deficient cells exhibit no obvious phenotype during exponential growth, such strains have reduced viability and are phenotypically similar to surE mutants in stationary phase. In addition, pcm mutants have an increased sensitivity to oxidative stress. Presumably, damaged proteins accumulate in stationary phase and are more prone to denaturation under stress conditions. 35 This cluster of survival genes seems to have been conserved during evolution. The equivalent portion of the Er. carotovora and Ps. aeruginosa chromosomes contain pcm, a nlpD homolog and rpoS, and open reading frames with homology to surE and nlpD (IppB) are linked in Haemophilus somnus. 35 This suggests that it has been important to preserve this region of the bacterial chromosome. In the case of H. influenzae Rd strain, the nlpD gene is adjacent to mutS; the two have striking homology to the same genes in E. coli. 29However, rpoS and three other genes that lie between mutS and nlpD in E. coli are not present in this nonpathogenic, laboratory Rd strain; perhaps these genes were deleted during numerous laboratory transfers.

Transcription of the rpoS gene Transcription of the rpoS gene is complex. Although no promoters have been detected in the 64 base pair nlpD-rpoS intergenic region, there are at least three promoters further upstream of rpoS that bring about transcription of this geneY '36 Two of the promoters that

Role of rpoSin ROS defense

979

02" NO

onROS

Environmental Stimuli

osmosis

/

cell density

SIGNAL MOLECULES

/

ppGpp

Transcription

heat

UDP Glucose

Na+/K+

starvation

"--. homoserine lactone

Fe2÷/Fe3+/Fe-S

cAMP [sure

[

pcm

[

I

nlpD

Translation

I

rpoS

~ Sigma 38

Protein Stablilization

See Targets on Table 1

~

nNs

active RNA polymerase

Fig. 1. RpoS induction--environmental stimuli trigger signal molecules to initiate rpoStranscription. Protein stability within the cell is dependent upon other proteins, such as HNS, ClpP/ClpX, and possibly Fur. This figure illustrates that the orchestration of the production of the rpoS protein is regulated at transcription, translation, and protein stabilization levels.

contribute to rpoS transcription are the constitutive sigma-70 type promoters, which direct expression of nlpD. Because there is no apparent transcriptional terminator between nlpD and rpoS, the nlpD promoters probably contribute to the low level of sigma-38 expression observed during exponential growth. A major promoter for rpoS transcription is located within the nlpD structural gene. Using multicopy gene fusions, the activity of this promoter was shown to increase two to threefold upon entry into stationary phase in rich media. A similar increase in transcription in rich media was obtained by Lange and Hengge-Aronis 34'37 in a study using single-copy transcriptional fusions. However, the authors noted that there was no increase in transcription when cells grown in minimal glucose medium entered stationary phase (although the level of sigma-38 increased significantly during this time). This finding illustrates the complexity of rpoS expression. Under different growth conditions various alternative

mechanisms or pathways are employed to transduce the signal to increase sigma-38 levels in the cell. 41-44There is some discrepancy as to whether there are three additional weak rpoS promoters within the nlpD gene.

Posttranscription regulation of RpoS synthesis The dramatic elevation in sigma-38 concentration in the cell during stationary phase, without a large increase in rpoS transcription, indicates that posttranscriptional mechanisms may be responsible for these changes. ~'38"43~4 In studies aimed at dissecting these mechanisms, two levels of regulation have been noted. First, as cells grown in rich media enter stationary phase, rpoS translation increases greater than 10-fold. Then to further increase the concentration of sigma-38 in stationary phase, the stability of the protein increases 10-fold. 38 Although the precise mechanisms that govern these changes are not known, the nu-

980

A. EISENSTARKet al.

cleoid-associated protein H N S 41'45-53 and the heat shock protease ClpPX have been implicated as two possible participants. 44 Mutations in HNS cause a 10-fold increase in the translation of rpoS mRNA during exponential growth.41"5°Therefore, in the wild-type situation, HNS or an HNS-dependent protein represses rpoS translation during log phase, but the inhibition is alleviated in stationary phase. The observation that HNS can inhibit in vitro protein-synthesizing systems suggests that although primarily considered a DNA-binding protein, HNS may inhibit translation via an RNA mediated interaction either with rpoS mRNA or with rRNA. Lee et al.44 have described experiments that would account for low levels of sigma-38 during the growth phase. They conclude that proteases ClpP and ClpX degrade sigma-38 upon rapid growth. In null mutants of these proteases, sigma-38 remained high in both stationary and growth phases. This may clarify observations in previous reports dealing with posttranscription regulation by rpoS. 5'4s It is interesting to note that mutations in hns also increase the stability of sigma-38 during exponential growth. Mutations in hns and clpPX have similar consequences with respect to sigma-38 concentration. Whether the actions of HNS and ClpPX protease are interrelated in sigma-38 turnover is an intriguing possibility that awaits further characterization.

