Systematic review of the Listeria monocytogenes σ^B regulon supports a role in stress response, virulence and metabolism

Importance of Listeria monocytogenes as a foodborne pathogen & model organism

Listeria monocytogenes is a foodborne pathogen of concern for food safety and public health worldwide. This Gram-positive pathogen causes the serious infection, listerosis, in susceptible populations, such as the immunocompromised, pregnant women, newborns and the elderly. This bacterium challenges food safety because it is commonly present in farm and food-processing environments and can utilize various food vehicles to transmit to a human host. L. monocytogenes’ ability to rapidly adapt to changing environmental conditions enables it to survive harsh environments during food processing, as well as to adapt to intracellular growth and proliferation within a host. This bacterium is able to survive in a wide range of temperature and pH as well as high osmotic pressure and limited nutrient availability [1].

Several characteristics of L. monocytogenes’ physiology have made it a unique model in infection biology, cell biology and fundamental microbiology. This successful intracellular pathogen can infect human and animal host cells, survive and grow under different conditions, and overcome important pathophysiological barriers, such as the intestinal epithelium, the blood–brain barrier and the placenta. Decades of studies have revealed important mechanisms of transmission, invasion, motility and resistance in L. monocytogenes; these findings support the consideration of this pathogen as a multifaceted model for the study of host–pathogen interactions, intracellular growth and pathogenic mechanisms (for a recent review, see [2]). L. monocytogenes is also an emerging model in prokaryotic transcriptomic studies and in environmental studies [3,4].

Importance of alternative sigma factor B in L. monocytogenes’ gene regulation

Overview of the role of σ^B in L. monocytogenes’ gene regulation

To overcome changing conditions, gene expression in L. monocytogenes is tightly regulated by transcriptional regulators including alternative sigma (σ) factors, transcriptional activators and repressors [5]. Among the alternative sigma factors, σ^B has the largest regulon in L. monocytogenes [6]. The complex and fine-tuned interplay between the regulatory circuits of σ^B, other transcriptional regulators and other sigma factors is the key for successful infection and survival under different conditions [7], including the transition from a saprophytic to a host-associated life stages of L. monocytogenes [8]. σ^B and other sigma factors such as σ^A and σ^H have overlapping regulons, which play important roles in metabolism and the transition to a pathogenic lifestyle [6]. In addition, σ^B and σ^B-regulated regulators, such as PrfA and Hfq, together coordinate the response to the stresses encountered during invasion and intracellular growth [9]. Deletion of sigB makes L. monocytogenes more susceptible to environmental stresses and attenuated in virulence [10–12], with transcriptomic and proteomic data showing significant changes in the gene expression profile of these mutants [9,13,14]. Based on these studies, σ^B is currently considered as the regulator of general stress response, virulence and resilience [7]. Reviewing studies on the σ^B regulon not only provides an overview of σ^B regulation in L. monocytogenes but also highlights the importance of future research on σ^B-dependent genes carrying out important functions for this pathogen.

The σ^B regulon in L. monocytogenes

Several studies on the σ^B regulon in a single L. monocytogenes strain have suggested more than 200 genes to be directly regulated by σ^B [9,13,15,16]. However, as a pathogen with global distribution, multiple L. monocytogenes strains and isolates have been used in various studies worldwide. The most common lab strains of L. monocytogenes are 10403S (serotype 1/2a), EGDe (serotype 1/2a) and EGD (serotype 1/2a) [17]; other strains used less frequently include LO28 (serotype 1/2c), H7858 (serotype 4b) and clinical isolates [18,19]. Interestingly, even the commonly used lab strains have diverse genomic characteristics. While the names of EGD and EGDe imply a similar origin, these two strains are genetically highly distinct from each other with approximately 30,000 SNP differences [17]. Additionally, the commonly used L. monocytogenes strains do not share all genes, making it difficult to use the regulon of one strain to represent all. Previous studies have revealed that the σ^B regulon in L. monocytogenes can vary in different lineages and strains [16,20]. In this review, we searched for and summarized σ^B regulon members in different strains of L. monocytogenes.

Methods

Systematic review of literature on σ^B in L. monocytogenes

A systematic literature search was performed guided by preferred reporting items for systematic reviews and meta-analyses (PRISMA) [21] (Figure 1).

