Metabolic reprogramming in the food-borne pathogen 
Listeria monocytogenes is a well-studied member of the bacterial phylum Bacillota that is of significant interest to researchers because it can cause potentially life-threatening food-borne infections and because it is a model intracellular pathogen, capable of invading and growing within cells of the host (Freitag et al. ). It is a hardy bacterium that colonizes a wide variety of niches in the natural environment, making it very difficult to eliminate completely from the food chain. It has multiple traits that make it capable of withstanding the harsh conditions found in some foods and food processing environments, including an ability to grow at refrigeration temperatures, a tolerance to low water activity, and an ability to survive in low-pH environments (Osek et al. ). Since normal cooking temperatures readily kill this pathogen, the primary concern of food producers is in relation to ready-to-eat (RTE) foods, such as dairy products, processed cold meats, salamis, fruits, and salads (Ravindhiran et al. ). Many of these foods have an acidic pH that helps to limit the growth of pathogens and spoilage microorganisms. As an acid-tolerant bacterium, L. monocytogenes can persist in these foods and then its robust response to severe acid stress can aid its transit through the stomach of its host. In recent years, much has been learned about the cellular mechanisms involved in protection against low pH in representative strains of this pathogen (Arcari et al. ), although doubtless, there is still much to learn. The regulatory processes that allow the bacterium to sense and respond to acid are less well understood, but some recent advances will be discussed here. It is expected that understanding the mechanisms that contribute to acid tolerance (persistence or growth at moderately low pH; pH 4.0–6.0) and acid resistance (survival at extremely low pH; pH 2.0–4.0) will prove to be useful in assessing the risks presented by this organism in food and food processing environments. It may also aid the development of new food formulations that reduce the risk of growth of L. monocytogenes during storage and potentially even limit its intragastric survival. While the acid stress response is a complex phenotype that involves changes at multiple levels (Arcari et al. ), herein we summarize the metabolic reprogramming that occurs when L. monocytogenes encounters acid stress. Many of the reactions activated serve to buffer the cytoplasmic pH against the elevated proton concentration in the acidic extracellular milieu. Metabolism of the amino acids arginine and glutamate is critical for survival in response to extremely low pH. Thus far, three principal regulators are known to play important roles in the transcriptional reprogramming required for these metabolic changes: the alternative sigma factor Sigma B (σ B ), the RofA-like transcriptional regulator GadR, and the arginine repressor ArgR. The minireview focuses on recent developments in this field and highlights areas where further research is needed. 

Glutamate decarboxylation The best studied metabolic reaction that contributes to acid stress in L. monocytogenes is glutamate decarboxylation (Arcari et al. ). The pyridoxal 5′-phosphate (vitamin B6)-dependent glutamate decarboxylase (GadD) catalyses the decarboxylation of glutamate, which is coupled to proton consumption and CO 2 production, and thus contributes to neutralizing the intracellular pH (Fig. ) (De Biase and Pennacchietti ). The decarboxylation product, gamma-aminobutyrate (GABA) can be exported through a glutamate/GABA antiporter (GadT) or consumed through the GABA shunt, which bypasses the two reactions in the tricarboxylic acid (TCA) cycle that L. monocytogenes cannot carry out (Fig. ) (Glaser et al. , Feehily and Karatzas , Feehily et al. , ). Most L. monocytogenes genomes encode a complete GAD system, including a glutamate decarboxylase (GadD2/Lmo2363) coupled with a glutamate/GABA antiporter (GadT2/Lmo2362) and a stand-alone decarboxylase (GadD3/Lm02434) (Cotter et al. , Feehily et al. ). Less than half of L. monocytogenes isolates also encode an additional decarboxylase/antiporter operon gadD1T1 ( lmo0447 – 0448 ) in a genetically variable locus termed the stress survival islet-1, which contributes to growth under mildly acidic conditions (Cotter et al. , Ryan et al. , Lakicevic et al. ). Both GadT2D2 and GadD3 were shown to support the survival of this bacterium when confronted with a lethal acid challenge (Cotter et al. , , Feehily et al. , Fang et al. , Wu et al. ), with the former providing the most significant protection (Fang et al. ). Arginine/agmatine deimination Another amino acid-based proton-consuming acid stress response mechanism is the arginine/agmatine deimination system (ADI/AgDI). The ADI pathway involves an arginine deimination step catalysed by ArcA (Lmo0043), producing citrulline and NH 3 . Carbamoyltransferase ArcB (Lmo0036) then converts citrulline to ornithine and carbamoyl∼P (Fig. ) (Cunin et al. ). In the final step, carbamoyl∼P is broken down by ArcC (Lmo0039) producing NH 3 and CO 2 with the generation of ATP (Cunin et al. ). The ornithine produced can be exported by the arginine/ornithine antiporter ArcD (Lmo0037) in exchange for extracellular arginine (Cunin et al. ). AgDI functions via a similar mechanism in which agmatine is fed into the AguA1-mediated deimination reaction with the production of carbamoyl putrescine (Fig. ) (Cunin et al. , Cheng et al. ), which is subsequently cleaved to produce putrescine and carbamoyl phosphate. The latter two steps share the same enzymes (and presumably transporter) with the ADI pathway (Fig. ) (Chen et al. ). The first and third steps of these reactions produce NH 3 , which sequesters protons and thereby helps to neutralize the