Search for a signal (sensor) molecule The RpoS concentration is initially altered by an environmental (extra- or intracellular) change recognized by a signal molecule. Key to understanding how a cell recognizes the need for active RpoS is to identify signal molecules. To date, three candidates have been proposed. Huisman and Kolter54 reported that homoserine lactone, a metabolite synthesized from intermediates in threonine biosynthesis might be this signal molecule. The accumulation of homoserine lactone to a critical concentration may trigger an increase in rpoS expression so that its various genetic targets can be induced. The production of homoserine could be through a pathway involving ppGpp. This would correlate the induction by both ppGpp and homoserine lactone, the latter being the direct signal. Bohringer et al. ss proposed that UDP-glucose might have the role of an intracellular signal molecule to control expression of rpoS and rpoS-dependent genes. Manipulation of its intracellular levels, mainly by use of different concentrations of glucose in media, certainly influences the rpoS switch. Their observations may help our understanding of the effect of high glucose concentrations in drastically

lowering catalase activity and in making cells sensitive to H202 and near-UV. ~2'13'21 In a sense, high glucose turns a wild-type cell into a phenotypic rpoSminus cell. 21'5s A particularly attractive candidate as a signal molecule is guanosine tetraphosphate (ppGpp). 56-59Levels of ppGpp increase in response to amino acid, carbon source, or phosphate starvation and induce the stringent response. 56 In the case of amino acid starvation, increased ppGpp synthesis is brought about by activation of ribosome-associated RelA protein by uncharged tRNA. Although the most striking feature of the stringent response is the shut down of stable RNA synthesis, 2D gel analysis revealed that many proteins are positively controlled.56 Because mutants defective in ppGpp synthesis have pleiotropic phenotypes that overlap with those exhibited by rpoS mutants, Gentry et al. 56investigated the possibility that ppGpp is a positive regulator for RpoS synthesis. The results from this study support this concept. Strains deficient in ppGpp synthesis fail to increase levels of RpoS upon entry into stationary phase, and strains with artificially elevated ppGpp levels during exponential growth have a 60-fold increase in RpoS. 56 This observation makes ppGpp a particularly attractive candidate as the signal molecule for RpoS synthesis. Apparently, elevated levels of RpoS are due to effects of ppGpp on transcription, and not on translation or protein stability. A recent report suggests that the effect of ppGpp on rpoS mRNA synthesis occurs primarily at the level of transcript elongation, but also an as yet unidentified posttranscriptional process. 45'58

Dual regulation by SIGMA-38 and SIGMA-70 Genes regulated by both oxyR/Sigma-70 and Sigma38. The oxyR gene, located at 89.6 min on the E. coli K12 chromosome, encodes a 306 amino acid member of the LysR family of proteins. 11'14'15'6°-63 The oxyR protein is a transcriptional regulator that activates the expression of antioxidant defense genes under oxidizing conditions. When oxidized, OxyR polypeptides assemble into a tetramer, which binds to four ATAGxt motifs in the major groove of the DNA helix. 11'15'6° Upon OxyR binding to a specific promotor, rapid transcription is allowed by the sigma-70 form of RNA polymerase holoenzyme, through a conformational change in the promotor. ~'15'59In addition to its function as a transcriptional activator, OxyR negatively regulates its own expression. The ability of OxyR to upregulate the expression of genes under oxidizing conditions has been established for the genes encoding alkyl hydroperoxidase (ahpCF), glutathione oxidoreductase (gor), hydroperoxidase I

Role of rpoS in ROS defense

981

Stationary Phase Log Phase

IHF t -

/N

/l\

oxyS ahp dnaK

j ~ I .s.**,r i

.... •- katG " ~ //,t t " """~'- gor GSSH

~

GSH

11

1t I

.//

/

/

jP"

~/

xthA

'~- dps/pex ~ /

dps mRNA

.f

i

katE ~

n2o2

C starvation

u2o÷02 E70

......... N starvation

E38

dps protein A

~H202

resLstancein stationary phase

oxyR

*fJ. . . . .

genes reg by oxyR/E 70 or E 38

,~~.~. . . . . .

negative regulation

Fig. 2. Genes regulated by oxyR/Esig70 and Esig38--response of the E. coli cell to oxidative stress is regulated differently in log phase versus stationary phase. In log phase, OxyR with sigma-70 regulates the genes oxyS, dnaK, ahp, katG, gor, and dps/ pex. In stationary phase, sigma-38 regulates the genes ihf xthA, katE, katG, gor, and dps/pex. A unique balance of regulation occurs between OxyR/sigma-70 and sigma-38 on the katG, gor, and dps/pex genes. In the case of dps, if cells are starved of carbon, transcription is increased. Nitrogen starvation leads to the negative regulation of Dps protein production. The Dps protein protects cells from H=O=stationary phase stress.

(katG), an abundant DNA-binding protein (dps), and a small untranslated R N A of unknown function (oxyS) that m a y act on or repress numerous other E. coli genes. 11'61~3 Two-dimensional polyacrylamide gel analysis reveals that O x y R regulates a total o f nine proteins upon H=O= induction. The same studies reveal that there are an additional twenty proteins that are induced by oxidative stress, but are not part o f the oxyR regulon. It will be of interest to learn how these oxyR-independent genes are regulated. To date, oxyR homologues have only been identified in the gram-negative bacteria Er. carotovora (manuscript in preparation), S. typhimurium, 61 and H. influenzae. 29 There is interesting interplay between OxyR/Esig70 and Esig38 at the katG, dps, and gor promoters (Fig. 2). The katG gene encodes a hydroperoxidase/catalase (HPI).6 Initially, this gene was thought to be transcribed solely as part o f the oxyR regulon by Esig70 and OxyR.

However, Ivanova et a l . 62 noted that strains with lesions in both rpoS and oxyR possessed significantly less HPI than an oxyR single mutant. Also, a second site suppressor of an oxyR mutant, that had elevated levels o f HPI and exhibited resistance to H202 and menadione, lost these properties when rpoS was inactivated. The effect o f rpoS on HPI synthesis was shown to be at the transcriptional level as the activity of katG::lacZ promoter fusions was significantly reduced in various rpoS mutants. Interestingly, both Esig70 and Esig38 initiate transcription from the same start site in the katG promoter, yet not all sigma-70 type promoters are recognized by Esig38 and vice v e r s a . 65'66 Until the elements that determine promoter specificity for each sigma factor are known, the extent to which this dual specificity occurs will have to be determined empirically for each promoter. Studies with purified R N A polymerase had indicated