Search strategy

PubMed and Web of Knowledge databases were searched for relevant articles. Broad search terms for Listeria monocytogenes, σ^B regulation, stress response and transcriptional studies were used. Specifically, the Medical Subject Headings (MeSHs) in PubMed search as well as the text-word searching was employed. MeSH terms covered the ones linked to L. monocytogenes, σ^B and methods (Table 1). Text-words included ‘Listeria monocytogenes’, ‘listeriosis’, ‘SigB’, ‘Sigma B’, ‘SigmaB’, ‘stress response’ and ‘virulence gene’. The final search was conducted on 29 October 2017. A list of 291 publications was generated through the search.

Inclusion & exclusion criteria

Articles were reviewed through an abstract screen and a subsequent full-text screen. The inclusion criteria for abstract screen were that the abstract: mentioned σ^B-dependent promoter, σ^B regulon, σ^B-dependent genes with transcriptional or translational evidence in L. monocytogenes; or was a transcriptional/proteomic study with transcription start site and/or promoter region prediction. The exclusion criteria for abstract screen were that the abstract was: not a study in L. monocytogenes (e.g., Bacillus); not focusing on the genes regulated by σ^B (e.g., growth of ΔsigB mutant under stress condition); on regulation of other regulators except σ^B (e.g., PrfA); on phylogenetic studies using sigB to subtype Listeria; a study of the mechanism of σ^B activation. Abstracts meeting the inclusion criteria were then screened through full-text screening.

The inclusion criteria for full-text screen were that: the article identified σ^B-dependent genes that showed evidence for higher expression levels in the presence of σ^B as compared with an isogenic null mutant; or the article identifies σ^B-dependent promoters or transcription units (TUs). Only papers with full text in English were included. No further restrictions were placed on study design, lineages and strains, study location or technique methods (e.g., transcriptomic or proteomic).

With these inclusion and exclusion criteria, 42 publications that reported σ^B regulon members in L. monocytogenes or reported TUs by genome-wide analyses were identified (Figure 1). An additional dataset [22], which was unpublished at the time of the search, was also included as it consisted of a transcriptomic study in wild-type and isogenic ΔsigB mutants of L. monocytogenes under stress. Data were extracted from these 43 publications (Supplementary Table 1) to summarize the σ^B regulon in L. monocytogenes.

Compilation of the L. monocytogenes σ^B regulon

Summary of the σ^B regulon in L. monocytogenes strains

A recent study identified 127 TUs, which had upstream σ^B-dependent consensus promoters and encompassed 240 genes, as the σ^B regulon in L. monocytogenes strain 10403S [15]. The systematic literature review described above was used to: verify and expand this regulon for strain 10403S and identify additional TUs with upstream σ^B-dependent consensus promoters in other strains, including EGDe, EGD, H7858, LO28 and others. Overall, these efforts identified 171 TUs that encompassed 304 genes (Table 2).

Function search strategy & reference categories

Genes of interest were searched individually for studies on their functions in L. monocytogenes and other bacteria. Evidence for functions was classified into four categories: Functions with experimental evidence in L. monocytogenes; Functions with experimental evidence in Bacillus, other firmicutes or any bacteria; Functions suggested by GO terms and conserved amino acid motif; and Unknown function. Functions not based on references in category 1 need to be further explored with complementary experiments. Searches for studies on gene functions were performed through Google scholar and Pubmed, while the databases Subtiwiki, QuickGO, and Uniprot were searched for functions in Bacillus, GO terms and homology in protein products. Gene names, as well as locus name in 10403S (LMRG) and EGDe (lmo) were used as keywords in searching. A snowballing approach [23] was also used to find references on gene functions.

Functional groups

While specific functions of σ^B-dependent genes were found during the function search, broader functional groups could provide insights in the connections and cooperation of these genes and allow easier classification. Generally, gene functions were divided into five general categories based on known roles of σ^B, including stress response, virulence, metabolism, other functions and unknown function. While more than 100 genes with unknown functions were found, most of the genes with identified putative functions were classified into the ‘stress response,’ ‘virulence’ and ‘metabolism’ categories. Some genes were classified into more than one general category (Table 3).