intracellular pH (Fig. ). Both ADI and AgDI contribute to surviving lethal acidic pH, while ADI also contributes to growth under mildly acidic conditions (Ryan et al. , Chen et al. , Cheng et al. ). Glutamate and arginine biosynthesis Theoretically, the GAD system functions in a way that solely depends on extracellular glutamate without the need to exploit existing intracellular glutamate pools. However, the presence of an orphan glutamate decarboxylase GadD3 suggests that intracellular glutamate might also be important, independently of the two Glu/GABA antiporters. Indeed, Feehily et al.  reported that in strain EGD-e, which has lost its primary GAD activity by GadT2D2, GadD3 is responsible for intracellular accumulation of GABA. This observation suggests that intracellular glutamate pool is important for the GadD3-catalysed GAD reaction. Indeed, it hints that there may be spatial coupling of the Glu/GABA transporters with their cognate glutamate decarboxylases, despite the lack of direct evidence for this at present. Ryan et al.  showed that arginine biosynthesis accounts, at least partially, for providing substrate for the ADI pathway. In line with this, transcriptional upregulation of genes from the arginine biosynthetic pathway following a treatment at pH 5.0 for 15 min was recently observed (Wu et al. ). The roles of de novo glutamate and arginine biosynthesis in acid stress have not been specifically addressed experimentally to date, but it is worth noting that arginine biosynthesis is dependent on glutamate as a precursor (Fig. ). Acetoin production Another proton-consuming mechanism that contributes to the acid stress response in L. monocytogenes is acetoin production (Bowman et al. , Stasiewicz et al. , Madeo et al. , Horlbog et al. ). This pathway feeds on pyruvate and generates α-acetolactate, and the subsequent decarboxylation of α-acetolactate produces acetoin (Fig. ). Each one of these decarboxylation steps requires thiamine (vitamin B1) as a cofactor and each consumes one proton (Romick and Fleming ). In line with this, thiamine uptake is also critical for acid stress response in L. monocytogenes (Madeo et al. ). One additional consequence of fermentative acetoin production is the reduced amount of pyruvate that flows into acetate or lactate fermentation (Stasiewicz et al. ). This may avoid further acidification of the environment as L. monocytogenes grows in the presence of acid stress. Regulators that influence acid resistance SigB The first phenotype associated with loss of the stress-inducible sigma factor SigB (σ B ) was an extreme sensitivity to low pH (Wiedmann et al. ). Soon after that it was shown that the GAD system plays a critical role in allowing L. monocytogenes to survive in gastric fluid and in foods (Cotter et al. , , ) and that σ B controls the transcriptional induction of gadD3 in response to acidification of the medium (Fig. ) (Kazmierczak et al. , Wemekamp-Kamphuis et al. ). Interestingly, the lmo0913 gene (also called gabD ) encoding succinic semialdehyde dehydrogenase (Fig. ), a key component of the GABA shunt (Feehily et al. ), is also induced by acid in a σ B -dependent manner (Abram et al. , Bowman et al. ). Deletion of lmo913 produces an acid survival phenotype in L. monocytogenes , but the precise nature of the effect is strain dependent (Abram et al. , Feehily et al. ). In response to mild acidification, the activity of σ B is increased through a post-translational regulatory mechanism that involves a high-molecular weight sensory hub called the stressosome (Guerreiro et al. ). Recently, it has been shown that low pH signals, the nature of which are not yet known, are sensed by the stressosome, resulting in a regulatory cascade that liberates σ B from an antisigma factor called RsbW (Fig. ). The signal transduction is dependent on the phosphorylation of two proteins in the stressosome, RsbR and RsbS, by another stressosome protein, the kinase RsbT (Fig. ) (Guerreiro et al. ). This signal transduction mechanism is required for the σ B -dependent transcriptional activation of gadD3, arcA (arginine deiminase), and aguA1 (agmatine deiminase) in response to acidification of the medium (Guerreiro et al. ). In wild isolates of L. monocytogenes , heterogeneity in the activity of σ B appears to be an important determinant of acid resistance (Wu et al. ). Mutations that negatively affect σ B activity arise comparatively easily (Guerreiro et al. , , Wu et al. ), suggesting that there is a selective advantage to loss of σ B under some environmental conditions. Thus, it appears that a trade-off exists between the general stress response, which is σ B mediated and confers protection against environmental stressors, and achieving maximal growth rates, an idea that has been explored more fully in a recent review (Abram et al. ). GadR Recently, a RofA-family transcriptional regulator was identified and named GadR, which specifically controls the expression of gadT2D2 (Wu et al. ). GadR positively influences gadT2D2 expression during the stationary phase and upon exposure to acidic stress, and thereby confers acid resistance (Fig. ) (Wu et al. ). Importantly, GadR-mediated gadT2D2 upregulation is also the most significant transcriptional change upon exposure to pH 5 acidic stress (Wu et al. ), a treatment that induces the adaptive acid tolerance response (Davis et al. ). The discovery of this regulator to a large extent explains the intraspecies variations in acid resistance as gadD2T2 accounts for the most acid resistance in L. monocytogenes (Cotter et al. , Fang et al. ) and loss-of-function mutations in gadR are prevalent (Wu et al. ). Interestingly, gadR transcription appears to remain stable across different conditions suggesting that the transcriptional upregulation of gadT2D2 may involve the post-translational modification