REVIEW For reprint orders, please contact: [email protected]

Elucidating the function of the RpoS regulon

Herb E Schellhorn*

ABSTRACT: Bacterial adaptation to suboptimal nutrient environments, including host and/or extreme environments, is subject to complex, coordinated control involving many proteins and RNAs. Among the γ-proteobacteria, which includes many pathogens, the RpoS regulon has been a key focus for many years. Although the RpoS regulator was first identified as a growth phase-dependent regulator, our current understanding of RpoS is now more nuanced as this central regulator also has roles in exponential phase, biofilm development, bacterial virulence and bacterial persistence, as well as in stress adaptation. Induction of RpoS can also exert substantial metabolic effects by negatively regulating key systems including flagella biosynthesis, cryptic phage gene expression and the tricarboxylic acid cycle. Although core RpoS-controlled metabolic functions are conserved, there are substantial differences in RpoS regulation even among closely related bacteria, indicating that regulatory plasticity may be an important aspect of RpoS regulation, which is important in evolutionary adaptation to specialized environments. Adaptive modulation is a hallmark of bacterial gene expression with groups of genes coordinately regulated in response to changing environmental conditions, including host adaptation. In Escherichia coli, genes are expressed by RNA polymerase (RNAP) complexed with one of seven sigma factors (Table 1) that confer promoter recognition, sometimes in the presence of accessory regulators. Most metabolic and transcription/translational genes expressed under fast growth conditions are controlled by RpoD, the main vegetative sigma factor. Other E. coli sigma factors have more specialized roles (Table 1) . Sigma factors interact with core polymerase to direct the expression of specific sets of genes. Owing to the amount of core polymerase being limited in the cell, multiple sigma factors must compete for binding and this results in an epistatic form of control of gene expression that can have important phenotypic effects; loss of or attenuation in the activity of a sigma factor may result in an increase in the expression of other, noncognate genes. Starvation and/or stress conditions create new metabolic imperatives for the cell that require the programmed cessation of RpoD activity (mainly through the action of antisigma factors) and a coordinated increase in the expression of a class of adaptive functions under the control of a second vegetative sigma factor, RpoS, which is an RpoD paralog that arose during evolution of the major groups of proteobacteria (Figure 1) . Over the last three decades, the RpoS regulator has emerged as a key conserved regulator for many genes in E. coli and other related bacteria. Initially identified as a regulator of catalase synthesis (katE) in E. coli, RpoS was subsequently implicated in the regulation of other genes including otsAB, osmY and poxB. The regulation of RpoS is complex and has been critically and extensively reviewed elsewhere [1,2] . This article will focus on the role of the regulon with some perspectives on recent research on regulation of RpoS action, particularly involving the Crl protein.

KEYWORDS 

• adaptation • evolution • gene regulation • persistence • regulon • starvation • stationary phase • stress • viable but

not culturable

part of

*Department of Biology, McMaster University, Hamilton, ON L8S 4K1, Canada; [email protected]

10.2217/FMB.14.9 © 2014 Future Medicine Ltd

Future Microbiol. (2014) 9(4), 497–507

ISSN 1746-0913

497

Review Schellhorn Table 1. Properties of Escherichia coli sigma factors. Sigma factor

Approximate number of controlled genes

Activated by

Function

Required for

σ70(RpoD)

>2000

High growth rate

Essential

σ38 (RpoS)

Approximately 400

Stationary phase stress

Main vegetative sigma factor Second alternative sigma factor

σ32 (RpoH)

Approximately 129

Transient heat shock

Heat shock adaptation

σ19 (FecI) σ24 (RpoE)

1 Approximately 129

σ54 (RpoN)

Approximately 40

Iron limitation Extreme heat shock, other stresses Nitrogen limitation

σ28 (RpoF)

Approximately 30

– Extracytoplasmic stress protection Allows the cell to use nonoptimal nitrogen sources Flagellum biosynthesis Controls programmed synthesis of flagellum

RpoS mutants are sensitive to many stresses including starvation [15] , heat [16] , low pH [17] , near UV [18] , bile salts [19] and oxidative stress [20] . RpoS mutants also exhibit elevated levels of replication-dependent mutagenesis in stationary phase [21] owing to increased transcription of dinB-encoded PolIV DNA polymerase [22] . Although RpoS is generally regarded as a stress-activated sigma factor, it may be as important in the transient induction of key adaptive functions, as well as the sustained expression of stationary phase genes. Induction of RpoScontrolled genes at the transcriptional level is initiated at least two generations prior to stationary phase, suggesting that the cell ‘anticipates’ the need for genes to cope with stresses associated with dormancy. It is well-established that the RpoS protein is unstable in the exponential phase (half-life = 1.5 min), but it is relatively stable in the stationary phase (half-life = 20 min) [23,24] . However, even in stationary phase, in the absence of de novo synthesis, RpoS protein levels likely fall rapidly within a few hours, a prediction that we have verified by immunoblot analysis for pathogenic E. coli [8] . Similarly, the expression of RpoS-dependent genes is maximal in early stationary phase (optical density at 600 nm = 1.5–2.0), after which they decline [25] . These data support the idea that RpoS acts as a transient regulator, which is induced and is functional mainly during starvation adaptation. The expression of rpoS is thus usually studied in the context of growth-phase-dependent gene regulation in E. coli. While it is well-established that RpoS is required for the full expression of

498

Future Microbiol. (2014) 9(4)

Ref.   [4]

Nonessential, required for stress, nutrient scavenging and stationary phase adaptation Nonessential but mutants unable to grow at 37°C – Nonessential, required for virulence –

[5–8]

[9] [10] [11] [12,13]