Results & discussion

Functions associated with the σ^B regulon members

Stress response (including osmotic, oxidative, acid, antibiotic, bile, alkaline & other stresses)

Since the first report of σ^B in L. monocytogenes in 1998 [10], modulation of stress response has been recognized as a key role of σ^B in this organism, consistent with the role of σ^B in other firmicutes, including Bacillus and Staphylococcus spp. [7]. Transcriptional, translational and phenotypic data support contribution of σ^B to this bacterium’s ability to survive and grow under different stresses outside a host, such as osmotic stress, as well as specific stresses inside a host, such as bile stress [12,24–27]. In this review, we identified 73 σ^B regulon members that are involved in different aspects of stress response and survival, including osmotic, oxidative, acid, alkaline and bile stress, or are involved in antibiotic resistance. Identification of these 73 σ^B regulon members in response to different stress conditions confirms the role of σ^B as the general stress response regulator in L. monocytogenes, allowing this pathogen to cope with host defense mechanisms and to survive under stress conditions.

The σ^B regulon includes 18 members involved or putatively involved in osmotic stress response

Soon after its initial discovery, σ^B has been reported to contribute to the osmotic stress resistance of L. monocytogenes, as supported by a variety of phenotypic [11,20,28] as well as gene expression studies [29,30]. The 18 σ^B regulon members identified with known or putative roles in osmotic stress resistance include eight genes that represent three well-studied σ^B-dependent osmotic stress resistance systems, including OpuC (opuCABCD), Gbu (gbuABC) and BetL (betL). All three of these systems facilitate accumulation of compatible solutes under high osmolarity conditions. Specifically, OpuC is required for carnitine accumulation while Gbu and BetL are two betaine transporters. These systems transport carnitine and glycine betaine into the cytoplasm from outside of the cell membrane to combat high salt stress [29,31]. An additional eight genes that are part of the σ^B regulon encode proteins with putative roles in osmotic stress resistance. For three of these genes (LMRG_00884, LMRG_00583 and LMRG_01600) transposon insertions affected the growth of L. monocytogenes LO28 under high salt conditions [32]; however, mechanisms and specific functions of these genes have not yet been reported. LMRG_01658 as well as three genes (LMRG_00208, LMRG_00211-00212) in a five-gene TU are σ^B regulon members that have been reported as induced by salt stress; Ribeiro et al. hypothesized that under salt stress, expression of these genes allowed for enhanced synthesis of exopolysaccharides in response to osmolarity changes [33]. While transcriptomic data indicate a contribution to osmotic stress response by these genes, further physiologic studies are needed to clearly define their functions.

We identified three genes (LMRG_00672, hfq and dtpT) that are also involved in other functions in addition to osmotic stress response. Phenotypic characterization of LMRG_00672 transposon insertion mutants has revealed that this gene supports growth of L. monocytogenes in sodium chloride, and it is involved in resistance to streptomycin as well as attachment and virulence [34]. Furthermore, two σ^B regulon members (hfq, dtpT) have been reported to play important roles in L. monocytogenes’ osmotic stress resistance as well as other stress responses. Hfq is a regulator binding to small RNAs (sRNAs) during intracellular growth [35]. Hfq mutants show attenuated virulence and impaired resistance to osmotic stress and ethanol stress [36]. The di- and tripeptide transporter dtpT is involved in salt stress protection as well as in virulence, as shown in a mouse infection model [37]. Overall, the apparent involvement of at least 18 σ^B regulon members in osmotic stress further supports a key role of σ^B in L. monocytogenes’ resistance and response to osmotic stress, a stress that this pathogen is likely to encounter in a variety of environments, including but not limited to certain foods (e.g., food preserved with salt, such as many cheeses and smoked seafood) as well as the environment encountered in the small intestine [38].

The σ^B regulon includes 14 members involved or putatively involved in oxidative stress response