982

A. EISENSTARK et al.

that overlapping specificities may occur in vivo. Tanaka et al. 64 and Nguyen et al. 42 noted that in vitro certain promoters could be recognized by more than one form of RNA polymerase holoenzyme. Since this initial observation, two other members of the oxyR regulon, dps and gor, have been shown to have both an OxyR/sigma-70 and sigma-38 component. In the case of Dps expression, the different forms of RNA polymerase may interact with the dps promoter exclusively at distinct stages in the life cycle. Thus, the sigma-38 dependent expression occurs only late in stationary phase or in response to starvation and the oxyR/sigma70 transcription is only manifested during exponential growth and under oxidative stress. In each of these situations there appears to be relatively little, if any, contribution from the other RNA polymerase holoenzyme. The gor gene encodes the enzyme, glutathione oxido reductase, which catalyses the reduction of oxidized glutathione via NADPH oxidation. 11"61'67~8The role of glutathione in most cells is to protect essential cellular components from oxidative damage. In E. coli the role of glutathione has been specifically linked to the protection of the beta-1 subunit of ribonucleotide reductase. It has been known for some time that gor requires OxyR for its maximal expression, but recent experiments from our laboratory have shown that the gor promoter is also regulated (directly or indirectly) by sigma38. 67 In this study, Gor levels were found to be much lower in oxy rpoS double mutants than in oxyR single mutants. This result explains a previous report that the level of glutathione increases as cells reach stationary phase without concomitant elevation in the levels of glutathione synthesizing enzymes.

Dual regulation by ada/Sigma-70 and Sigma-38 Dual specificity has also been described for the aidB promoter where sigma-38 or sigma-70 plus the transcriptional activator, Ada, function with RNA polymerase, depending on the conditions. 8 The aidB gene is induced as part of the adaptive response to alkylating agents to repair alkyl-damaged DNA. The adaptive response is mediated by an activated Ada protein, which induces transcription of the ada regulon by the sigma-70 form o f R N A polymerase. However, during anaerobic growth, transcription of aidB is induced from the same promoter but in an rpoSdependent, ada-independent manner. The AidB protein has isovaleryl acetyl coA dehydrogenase activity. 8'69'7° Mutants of aidB have increased mutation rates, further evidence of Ada involvement. Whether such dual regulation of single promoters by different sigma factors is widespread has yet to be determined.

INTERACTION WITH OTHER REGULATORS

As noted above, the complex regulation of rpoS expression responds to diverse environmental and nutritional signals. Although a number of factors modulate the expression of rpoS, for example, CRP-cAMP, HNS, and ppGpp, further complexity can be introduced at any given rpoS-dependent promoter by the interaction of RNA polymerase containing sigma-38 with additional regulators. 7° When the target promoter encodes a regulator itself, for example, bolA and appY, then yet more opportunity for fine tuning or signal amplification exists, because the target genes for these regulators may be modulated by protein factors. 63 The examples of regulators given below include a group of abundant DNA binding proteins that are collectively referred to as the histone-like, or nucleoidassociated proteins. 71-77 Fur may also have a sigma-38 regulatory role (manuscript in preparation).

Histone-like proteins of E. coli One of the emerging themes in the regulation of

rpoS and the expression of rpoS-dependent genes is the role of the nucleoid associated or histone-like proteins. This group of proteins includes H U , 71 HNS, 41"45'46'48-53 integration host factor ( I H F ) , 63'71'73 Fis, 71'74-76 and Dps. 16'63'77 Because of their direct contact with DNA, histone-like proteins may also have structural roles in defense against oxidative damage. Although many of these proteins participate in the structural organization of the chromosome, most are involved in the regulation of gene expression. With the exception of HU, all of these proteins are involved in transcriptional regulation pertaining to rpoS in E. coli.

HNS (histone-like nucleoid binding protein). HNS is the second most abundant, after HU, of the nucleoidassociated proteins and appears to exist as three isoforIrlS. 41'45'51 Although this neutral protein may play a role in the compacting of DNA, the varied effects of HNS on gene expression have recently become the focus of increased study. That HNS can negatively regulate transcription at sigma-70-dependent promoters, has been known for some time, but recent studies have shown that various sigma-38 dependent promoters are also targets for inhibition (Fig. 3). In addition, there is evidence that HNS is an important determinant of the level of sigma-38 within the cell. Somewhat surprising is the result that HNS-mediated regulation of rpoS concentration operates at the level of translational inhibition of sigma-38 synthesis and increased degradation of RpoS. s° Two effects of HNS on DNA have been described.

Role of rpoS in ROS defense

One key observation is that DNA from hns mutants has a different superhelical density than that isolated from wild-type cells. Because many promoters are sensitive to changes in supercoiling, HNS may regulate genes by changes in this parameter. 41'45Direct repression of transcription by HNS has also been reported. 41'45'46'49 It is quite possible that HNS repression comprises contributions from both of these mechanisms. A clue to how rpoS-dependent promoters can be targets for HNS repression comes from the observation that such promoters frequently contain curved DNA sequences. Although it has not been possible to identify a typical consensus sequence for HNS binding to DNA, a preference for curved DNA has been reported. 41'45 The rpoS dependent promoters can be divided into three classes based on their properties in hns mutants. 4~ Class I promoters show increased activity in hns mutants but remain rpoS dependent. This class includes the promoters for osmY and bolA, and probably those for some of the 20 protein spots on 2D gel protein profiles that are greatly increased in hns mutants. 45 Whether the increase in activity from these promoters is due to an alleviation of repression by HNS or due to the increase in cellular sigma-38 levels that has been reported in hns mutants is not known. The second class of promoters includes those that are rpoS dependent in wild-type cells, but that can become sigma-70 depen-

