Introduction

One mechanism of stress adaptation used by bacteria is activation of alternative sigma factors to change the transcriptional profile of cells in order to overcome growth limiting conditions. SigB, the general stress response sigma factor, is conserved across Firmicutes species and regulates the expression of hundreds of genes when cells experience environmental, oxidative and energy stress (Hecker et al. 2007; Price 2011). SigB is tightly regulated so that induction happens during conditions of stress and is maintained in an inactive state until stress is sensed (Benson and Haldenwang 1993). Then SigB binds to RNA polymerase recruiting it to the promoters of target genes collectively called the SigB regulon (Boylan et al. 1993; Petersohn et al. 1999, 2001; Price et al. 2001). These genes then confer resistance to a wide array of stressors such as ethanol, osmotic stress, heat shock and low pH (Hecker and Völker 1998; Hoper et al. 2005). Additionally, SigB is induced by oxidative stress and can provide cells with cross-protective properties that increase cell survival (Engelmann and Hecker 1996; Helmann et al. 2003; Reder et al. 2012). More recently, our laboratory showed that SigB activation by environmental and energy stress could also protect against other oxidants such as diamide and sodium nitroprusside that cause disulfide and nitrosative stress respectively, arguing for broad antioxidant properties encoded by SigB regulated genes (Tran et al. 2019).

Still, little attention has been paid to the mechanism of SigB activation by oxidative stress or the physiological significance of the genes induced; likely due to the presence of major oxidative stress systems that directly sense the stress and regulate a significant transcriptional network with defined antioxidant biochemical functions (Antelmann and Helmann 2011; Mongkolsuk and Helmann 2002; Zuber 2009). This review will focus on the lesser understood, predicted SigB antioxidant functions to understand the contributions of SigB to oxidative stress protection. We broadly defined antioxidant functions as the reactions carried out by gene products such as enzymes, the metabolites they produce or the modifications they promote; that provide a protective role in the presence of oxidative and electrophilic reactive species so that cells can (1) prevent formation of reactive molecules, (2) eliminate oxidative compounds, and (3) repair damaged cellular components. In our definition, SigB regulated genes with antioxidant functions should be induced during oxidative conditions. We performed a survey of the literature of SigB regulon members with increased expression during oxidative conditions in three Gram-positive model bacteria Bacillus subtilis, Staphylococcus aureus and Listeria monocytogenes. We systematically analyzed the literature on the transcriptional and proteomic responses regulated by SigB in the presence of hydrogen peroxide, diamide, methylglyoxal, allicin and hypochlorite to understand the protective functions induced due to reactive oxygen species (ROS) and reactive electrophilic species (RES). These bacterial species were chosen as they are powerful model organisms specifically the pathogens S. aureus and L. monocytogenes where the general stress response contributes to virulence (Jenul and Horswill 2018; Oliver et al. 2010). Knowledge gained about these pathogens’ antioxidant defense mechanisms will aid in understanding the role of SigB in virulence.

SigB activation by environmental and energy stress

Two pathways control SigB activation in B. subtilis, the stressosome that transduces environmental stress (osmotic, temperature, pH stress) and the RsbP/RsbQ complex that communicates energy stress (conditions that lower ATP levels) (Akbar et al. 2001; Brody et al. 2001; Vijay et al. 2000) (Fig. 1). Under non-inducing conditions, SigB is kept in an inactive form bound to an anti-sigma factor, RsbW (Benson and Haldenwang 1993). When inducing conditions are encountered, SigB is freed from its interaction with RsbW by the anti-anti-sigma factor RsbV through a partner-switching mechanism. Specifically, dephosphorylated RsbV favors RsbW binding causing RsbW to switch partners and release SigB (Alper et al. 1996). This partner-switching mechanism of SigB activation is conserved in all three species which all encode the regulatory proteins in an operon with sigB. During environmental stress, activation of the stressosome causes the phosphatase activity of RsbU to dephosphorylate RsbV (Yang et al. 1996). And during low energy conditions, RsbP phosphatase activity is regulated by its partner RsbQ leading to RsbV dephosphorylation (Brody et al. 2001). Ultimately, both pathways converge on the anti-anti-sigma factor RsbV promoting SigB activity. Even though both stresses induce SigB in all three species, the signaling cascades differ amongst them (Fig. 1). The most upstream regulators of SigB in S. aureus remain unknown although the RsbU ortholog is required for physical stress and stationary phase SigB dependent gene induction (Pane-Fare et al. 2006). In L. monocytogenes which has more components in common with B. subtilis, namely the stressosome complex, RsbT and RsbU, both energy and environmental stress are sensed through the stressosome signaling cascade (Chaturongakul and Boor 2004; Martinez et al. 2010).