Nonessential, mutants are nonmotile

[14]

several hundred genes of E. coli during entry into stationary phase, it is not clear how important continued expression of this regulon is in surviving cells following entry into stationary phase. Many RpoS-directed transcripts appear to drop after entry following stationary phase adaptation [25] , suggesting that there may be a high metabolic cost in continued expression of these genes in senescent cells beyond the transition adaptation period. Identifying the core RpoS regulon is critical if we are to fully understand RpoS function. It may be useful to think of RpoS as controlling an RpoD-independent means by which the cell can attenuate the expression of genes associated with high-growth, such as ribosomal RNA, and RNA proteins and translation factors, by switching the specificity of core polymerase. As RpoS levels are lower than those of RpoD even in stationary phase [26] , it is probable that the sequestration of RpoD combined with the stabilization of RpoS is required for the cessation of ribosome synthesis and translation, and the induction of the RpoS regulon. Although analogous stationary phase adaptation regulatory processes exist in other bacteria (e.g., the SigB regulon in Bacillus sp.), this article will focus on RpoS regulation in enteric bacteria, particularly E. coli. RpoS regulon The large potential size of the regulon made this system an ideal candidate for mutant screening using promoter fusions, and this led to the identification of many other RpoS-controlled genes, which were subsequently confirmed in

future science group

Elucidating the function of the RpoS regulon  microarray studies. The many genes that are controlled by RpoS in stationary phase have been described in several studies [5–8] and these consist of many regulators, their cognate systems and other structural genes that are directly controlled by RpoS. Although several hundred genes are at least partially dependent on RpoS for full expression, it appears that the number of genes that are highly dependent on RpoS expression is much smaller – perhaps only a few dozen (less than fourfold difference in expression between wild-type and an rpoS mutant [5]). RpoS also controls programmed induction of specific regulatory systems in both laboratory E. coli and pathogenic strains – in human pathogen E. coli O157:H7, 19 regulators (and presumably their downstream targets) are controlled by RpoS [8] . While it is beyond the scope of this article to exhaustively discuss the composition of the RpoS regulon, a few general observations can be made. Genomic sequencing has revealed that E. coli contains many paralogous gene systems, including a nitrate reductase operon [27,28] , lysine decarboxylase (ldcC), a polyamine uptake operon [25] , a two member drug efflux pump [25] and a transaldolase/transketolase operon (talAtktB) [5] . RpoS thus seems, in part, to control a possibly redundant portion of E. coli’s metabolic

Review

repertoire whose function must be sufficiently important to warrant continued expression under nonoptimal conditions. The composition of the RpoS regulon and degree of RpoS-mediated control among even closely related bacteria such as E. coli K12 and O157:H7 may differ substantially owing to variable genome composition, possible attenuation of RpoS control in domesticated strains such as K12 and the need to express horizontally transferred virulence genes (O-islands) in pathogenic strains under RpoS control in suboptimal conditions. Promoter recognition by RpoS Many genes are expressed by RpoD and RpoS, and as a consequence, many of the promoters recognized by these two vegetative sigma factors share common recognition motifs at the -35 and -10 sites [29] . RpoS promoters may also possess suboptimal spacing (for RpoD recognition) between the -10 and -35 regions [30] . Recently, in vitro studies have confirmed that RpoS promoters have reduced conservation of the consensus -35 sequence, but often possess a conserved C at the -13 position and lack a conserved UP element found upstream of many RpoD promoters [31] .

Ancestral sigma factor

Bacterial RpoS Duplication-loss of region 1

Spirochaete RpoD

Spirochaete RpoS Chlamydia, cytophaga, green sulfur RpoD Duplication-loss of region 1 δ-proteobacterial RpoS Loss in α- and ε-proteobacteria

α-proteobacteria

δ- and ε-proteobacterial RpoD α-proteobacterial RpoD

β- and γ-proteobacterial RpoS β- and γ-proteobacterial RpoD

Figure 1. Evolution of the RpoS and RpoD sigma factors. The RpoS sigma factor derived from RpoD through successive duplication and deletion of region 1.1 of the ancestral sigma factor. Adapted with permission from [3] © Springer Science and Business Media (2010).

future science group

www.futuremedicine.com

499

Review Schellhorn Expression in exponential phase Most members of the RpoS regulon achieve maximal levels shortly after entry into stationary phase, and this is due to the increased intra­cellular concentration of the RpoS protein, primarily through protein stabilization, of at least two orders of magnitude during the transition from exponential phase. Despite extremely low levels of the cognate regulator, some regulon members expressed in exponential phase are attenuated in expression in an rpoS mutant (see Table 2 in reference [25] for example). Microarray [32] and proteomics studies [33] reveal that new members of the RpoS regulon are controlled in the exponential phase, in addition to a subset of the stationary phase regulon, indicating that RpoS can exert regulatory effects at extremely low concentrations and suggesting that additional modulating factors may be relevant to RpoS action. One such regulator, the Crl protein [34] , specifically increases binding of RpoS to core polymerase [35] through region 2 [36] . This activation is thus likely independent of RpoS-complexed promoter recognition at promoter sites in most cases, although it does play a role in recognition of some promoters [37] . In addition to the exponential phase, Crl is required for full RpoS-dependent expression of a subset of the RpoS regulon in early stationary phase [35] . Crl functions at multiple levels by: directly facilitating assembly of the RpoS-RNAP holozyme; stimulating the RpoS-dependent expression of RssB, a protein that helps target RpoS for degradation by the ClpXP degradosome; and protecting free RpoS from ClpXP degradation through holozyme formation. These distinct effects help explain how RpoS, with low intrinsic affinity for holozyme and low intracellular concentration even in stationary phase, can be an effective global regulator. Since Crl effects are most pronounced at low RpoS concentrations and in the presence of RpoD [35] , it is likely relevant in the exponential phase and may help explain why many genes are controlled by RpoS at this stage of growth [37] . Crl-mediated control of exponential phase genes may be relevant for pathogenesis, as RpoS levels are much higher in most O157:H7 strains in all phases of growth [38] , although rpoS mutations appear common in many strains of this pathogenic serotype [39] . Negative control of gene expression Negative control of genes by RpoS was first identified in Salmonella sp. by examining the effect