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dent in the absence of HNS and sigma-38. The promoter for two genes involved in curli synthesis (csgAB) 48'53 and that for the hdeAB genes (the functions of which are unknown) are examples. The otsA promoter also has increased activity in the absence of HNS, but rpoS still contributes slightly to transcription from this promoter. Class III promoters show little if any change in activity in hns mutants compared to wild-type cells. The promoters for csiD and csiE (genes of unknown function) are typical of this class. 37 These examples show the complexity of HNS interaction at rpoS promoters. At certain promoters, HNS appears to prevent transcription by sigma-70 (class II), whereas at other promoters, HNS appears to reduce expression, either by inhibiting transcription, limiting the cellular concentration of sigma-38, or perhaps by both. 41'45 The effects of HNS on promoter activity in vivo, may account for some of the anomalies from in vitro studies of promoter specificity. A number of reports have noted that RNA polymerase core protein reconstituted with purified sigma-38 or sigma-70 showed different promoter specificities than those deduced from studies in vivo. Because Hns appears to contribute to sigma factor specificity at some promoters, any HNSdependent constraints on selectivity would not operate in v i t r o . 41'45

csgA hdeAB ctsA rpos mRNA

repression of __ -many sigma-70 targets

~ ( repressionof ~ k~ proV

NS

~

)

rpoS protein clpP/clpX

r

osmY bolA

stability

Fig. 3. The role of HNS in the regulation of RpoS transcription--Hns has many roles in the bacterial cell. It blocks transcription of csgA, hdeAB, and the ctsA genes by sigma-70. It represses proV and many sigma-70 transcribed genes. It negatively regulates osmY and bolA. One key role is to block the translation of rpoS and to decrease RpoS stability.

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A. EISENSTARKet al.

transcriptional repression and inhibition of rpoS translation

~. INT, TN,

,•

HNS~

Represson/Activator IHF eg. dps Feast/Famine /~(~rp many operons Leucine Response

osmY

/

sigma70

rpoS

(~ aldB

majority of log phase mRNA transcription

ethanol Fig. 4. Factors acting at single promotors--this figure demonstrates the complex nature of transcriptional factors acting on one single gene and their inter-relationship. The osmY gene is negatively regulated by HNS, IHF, Lrp, and cAMP, while it is positively regulated by RpoS. RpoS and cAMP positively regulate AIdB. AldB is also upregulated by ethanol. Fis negatively regulates this gene.

IHF (integration host factor) The basic protein, integration host factor (IHF), is a heterodimer of the products of the related himA and hip genes. In addition to a role in site-specific integration and transposition, IHF also functions in the positive and negative control of gene expression (Figs. 2 and 4). Many, if not all, of these functions are a consequence of strong DNA bending that is induced upon IHF binding to a consensus recognition sequence. Although IHF is abundant during exponential growth, there is a 5-10-fold increase upon entering stationary phase. This result suggests that IHF is important in this growth phase. 37'59"73 The number of IHF-regulated genes could exceed 100, based on the number of binding sites already identified and the abundance of the protein. IHF has been reported to influence the expression of two RpoS-regulated genes, although its effects on transcription were very different in each case. IHF has no effect on expression of rpoS itself, but it is essential for the sigma38 dependent expression of the DNA binding protein, Dps, in stationary phase. 77 However, IHF is not needed

for an alternative, HzO2-induced expression of dps that is dependent on oxyR/sigma-70. 63 A report of IHF as a negative regulator of rpoSmediated transcription comes from a study of the complex regulation at the osmY promoter. 37 Transcription from this promoter was increased 50% in IHF mutants. Although these are the only reports of IHF interplay with rpoS, there are numerous examples of IHF regulation of sigma-70 mediated expression. Indeed, the residual rpoS-independent transcription from the osmY promoter is also increased in IHF mutants. When the signals to which IHF levels respond are better understood, it may become apparent why dps and osmY are regulated differently by IHF in stationary phase.

FIS (factor for inversion stimulation) Fis is a small homodimer which, like IHF, also causes strong DNA-bending at a target sequence. 74 Although originally described for its contribution to sitespecific inversions, Fis has since been shown to positively or negatively regulate at least 40 genes. Much of

Role of rpoSin ROS defense this regulation may be at the transcriptional level, because Fis binding sites are present in promoter regions. The concentration of Fis in the cell must be important for its physiological function, because dramatic changes occur during growth. There is a 500-fold increase in Fis following a nutritional upshift, but once cells pass through log phase the concentration drops until there is less than 1% of the maximal level remaining in stationary p h a s e . 69'75'76 A recent study of promoters that were repressed by Fis, revealed that rpoS could act as either a positive or negative regulator of such promoters. For example, the promoter of the acetaldehyde dehydrogenase gene, aldB, was identified as being transcriptionally dependent upon sigma-38 and repressed 10-fold by F i s . 69 Because the Fis concentration is so low in stationary phase, this repression is quite striking. Expression of aldB is further complicated by the action of CRP, which was shown to increase transcription 20-fold by binding to the aldB promoter. 74 A somewhat surprising result was that several Fis repressed promoters were three- to sevenfold more active in rpoS mutants. Furthermore, the inhibitory contributions of Fis and RpoS were independent of each other. Whether rpoS itself or an rpoS regulated gene product is functioning to limit the synthesis of certain gene products as cells enter stationary phase awaits further analysis.