Fig. 1
figure 1

SigB signaling cascade during environmental and energy stress. The two branches of the general stress response, environmental and energy/starvation stress, are shown along with the corresponding Regulators of Sigma B genes (Rsb). All three species share the anti-anti sigma factor RsbV, the anti-sigma factor RsbW and SigB. B subtilis uses two different complexes, the stressosome with RsbT and RsbU, and the RsbP/Q complex. L. monocytogenes uses the stressosome, RsbT and RsbU to transduce both types of stress but contains no RsbP/Q. S. aureus’s upstream regulators are unknown but RsbU is conserved and required for both environmental stress and for SigB induction during early stationary phase

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Possible mechanisms of SigB activation by oxidative stress

How is oxidative stress transduced to SigB? What is the signal? What is the sensor? It is possible that oxidative stress is transmitted through one of the two pathways although the experimental evidence needed to answer these questions is minimal. Interestingly, the energy stress phosphatase RsbP contains a conserved Per-Arnt-Sim (PAS) domain, which typically binds ligands such as heme groups and flavin adenine dinucleotide (FAD) that could serve as redox sensors during oxidative stress (Henry and Crosson 2011). In support of RsbP sensing oxidative stress, when B. subtilis was treated with nitric oxide in aerobic conditions, SigB induction was observed and was dependent on RsbP but no mechanism was determined (Moore et al. 2004). On the other hand, in the same study RsbU was required for SigB activation during stress induced by sodium nitroprusside, suggesting that both branches of the SigB regulatory circuit could be direct sensors of nitrosative stress, although through different molecular mechanisms.

A possible mechanism of oxidative stress sensing by the environmental stress branch could be envisioned through the components of the stressosome, RsbRA, RsbRB, RsbRC, RsbRD and YtvA. RsbR paralogs contain an N’ terminal non-heme sensing domain, and YtvA has a specific type of PAS domain, a light-oxygen-voltage sensing domain (LOV), although it has been shown to be a light sensor instead (Akbar et al. 2001; Avila-Perez et al. 2006; Henry and Crosson 2011). RsbR proteins could bind a ligand created during oxidative stress or interact with another protein that directly senses oxidative stress.

While the B. subtilis scenarios are speculative, recent experimental evidence implicated the stressosome in L. monocytogenes in oxidative stress sensing through its direct interaction with a transmembrane miniprotein, Prli42. This suggests that oxidative stress can be sensed via the stressosome (Impens et al. 2017). Since the upstream regulatory proteins are not conserved, such as the stressosome or RsbP/Q, a conserved mechanism for oxidative stress signal transduction to SigB is unlikely. Still, induction by oxidative stress of the regulon happens in all three species. Therefore, we analyzed the published data in B. subtilis, S. aureus and L. monocytogenes to summarize the shared characteristics of the physiological responses by SigB during oxidative stress.

Sources of oxidative stress and commonly used compounds to study them

Metabolic reactions in the presence of oxygen have the potential to create stress by the essential reductive/oxidative (redox) reactions that are carried out inside the cell (Imlay 2019). Pathogenic bacterial species encounter strong oxidative stress when faced with the oxidative burst imposed by the immune system of the host. Phagocytes activate the production of reactive compounds such as hydrogen peroxide, nitric oxide, and hypochlorite as strong oxidants to kill the invading bacteria (Hurst 2012). Additionally, bacteria such as Bacilli species are widely used in industry for the synthesis of vitamins, detergent agents and other bio-medically relevant products and must successfully cope with oxidative stress during these energy demanding processes (Outtrup and Jorgensen 2008). The compounds chosen in this review are those most commonly used to induce different types of oxidative damage.