While L. monocytogenes can survive under oxidative stress conditions encountered inside macrophages, the role of σ^B in regulation of oxidative stress response remains controversial. Previous phenotypic studies have reported results ranging from hypersensitivity to hyper-resistance for sigB null mutants, suggesting a complex σ^B-regulated network responding to different oxidative conditions (recently reviewed in [39]). In our systematic review, 14 σ^B regulon members are classified into the subgroup of oxidative stress response. Oxidative stress caused by products such as reactive oxygen species is a critical part of host defense mechanisms, so it is not surprising that 6 of 14 genes are also grouped into virulence. The four-gene TU qoxABCD encodes QoxAB, an aa₃-type oxidase, which is required for aerobic growth of Bacillus [40]. In L. monocytogenes, QoxAB has been found to contribute to aerobic respiration and intracellular replication, as well as the initial stage of murine infection in experiments using a ΔqoxAB mutant [41]. The σ^B regulon member SpxA, encoded by LMRG_01641, is required for pathogenesis and oxidative stress response in L. monocytogenes as an spxA mutant showed impaired ability of vacuolar escape and increased sensitivity to both peroxide and disulfide stress [42]. In addition, the superoxide dismutase (Sod), encoded by the σ^B regulon member LMRG_00891, protects organisms against superoxides and reactive oxygen species. Deletion of sod results in impaired survival within macrophages and mice, indicating its contribution to virulence [43]. In addition, two σ^B regulon members, uspA and yqhD, may be involved in oxidative stress response as well as other functions. The universal stress protein A (UspA), encoded by LMRG_00196, contributes to extracellular survival of L. monocytogenes under acid stress and oxidative stress [44]. In E. coli, yqhD encodes a NADPH-dependent aldehyde reductase, which is part of a glutathione-independent mechanism in response to oxidative stress [45]. In addition, yqhD has been reported to contribute to isobutanol production in E. coli [46]. The role of yqhD in L. monocytogenes has not been fully confirmed.

Other σ^B regulon members in the oxidative stress response group include five genes and one noncoding RNA (ncRNA). The σ^B-dependent TU LMRG_02643-02644 contains hlsO and coaX. The heat-shock proteins HslO is redox regulated and protects both thermally and oxidatively damaged proteins from irreversible aggregation [47]. In Bacillus, coaX encodes a type III pantothenate kinase that is responsible for the conversion of pantothenate into Coenzyme A [48]. LMRG_00885, encoding a glutathione reductase, has also been reported to play roles in oxidative stress resistance in E. coli [49]. Transcriptional studies have revealed upregulation of putative oxidoreductase-encoding genes including LMRG_00357 [24] and LMRG_02813, when L. monocytogenes is exposed to oxidative stresses such as chlorine dioxide [50]. The ncRNA sbrE contributes to the expression of TU LMRG_00319-00320 and has been reported to be upregulated when L. monocytogenes is exposed to oxidative stress [51]. Further studies are needed to define the roles of these six σ^B regulon members in L. monocytogenes’ defense against oxidative stress. Taken as a whole, the regulation of these genes and ncRNA suggests important contributions of σ^B to L. monocytogenes’ response to endogenous oxidative stress in an aerobic environment, and exogenous oxidative stress caused by host immune system as it has been shown in Staphylococci [52].

The σ^B regulon includes 12 members involved or putatively involved in acid stress response

σ^B directly regulates the transcription of 12 genes contributing to acid resistance. The glutamate decarboxylase (GAD) system is one of the principal systems for L. monocytogenes to withstand acid stress. Among genes encoding components of the GAD system, gadB, gadC and gadD belong to σ^B regulon [53,54]. The GAD system consists of a glutamate decarboxylase enzyme and a glutamate-γ-aminobutyrate (GABA) antiporter. This system produces accumulated GABA and collaborates to reduce acidification inside the cell. In addition, gadD is involved in GABA metabolism via the GABA shunt pathway. Deletion of gadD increased survival of L. monocytogenes under acid treatment by increasing accumulation of GABA [55]. Another crucial mechanism activated by σ^B under acid stress involves putative arginine deiminase genes, which have been shown to play a role in the acid tolerance of other bacterial genera. A σ^B-dependent putative regulator ArgR, regulates transcription of the arcA, arcB, arcDlocus where an additional putative σ^B-dependent promoter was identified upstream [19]. In addition to these two systems, uspA is crucial for L. monocytogenes’ resistance to acid stress, and as discussed above, this gene is also involved in oxidative stress [44]. Other σ^B regulon members identified to contribute to acid resistance of L. monocytogenes include LMRG_00484 and LMRG_02736. Importance of these genes in acid resistance has been confirmed in gene knockout experiments, but their function remains unknown [56]. LMRG_02736 and LMRG_01387 have putative roles in acid stress as suggested by their homology to B. subtilis general stress proteins and induction under acid stress [57]. Overall, these 12 genes reveal important σ^B-dependent systems responsible for L. monocytogenes’ adaption to acid stress.