of GadR. Based on functional domain predictions, GadR is classified as a RofA-like protein, and they are collectively assigned to the recently defined PRD (phosphotransferase regulatory domain)-containing virulence regulator (PCVR) family (Beckert et al. , Rom et al. ). PCVRs were predominately found in Streptococcus previously and GadR represents the first PCVR described in L. monocytogenes . Interestingly, the previously identified transcriptional activator of gadD1T1 , Lmo0445 (Ryan et al. ), is also classified as a PCVR by definition. The presence of two PRDs suggests that the activation of these regulators might be tightly associated with phosphorylation by PTS transporters and the histidine phosphocarrier protein Hpr, as has been reported in other PCVR homologues (Fig. ) (Galinier and Deutscher , Rom et al. ). Interestingly, trehalose metabolism was shown to result in gadT2D2 upregulation in L. monocytogenes , contributing to survival at lethal acidic pH (Wu et al. ). This observation suggests that carbohydrate metabolism might influence the regulation of GadR activity. ArgR ArgR modulates the expression of enzymes involved in arginine biosynthesis and arginine deimination pathways in L. monocytogenes (Ryan et al. , Cheng et al. ). It is homologous to the well-studied Bacillus subtilis arginine repressor/activator AhrC (Cheng et al. ). Purified ArgR multimerizes in vitro (forming a tetramer and hexamer) and its highly conserved S42 and A43 residues are critical for the DNA-binding activity (Cheng et al. ). ArgR directly binds to the promoter regions of genes encoding arginine biosynthetic enzyme and ADI enzymes (Cheng et al. ). It represses the expression of these genes while it can also activate the expression of arc operon, likely in the presence of arginine (Fig. ) (Ryan et al. , Cheng et al. ). In addition to arginine metabolic pathways, ArgR was also shown to repress the expression of σ B (Cheng et al. ). Ryan et al.  reported that Δ argR strain displayed reduced ADI activity, retarded growth under sublethal acidic conditions, and compromised survival under lethal acidic conditions. Interestingly, Cheng et al.  reported that Δ argR survived better under lethal acidic condition, possibly attributed to different media or strains used [10403S grown in BHI in Cheng et al.  versus LO28 grown in TSB-YE in Ryan et al. ]. Although multiple studies have addressed the importance of the ADI/AgDI systems in the acid stress response, further elucidation is required to understand how both systems are regulated in response to environmental pH or arginine/agmatine availability and whether these systems are differently regulated among different strains of L. monocytogenes . Role of acid resistance systems in food persistence Food and food processing environments serve as important means of transmission of L. monocytogenes to humans. In these environments, the bacteria are often exposed to various physical and chemical challenges including low pH, low water activity, low storage temperature, industrial cleaning agents, and antimicrobial packaging materials (NicAogáin and O’Byrne , Jordan and McAuliffe ). Listeria monocytogenes is more tolerant to these stresses than many other bacteria, making it challenging to control in the food chain (Wiktorczyk-Kapischke et al. ). To circumvent the diverse stresses encountered in food matrices and food processing environments, several of the protective mechanisms described above contribute to the survival and growth, leading to a persistence in the food product and raising a potential public health risk. Acid adaptation at mild acidic pH (5.5) is known to aid L. monocytogenes in surviving in acidic foods, including fermented dairy products (Gahan et al. ). The glutamate decarboxylase system of L. monocytogenes plays an important role in facilitating survival in acidic foods like fruit juices, mayonnaise, and fermented dairy products (Cotter et al. , Collins et al. ). Mutants lacking both the gadD1 and gadD2 genes were found to survive poorly in foods in modified atmosphere packaged food (Francis et al. ). The role of other acid resistance systems in food matrices remains to be explored. Recently, transcriptomic analyses of L. monocytogenes growing on various food matrices reveal a complex picture that is both food and strain dependent. The transcription of gadT2D2 was found to be very high in cold smoked salmon (Tang et al. ) but reduced significantly in cantaloupe relative to a BHI-grown control (Kang et al. ), presumably reflecting differences in the physical and chemical properties of these foods. Interestingly, the transcription of GAD genes in meat-based food matrices is highly strain dependent (Rantsiou et al. ), although this effect can be partly explained by the inclusion of EGD-e in the study, which is now known to have a gadR − genotype, and therefore defective for gadT2D2 transcription (Wu et al. ). It is worth noting that organic acids (e.g. lactic acid and acetic acid) are often present in acidic food products, but limited information is available on how metabolic reprograming contributes to protection against organic acid stress (Heavin et al. , Stasiewicz et al. ). Additionally, most research on acid resistance mechanisms has been carried out at the host-related temperature (37°C) instead of lower temperatures, which are more reflective of the conditions used to store RTE food products. Role of acid resistance systems in host persistence and virulence The σ B -mediated general stress response has been shown to play an important role in the gastrointestinal stages of infection, although it appears largely dispensable during later stages of the infectious cycle (Garner et al. , Toledo-Arana et al. ). Since mutants lacking sigB are sensitive to gastric acid (Wiedmann et al. , Ferreira et al. ), it seems