Hydrogen peroxide causes cytotoxicity via its potential to generate powerful reactive oxygen species like hydroxyl radicals (Juven and Pierson 1996). These free radicals can induce damage on DNA, proteins and lipids. Diamide increases intracellular disulfide crosslinking in proteins and readily reacts with low molecular weight thiols (Cumming et al. 2004). This results in protein misfolding and loss of function. Methylglyoxal has a variety of deleterious effects in cells. It promotes generation of advanced glycation end products in proteins via Millard reactions, which could impair functions of essential proteins. Moreover, methylglyoxal causes crosslinking between amino acids or between polymers, and it induces free radical formation such as superoxide anion (Chakraborty et al. 2014). The antibacterial characteristic of allicin lies in its interaction with thiol-containing proteins via formation of S-allylmercaptoglutathione, which could impair vital enzymes involved in metabolism and antioxidant mechanism in microbes (Muller et al. 2016). Additionally, evidence suggests DNA, protein, and RNA synthesis is inhibited by allicin (Ankri and Mirelman 1999). Finally, sodium hypochlorite oxidizes thiol groups on enzymes similar to allicin; and forms chlorinated amino acids which damage DNA (Fukuzaki 2006). It is possible that the choice of oxidants found in the literature caused bias in the observed sets of genes, since diamide, methylglyoxal and allicin can cause disulfide stress amongst other types of oxidative damage. A similar bias could be inferred from the transcriptomic data in S. aureus and L. monocytogenes giving us an incomplete view of the conserved antioxidant capacities of the SigB regulon in those species.

Literature search and selection criteria of oxidative stress induced genes

Google Scholar and PubMed were used to search the literature through a combination of the following keywords: Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes; transcriptome, transcriptomic; proteome, proteomic; SigB, Sigma B; oxidative stress, oxidation; hydrogen peroxide, diamide, methylglyoxal, hypochlorite and allicin. In addition, the databases SubtiWiki (Zhu and Stulke 2018), AureoWiki (Fuchs et al. 2018) were used to find information on nomenclature, gene function and regulation. Each article was screened for transcriptomic and proteomic data that were presented as microarray, RNA-seq or two-dimensional gel electrophoresis (2D-PAGE) followed by Mass Spectrometry (MS) under different oxidative stress treatments. All genes were chosen based on the original fold-change criteria set by the authors of each study to maintain fidelity. Among the genes and proteins identified, only those that met the following criteria were chosen. Firstly, genes must be members of SigB regulon. Secondly, genes must have been upregulated in samples with oxidant treatment in comparison to control samples since protective functions are unlikely to be downregulated during oxidative stress conditions. SigB-dependent genes whose expression was downregulated were left out since they likely do not provide protective functions. Although they are still interesting and represent clues into potentially damaging conditions that must be repressed during oxidative stress.

Bacillus subtilis. Six articles were used to identify genes upregulated in the five oxidants. Specifically, Helmann et al. (2003) used microarrays to measure gene expression in the presence of 8 and 58 µM hydrogen peroxide exposure (Helmann et al. 2003; Mostertz et al. 2004) found additional genes to be upregulated under 58 µM hydrogen peroxide (Mostertz et al. 2004). SigB-regulated genes were identified by Chi et al. (2019) via microarray analysis using 90 µM allicin treatment for 30 min (Chi et al. 2019). Chi et al. (2011) measured microarray data in a 10-min treatment with 50 µM sodium hypochlorite (Chi et al. 2011). Genes induced by methylglyoxal were identified by Nguyen et al. (2009) in a 10-min treatment under 2.8 and 5.6 mM methylglyoxal (Nguyen et al. 2009). In addition, microarray data from 1 mM diamide treatment for 5- and 15-min published by Leichert et al. identified diamide induced genes (Leichert et al. 2003). The SigB regulon was defined using the SubtiWiki database (Zhu and Stulke 2018).

Staphylococcus aureus. Genes induced under sodium hypochlorite and allicin were determined using 150 µM and 1 mM sodium hypochlorite and 300 µM allicin for 30 min (Loi et al. 2018, 2019). Chang et al. found genes induced in the presence of 10 mM hydrogen peroxide upon 10- and 20-min treatments (Chang et al. 2006). Two proteins were also identified by 2D-PAGE under 10 mM hydrogen peroxide and 1 mM diamide treatments (Posada et al. 2014; Wolf et al. 2008). Genes were determined by the authors in each individual publications, and cross referenced using the AureoWiki database and published SigB regulon members (Pane-Farre et al. 2006).

Listeria monocytogenes. Two articles were used to catalog SigB regulated genes with antioxidant function using our approach. Liu et al. identified genes in a thorough review of the literature based on phenotypic data (Liu et al. 2019), and Cortes et al. reported twenty-one SigB regulon genes with upregulation under 0.01 % hydrogen peroxide for 30 min (Cortes et al. 2019). We also used the core regulon identified by Oliver et al. and new members by Liu et al. 2017 to make sure we did not miss any genes (Liu et al. 2017; Oliver et al. 2010).