500

Future Microbiol. (2014) 9(4)

of RpoS on the expression of starvation-induced transcriptional fusions [40] and later in the expression of repair genes in E. coli [41] ; however, the significance and magnitude of negative regulation could not be determined until microarrays were employed to assess global gene expression. Negative regulation or repression by RpoS affects the expression of many genes in E. coli including those encoding functions of the tricarboxylic acid (TCA) cycle, flagella biosynthesis and cryptic phage genes [5] . This unexpected finding might be explained by sigma factor competition (as RpoS-complexed RNAP reduces the transcription of other genes) or it may be due to positive regulation of a repressor by RpoS. The latter may be particularly relevant for RpoS control of the anaerobic repressor/activator Fnr that attenuates the expression of many oxidative genes including the TCA cycle members [42] . Evolution of the RpoS sigma factor As a conserved regulator, RpoS is present in many bacteria, particularly those associated with human, animal and plant disease. Structurally, RpoS is related to RpoD, but it lacks region 1, making it approximately half the size of the housekeeping sigma factor. RpoS is found primarily in many proteobacteria and is thought to be phylogenetically coherent and distinct from RpoD based on reciprocal BLAST hit analysis [3] , gene synteny and insertion/deletion analysis [3] . RpoS duplicated from RpoD prior to the proteobacteria divergence from the last common ancestor. The spirochete RpoS (found in Borrelia) was likely the first (that is, most ancient) duplication, while the RpoS duplication found in proteobacteria followed later. Accordingly, loss of region 1 occurred in these two groups independently. Among the Spirochaetes, RpoS has been lost through reductive deletion in Treponema sp. [43] . In proteobacteria, RpoS was subsequently lost from the ε- and α-proteobacteria. It is thus found primarily in present day β- and γ-proteobacteria and a related ortholog in spirochetes. Only Neisseria gonorrhea among the β-proteobacteria lacks an RpoS homolog – this may be attributable to an isolated gene loss [3] . Given the importance of RpoS in controlling the expression of large numbers of genes, is the RpoS regulon conserved among modern-day bacteria? A comparative analysis of microarray studies of regulons found in Pseudomonas aeruginosa and E. coli indicates that, while both

future science group

Elucidating the function of the RpoS regulon  bacteria have more than 200 RpoS-dependent genes of the regulon, they share only 12 members [3] . In addition, Pseudomonas seems to lack many of the prototypical highly RpoS-dependent genes found in E. coli, including osmY, katE, otsAB and poxB. This may reflect the fact that RpoS controls nonessential genes that are largely involved in stress and host adaptation. Consistent with this idea, the role of RpoS in pathogenic proteobacteria that are involved in human, plant and animal disease is species-specific and may further indicate that RpoS is important to niche adaptation where nonorthogolous proteins, including virulence factors, adhesins and host adaption metabolic functions, are important for virulence but are not essential outside the host environment. In fact, the TCA cycle is negatively regulated in E. coli [5] , while it is positively controlled in Geobacter sp. [44] , suggesting that selection and/or modulation of metabolic capacity can be incorporated into the RpoS regulon in very different ways. Role of RpoS mutations: positive & negative selection Polymorphisms, including losses of function in the rpoS gene, have clearly been selected for and maintained in many laboratory strains of E. coli – this has been a longstanding issue for the field as potential roles for RpoS in physiology have been inadvertently overlooked as many laboratory strains carry an amber mutation in RpoS [45] . In addition, many studies indicate that starvation and the availability of nonoptimal nutrient sources may select for loss of rpoS function. These observations indicate that RpoS, while an important regulator of adaptive genes, may be deleterious under some circumstances. Given its role in adaptation, it is perhaps not surprising that rpoS mutations are common among laboratory strains [46,47] , pathogens [38,48] and natural isolates [47,49] . RpoS mutants arise readily during prolonged incubation and nutrient starvation. Such mutants can, paradoxically, compete effectively with wild-type strains and can quickly predominate in a culture. This type of selective advantage was initially surprising because it has long been known that rpoS mutants, growing as monocultures, are much less viable upon prolonged incubation than wild-type strains. In co-culture experiments and among newly arising mutants within a culture, rpoS mutants can selectively scavenge nutrients

future science group

Review

and form a major class of the so-called growth advantage in stationary phase (GASP) mutants (for review see [50]). The need to express adaptive functions under stress conditions (i.e., those controlled by RpoS) and the ability to scavenge nutrients released from cells during high density stationary phase creates a metabolic dichotomy, which apparently accommodated a single global regulatory system. This dual selective process is termed the self-preservation and nutritional competence, or SPANC balance [51,52] . This idea posits that populations of cells are a type of growth/mutation equilibrium that combines the mutational process with distinct and changing selective forces (e.g., nutritional) – a phenomenon in which rpoS mutations are a common modifier. Given the high frequency of rpoS mutants among laboratory cultures and the strong selection for loss of function during cultivation, how common are these mutations in natural E. coli isolates prior to laboratory manipulation? Surveys of minimally handled independent natural isolates indicate that approximately 0.3–2.0% strains carry rpoS mutations [49,53] , which is much higher than the spontaneous mutational frequency of approximately 10 -6 per cell for an average E. coli gene. Conditions favoring neutral mutations in rpoS are likely stress-related for gain-of-function mutations and enhanced metabolism for nonpreferred carbon sources for loss-of-function mutations. Consistent with the latter, growthenhanced mutants that have acquired rpoS mutations in bacteria can be readily selected on succinate [54] , acetate [55] and presumably other poor carbon sources and these, when selected for reversion, often restore rpoS function (rather than being pseudo-reversions in other genes), forming a metabolic switch by mutation [54] that supports the SPANC concept. This type of selection of loss of rpoS function occurs in pathogenic [48] and feral environmental isolates of E. coli [49,56] , extending this laboratory-based regulatory paradigm to the field. Persistence, biofilm, the viable but not culturable state & adaptive mutations Studies on gene expression in E. coli have mainly been performed using exponential or early stationary phase planktonic cultures; however, there are important characteristics of mature cultures such as cell aggregation and differentiation into physiologically distinct subpopulations that have received relatively little attention.