likely that passage through the mammalian stomach will be influenced by σ B -dependent acid resistance mechanisms, including the GadD3 glutamate decarboxylase (Feehily et al. ). Indeed, spontaneous mutants of L. monocytogenes that have acquired increased resistance to acid demonstrate increased lethality for mice and reach higher numbers in their spleens (O’Driscoll et al. ). There is some evidence for both GadD1 and GadD3 playing a role in colonization of the liver and spleen in mice, but these effects are rather small and were observed in a strain background that lacked a full-length gadR (Feehily et al. , Wu et al. ). When a gadR + strain was tested, the same authors found that loss of gadD2 causes a significant increase in the lag time for intracellular growth in macrophages, although the final bacterial cell numbers reach similar levels (Feehily et al. ). Mutants lacking rsbX (a negative regulator of σ B ) display increased σ B activity, but this does not alter the virulence and infectivity to a significant degree, at least in a chick embryo model (Oliveira et al. ). The arginine deimination system has been less well studied in the context of virulence within the host. Mutants lacking the arginine deiminase enzyme (Δ arcA ) are compromised for survival under acidic conditions. These mutants are not compromised for intracellular growth in macrophages, but fail to reach the same numbers as the parental strain in the murine spleen following intraperitoneally inoculation (Ryan et al. ). Further studies are needed to conclusively determine whether gastric passage is aided by the ADI system. Future perspectives While a great deal has been learned about the principal mechanisms that L. monocytogenes deploys to counter the damaging effects of acidic pH, a number of challenges remain in this field. Chief among these is understanding how this pathogen senses acid stress. While it is clear that the stressosome plays a critical role in sensing acid stress and activating the general stress response (Guerreiro et al. ), neither the precise mechanism of sensing nor the nature of the signal that is sensed (it could be proton concentration and/or some secondary effect of reduced pH) has been elucidated. Likewise, although GadR is now known to be a key transcriptional regulator of adaptive acid resistance (Wu et al. ), in this pathogen the post-translational signal transduction mechanisms that activate this regulator remain to be identified. As a member of PCVR family of regulators, it seems likely that interactions with the PTS system will play a part in the regulation of GadR activity (Fig. ). The heterogeneity of acid resistance as a phenotype within the species also presents a significant challenge to food producers and regulators in assessing the risk presented by individual strains and clonal complexes. A better understanding of how genetic variations contribute to acid resistance will be needed to make more informed risk assessments and to develop better control strategies for this pathogen. Larger-scale genome-wide association studies (GWAS) that exploit the availability of large fully sequenced strain collections will play an important role in helping to identify key genetic markers that underpin acid resistance (Lees and Bentley ). While the power of bacterial GWAS is somewhat hampered by the highly clonal population structure of L. monocytogenes (Hingston et al. , Myintzaw et al. ), transposon-directed insertion sequencing might be a suitable complementary method to uncover strain-specific features in acid stress response (Wu et al. ). By carefully harnessing available strain collections and exploiting integrative omics approaches (e.g. transcriptomics, proteomics, and metabolomics), in the future new insights are likely to be uncovered in the sensory, regulatory, and homeostatic mechanisms that combine to influence acid resistance of individual strains (Abram et al. , Toledo-Arana et al. , Maury et al. , Impens et al. , Zhou et al. ). The trade-offs that likely exist between acid resistance and other cell properties may also be better understood using these approaches (Abram et al. ). While more fundamental studies on the mechanistic basis of acid resistance are still needed, there is also a need for additional research on the role of the individual acid resistance systems in persistence and growth in food products where organic acid may be encountered. Understanding which mechanisms contribute to growth and survival in different food products will be essential to improve food safety through a more informed design of food formulations. Ultimately, it may even be possible to design foods that specifically inhibit components of the cells’ acid resistance machinery, thereby limiting the growth and survival of this pathogen in food and in the stomach. For example, maleic acid has been shown to inhibit the GAD system in L. monocytogenes and its presence leads to increased acid sensitivity even in cells growing as biofilm (Paudyal et al. ). Although maleic acid is not permitted as a food additive, it could potentially be used in combination with acidic cleaning agents in food production settings. Manganese was recently shown to be required for the growth of L. monocytogenes at low pH (Wu et al. ). It is known that manganese limitation in cottage cheese, which has an acidic pH, restricts the growth of Listeria (van Gijtenbeek et al. ) and also protects against fungal spoilage in yogurt (Siedler et al. ). Formulating acidic foods with limited manganese could therefore be a potential means of limiting the growth of this pathogen and spoilage organisms. Thus, approaches based on a mechanistic understanding of the underlying stress responses have the potential to limit pathogen growth and extend shelf life, ultimately enhancing food safety and reducing food waste. 