Summary of genes induced under multiple ROS and RES conditions

We compared the gene expression patterns for each species with every oxidant from the literature. They are organized in Supplementary Tables 1, 2 and 3. In B. subtilis, five genes were induced under all conditions: clpC, mcsB, gabD, trxA and yraA; and four genes induced under four out of five conditions: ctsR, spx, mcsA and ygvN. The analysis of S. aureus SigB regulon members is complicated by the multiple strains found in the literature, so we standardized the nomenclature and listed gene names based on predicted homolog names from AureoWiki (Fuchs et al. 2018). We found two genes, hchA and SACOL2114, were induced by three oxidants (NaOCl, diamide and allicin), and eleven genes induced by both hypochlorite and allicin only: hxlA, hxlB, ktrB, rbfa, ribC, yvdD, yvgN, yceI, yflT, SACOL2114 and SACOL2132. The SigB regulon differs in L. monocytogenes strains (Oliver et al. 2010). Therefore our analysis only applies to the strains used in the literature. Cortes et al. used strains 6179 and R479a to perform a transcriptomic study in the presence hydrogen peroxide and found twenty-one SigB-dependent induced genes and we added them to the list compiled by Liu et al. 2019. Given the minimal overlap between the regulons of each species, we discuss the antioxidant capabilities of SigB regulated genes by functional categories organized in Table 1. Categories like virulence, membrane transport and transcriptional regulation are not discussed in order to focus on oxidoreductases, protein quality and metabolism.

Table 1 Functional categories of SigB induced genes after oxidative stress
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Antioxidant functional categories regulated by SigB

Oxidoreductases

Broadly speaking, proteins involved in redox reactions constitute the largest category, after genes of unknown function, of SigB regulated genes induced by ROS and RES in B. subtilis, S. aureus and L. monocytogenes. This suggests that one main function of SigB during oxidative stress is prevention and management of oxidative damage through reductases and dehydrogenases to maintain an intracellularly balanced redox state. The specific activity of many SigB regulated reductases are untested and open to exploration. Dehydrogenases and reductases such as aldY, ydaD, yfkM, yvgN, yhdN, ytxJ, and yxnA, were induced by hypochlorite, hydrogen peroxide and diamide in B. subtilis (Table S1). Since all these oxidants cause multiple types of damage it is difficult to assign a specific function to each enzyme. However, through genetic analysis, sensitivity to oxidative compounds has been observed for mutants of some of these genes. Chandrangsu et al. found that cells lacking yhdN were more sensitive to methylglyoxal toxicity than wild type, while single mutants in yfkM and yvgN, were not affected (Chandrangsu et al. 2014). Deleting them in combination caused cells to become more sensitive suggesting redundant antioxidant pathways. Methylglyoxal causes lipid, protein and DNA oxidation (Lee and Park 2017), therefore each gene product could remediate a specific damage or degrade methylglyoxal intermediates through different mechanisms. The lack of transcriptional induction during methylglyoxal exposure complicates the interpretation. The difference between gene expression and sensitivity in the literature could be explained by the different growth conditions used in both experiments.

In. B subtilis, the aldo-keto reductase yvgN was induced in four out of the five oxidants from the literature, except methylglyoxal, suggesting antioxidant functions capable of reducing multiple substrates during exposure to oxidative stress (Lei et al. 2009). Similarly, in S. aureus SACOL2114, a predicted NAD + dependent aldehyde dehydrogenase, was induced under hypochlorite, diamide and allicin exposure. All three compounds are known to react with thiol-containing proteins so the reversal of this damage could require SACOL2114. In L. monocytogenes putative oxidoreductases, namely ywnB, yqhD, LMRG_02813, lmo0669, lmo2230 were induced by hydrogen peroxide yet each is predicted to carry out a different biochemical activity, so their molecular function remains unknown (Cortes et al. 2019; Liu et al. 2019). SigB regulated genes may not directly contribute to detoxification of damaged DNA or proteins, but instead could be responsible for maintaining the total antioxidant capacity of the cell through NAD(P)H-dependent dehydrogenases such as ydaD, aldY, and yjgC in B. subtilis. NAD(P)H is a coenzyme essential for cellular processes from metabolism to the degradation of oxidative compounds, and therefore contributes to the total antioxidant capacity of cells (Selles Vidal et al. 2018).