The σ^B regulon includes six members involved or putatively involved in antibiotic resistance

While previous studies report a low prevalence of resistant strains [58,59], it is likely that L. monocytogenes is exposed to antibiotics in different environmental niches. There are six σ^B regulon members involved in antibiotic resistance mechanisms, with five of them also having additional functions. The TU mpoABCD is involved in carbon metabolism and resistance to class IIa bacteriocins in L. monocytogenes [60]. This TU encodes a mannose PTS permease similar with that encoded by the mpt operon. Mutants of mpo showed an intermediate resistance to class IIa bacteriocins. In 2011, characterization of mpo mutant also showed that this PTS system plays an important role in PrfA inhibition [61]. The stress response gene LMRG_00672 is a drug exporter of the RND superfamily, responsible for the streptomycin resistance exhibited by L. monocytogenes strain 10403S [34]. The overlap of antibiotic resistance and other functions is further supported by the upregulation of stress and virulence genes under subinhibitory concentrations of antibiotics [62]. The fosfomycin resistance protein (FosX) encoding gene is also part of σ^B regulon, and it helps L. monocytogenes to respond to antibiotic stress by catalyzing the hydration of fosfomycin [63]. These six σ^B regulon members contributing to antibiotic resistance in L. monocytogenes point to a future direction of work on the role of σ^B in the regulation of antibiotic resistance in L. monocytogenes.

The σ^B regulon includes three members involved or putatively involved in bile stress response

In host animals, L. monocytogenes is typically exposed to a bile stress environment with more than 0.3% (wt/vol) bile salts [64]. Three important genes in bile resistance are directly regulated by σ^B: bsh, bilEA and bilEB.bsh encodes a bile-salt hydrolase, and the TU of bilEAB (opuB) encodes a bile-exclusion system [65]. Bsh is the only bile-salt hydrolase in L. monocytogenes, and deletion of bsh results in decreased resistance to bile and reduced virulence and liver colonization in mice [66]. BilE, a transporter similar to the OpuC protein, was believed to be a weak regulator of osmotic stress response, but phenotypic experiments have supported its specific role in bile stress response and intestinal colonization of L. monocytogenes [65]. These three genes have also been reported to be directly regulated by PrfA and, therefore, are considered virulence-related [67]. Regulation of bile stress response by σ^B and PrfA is an important example of the regulatory networks that govern the transition between environmental stresses in L. monocytogenes’ passage through the digestive tract.

The σ^B regulon includes 24 members involved or putatively involved in other stress responses

A σ^B-dependent positive feedback loop regulates transcription of the sigB and rsb genes in the σ^B operon [7]. As the σ^B operon has a σ^B-dependent promoter, this four-gene TU of rsbV-rsbW-sigB-rsbX is included in the σ^B regulon. This operon is also crucial for the post-transcriptional regulation of σ^B activity. Briefly, the activation of σ^B is allowed when the anti-sigma factor RsbW binds to anti-anti-sigma factor RsbV, and σ^B is released from the anti-sigma factor to initiate the transcription [68]. Other stress response genes in σ^B regulon include two involved in alkaline stress, one involved in cold stress and 21 with roles in general stress response. LMRG_00583 and LMRG_01600 are involved in alkaline stress and osmotic stress [32]. σ^B has been reported to be required for L. monocytogenes to grow at low temperature with nutrition restriction, a gene essential for this bacterium to grow at cold, ltrC, is σ^B-dependent [69–71]. Among the σ^B-dependent general stress response genes, five genes are also virulence-related, including htrA, fri and the TU of clpC-mcsAB. HtrA is a serine protease required for optimal growth of L. monocytogenes under stress conditions including salt, heat and oxidants [72]. Fri promotes tolerance to stresses such as iron limitation and oxidative stress, as well as contributes to intracellular growth in L. monocytogenes [73]. The clpC operon is involved in response to stresses including iron limitation, heat and osmotic stress, as well as in virulence [74]. The other 12 genes were identified as putatively involved with general stress response as homology to stress response genes in other species was found. Further studies in L. monocytogenes are needed to define the roles of these putative stress response genes.