Dps (DNA-binding protein starvation) Dps is a 17 kDa DNA binding protein that is expressed at a very low level during exponential growth but that is greatly induced in stationary phase or in response to carbon or nitrogen starvation. 16'63 The increase in Dps concentration that is induced by entry into stationary phase or by carbon starvation is largely due to elevated levels of RpoS. However, the increase in Dps in response to nitrogen limitation proceeds by a posttranscriptional mechanism that has not been characterized. Null mutants of dps are viable and have wild-type growth rates. However, in the stationary phase there are clear differences between dps mutants and wildtype cells. Two-dimensional gel analysis indicate that dps mutants have altered protein profiles in stationary phase. This suggests that Dps may be a regulator of transcription in this stage of the life cycle. The only phenotype reported for dps mutants is their increased sensitivity to H202 in the stationary phase. Whether binding of Dps to DNA provides direct protection against H202 or whether Dps plays a regulatory role is not known at present. It should be emphasized that other cellular targets for oxidative damage besides

985

DNA are also present in the cell and whether these are damaged in dps mutants awaits further analysis. The control of dps expression is further complicated in that the dps gene can be induced in exponential growth by oxidative stress as part of the oxyR regul o n . 63 If further studies reveal that Dps is, indeed, a regulatory protein, the next step would be to identify the targets for regulation under conditions of oxidative stress and stationary phase, and to determine whether they are transcribed by sigma-38, sigma-70, or both forms of RNA polymerase.

LRP (leucine responsive protein) Although not strictly a histone related protein, the global regulator Lrp is included here because of its abundance (3,000 copies), size (19 kDa), and basic charge. TM Lrp binding to a cognate DNA sequence causes DNA bending. The physiological role of Lrp may be to positively regulate genes that function in famine and negatively regulate those needed in a feast (many of the target genes are involved in metabolism of amino acids). The most complicated facet of Lrp is that there are many modes of regulation of target genes. Thus, at a specific promoter, Lrp can either activate or repress transcription but in each situation leucine can either potentiate, antagonize, or have no discernible effect. 78 At this point it is not clear why the activity of Lrp is sensitive to leucine at some promoters and not at others. The only rpoS-dependent promoter that has been reported as being under Lrp regulation is that for osmY. 37 Transcription from the osmY promoter increases as cells enter stationary phase and is largely rpoS dependent. However, there is a low level of sigma-70-mediated activity in rpoS mutants, which also increases in stationary phase. Transcription of osmY was elevated 2.5-fold in lrp mutants and the low level of sigma-70dependent transcription was constitutively expressed at the stationary phase level in these mutants. Thus, Lrp relieves osmY repression as cells enter stationary phase. Repression of osmY by CRP and IHF has also been noted, so there may be a concerted effort to limit the synthesis of OsmY. Presumably, the involvement of numerous regulators allows the cell to fine tune the activity of the osmY promoter in response to different nutritional or environmental stimuli.

Regulation by cAMP-CRP Adding cAMP to growing culture of a Acya mutant decreases rpoS expression. 5 As noted, adenyl cyclase also influences expression of some rpoS-involved genes, including aldB TM and o s m Y 37 (Fig. 4). aldB mes-

A. EISENSTARKet al.

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sage level is lowered in rpoS mutants, but also decreased in fis mutants. A Crp binding site has been located in the aldB promoter region, thus establishing its role for transcription. 74 Lange et al. 37 showed a consensus for CRP within the osmY transcriptional start region.

Regulation of oxidative stress proteins by fur (ferric uptake regulation) E. coli fur mutants are far more sensitive to nearUV and H202 than wild-type strains; also, the addition of nontoxic levels of H202 just prior to irradiation increased the sensitivity of fur mutants further. These observations raise the question as to whether f u r has an association with rpoS. A number of genes negatively regulated by fur, including the gene for Mn-superoxide dismutase synthesis j-3 and fur itself, have fur box sequences in the promoter regions. 1'2 Although f u r boxes have not yet been experimentally identified in katE and katG, we have observed that both HPI and HPII catalase activities are reduced in a f u r mutant. 79 The reduction in activity in fur mutants is at least partly due to the reduced transcription of katE and katG. This suggests that Fur protein may regulate the transcription of rpoS, because Fur-deficient cells exhibit significantly reduced transcription from the rpoS promoter, particularly in early stationary phase. However, alternative scenarios could be envisaged whereby a defect in Fur protein might indirectly effect rpoS and katG transcription. 79 A recent report by Touati et al. 1 demonstrated the importance of Fur in E. coli. In the absence of Fur, the deregulated uptake of iron can cause oxidative stress and ultimately lead to lethal DNA damage, especially under low doses of near-UV or H202, 79 especially under low doses of near-UV or H202 .79 UNIQUE PROPERTIES OF rpoS

Studies of the rpoS gene have received considerable attention because of the numerous functions subject to RpoS control. Several of these studies have provided insight into fundamental cellular processes.

Stationary phase death of RpoS mutants (genes needed during dormancy) Perhaps the most striking characteristic phenotype of rpoS mutants is the rapid death of cells soon after reaching dormancy. ~2'13'~6'21This is in contrast to wildtype " a g e d " cultures that may remain viable for decades when properly sealed and stored in agar stabs, zl It is not known whether there is a crucial rpoS-regulated