www.futuremedicine.com

501

Review Schellhorn Beyond entry into stationary phase, cells can form aggregates or biofilms that are physiologically distinct from planktonic cells. This requires the coordinated production of an extracellular matrix of polymers and proteins that facilitate aggregation and adhesion [57] . The involvement of RpoS in biofilm formation is likely due to the role of the sigma factor in controlling csgD, a regulator of curli and biofilm formation. The expression of csgD is modulated by an antisense regulator, RprA, which attenuates expression of csgD during exponential planktonic growth when levels of csgD message are low [58] . RpoS-dependent increases in csgD overcome this control and allow biofilm formation [58] . Balancing this antagonistic control may be markedly affected by the nature of the bacterial strain tested and by culture conditions – RpoS has been found to be a negative [59,60] and positive [61] regulator of factors affecting biofilm formation depending on experimental context [62] . Further complicating analysis, expression of rpoS [63] and csgD [64] among cells within biofilms is heterogeneous and this is likely important in biofilm formation and differentiation. During prolonged starvation and even in exponential phase cultures, a small number of individual cells may become persister cells – metabolically quiescent cells that are refractile to antibiotics and other bacteriocidal agents, but are capable of outgrowth when placed in fresh media. Bacterial persistence – the nonheritable survival of the quiescent yet viable fraction of bacterial populations exposed to antibiotics or other inhibitory agents – is a phenomenon that was recognized during the first use of antibiotics. The failure of human bactericidal antibiotics to completely eliminate bacterial infections can obviously cause problems during treatment of chronic disease as surviving cells have the ability to regain proliferation capability. Although persistent cells within a population are relatively rare, they allow populations to survive starvation and/or antimicrobial agents. The nature of persistence has long been known; however, few genetic loci have been identified that contribute to this phenotype. Disruption of the hipA gene leads to an increased portion of persisters in a population [65] . HipA is a member of the type II toxin–antitoxin (TA) family – each of these encode a bicistronic autoregulated mRNase coupled to a repressor RNA [66] . This suggests that increased mRNA turnover may be a genetic

502

Future Microbiol. (2014) 9(4)

determinant for persistence. Recently, other bacterial TA systems have been implicated in persistence [67] . This may be strain-specific as combinatorial deletion of TA modules is necessary in other laboratory strains (specifically MG1655 [67] ). Transcriptome analysis reveals that many stress function mRNAs (e.g., osmY ) are reduced as a consequence of TA expression, including many RpoS-controlled functions. Reduction of RpoS-controlled functions is thought to render the cell sensitive to growth inhibition caused by stress, including exposure to antibiotics. Thus, rpoS mutant cultures have 10,000-fold higher levels of persister cells, which may help to partially explain why rpoS mutants accumulate in stationary phase cultures. However, additional experiments will be needed to test this idea. In contrast to persistence, the viable but not culturable state appears to depend on RpoS, as rpoSnegative strains lose culturability much faster than the wild-type strain [68] – reconciling this apparent paradox will probably require a fuller characterization of factors involved in the two phenomena. In addition to persistence and SPANC, RpoS causes general increase in adaptive stationary phase-associated mutations through increased expression of error-prone PolIV [21,69] and PolII [69] . This can have important consequences as sublethal levels of antibiotics, which cause an increase in RpoS levels, can contribute to increased frequency of antibiotic resistant mutants [70] . RpoS as a virulence factor Among the proteobacteria, there are many important pathogens that can infect plants, animals and insects – RpoS has been examined for potential roles in the multistep infection and colonization for many pathogens in their respective hosts (for a review see [71]). Although it is a nonessential regulator, RpoS is an excellent potential regulatory candidate for controlling virulence factors, particularly those that are horizontally transferred and may have suboptimal (for E. coli) heterologous promoters. Infection in a given host may involve many distinct processes including attachment, colonization, evasion of host defense mechanisms and the production of host modification factors. The role of bacterial factors, including regulators, needed for successful infection can be complex and, in the case of RpoS, studies may yield inconsistent results as disease etiology, even among closely

future science group

Elucidating the function of the RpoS regulon  related strains, may be quite different. Among the many functions controlled by RpoS, it is likely that genes protecting against oxidative stress (e.g., katE [catalase], sodC [superoxide dismutase]), maintaining osmotic equilibrium (e.g., otsAB [trehalose synthesis]) and balancing the activities of major metabolic pathways (e.g., talA tktB [transaldolase/transketolase]) aid the cell during host adaptation in many bacterial pathogens. In addition, there are many cases where RpoS has been more directly implicated in the expression of pathogenic factors. In Salmonella sp, RpoS is required for full expression of the plasmid-borne spvR (encoding a LysR-type regulator) and spvABCD, whose encoded functions are required for intracellular growth and infection [72,73] . RpoS-attenuated virulent Salmonella serovars are unable to cause infection in a mouse model when administered orally or by intraperitoneal injection [74] . In mouse intestinal colonization competition experiments, E. coli rpoS mutants can outcompete wild-type strains [75] , suggesting that the expression of the regulon may be deleterious under some conditions. We have found that many pathogenic strains of E. coli, such as laboratory strains, readily yield fast-growing rpoS mutants on succinate [48] , a suboptimal carbon source. Selection by enhanced growth on certain nutrients available may be an important factor in maintaining (and losing) RpoS as has been proposed for the GASP [76] and SPANC phenotypes [77] . In enterohemorrhagic E. coli strain O157:H7, RpoS is required for passage through the bovine GI tract, probably because RpoS controls expression of acid resistance functions (e.g., glutamate decarboxylases A and B) that help to counteract stresses in this environment [78] . More specifically, RpoS regulates production of a fibronectin and laminin-binding host attachment factor, curli, through control of the CsgD regulator [34] . Through expression of a specific set of pathogenicity island-associated genes – the locus of enterocyte effacement (LEE) – O157:H7 Shiga toxin E. coli strains produce characteristic attachment and effacing lesions [79] . The LEE operon, encoding a type III secretion system controlled by the Ler regulator, is required for full virulence [79] . The possibility of RpoS control of LEE has been examined by several groups and the results are somewhat mixed – RpoS was been found to be a negative regulator of Ler and LEE gene expression under