Virulence

Overall, 51 σ^B regulon members were classified in the ‘virulence’ group. The 51 σ^B regulon members in the ‘virulence’ group included: 23 genes also classified into the group ‘stress response,’ four genes also classified into the group ‘metabolism’ and 24 genes classified into the ‘virulence’ group only. Genes classified into both ‘virulence’ and ‘stress response’ groups are typically required for survival and multiplication under host-related conditions such as bile and acidic pH in the gastric environment or oxidative stress in the host–cell phagosome.

Among the 24 σ^B-regulated, virulence-specific genes, we identified the key transcription activator of L. monocytogenes’ virulence gene expression, positive regulatory factor A (PrfA). PrfA can be transcribed from multiple promoters, regulated by σ^B, σ^A and PrfA itself [67]. The tightly regulated expression of PrfA is required for optimal growth inside and outside the host, as virulence gene expression is costly under limited resources [75]. Meanwhile, several virulence-specific genes are under coregulation of PrfA and σ^B, including internalin-encoding genes inlA and inlB, which are required for adhesion and invasion of L. monocytogenes into host cells [76]. Other σ^B-dependent internalin genes include inlC2, inlD and LMRG_00293, as well as H7858 specific σ^B regulon members LMOh7858_2920, LMOh7858_0394 and LMOh7858_0527. In addition, srtA, which is involved in proteolysis and processing of internalin proteins [77], is required for infection. Two virulence genes chiA and LMRG_02354 encode proteins in chitin catabolic process. Chitinase expressed by bacterial pathogens have proven to be crucial for infecting animals and humans, and in L. monocytogenes, the mutants lacking chiA were defective for growth in the livers and spleens of mice [78,79]. Other important virulence genes include mogR, which regulates flagellar motility genes as a transcriptional repressor [80]; lapB, which is required for entry into eukaryotic cells [81]; mecA, which regulates virulence genes needed for escape from phagosomes [82]; and LMRG_02556, which is involved in cell morphogenesis and intracellular replication [83]. Three σ^B-dependent ncRNAs also contributing to pathogenesis were identified: Lysine, strain-specific lsiIA and rili33 [84–86].

It is worth noting that specific metabolism activities (e.g., glycolysis, glycerol metabolism) are involved in the pathogenesis of intracellular pathogens like L. monocytogenes. A well-studied example highlighting the overlap between metabolism and virulence is the interaction between the PTS system and PrfA [87,88], which shows a close link between the glucose-, mannose- and cellobiose-specific PTS permeases and the modulation of the PrfA activity in L. monocytogenes [87,88]. There are four σ^B regulon members involved in both virulence and carbon metabolism. LMRG_01789 encodes glyceraldehyde 3-phosphate dehydrogenase (Gap), one of the most important enzymes in glycolysis, while LMRG_01793 encodes enolase (Eno). In L. monocytogenes, Gap and Eno have been shown to also bind to human plasminogen, suggesting a role in virulence [89]. In addition, lacD and pfk have been identified as virulence-related in other Firmicutes; in Streptococcus pyogenes, lacD is responsible for lactose and galactose utilization as well as virulence gene regulation [90], and the glycolytic gene pfk is also a putative virulence-related gene in Bacillusanthracis [91]. Together, σ^B regulon members, including transcriptional regulators, expose important features of virulence regulation by σ^B in L. monocytogenes, such as the regulation of metabolism and stress response genes contributing to infection.

Metabolism (including carbon, nucleotide, ion, vitamin, protein & others)

Accumulating evidence suggests that σ^B is also involved in regulation of metabolism-related functions in L. monocytogenes. In particular, σ^B-regulated carbon metabolism (e.g., PTS and glycerol metabolisms) has been well established [56,87,92]. Changes in carbon metabolism do not only provide energy to the bacterium under conditions with limited nutrients but also allow indirect regulation of other genes (e.g., glycerol metabolism affects PrfA activity). Other metabolic activities are also important for L. monocytogenes’ homeostasis and pathogenesis; for example, modulation of ion uptake is crucial for intracellular growth as host immune systems could reduce the bioavailability of ions to invading microbial pathogens [93]. Overall, the involvement of more than 100 σ^B regulon members in metabolism confirms a key role of L. monocytogenes σ^B in regulation of metabolic functions, particularly in carbon metabolism, which involves 73 σ^B regulon members. Since the role of σ^B-regulated genes in the transition of carbon metabolism contributes to intracellular growth, together with metabolism of other macronutrients, micronutrients and cell wall, it seems feasible that σ^B assists the cell to maintain homeostasis during growth and adaptation to changing conditions.