gene or whether the loss of viability of rpoS mutants culminates from incremental contributions from each of the many RpoS-regulated genes. Death of rpoS mutant cells could be failure to exclude certain growth phase molecules that are no longer required resulting in a burdensome extra metabolic load. Death could also be by starvation, synthesis of a toxic product (including acid), failure to achieve a morphological spherical state, or by some other mechanism. 8°-88 One interesting property of cells grown on solid media is that the crowding of cells in a colony might obstruct the dissemination of toxic products (including endogenous H202), resulting in undesirable accumulation. 89 It is known that several sensing systems operate in bacteria to monitor cell density and bring about changes in gene expression to respond to "crowding." It may be more than coincidental that homoserine lactone derivatives have been reported as signal molecules in a number of these systems and have also been proposed as a signal for rpoS induction. 54'9° It will be interesting to determine whether any rpoS-regulated genes allow cells to survive better at high cell density. We have explored the possible genetic relationship between "stationary phase death" and "near-UV (oxidative) death" and the role of rpoS in each] 2'13'21 Exposure of cells to near-UV induces the synthesis of a set of proteins that overlaps those induced by certain other stresses (i.e., heat shock, oxidative stress, superoxide anion stress). 61 A striking effect of near-UV is the abrupt cessation of amino acid uptake; 12'13 this resembles cessation of growth when cells enter stationary phase. It is unknown whether similar signalling pathways or molecules are involved in both cases but many mutants are sensitive to near-UV and starvation. Furthermore, at least two of these genes, xthA and dps, 19.92 are RpoS regulated. Knowledge of the biology of dormant bacterial cells (and the emergence from dormancy) 83 should have important practical value, especially in this age of genetically engineered microorganisms. For example, insights on the biology of dormant cells could lead to the development of new antimicrobial agents. 93-96 Most of the currently favored antibiotics only inhibit growing cells. Cells that are resting in a dormant state may be refractory to bacteriocidal agents and may emerge to reproduce and cause infection once the antibiotic has been cleared from the body. 95-98 It is interesting that cells enriched in catalase are sensitive to the toxicities of bleomycin, adriamycin, and paraquat. 97 An understanding of viability during dormancy would be beneficial in the development of vaccines against bacteria. 98 In this context, the use of attenuated bacteria that are unable to survive for prolonged periods of time might be feasible, provided the inoculum

Role of rpoS in ROS defense survived for sufficient time to stimulate an antibody response. A Salmonella vaccine strain is in fact such a rpoS mutant. 98 Similarly, in agricultural and environmental practices (e.g., cleaning up of dump sites and groundwaters), 99-1°2 the use of strains that are compromised in their survival capabilities might be desirable as a method of containment, rpoS mutants or cells with lesions in other putative RpoS-regulated survival genes may be useful in this respect.

Mutation rate ROS may damage many cellular targets. When the target is a molecule that is turned over during growth, the cell can recover from such oxidative insult once the oxidant has been detoxified. However, when the target is DNA, oxidative damage can lead to mutation. 4'1°3 Redox changes cause shifts in contact points between OxyR and D N A ) °4 Thus, there may be differences in promoter selection. The relation between this and mutation frequency is an area to be explored. OxyR deficient cells have an elevated mutation rate, presumably because of their inability to induce high level synthesis of HPI. 1°5 Abril and Pueyo 1°3 showed that strains completely lacking catalase due to mutations in katG, katE, and katF (rpoS) had higher rates of spontaneous mutation as well as enhanced mutagenic effects of known mutagens, including H202. rpoS also regulates noncatalase genes, which could lead to a complexity in mutagenesis studies. The rpoS-regulated enzyme exonuclease III is required for the processing of oxidatively damaged D N A (which results in mutation), and xthA mutants do have a lower mutation rate than wild-type alleles .4.12-13

Variability in RpoS sequence There are data that suggest that the rpoS gene itself is highly mutable and that this may provide a means of adaptation to stress. The first suggestion, that there is some plasticity in the rpoS sequence, came from a survey of rpoS sequences from different E. coli strains.I°6 In this study, the variation included single nucleotide differences that did not change the amino acid sequence of sigma-38 and substitutions that caused the synthesis of sigma-38 molecules with single amino acid alterations. The finding that there are rpoS variants in E. coli is somewhat surprising, because evolutionarily the gene has been highly conserved in enterobacteria. However, the most dramatic manifestation of variation is a 46 nucleotide duplication close to the 3' end of rpoS, which results in a deduced protein sequence containing additional amino acids at the carboxy-terminUS. 16'77'1°6 Cells containing the altered rpoS gene out-

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grow wild-type cells so that when grown together in mixed culture the mutant bacteria exhibit a "killer" phenotype. 77 This is the clearest example of rpoS variation resulting in an increased ability to contend with adverse conditions such as high cell density or starvation. There are two recent reports that show that mutations in rpoS can occur at high frequency in certain genetic contexts. 8'68 Volkert et al. 8'68 noted that mutations in an unknown gene that increased aidB gene expression were unstable and that suppression by a second mutation occurred at high frequency. The second site mutations were mapped to the rpoS gene. At the present time, it is not known whether the high level of suppressor accumulation is due to strong selective pressure because of AidB overexpression or due to the possibility that rpoS is prone to high mutation rate. Recently, Barth et al. 45 reported that hns mutants frequently acquire second site mutations that reside either in or close to the rpoS gene. In most of the cases studied, the cellular content of sigma-38 was reduced but not abolished. These findings support the notion that under certain conditions the rpoS gene may be a hot spot for mutation and illustrates the underlying importance of this gene in the ability of cells to adapt to a given situation. A topic of debate is whether such mutation is Darwinian (mutation/selection) or Cairnsian (adaptive). l°7

rpoS ROLE IN BACTERIAL DISEASE Because rpoS regulates several functionally diverse genes in E. coli, and because rpoS homologues have been found in several important pathogens, RpoS might regulate some pathogenicity determinants. In at least three enterobacterial pathogens this has proved to be so. A number of investigators have reported that RpoS can regulate virulence factors, particularly in Salmonella22-24,49,1o8-114and Shigella. 115Mutation in S. typhimurium rpoS drastically increases the LDs0 in infected mice. 116 It is important, however, to distinguish between true virulence determinants or genes in which a mutation would reduce the general viability of the pathogen in particular environments. Mutants of rpoS have difficulty staying alive under a number of different environments, especially that of low pH and, thus, would have difficulty surviving in the stomach and intestines. 1°3-1°4'1t4-115'117 This is true of cells in a laboratory broth culture as well as inside a host. In a detailed study of the role of RpoS in defense against pH extremes in Shigellaflexneri, Small et al., 1~5 and in Salmonella, Lee et al. 114point out that rpoS-regulate alkaline/acid resistance may have clinical signifi-