future science group

Review

LEE inducing-conditions [8,80–82] , but a positive regulator of promoter fusions to LEE genes in other studies [83] . The levels of and growth phase-dependent nature of RpoS may vary widely; however, we have found that the type O157:H7 EDL933 strain exhibits growth phasedependent expression (low in exponential phase and high in stationary) of RpoS, which is similar to that of laboratory strains [8] . A recent survey of many Shiga toxin E. coli strains indicates that levels of RpoS in exponential phase may be a third of the levels expressed in stationary phase [38] . Additional comparative studies will be needed to resolve these differences. It may be that the strain-specific expression of RpoS is sufficiently flexible to allow modulation of expression of important virulence factors. What is the function of the RpoS regulon? Many of the features of the RpoS regulon have been elucidated from laboratory strains of bacteria, mainly E. coli and Salmonella sp. These have inherent limitations because such strains have adapted to nonphysiological conditions that may be markedly different from their wild states. As such, considerable validation of qualitative relationships determined in the rate for the regulation of RpoS and the expression of its regulon must be performed to fully understand how RpoS functions in particular contexts. RpoS and its associated activators/inhibitors can be thought of as an ancillary, reconfigurable regulatory system that allows the cell to sense inputs to RpoS modulators through environmental stimuli, changing conditions that require recalibrated expression of a large, plastic repertoire of genes that may be paralogs, horizontally transferred genes, stress adaptation functions and genes encoding ancillary, nonessential metabolic and structural functions. In maintaining a permissive expression environment, the RpoS system allows specific niche adaptations that impose new metabolic imperatives upon the bacterial cell. Conclusion & future perspective The complex regulatory circuitry of the RpoS regulon has largely been determined using laboratory strains that have undergone many changes, which may have impacted regulation. Thus, while regulatory studies have been useful in qualitatively elucidating the nature of a remarkable network of proteins and RNAs that control RpoS levels and activities, these

www.futuremedicine.com

503

Review Schellhorn interactions must be quantified and validated in feral and pathogenic strains of E. coli to properly understand RpoS regulon function. The development and use of exhaustive tools, such as ChIP, Tn-seq, proteomic analysis coupled with mass spectrometry analysis and genomic sequencing, will help to extend observations based on laboratory strains to ‘wild-type’ E. coli. Understanding selective pressures that favor rpoS gain and loss of function is needed. Although recent studies have identified some factors required for long-term growth phenomena including the GASP phenotype, SPANC, persistence and the viable but not culturable state, it is likely that there are additional undiscovered connections between these phenotypes that may reveal how E. coli survives acute and chronic stresses including antimicrobials, such as antibiotics. This information may be helpful in the development of new, more

effective antimicrobial agents that are applicable to other proteobacterial pathogens, as well as pathogenic E. coli. Acknowledgments The author would like to thank present and past members of his laboratory for helpful discussion and contributions, as well as innumerable colleagues for fruitful and stimulating discussions over many years.

Financial & competing interests disclosure Current funding is provided by the Natural Sciences and Engineering Research Council of Canada (NSERC). The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

EXECUTIVE SUMMARY RpoS regulon ●●

RpoS controls a large regulon of adaptation functions in E. coli. While RpoS itself is nonessential, many of the genes it controls are.

Evolution of the RpoS sigma factor ●●

RpoS originated by gene duplication and has evolved through selective loss, truncation and retention among individual groups of the proteobacteria and is now found primarily in present β- and γ-proteobacteria.

Persistence & the viable but not culturable state ●●

RpoS plays important but incompletely characterized roles in bacterial persistence and the viable but not culturable state. RpoS-dependent adaptive mutations contribute to selection for antibiotic resistance.

RpoS as a virulence factor ●●

The RpoS regulon is required for many aspect of bacterial virulence in the expression of the specific virulence factors and host specific adaptation.

References

4

Paget MS, Helmann JD. The sigma70 family of sigma factors. Genome Biol. 4(1), 203 (2003).

5

Patten CL, Kirchhof MG, Schertzberg MR, Morton RA, Schellhorn HE. Microarray analysis of RpoS-mediated gene expression in Escherichia coli K-12. Mol. Genet. Genomics 272(5), 580–591 (2004).

Papers of special note have been highlighted as: • of interest 1

Hengge R. Proteolysis of sigmaS (RpoS) and the general stress response in Escherichia coli. Res. Microbiol. 160(9), 667–676 (2009).

2

Battesti A, Majdalani N, Gottesman S. The RpoS-mediated general stress response in Escherichia coli. Annu. Rev. Microbiol. 65, 189–213 (2011).

3

504

Chiang SM, Schellhorn HE. Evolution of the RpoS regulon: origin of RpoS and the conservation of RpoS-dependent regulation in bacteria. J. Mol. Evol. 70(6), 557–571 (2010).

•

This study, along with [16] and [17] employed transcriptomic analyses to define the extent of RpoS control of gene expression in Escherichia coli which, while confirming that stress genes are part of the regulon, also revealed the extent of regulation of paralogous genes and negative control by RpoS.

Future Microbiol. (2014) 9(4)

6

Lacour S, Landini P. SigmaS-dependent gene expression at the onset of stationary phase in Escherichia coli: function of sigmaS-dependent genes and identification of their promoter sequences. J. Bacteriol. 186(21), 7186–7195 (2004).

•

This study, along with [15] and [17] , employed transcriptomic analyses to define the extent of RpoS control of gene expression in Escherichia coli which, while confirming that stress genes are part of the regulon, also revealed the extent of regulation of paralogous genes and negative control by RpoS.

7

Weber H, Polen T, Heuveling J, Wendisch VF, Hengge R. Genome-wide analysis of the

future science group

Elucidating the function of the RpoS regulon  general stress response network in Escherichia coli: sigmaS-dependent genes, promoters, and sigma factor selectivity. J. Bacteriol. 187(5), 1591–1603 (2005). •

8

9

This study, along with [15] and [16] , employed transcriptomic analyses to define the extent of RpoS control of gene expression in Escherichia coli which, while confirming that stress genes are part of the regulon, also revealed the extent of regulation of paralogous genes and negative control by RpoS.