The σ^B regulon includes 73 members involved or putatively involved in carbon metabolism

Previous studies clearly suggest a major role of σ^B in regulating carbon and energy metabolism presumably allowing L. monocytogenes to utilize the limited nutrients available in the changing environments [94,95]. With 73 σ^B regulon members, carbon metabolism is the largest functional subgroup. The full list of 73 σ^B regulon members is provided in Supplementary Table 2. Out of the 73 σ^B regulon members assigned to carbon metabolism, five are also involved in stress response, four are also involved in virulence and the remaining 64 are carbon metabolism-specific genes directly regulated by σ^B. Among the 64-carbon metabolism-specific genes, there are three regulators of carbon metabolism, CggR, LMRG_02213 and NagR. CggR is the central glycolytic gene regulator controlling the transcription of the gapA operon in Bacillus [96]. LMRG_02213 is involved in the phosphoenolpyruvate-dependent sugar phosphotransferase system. NagR acts as a transcriptional inhibitor in the absence of N-acetylglucosamine (GlcNAc) by repressing the expression of nagA and nagB and is also a gene product of the three-gene TU nagABR [97]. NagA and NagB are two deaminases responsible for GlcNAc degradation while NagA is also essential for cell wall components biosynthesis [98]. As a major component of chitin, GlcNAc can be utilized by Listeria species as the source of carbon and nitrogen in a temperature-dependent way in the absence of glucose [99]. The ability of utilizing GlcNAc may contribute to the saprophytic lifestyle of L. monocytogenes in soil [99]. Another TU, LMRG_00594-00611, contains 18 genes that are involved in 1,2-propanediol utilization via a pduD-dependent pathway [100]. 1,2-propanediol is an important intracellular carbon source during infection for bacterial pathogens like Salmonella [101] and has been reported to be degraded by L. innocua [100]. Another confirmed σ^B-dependent multigene TU involved in carbon metabolism is LMRG_01431-01432 encoding a glycerol kinase and a glycerol uptake facilitator involved in glycerol metabolism [87]. Several genes involved in pyruvate metabolism are directly regulated by σ^B, such as the pyruvate oxidase gene (LMRG_00411) and the acetolactate decarboxylase gene (LMRG_00142). Other σ^B regulon members with confirmed carbon metabolism functions in L. monocytogenes include LMRG_01627 (gpmA), which is involved in glycolysis, and LMRG_00977 [56]. Functions of these genes involved in carbon metabolism support that the transition of metabolism, including utilization of alternative carbon sources, is regulated by σ^B. While not directly related to virulence functions such as invasion, the transition of carbon source utilization and energy metabolism is required for optimal intracellular growth [92]. Additionally, as 38 genes with putative roles in carbon metabolism do not have sufficient data in L. monocytogenes to support a clear function, σ^B regulation of carbon metabolism could extend beyond the intracellular life.

The σ^B regulon includes nine members involved or putatively involved in nucleotide metabolism

A nine-gene σ^B-dependent TU (LMRG_00978-00985) is involved in nucleotide metabolism. Genes in this TU encode a set of enzymes involved in a pyrimidine ribonucleotide de novo biosynthesis pathway. In L. monocytogenes, pyrE is required for intracellular proliferation of L. monocytogenes, suggesting that pyrimidine is not provided by the host cell, but must be synthesized by the bacterium machinery [83,102]. The reported studies support σ^B as one of the key regulators for nucleotide metabolism responsible for L. monocytogenes to survive and thrive in the intracellular environment.