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cance. Enteric bacteria make a sudden shift from a stomach pH o f 1 - 2 to pH 9 - 1 0 secretions of the pancreatic duct just a few centimeters below the pylorus. As noted, a Salmonella wild-type strain is substantially more lethal to mice than is the rpoS mutant; 115 thus, genes other than changing acid/base sensitivity may be involved. While acid may kill the rpoS mutant, there is evidence that RpoS directly regulates certain genes that directly initiate cellular infectivity. One key virulence determinant that is regulated by Sigma-38 is the transcriptional activator S p v R . 24"49'109-110'113 The spvR gene is the first in a cluster of four plasmid encoded determinants that are essential for virulence. A virtually identical cluster is present in several Salmonella servovars that cause systemic disease in animals. RpoS is critical for the expression of these determinants, because SpvR, which appears to be directly under RpoS control, is needed to activate transcription of the other spv genes. 113 The rpoS gene of Yersinia enterocolitica has been cloned and sequenced. 26 A few virulence determinants have also been described for this pathogen including the heat-stable enterotoxin that causes diarrhea. The enterotoxin gene, yst, which is transcribed when cells reach stationary phase, appears to be regulated by sigma-38, because rpoS mutants produce sixfold less enterotoxin. However, several other virulence determinants in Y. enterocolitica are not subject to RpoS regulation; it is not known how important a contribution RpoS makes to the development of gastroenteric disease. As noted above, investigators with interest in epidemiology and public health have assessed the importance of the rpoS gene in the virulence of enteric bacteria in water supplies] °°'1°2 The catalases in certain bacterial pathogens may have roles in determining infection. Hydroperoxidase II is present in H. influenzae, but no rpoS gene has been identified. 29"3° It will be interesting to observe the unraveling of how this heme-requiring organism regulates the gene products needed to deal with ROS and other adverse environmental conditions. Another example of a catalase role in a clinical situation is drug susceptibility of Mycobacterium tuberc u l o s i s . 117 Catalase HPI is needed for isoniazide and related drugs to be effective; some katG mutants are resistant to these drugs. Apparently, catalase alters the drug to make it effective against M. tuberculosis. Presently, the sigma factor responsible for transcription of the katG gene is not known, but it is interesting to note that M. smegmatis has been reported as having at least two major sigma factors. 117

Penicillin-binding proteins One E. coli rpoS phenotype is the failure of the mutant to make the morphological shift from rod to ball shape as cells enter stationary phase. Because the penicillin-binding proteins (PBPs) are important determinants of morphology, in addition to being targets for /3-1actam antibiotics, PBPs have been studied in rpoS mutant cells. 98 Of the six PBPs studied, differences in the patterns of expression of PBP3 and PBP6 were observed between wild-type and mutant bacteria. The increase in PBP6 that is normally seen as wild-type cells enter stationary phase was not seen in rpoS mutants. Disruption of RpoS also abolished the decrease in PBP3 content that accompanies the transition into stationary phase in wild-type cells. That RpoS can affect the expression of antibiotic target sites has important implications in development of new antibacterial drugs, especially because there is need for drugs that are effective on stationary phase cells. Wang and Cronan 118 noted that the growth phasedependent synthesis of cyclopropane fatty acids in E. coli is the result of a RpoS-dependent promoter plus enzyme instability. This could contribute to understanding the role of RpoS in cell surface metabolism and antibiotic resistance.

Relation to Apoptosis--life and death Programmed cell death is important in the study of cancer, and a question arises as to whether there is apoptotic-like activity in the bacterial cell cycle. As noted by Naito et al. 119 and Yarmolinsky, 12° bacterial populations may undergo strategic apoptotis by switching between genic and epigenetic events. Earlier in this review, it was noted that cells defective in rpoS may be at a disadvantage to wild-type cells within a population. Because a population of cells would always be a mixture of genotypes as a result of mutation, the death of a subpopulation may be part of nature. Naito et al. 119 describe a "selfish gene" mechanism of programmed cell death in E. coli in which cell death may result upon plasmid loss in a subpopulation, especially if the plasmid contains restriction-modification genes. A particularly interesting example of a relationship between apoptosis and ROS is that described by Steinman.121 He found that expression of the bcl-2 oncogene, which exerts both antiapoptotic and antioxidant action, when transformed into superoxide dismutase-defective E. coli cells, results in increase transcription of katG and resistance to H202. Both katG and oxyR are required for aerobic survival of bcl-2 containing cells. Steinman's data indicate that Bcl-2 influences levels of reactive oxygen intermediates that induce endogenous

Role of rpoS in ROS defense

cellular antioxidants. Yet to be explored is whether RpoS could contribute to understanding the relationship between bcl-2 oncogene and ROS. SUMMARY AND PERSPECTIVES