The best studied metabolic reaction that contributes to acid stress in L. monocytogenes is glutamate decarboxylation (Arcari et al. ). The pyridoxal 5′-phosphate (vitamin B6)-dependent glutamate decarboxylase (GadD) catalyses the decarboxylation of glutamate, which is coupled to proton consumption and CO 2 production, and thus contributes to neutralizing the intracellular pH (Fig. ) (De Biase and Pennacchietti ). The decarboxylation product, gamma-aminobutyrate (GABA) can be exported through a glutamate/GABA antiporter (GadT) or consumed through the GABA shunt, which bypasses the two reactions in the tricarboxylic acid (TCA) cycle that L. monocytogenes cannot carry out (Fig. ) (Glaser et al. , Feehily and Karatzas , Feehily et al. , ). Most L. monocytogenes genomes encode a complete GAD system, including a glutamate decarboxylase (GadD2/Lmo2363) coupled with a glutamate/GABA antiporter (GadT2/Lmo2362) and a stand-alone decarboxylase (GadD3/Lm02434) (Cotter et al. , Feehily et al. ). Less than half of L. monocytogenes isolates also encode an additional decarboxylase/antiporter operon gadD1T1 ( lmo0447 – 0448 ) in a genetically variable locus termed the stress survival islet-1, which contributes to growth under mildly acidic conditions (Cotter et al. , Ryan et al. , Lakicevic et al. ). Both GadT2D2 and GadD3 were shown to support the survival of this bacterium when confronted with a lethal acid challenge (Cotter et al. , , Feehily et al. , Fang et al. , Wu et al. ), with the former providing the most significant protection (Fang et al. ). 

Another amino acid-based proton-consuming acid stress response mechanism is the arginine/agmatine deimination system (ADI/AgDI). The ADI pathway involves an arginine deimination step catalysed by ArcA (Lmo0043), producing citrulline and NH 3 . Carbamoyltransferase ArcB (Lmo0036) then converts citrulline to ornithine and carbamoyl∼P (Fig. ) (Cunin et al. ). In the final step, carbamoyl∼P is broken down by ArcC (Lmo0039) producing NH 3 and CO 2 with the generation of ATP (Cunin et al. ). The ornithine produced can be exported by the arginine/ornithine antiporter ArcD (Lmo0037) in exchange for extracellular arginine (Cunin et al. ). AgDI functions via a similar mechanism in which agmatine is fed into the AguA1-mediated deimination reaction with the production of carbamoyl putrescine (Fig. ) (Cunin et al. , Cheng et al. ), which is subsequently cleaved to produce putrescine and carbamoyl phosphate. The latter two steps share the same enzymes (and presumably transporter) with the ADI pathway (Fig. ) (Chen et al. ). The first and third steps of these reactions produce NH 3 , which sequesters protons and thereby helps to neutralize the intracellular pH (Fig. ). Both ADI and AgDI contribute to surviving lethal acidic pH, while ADI also contributes to growth under mildly acidic conditions (Ryan et al. , Chen et al. , Cheng et al. ). 

Theoretically, the GAD system functions in a way that solely depends on extracellular glutamate without the need to exploit existing intracellular glutamate pools. However, the presence of an orphan glutamate decarboxylase GadD3 suggests that intracellular glutamate might also be important, independently of the two Glu/GABA antiporters. Indeed, Feehily et al.  reported that in strain EGD-e, which has lost its primary GAD activity by GadT2D2, GadD3 is responsible for intracellular accumulation of GABA. This observation suggests that intracellular glutamate pool is important for the GadD3-catalysed GAD reaction. Indeed, it hints that there may be spatial coupling of the Glu/GABA transporters with their cognate glutamate decarboxylases, despite the lack of direct evidence for this at present. Ryan et al.  showed that arginine biosynthesis accounts, at least partially, for providing substrate for the ADI pathway. In line with this, transcriptional upregulation of genes from the arginine biosynthetic pathway following a treatment at pH 5.0 for 15 min was recently observed (Wu et al. ). The roles of de novo glutamate and arginine biosynthesis in acid stress have not been specifically addressed experimentally to date, but it is worth noting that arginine biosynthesis is dependent on glutamate as a precursor (Fig. ). 

Another proton-consuming mechanism that contributes to the acid stress response in L. monocytogenes is acetoin production (Bowman et al. , Stasiewicz et al. , Madeo et al. , Horlbog et al. ). This pathway feeds on pyruvate and generates α-acetolactate, and the subsequent decarboxylation of α-acetolactate produces acetoin (Fig. ). Each one of these decarboxylation steps requires thiamine (vitamin B1) as a cofactor and each consumes one proton (Romick and Fleming ). In line with this, thiamine uptake is also critical for acid stress response in L. monocytogenes (Madeo et al. ). One additional consequence of fermentative acetoin production is the reduced amount of pyruvate that flows into acetate or lactate fermentation (Stasiewicz et al. ). This may avoid further acidification of the environment as L. monocytogenes grows in the presence of acid stress. 