Escherichia coli and Shigella flexneri: role of rpoS and growth pH. J. Bacteriol. 176(6), 1729–1737 (1994).

Zhao K, Liu M, Burgess RR. The global transcriptional response of Escherichia coli to induced sigma 32 protein involves sigma 32 regulon activation followed by inactivation and degradation of sigma 32 in vivo. J. Biol. Chem. 280(18), 17758–17768 (2005).

21 Layton JC, Foster PL. Error-prone DNA

defense and deterioration of growth-arrested Escherichia coli cells. J. Biol. Chem. 274(37), 26027–26032 (1999). polymerase IV is controlled by the stress-response sigma factor, RpoS, in Escherichia coli. Mol. Microbiol. 50(2), 549–561 (2003). 22 Storvik KA, Foster PL. RpoS, the stress

response sigma factor, plays a dual role in the regulation of Escherichia coli’s error-prone DNA polymerase IV. J. Bacteriol. 192(14), 3639–3644 (2010).

10 Maeda H, Jishage M, Nomura T, Fujita N,

is located in an operon with rpoS on the Escherichia coli chromosome and encodes a novel lipoprotein with a potential function in cell wall formation. Mol. Microbiol. 13(4), 733–743 (1994).

Characterization of the Escherichia coli sigma E regulon. J. Biol. Chem. 276(24), 20866–20875 (2001).

concentration of the sigma S subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation, and protein stability. Genes Dev. 8(13), 1600–1612 (1994).

13 Dong T, Yu R, Schellhorn H. Antagonistic

•

14 Zhao K, Liu M, Burgess RR. Adaptation in

17 Small P, Blankenhorn D, Welty D, Zinser E,

Slonczewski JL. Acid and base resistance in

future science group

30 Typas A, Stella S, Johnson RC, Hengge R.

The -35 sequence location and the Fis-sigma factor interface determine sigmas selectivity of the proP (P2) promoter in Escherichia coli. Mol. Microbiol. 63(3), 780–796 (2007). 31 Maciag A, Peano C, Pietrelli A, Egli T, De

Bellis G, Landini P. In vitro transcription profiling of the Sigma(S) subunit of bacterial RNA polymerase: re-definition of the Sigma(S) regulon and identification of Sigma(S)-specific promoter sequence elements. Nucleic Acids Res. 39(13), 5338–5355 (2011). 32 Dong T, Kirchhof MG, Schellhorn HE. RpoS

regulation of gene expression during exponential growth of Escherichia coli K12. Mol. Genet. Genomics 279(3), 267–277 (2008).

This key early study revealed the complexity of RpoS regulation at multiple levels and formed the basis for subsequent investigations identifying the many proteins and regulatory RNAs that modulate RpoS effects.

33 Rahman M, Hasan MR, Oba T, Shimizu K.

Effect of rpoS gene knockout on the metabolism of Escherichia coli during exponential growth phase and early stationary phase based on gene expressions, enzyme activities and intracellular metabolite concentrations. Biotechnol. Bioeng. 94(3), 585–595 (2006). 34 Olsen A, Arnqvist A, Hammar M, Sukupolvi

S, Normark S. The RpoS sigma factor relieves H-NS-mediated transcriptional repression of csgA, the subunit gene of fibronectin-binding curli in Escherichia coli. Mol. Microbiol. 7(4), 523–536 (1993).

Identification of conserved, RpoS-dependent stationary-phase genes of Escherichia coli. J. Bacteriol. 180(23), 6283–6291 (1998). 26 Jishage M, Ishihama A. Regulation of RNA

35 Typas A, Barembruch C, Possling A, Hengge

polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of sigma 70 and sigma 38. J. Bacteriol. 177(23), 6832–6835 (1995).

16 Hengge-Aronis R, Klein W, Lange R,

Rimmele M, Boos W. Trehalose synthesis genes are controlled by the putative sigma factor encoded by rpoS and are involved in stationary-phase thermotolerance in Escherichia coli. J. Bacteriol. 173(24), 7918–7924 (1991).

basis of selective promoter activation by the sigmaS subunit of RNA polymerase. Mol. Microbiol. 63(5), 1296–1306 (2007).

25 Schellhorn HE, Audia JP, Wei LI, Chang L.

15 Lange R, Hengge-Aronis R. Identification of a

central regulator of stationary-phase gene expression in Escherichia coli. Mol. Microbiol. 5(1), 49–59 (1991).

29 Typas A, Becker G, Hengge R. The molecular

24 Lange R, Hengge-Aronis R. The cellular

regulon analysis of nitrogen assimilation factor, sigma54, reveal alternative strategy for E. coli MG1655 flagellar biosynthesis. Nucleic Acids Res. 38(4), 1273–1283 (2010).

bacterial flagellar and motility systems: from regulon members to ‘foraging’-like behavior in E. coli. Nucleic Acids Res. 35(13), 4441–4452 (2007).

SM et al. The rpoS-dependent starvationstress response locus stiA encodes a nitrate reductase (narZYWV ) required for carbon-starvation-inducible thermotolerance and acid tolerance in Salmonella typhimurium. Microbiology 145(Pt 11), 3035–3045 (1999).

23 Lange R, Hengge-Aronis R. The nlpD gene

11 Dartigalongue C, Missiakas D, Raina S.

regulation of motility and transcriptome expression by RpoN and RpoS in Escherichia coli. Mol. Microbiol. 79(2), 375–386 (2011).

28 Spector MP, Garcia Del Portillo F, Bearson

Casadesus J. Adaptation and preadaptation of Salmonella enterica to Bile. PLoS Genet. 8(1), e1002459 (2012). 20 Dukan S, Nystrom T. Oxidative stress

12 Zhao K, Liu M, Burgess RR. Promoter and

Schellhorn HE. Expression of the Escherichia coli NRZ nitrate reductase is highly growth phase dependent and is controlled by RpoS, the alternative vegetative sigma factor. Mol. Microbiol. 34(4), 756–766 (1999).