The σ^B regulon includes eight members involved or putatively involved in ion transport

We identified eight σ^B-dependent genes that have a confirmed or putative function in ion transport, including iron (hmoB and dhbA), cation (LMRG_01601), magnesium (LMRG_00335), sulfate (LMRG_00205), arsenite (LMRG_00750) and phosphate ion (LMRG_00098 and phoU). In L. monocytogenes, HmoB is involved in degradation of hem, the most abundant source of iron in the human body [103]. In Bacillus subtilis, DhbA is directly regulated by the ferric update regulator and is involved in synthesis of the siderophore bacillibactin, which contributes to iron uptake, especially under iron-deficient conditions [104]. While L. monocytogenes has never been found to synthesize siderophores, it can acquire iron by transporting ferric siderophores produced by other microbes [105]. LMRG_01601 encodes a cation efflux family protein similar to czcD, a proton-driven transporter [95,106]. In E. coli, phoU mutation causes accumulation of inorganic polyphosphate [107]. The last four σ^B-dependent genes (LMRG_00098, LMRG_00205, LMRG_00335 and LMRG_00750) have putative roles in ion transport as suggested by their GO annotations. Overall, these eight members in σ^B regulon indicate that several transport systems for different inorganic ions in L. monocytogenes are controlled by σ^B. This ability of σ^B to regulate ion concentrations suggests a role in resilience under imbalanced ion challenges.

The σ^B regulon includes five members involved or putatively involved in vitamin metabolism

Five genes encoded in two σ^B-dependent TUs are involved in vitamin metabolism. A four-gene TU (LMRG_01077-01080) contributes to menaquinone (vitamin K2) biosynthesis [108], as well as folic acid biosynthesis [109] in B. subtilis. The gene LMRG_02696 is putatively involved in the riboflavin (vitamin B2) biosynthetic process suggested by its GO annotation. Although additional experiments in L. monocytogenes are required to confirm the function of these genes, their proposed function suggests that σ^B also regulates metabolisms of micronutrients like vitamins.

The σ^B regulon includes four members involved or putatively involved in protein metabolism

Four members of the σ^B regulon have protein metabolism-related functions. LMRG_02611 encodes DapE, an enzyme that contributes to lysine metabolism and cell wall metabolism in E. coli [110]. GO annotations suggest that three more σ^B-dependent genes are involved in protein metabolism: LMRG_00826 encoding a peptidase, hpf encoding ribosome hibernation promoting factor and LMRG_01786 encoding a dipeptidase. Future work is needed to better define a possible regulatory role of σ^B in protein metabolism.

The σ^B regulon includes four members involved or putatively involved in other metabolisms

Three σ^B regulon members are involved in cell wall metabolism, murC, nagA and dapE. Cell wall metabolism is crucial for bacterial cell growth, cell division and sensitivity to antibiotics, however, the role of σ^B-dependent regulation of these genes in specific cell wall metabolism processes is not understood. MurC and NagA are both involved in peptidoglycan biosynthesis of the bacterial cell wall. MurC is a L-alanine ligase in Mycobacterium [111], while NagA is essential for L. monocytogenes to maintain its envelope shape and to divide normally [98]. DapE is upregulated in stationary phase and within host cells, with a potential role in anchoring surface proteins [14]. Additionally, another σ^B-dependent gene involved in metabolism is LMRG_00273, which is suggested by GO annotation to be part of lipid metabolism.

Other functions & unknown functions

The nine σ^B regulon members involved in ‘other functions’ include LMRG_01950, which is homologous to the competence gene tfoX in Vibrio cholera [112]. While L. monocytogenes has never been reported as naturally competent, it possesses conserved mechanisms for regulation of its competence components [113], and these competence components have been proposed to contribute to other functions such as virulence [114]. Additionally, eight genes that encode proteins homologous to transcriptional regulators with unknown function are also included in this category. Furthermore, a total of 102 genes in the σ^B regulon have unknown function, therefore, future studies are required to identify the functions of this a third of the σ^B regulon.

Conclusion

In this review, previous transcriptomic and proteomic studies were summarized to generate an overall list of σ^B regulon members in a reproducible way. Based on data for 9 L. monocytogenes strains, we show that the σ^B pan regulon consists of 304 genes, including 102 genes with unknown function; the function of more than 30% of the σ^B regulon thus still remains to be elucidated. Grouping σ^B regulon members into functional groups, helped improve our understanding of the roles of this important alternative sigma factor in L. monocytogenes. While most of the functions conferred by σ^B regulon members confirm the well-known role of σ^B in virulence and stress response, new insight into novel roles for σ^B in metabolism and overall resilience of L. monocytogenes were also identified. Experimental work to define the functions of a number of specific σ^B-regulated genes is still needed though to provide a more thorough understanding of the regulatory networks governed by σ^B in L. monocytogenes.