SigB The first phenotype associated with loss of the stress-inducible sigma factor SigB (σ B ) was an extreme sensitivity to low pH (Wiedmann et al. ). Soon after that it was shown that the GAD system plays a critical role in allowing L. monocytogenes to survive in gastric fluid and in foods (Cotter et al. , , ) and that σ B controls the transcriptional induction of gadD3 in response to acidification of the medium (Fig. ) (Kazmierczak et al. , Wemekamp-Kamphuis et al. ). Interestingly, the lmo0913 gene (also called gabD ) encoding succinic semialdehyde dehydrogenase (Fig. ), a key component of the GABA shunt (Feehily et al. ), is also induced by acid in a σ B -dependent manner (Abram et al. , Bowman et al. ). Deletion of lmo913 produces an acid survival phenotype in L. monocytogenes , but the precise nature of the effect is strain dependent (Abram et al. , Feehily et al. ). In response to mild acidification, the activity of σ B is increased through a post-translational regulatory mechanism that involves a high-molecular weight sensory hub called the stressosome (Guerreiro et al. ). Recently, it has been shown that low pH signals, the nature of which are not yet known, are sensed by the stressosome, resulting in a regulatory cascade that liberates σ B from an antisigma factor called RsbW (Fig. ). The signal transduction is dependent on the phosphorylation of two proteins in the stressosome, RsbR and RsbS, by another stressosome protein, the kinase RsbT (Fig. ) (Guerreiro et al. ). This signal transduction mechanism is required for the σ B -dependent transcriptional activation of gadD3, arcA (arginine deiminase), and aguA1 (agmatine deiminase) in response to acidification of the medium (Guerreiro et al. ). In wild isolates of L. monocytogenes , heterogeneity in the activity of σ B appears to be an important determinant of acid resistance (Wu et al. ). Mutations that negatively affect σ B activity arise comparatively easily (Guerreiro et al. , , Wu et al. ), suggesting that there is a selective advantage to loss of σ B under some environmental conditions. Thus, it appears that a trade-off exists between the general stress response, which is σ B mediated and confers protection against environmental stressors, and achieving maximal growth rates, an idea that has been explored more fully in a recent review (Abram et al. ). GadR Recently, a RofA-family transcriptional regulator was identified and named GadR, which specifically controls the expression of gadT2D2 (Wu et al. ). GadR positively influences gadT2D2 expression during the stationary phase and upon exposure to acidic stress, and thereby confers acid resistance (Fig. ) (Wu et al. ). Importantly, GadR-mediated gadT2D2 upregulation is also the most significant transcriptional change upon exposure to pH 5 acidic stress (Wu et al. ), a treatment that induces the adaptive acid tolerance response (Davis et al. ). The discovery of this regulator to a large extent explains the intraspecies variations in acid resistance as gadD2T2 accounts for the most acid resistance in L. monocytogenes (Cotter et al. , Fang et al. ) and loss-of-function mutations in gadR are prevalent (Wu et al. ). Interestingly, gadR transcription appears to remain stable across different conditions suggesting that the transcriptional upregulation of gadT2D2 may involve the post-translational modification of GadR. Based on functional domain predictions, GadR is classified as a RofA-like protein, and they are collectively assigned to the recently defined PRD (phosphotransferase regulatory domain)-containing virulence regulator (PCVR) family (Beckert et al. , Rom et al. ). PCVRs were predominately found in Streptococcus previously and GadR represents the first PCVR described in L. monocytogenes . Interestingly, the previously identified transcriptional activator of gadD1T1 , Lmo0445 (Ryan et al. ), is also classified as a PCVR by definition. The presence of two PRDs suggests that the activation of these regulators might be tightly associated with phosphorylation by PTS transporters and the histidine phosphocarrier protein Hpr, as has been reported in other PCVR homologues (Fig. ) (Galinier and Deutscher , Rom et al. ). Interestingly, trehalose metabolism was shown to result in gadT2D2 upregulation in L. monocytogenes , contributing to survival at lethal acidic pH (Wu et al. ). This observation suggests that carbohydrate metabolism might influence the regulation of GadR activity. ArgR ArgR modulates the expression of enzymes involved in arginine biosynthesis and arginine deimination pathways in L. monocytogenes (Ryan et al. , Cheng et al. ). It is homologous to the well-studied Bacillus subtilis arginine repressor/activator AhrC (Cheng et al. ). Purified ArgR multimerizes in vitro (forming a tetramer and hexamer) and its highly conserved S42 and A43 residues are critical for the DNA-binding activity (Cheng et al. ). ArgR directly binds to the promoter regions of genes encoding arginine biosynthetic enzyme and ADI enzymes (Cheng et al. ). It represses the expression of these genes while it can also activate the expression of arc operon, likely in the presence of arginine (Fig. ) (Ryan et al. , Cheng et al. ). In addition to arginine metabolic pathways, ArgR was also shown to repress the expression of σ B (Cheng et al. ). Ryan et al.  reported that Δ argR strain displayed reduced ADI activity, retarded growth under sublethal acidic conditions, and compromised survival under lethal acidic conditions. Interestingly, Cheng et al.  reported that Δ argR survived better under lethal acidic condition, possibly attributed to different media or strains used [10403S grown in BHI in Cheng et al.  versus LO28 grown in TSB-YE in Ryan et al. ]. Although multiple studies have addressed the importance of the ADI/AgDI systems in the acid stress response, further elucidation is required to understand how both systems are regulated in response to environmental pH or arginine/agmatine availability and whether these systems are differently regulated among different strains of L. monocytogenes . 