19 Hernandez SB, Cota I, Ducret A, Aussel L,

Dong T, Schellhorn HE. Global effect of RpoS on gene expression in pathogenic Escherichia coli O157:H7 strain EDL933. BMC Genomics 10, 349 (2009).

Ishihama A. Two extracytoplasmic function sigma subunits, sigma(E) and sigma(FecI), of Escherichia coli: promoter selectivity and intracellular levels. J. Bacteriol. 182(4), 1181–1184 (2000).

27 Chang L, Wei LI, Audia JP, Morton RA,

18 Sammartano LJ, Tuveson RW, Davenport R.

Control of sensitivity to inactivation by H 2O2 and broad-spectrum near-UV radiation by the Escherichia coli katF locus. J. Bacteriol. 168(1), 13–21 (1986).

•

The concentration of sigma factors relative to the amount of core polymerase (RNA polymerase) was evaluated and this provided a basis for observed epistatic interactions that govern regulon expression.

Review

R. Stationary phase reorganisation of the Escherichia coli transcription machinery by Crl protein, a fine-tuner of sigmas activity and levels. EMBO J. 26(6), 1569–1578 (2007). •

Modulation of RpoS under conditions of low protein levels (e.g., exponential phase) requires the Crl protein that tunes for promoter specificity of RpoS-complexed polymerase.

www.futuremedicine.com

505

Review Schellhorn 36 Banta AB, Chumanov RS, Yuan AH et al.

Key features of sigmaS required for specific recognition by Crl, a transcription factor promoting assembly of RNA polymerase holoenzyme. Proc. Natl Acad. Sci. USA 110(40), 6 (2013). 37 Dudin O, Lacour S, Geiselmann J.

Expression dynamics of RpoS/Crl-dependent genes in Escherichia coli. Res. Microbiol. 164(8), 838–847 (2013). 38 Mand TD, Dopfer D, Ingham B, Ane C,

Kaspar CW. Growth and survival parameter estimates and relation to RpoS levels in serotype O157:H7 and non-O157 Shiga toxin-producing Escherichia coli. J. Appl. Microbiol. 114(1), 242–255 (2013). 39 Uhlich GA, Chen CY, Cottrell BJ et al.

Phage insertion in mlrA and variations in rpoS limit curli expression and biofilm formation in Escherichia coli serotype O157:H7. Microbiology 159(Pt 8), 1586-1596 (2013). 40 O’Neal CR, Gabriel WM, Turk AK, Libby

SJ, Fang FC, Spector MP. RpoS is necessary for both the positive and negative regulation of starvation survival genes during phosphate, carbon, and nitrogen starvation in Salmonella typhimurium. J. Bacteriol. 176(15), 4610–4616 (1994). 41 Tsui HC, Feng G, Winkler ME. Negative

regulation of mutS and mutH repair gene expression by the Hfq and RpoS global regulators of Escherichia coli K-12. J. Bacteriol. 179(23), 7476–7487 (1997). 42 Myers KS, Yan H, Ong IM et al. Genome-

scale analysis of Escherichia coli FNR reveals complex features of transcription factor binding. PLoS Genet. 9(6), e1003565 (2013). 43 Subramanian G, Koonin EV, Aravind L.

Comparative genome analysis of the pathogenic spirochetes Borrelia burgdorferi and Treponema pallidum. Infect. Immun. 68(3), 1633–1648 (2000). 44 Nunez C, Adams L, Childers S, Lovley DR.

The RpoS sigma factor in the dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens. J. Bacteriol. 186(16), 5543–5546 (2004). 45 Jishage M, Ishihama A. Variation in RNA

polymerase sigma subunit composition within different stocks of Escherichia coli W3110. J. Bacteriol. 179(3), 959–963 (1997). 46 Ferenci T, Zhou Z, Betteridge T et al.

Genomic sequencing reveals regulatory mutations and recombinational events in the widely used MC4100 lineage of Escherichia coli K-12. J. Bacteriol. 191(12), 4025–4029 (2009).

506

47 Spira B, De Almeida Toledo R, Maharjan RP,

Ferenci T. The uncertain consequences of transferring bacterial strains between laboratories – rpoS instability as an example. BMC Microbiol. 11, 248 (2011). 48 Dong T, Chiang SM, Joyce C, Yu R,

Schellhorn HE. Polymorphism and selection of rpoS in pathogenic Escherichia coli. BMC Microbiol. 9, 118 (2009). 49 Chiang SM, Dong T, Edge TA, Schellhorn

HE. Phenotypic diversity caused by differential RpoS activity among environmental Escherichia coli isolates. Appl. Environ. Microbiol. 77(22), 7915–7923 (2011). 50 Finkel SE. Long-term survival during

stationary phase: evolution and the GASP phenotype. Nat. Rev. Microbiol. 4(2), 113–120 (2006). 51 King T, Ishihama A, Kori A, Ferenci T. A

regulatory trade-off as a source of strain variation in the species Escherichia coli. J. Bacteriol. 186(17), 5614–5620 (2004). 52 Phan K, Ferenci T. A design-constraint

trade-off underpins the diversity in ecologically important traits in species Escherichia coli. ISME J. 7(10), 2034–2043 (2013). 53 Snyder E, Gordon DM, Stoebel DM.

Escherichia coli lacking RpoS are rare in natural populations of non-pathogens. G3 (Bethesda) 2(11), 1341–1344 (2012). 54 Chen G, Patten CL, Schellhorn HE. Positive

selection for loss of RpoS function in Escherichia coli. Mutat. Res. 554(1–2), 193–203 (2004). 55 Alvarez-Ordonez A, Begley M, Hill C.

Selection for loss of RpoS in Cronobacter sakazakii by growth in the presence of acetate as a carbon source. Appl. Environ. Microbiol. 79(6), 2099–2102 (2013). 56 Eydallin G, Ryall B, Maharjan R, Ferenci T.