The well characterized enzymes encoded by sodA, trxA and ohr are differentially regulated in these three species, arguing that their regulation by SigB has evolved separately. Thioredoxins in B. subtilis encoded by trxA was induced by all oxidants, and ydbP was induced by hypochlorite and diamide (Table S1), and trxA-3 in S. aureus is induced by hypochlorite supporting the general role that thioredoxins play in ROS and RES scavenging and protection of oxidized proteins by their disulfide reducing activity (Lu and Holmgren 2014). The hydroperoxide resistance protein Ohr family members were induced in B. subtilis (ohrB) and S. aureus (ohr). These proteins are directly involved in protection against peroxide anions (Volker et al. 1998); and were also induced by hypochlorite (Table S1, S2). The superoxide dismutase sodA gene was induced in both L. monocytogenes and B. subtilis. Superoxide dismutase is responsible for the reaction that converts superoxide ions to hydrogen peroxide (Fridovich 1995). Its expression in the presence of hydrogen peroxide was expected, but it was also induced in hypochlorite and allicin, suggesting superoxide anions may be formed by these oxidants.

Low molecular weight thiol metabolism

An interesting predicted function of the SigB regulons of all three species is the regulation of low molecular-weight (LMW) thiols specifically glutathione in L. monocytogenes and bacillithiol in B. subtilis and S. aureus. In L. monocytogenes lmo1433, a putative glutathione reductase, in S. aureus SACOL2717, a putative bacillithiol-transferase, bstA; and in B. subtilis ytxJ a putative bacilliredoxin are all regulated by SigB (Table 1). LMW thiols such as glutathione and bacillithiol are important during oxidative stress for their multiple functions as cofactors used by oxidorectases, in protection of thiol-containing amino acids by direct thiolation, and as oxidation buffers themselves (Loi et al. 2015). Recycling of bacillihtiol to its reduced form during oxidative stress by a bacilliredoxin (ytxJ) would be important in B. subtilis. Although no experimental evidence exists of this function for ytxJ, it does suggest that SigB could directly contribute to the maintenance of reduced bacillithiol which could explain the need for SigB during oxidative stress.

Bacillithiol is also used in multiple reactions in the bacteria that produce it. One of its functions is to aid in the direct degradation of toxins by direct bacillithiolation by bacillithiol-transferase enzymes (bst) that carry out these reactions (Perera et al. 2014). In S. aureus, SACOL2717 encodes a bacillithiol transferase supporting the direct degradation of oxidants by the SigB regulon. Similarly, in L. monocytogenes which uses glutathione, the pool of the reduced form would need to be maintained due to ROS and RES and lmo1433 through a glutathione reductase activity could directly promote this. Thus, SigB in all three species appears to be directly involved in the metabolism of antioxidant molecules such as bacillithiol and glutathione.

Control of protein quality

One main function of SigB during oxidative stress is preventing the accumulation of oxidized proteins through their degradation by proteases and chaperones (Hecker and Völker 1998; Kruger et al. 1994). This is expected since ROS and RES cause direct protein damage such as protein oxidation and protein unfolding, and was one of the first characteristics of the general stress response identified. In B. subtilis most oxidants caused induction of the clpC protease and its regulators ctsR, mcsA and mcsB which are all induced in an operon (Derre et al. 1999). yraA and yfkM that encode glyoxylase III- like proteases were also induced in B. subtilis. Specifically, yraA was induced in the presence of all five oxidants suggesting it has a general proteolytic role. In support of yraA and yfkM’s function during RES a double mutant of these genes was sensitive to formaldehyde and methylglyoxal treatment compared to wild type (Nguyen et al. 2009). In S. aureus hchA, a predicted chaperon protein in the glyoxylase III family, was induced in hypochlorite, diamide and allicin stress conditions, and clpL was induced in hydrogen peroxide and hypochlorite exposure (Table S2). It appears that glyoxylase III-like proteins are a conserved feature of the SigB regulon. In L. monocytogenes, SigB-regulated proteases include serine protease htrA induced during hydrogen peroxide (Cortes et al. 2019), and a redox sensitive chaperonin, hslO (Table S3). Regulation of protein quality through chaperones and proteases is a shared feature of the SigB regulon.