The first phenotype associated with loss of the stress-inducible sigma factor SigB (σ B ) was an extreme sensitivity to low pH (Wiedmann et al. ). Soon after that it was shown that the GAD system plays a critical role in allowing L. monocytogenes to survive in gastric fluid and in foods (Cotter et al. , , ) and that σ B controls the transcriptional induction of gadD3 in response to acidification of the medium (Fig. ) (Kazmierczak et al. , Wemekamp-Kamphuis et al. ). Interestingly, the lmo0913 gene (also called gabD ) encoding succinic semialdehyde dehydrogenase (Fig. ), a key component of the GABA shunt (Feehily et al. ), is also induced by acid in a σ B -dependent manner (Abram et al. , Bowman et al. ). Deletion of lmo913 produces an acid survival phenotype in L. monocytogenes , but the precise nature of the effect is strain dependent (Abram et al. , Feehily et al. ). In response to mild acidification, the activity of σ B is increased through a post-translational regulatory mechanism that involves a high-molecular weight sensory hub called the stressosome (Guerreiro et al. ). Recently, it has been shown that low pH signals, the nature of which are not yet known, are sensed by the stressosome, resulting in a regulatory cascade that liberates σ B from an antisigma factor called RsbW (Fig. ). The signal transduction is dependent on the phosphorylation of two proteins in the stressosome, RsbR and RsbS, by another stressosome protein, the kinase RsbT (Fig. ) (Guerreiro et al. ). This signal transduction mechanism is required for the σ B -dependent transcriptional activation of gadD3, arcA (arginine deiminase), and aguA1 (agmatine deiminase) in response to acidification of the medium (Guerreiro et al. ). In wild isolates of L. monocytogenes , heterogeneity in the activity of σ B appears to be an important determinant of acid resistance (Wu et al. ). Mutations that negatively affect σ B activity arise comparatively easily (Guerreiro et al. , , Wu et al. ), suggesting that there is a selective advantage to loss of σ B under some environmental conditions. Thus, it appears that a trade-off exists between the general stress response, which is σ B mediated and confers protection against environmental stressors, and achieving maximal growth rates, an idea that has been explored more fully in a recent review (Abram et al. ). 

Recently, a RofA-family transcriptional regulator was identified and named GadR, which specifically controls the expression of gadT2D2 (Wu et al. ). GadR positively influences gadT2D2 expression during the stationary phase and upon exposure to acidic stress, and thereby confers acid resistance (Fig. ) (Wu et al. ). Importantly, GadR-mediated gadT2D2 upregulation is also the most significant transcriptional change upon exposure to pH 5 acidic stress (Wu et al. ), a treatment that induces the adaptive acid tolerance response (Davis et al. ). The discovery of this regulator to a large extent explains the intraspecies variations in acid resistance as gadD2T2 accounts for the most acid resistance in L. monocytogenes (Cotter et al. , Fang et al. ) and loss-of-function mutations in gadR are prevalent (Wu et al. ). Interestingly, gadR transcription appears to remain stable across different conditions suggesting that the transcriptional upregulation of gadT2D2 may involve the post-translational modification of GadR. Based on functional domain predictions, GadR is classified as a RofA-like protein, and they are collectively assigned to the recently defined PRD (phosphotransferase regulatory domain)-containing virulence regulator (PCVR) family (Beckert et al. , Rom et al. ). PCVRs were predominately found in Streptococcus previously and GadR represents the first PCVR described in L. monocytogenes . Interestingly, the previously identified transcriptional activator of gadD1T1 , Lmo0445 (Ryan et al. ), is also classified as a PCVR by definition. The presence of two PRDs suggests that the activation of these regulators might be tightly associated with phosphorylation by PTS transporters and the histidine phosphocarrier protein Hpr, as has been reported in other PCVR homologues (Fig. ) (Galinier and Deutscher , Rom et al. ). Interestingly, trehalose metabolism was shown to result in gadT2D2 upregulation in L. monocytogenes , contributing to survival at lethal acidic pH (Wu et al. ). This observation suggests that carbohydrate metabolism might influence the regulation of GadR activity. 