The nature of laboratory domestication changes in freshly isolated Escherichia coli strains. Environ. Microbiol. 16(3), 813-828 (2013). 57 Prigent-Combaret C, Brombacher E, Vidal O

et al. Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene. J. Bacteriol. 183(24), 7213–7223 (2001). 58 Mika F, Busse S, Possling A et al. Targeting of

csgD by the small regulatory RNA RprA links stationary phase, biofilm formation and cell envelope stress in Escherichia coli. Mol. Microbiol. 84(1), 51–65 (2012). 59 Corona-Izquierdo FP, Membrillo-Hernandez

J. A mutation in rpoS enhances biofilm

Future Microbiol. (2014) 9(4)

formation in Escherichia coli during exponential phase of growth. FEMS Microbiol. Lett. 211(1), 105–110 (2002). 60 Sheldon JR, Yim MS, Saliba JH, Chung WH,

Wong KY, Leung KT. Role of rpoS in Escherichia coli O157:H7 strain H32 biofilm development and survival. Appl. Environ. Microbiol. 78(23), 8331–8339 (2012). 61 Adams JL, Mclean RJ. Impact of rpoS

deletion on Escherichia coli biofilms. Appl. Environ. Microbiol. 65(9), 4285–4287 (1999). 62 Collet A, Cosette P, Beloin C et al. Impact of

rpoS deletion on the proteome of Escherichia coli grown planktonically and as biofilm. J. Proteome Res. 7(11), 4659–4669 (2008). 63 Ito A, May T, Taniuchi A, Kawata K, Okabe

S. Localized expression profiles of rpoS in Escherichia coli biofilms. Biotechnol. Bioeng. 103(5), 975–983 (2009). 64 Serra DO, Richter AM, Klauck G, Mika F,

Hengge R. Microanatomy at cellular resolution and spatial order of physiological differentiation in a bacterial biofilm. MBio 4(2), e00103–e00113 (2013). 65 Moyed HS, Bertrand KP. hipA, a newly

recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. J. Bacteriol. 155(2), 768–775 (1983). 66 Fozo EM, Hemm MR, Storz G. Small toxic

proteins and the antisense RNAs that repress them. Microbiol. Mol. Biol. Rev. 72(4), 579–589 (2008). 67 Maisonneuve E, Shakespeare LJ, Jorgensen

MG, Gerdes K. Bacterial persistence by RNA endonucleases. Proc. Natl Acad. Sci. USA 108(32), 13206–13211 (2011). 68 Boaretti M, Lleo MM, Bonato B, Signoretto

C, Canepari P. Involvement of rpoS in the survival of Escherichia coli in the viable but non-culturable state. Environ. Microbiol. 5(10), 986–996 (2003). 69 Frisch RL, Su Y, Thornton PC, Gibson JL,

Rosenberg SM, Hastings PJ. Separate DNA Pol II- and Pol IV-dependent pathways of stress-induced mutation during doublestrand-break repair in Escherichia coli are controlled by RpoS. J. Bacteriol. 192(18), 4694–4700 (2010). 70 Gutierrez A, Laureti L, Crussard S et al.

Beta-lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity. Nat. Commun. 4, 1610 (2013). 71 Dong T, Schellhorn HE. Role of RpoS in

virulence of pathogens. Infect. Immun. 78(3), 887–897 (2010).

future science group

Elucidating the function of the RpoS regulon  72 Fang FC, Libby SJ, Buchmeier NA et al. The

alternative sigma factor katF (rpoS) regulates Salmonella virulence. Proc. Natl Acad. Sci. USA 89(24), 11978–11982 (1992). •

This early work indicated that RpoS control is critical for expression of virulence factors.

73 Norel F, Robbe-Saule V, Popoff MY,

Coynault C. The putative sigma factor katF (RpoS) is required for the transcription of the Salmonella typhimurium virulence gene spvB in Escherichia coli. FEMS Microbiol. Lett. 78(2–3), 271–276 (1992). 74 Coynault C, Robbe-Saule V, Norel F.

Virulence and vaccine potential of Salmonella typhimurium mutants deficient in the expression of the RpoS (sigma S) regulon. Mol. Microbiol. 22(1), 149–160 (1996). 75 Krogfelt KA, Hjulgaard M, Sorensen K,

Cohen PS, Givskov M. rpoS gene function is a disadvantage for Escherichia coli BJ4 during

future science group

competitive colonization of the mouse large intestine. Infect. Immun. 68(5), 2518–2524 (2000). 76 Farrell MJ, Finkel SE. The growth advantage

in stationary-phase phenotype conferred by rpoS mutations is dependent on the pH and nutrient environment. J. Bacteriol. 185(24), 7044–7052 (2003). 77 Ferenci T. Maintaining a healthy SPANC

balance through regulatory and mutational adaptation. Mol. Microbiol. 57(1), 1–8 (2005). 78 Price SB, Cheng CM, Kaspar CW et al. Role

of rpoS in acid resistance and fecal shedding of Escherichia coli O157:H7. Appl. Environ. Microbiol. 66(2), 632–637 (2000). 79 Elliott SJ, Sperandio V, Giron JA et al. The

locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEEand non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli. Infect. Immun. 68(11), 6115–6126 (2000).

Review

80 Iyoda S, Watanabe H. ClpXP protease

controls expression of the type III protein secretion system through regulation of RpoS and GrlR levels in enterohemorrhagic Escherichia coli. J. Bacteriol. 187(12), 4086–4094 (2005). 81 Tomoyasu T, Takaya A, Handa Y, Karata K,

Yamamoto T. ClpXP controls the expression of LEE genes in enterohaemorrhagic Escherichia coli. FEMS Microbiol. Lett. 253(1), 59–66 (2005). 82 Laaberki MH, Janabi N, Oswald E, Repoila

F. Concert of regulators to switch on LEE expression in enterohemorrhagic Escherichia coli O157:H7: interplay between Ler, GrlA, HNS and RpoS. Int. J. Med. Microbiol. 296(4–5), 197–210 (2006). 83 Beltrametti F, Kresse AU, Guzman CA.

Transcriptional regulation of the esp genes of enterohemorrhagic Escherichia coli. J. Bacteriol. 181(11), 3409–3418 (1999).

www.futuremedicine.com

507