RpoS is now recognized as a major regulatory force in the bacterial life cycle. Its general role may be to orchestrate transcription of stationary-phase genes, but it may also downregulate specific growth-phase genes. Because a major role of RpoS is to operate as cells enter and rest in dormancy, some metabolism probably occurs during dormancy, one product of which is H202. Oxidative damage in dormant cells could also be caused by other intracellular oxidants, including nitrous oxide. 122 As the number of recognized genes involved with RpoS regulation increases, it is likely that this may constitute a "super-regulon" of genes of diverse function. Because RpoS targets are regulated by additional DNA binding proteins such as HNS, Fis, and Lrp, this may be a complex way of modulating the activity of different genes in different fashions. Advances have also been made in identifying molecules that are important for determining the timing of rpoS expression in response to various stimuli. In addition to its role as a positive regulator of transcription, RpoS can reduce the expression of a set of genes. How this is accomplished and why it is important for the expression of these genes to be reduced remains to be determined. The presence of rpoS homologs in many enteric bacteria and at least one pseudomonad suggests that this gene may be widely distributed amongst gram-negative bacteria. Although RpoS has not been found in any gram-positive bacteria, a functionally related sigma factor may exist, particularly in the regulation of catalases.123 The realization that RpoS not only allows certain pathogens to survive better under adverse conditions but also is responsible for the transcription of key pathogenicity determinants is important and should provide further impetus for research in this area. Also, RpoS may have biological roles among plant pathogens and symbionts.28'~24'~25 The purification and successful reconstitution of Esig38 should facilitate further structural studies on the interaction of the holoenzyme with promoter DNA and provide a platform from which to probe structurefunction relationships between the constituent subunits. However, the complexity of regulation suggests that there may be additional DNA binding proteins at sigma-38 dependent promoters. This is especially true in reconciling in vitro results with apparently conflicting findings from in vivo studies. Although dozens of RpoS-regulated promoters have

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been identified and Esig38 has been functionally reconstituted, it is still unknown how any given promoter is recognized by the sigma-38 form of RNAP. Until extensive mutagenesis of promoter regions is carried out, it will be difficult to determine what the crucial determinants of promoter selectivity are (including intrinsic curvature, binding sites for other DNA binding proteins, etc). Perhaps powerful combinatorial approaches such as SELEX 126 can be applied to Esig38 to enable the rapid determination of sequences that can be bound by Esig38 as compared to Esig70. Further experiments could then be developed to determine whether any of these sequences are able to function as promoter elements. Phage display methodology127-129 is another powerful combinatorial approach that is currently being applied to purified sigma-38. This method allows the rapid identification of specific peptides from a vast random pool of sequences that are able to bind to sigma38. In addition to providing useful tools for the dissection of structure-function relationships, it is quite possible that this approach might enable the facile identification of peptide domains in E. coli proteins that interact with sigma-38 (e.g., protein:protein contacts within RNAP holoenzyme and between other DNA binding proteins and sigma-38). This review describes multilevel layers of regulation to sigma-38 synthesis itself and to sigma-38 regulated genes. ~3° It is a complex network that allows very fine tuning in response to different sets of adverse conditions. Unravelling these will be a challenge for the forthcoming years. There is particular need to identify and understand the molecular events that transpire during preparation for, survival during, and emergence from dormancy. Also, there is need to identify which RpoS-regulated genes are crucial for efficient survival under these conditions of low oxidative metabolism, and under various environmental stresses. TM Acknowledgements- The research from our laboratory was made possible by Grant ES04889 from the National Institute of Environmental Health Service, NIH. M.J.C. was supported by the Wayne Mountjoy Memorial Fellowship, and A.I. by the James Kidwell Memorial Fellowship. We thank Marnie Tutt for preparation of the manuscript, including tables and figures.

REFERENCES 1. Touati, D.; Jacques, M.; Tardat, B.; Bouchard, L.; Despied, S. Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: Protective role of superoxide dismutase. J. Bacteriol. 177:2305-2314; 1995. 2. Compan, I.; Touati, D. Interaction of six global transcription regulators in expression of manganese superoxide dismutase in Escherichia coli K-12. J. Bacteriol. 175:1687-1696; 1993.

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131. Badger, J. L.; Miller, V. L. Role of rpoS in survival of Yersinia enterocolitica to a variety of environmental stresses. J. Bacteriol. 177:5370-5373; 1995. ABBREVIATIONS (SEE TABLE 1 FOR GENE DESIGNATIONS)

2-D P A G E - - t w o - d i m e n s i o n a l polyacrylamide gel electrophoresis c A M P - - c y c l i c adenine monophosphate D N A - - d e o x y r i b o n u c l e i c acid D p s - - D N A - b i n d i n g protein during starvation E/70 & E 3 8 - - h o l o e n z y m e complex of R N A - p o l y m e r ase with sigma factor F I S - - f a c t o r for inversion stimulation HNS - - h i s t o n e - l i k e b i n d i n g protein I H F - - i n t e g r a t i o n host factor k D a - - k i l o d a l t o n s (molecular weight) L R P - - l e u c i n e response protein N A D P H - - n i c o t i n a m i d e adenine dinucleotide n e a r - U V - - n e a r - u l t r a v i o l e t radiation ( 3 0 0 - 4 0 0 n m ) P B P - - p e n i c i l l i n b i n d i n g protein p p G p p - - g u a n o s i n e tetraphosphate R e d o x - - oxidoreductive R N A - - r i b o n u c l e i c acid R N A P - - R N A - p o l y m e r a s e holoenzyme R O S - - r e a c t i v e oxygen species r p o S - - I n italics ( r p o S ) when designated as a gene, RpoS when designated as the protein product rRNA--ribosomal RNA S O D - - s u p e r o x i d e dismutase

NOTE ADDED IN PROOF

Since s u b m i s s i o n of this manuscript, a n u m b e r of r p o S - r e l a t e d articles have appeared. The following on post-transcription regulation and turnover of RpoS are pertinent to this review. Muffler, A.; Fischer, D.; Altuvia, S.; Storz, G.; Hengge-Aronis, R. The response regulator RssB controls stability of the crs subunit of RNA polymerase in Escherichia coli. EMBO J. 15:1333-1339; 1996. Brown, L.; Elliott, T. Efficient translation of the RpoS sigma factor in Salmonella typhimurium requires host factor I, and RNA-binding protein encoded by the hfq gene. J. Bacteriol. 178:3763-3770.