Control of metabolism

Given that cellular respiration and ATP production are affected by oxidative agents, regulation of enzymes involved in metabolism of alternate sources of energy, electron acceptors, donors and co-factors are appropriate responses by these organisms. Consistently, in B. subtilis gabD which encodes succinic semi-aldehyde dehydrogenase involved in gamma-amino butyric acid (GABA) (Belitsky and Sonenshein 2002) was induced in all five conditions suggesting a general antioxidant role. GABA has been shown to have multiple roles during acid and oxidative stress in bacteria either by its effect on intracellular pH or by the production of NADPH during its reaction affecting the cellular redox potential (Feehily and Karatzas 2013). This could be a generalized response and main reason for metabolic gene induction during ROS and RES.

In S. aureus, the genes hlxA, hxlB were induced in two oxidative conditions (hypochlorite and allicin). They are involved in formaldehyde assimilation which could be important during detoxification of damaged metabolic intermediates (Chen et al. 2016). Similarly, predicted functions such as fatty acid biosynthesis by fabG, FAD synthesis by ribC, amino acid synthesis by hutG, mevalonate metabolism by mvaK2, and pyruvate oxidation by cidC (SACOL2553) were all induced although by different oxidants (Table S2). The putative regulator of gluconeogenesis, yqfL, was found to be induced during oxidative stress in L. monocytogenes (Cortes et al. 2019), consistent with other metabolic genes regulated by SigB in B. subtilis and S. aureus. As respiration is affected by oxidative stress, genes involved in alternate pathways could be necessary to control glycolytic or other metabolic pathways to aid in maintenance of appropriate redox conditions.

Conclusions

SigB is known as the general stress sigma factor, but oxidative stress protection is also one of its roles as is seen by the frequent induction of SigB-regulated genes under oxidative conditions in many bacterial species. Direct regulation of antioxidant genes by SigB could have come from the overlap between environmental and energy stress with oxidative stress as cells evolved a response to the constantly fluctuating conditions of life in natural environments. In fact, SigB is thought to increase resilience and promote higher stress tolerance by allowing cells to adjust and perform better under continued stress exposure (Guldimann et al. 2016). Gram-positive bacteria are used industrially for large-scale production of vitamins, enzymes, amino acids, etc. SigB is found in Gram-positive species of industrial and biomedical interest such as Bacilli species (Outtrup and Jorgensen 2008). Understanding the biochemical functions of SigB regulated genes could provide application avenues such as manipulating the appropriate SigB target(s) to optimize production by promoting higher stress tolerance in industrial conditions.

Higher resistance to oxidative stress caused during fermentation could be exploited to increase production yield in industrially used Gram-positive species. In B. pumilis, SigB targets in each of the categories in Table 1, spxA, yfkM, trxA, ohrB, radA and the clp proteases, were induced by hydrogen peroxide (Handtke et al. 2014). On the other hand, increased stress tolerance could be problematic during food production. Food sanitation often uses heat treatment and disinfectants such as hydrogen peroxide but depending on the amount, duration and sequence of these treatments, SigB could be activated giving food-borne pathogens an advantage, such as in the case of L. monocytogenes. Importantly, since food preservation aims to minimize bacterial contamination while maintaining nutritional properties, understanding the general stress response controlled by SigB will be important for designing effective protocols that successfully inhibit bacterial growth but do not promote the enhanced resistance (Bucur et al. 2018).

SigB plays a role in pathogenesis in both S. aureus and L. monocytogenes (Jenul and Horswill 2018; Liu et al. 2019). In S. aureus sigB-deleted cells were less effective at chronic intracellular persistence than their wild type counterparts (Tuchscherr et al. 2017). Chronic bacterial infection is characterized by the presence of persister cells that are resistant to antibiotics and are therefore a major concern in medical settings. Chronic infection requires a transcriptional program that can adapt to the hostile, intracellular environment of the host and SigB could play this role through the promotion of bacterial stress resilience contributing to the persister phenotype. Given the biochemical protective pathways associated with the SigB regulon, it would be interesting to characterize these predicted enzymes and their role in the persister phenotype as potential drug targets. In L. monocytogenes, SigB was not found to be a significant contributor to the persister phenotype in a culture assay, but it played a minor role in killing rate during early stationary phase when SigB is known to be active (Knudsen et al. 2013). The implication that the general stress response induced by SigB could be important for bacterial persistence makes it an important area of research.