ArgR modulates the expression of enzymes involved in arginine biosynthesis and arginine deimination pathways in L. monocytogenes (Ryan et al. , Cheng et al. ). It is homologous to the well-studied Bacillus subtilis arginine repressor/activator AhrC (Cheng et al. ). Purified ArgR multimerizes in vitro (forming a tetramer and hexamer) and its highly conserved S42 and A43 residues are critical for the DNA-binding activity (Cheng et al. ). ArgR directly binds to the promoter regions of genes encoding arginine biosynthetic enzyme and ADI enzymes (Cheng et al. ). It represses the expression of these genes while it can also activate the expression of arc operon, likely in the presence of arginine (Fig. ) (Ryan et al. , Cheng et al. ). In addition to arginine metabolic pathways, ArgR was also shown to repress the expression of σ B (Cheng et al. ). Ryan et al.  reported that Δ argR strain displayed reduced ADI activity, retarded growth under sublethal acidic conditions, and compromised survival under lethal acidic conditions. Interestingly, Cheng et al.  reported that Δ argR survived better under lethal acidic condition, possibly attributed to different media or strains used [10403S grown in BHI in Cheng et al.  versus LO28 grown in TSB-YE in Ryan et al. ]. Although multiple studies have addressed the importance of the ADI/AgDI systems in the acid stress response, further elucidation is required to understand how both systems are regulated in response to environmental pH or arginine/agmatine availability and whether these systems are differently regulated among different strains of L. monocytogenes . 

Food and food processing environments serve as important means of transmission of L. monocytogenes to humans. In these environments, the bacteria are often exposed to various physical and chemical challenges including low pH, low water activity, low storage temperature, industrial cleaning agents, and antimicrobial packaging materials (NicAogáin and O’Byrne , Jordan and McAuliffe ). Listeria monocytogenes is more tolerant to these stresses than many other bacteria, making it challenging to control in the food chain (Wiktorczyk-Kapischke et al. ). To circumvent the diverse stresses encountered in food matrices and food processing environments, several of the protective mechanisms described above contribute to the survival and growth, leading to a persistence in the food product and raising a potential public health risk. Acid adaptation at mild acidic pH (5.5) is known to aid L. monocytogenes in surviving in acidic foods, including fermented dairy products (Gahan et al. ). The glutamate decarboxylase system of L. monocytogenes plays an important role in facilitating survival in acidic foods like fruit juices, mayonnaise, and fermented dairy products (Cotter et al. , Collins et al. ). Mutants lacking both the gadD1 and gadD2 genes were found to survive poorly in foods in modified atmosphere packaged food (Francis et al. ). The role of other acid resistance systems in food matrices remains to be explored. Recently, transcriptomic analyses of L. monocytogenes growing on various food matrices reveal a complex picture that is both food and strain dependent. The transcription of gadT2D2 was found to be very high in cold smoked salmon (Tang et al. ) but reduced significantly in cantaloupe relative to a BHI-grown control (Kang et al. ), presumably reflecting differences in the physical and chemical properties of these foods. Interestingly, the transcription of GAD genes in meat-based food matrices is highly strain dependent (Rantsiou et al. ), although this effect can be partly explained by the inclusion of EGD-e in the study, which is now known to have a gadR − genotype, and therefore defective for gadT2D2 transcription (Wu et al. ). It is worth noting that organic acids (e.g. lactic acid and acetic acid) are often present in acidic food products, but limited information is available on how metabolic reprograming contributes to protection against organic acid stress (Heavin et al. , Stasiewicz et al. ). Additionally, most research on acid resistance mechanisms has been carried out at the host-related temperature (37°C) instead of lower temperatures, which are more reflective of the conditions used to store RTE food products. 

The σ B -mediated general stress response has been shown to play an important role in the gastrointestinal stages of infection, although it appears largely dispensable during later stages of the infectious cycle (Garner et al. , Toledo-Arana et al. ). Since mutants lacking sigB are sensitive to gastric acid (Wiedmann et al. , Ferreira et al. ), it seems likely that passage through the mammalian stomach will be influenced by σ B -dependent acid resistance mechanisms, including the GadD3 glutamate decarboxylase (Feehily et al. ). Indeed, spontaneous mutants of L. monocytogenes that have acquired increased resistance to acid demonstrate increased lethality for mice and reach higher numbers in their spleens (O’Driscoll et al. ). There is some evidence for both GadD1 and GadD3 playing a role in colonization of the liver and spleen in mice, but these effects are rather small and were observed in a strain background that lacked a full-length gadR (Feehily et al. , Wu et al. ). When a gadR + strain was tested, the same authors found that loss of gadD2 causes a significant increase in the lag time for intracellular growth in macrophages, although the final bacterial cell numbers reach similar levels (Feehily et al. ). Mutants lacking rsbX (a negative regulator of σ B ) display increased σ B activity, but this does not alter the virulence and infectivity to a significant degree, at least in a chick embryo model (Oliveira et al. ). The arginine deimination system has been less well studied in the context of virulence within the host. Mutants lacking the arginine deiminase enzyme (Δ arcA ) are compromised for survival under acidic conditions. These mutants are not compromised for intracellular growth in macrophages, but fail to reach the same numbers as the parental strain in the murine spleen following intraperitoneally inoculation (Ryan et al. ). Further studies are needed to conclusively determine whether gastric passage is aided by the ADI system. 

While a great deal has been learned about the principal mechanisms that L. monocytogenes deploys to counter the damaging effects of acidic pH, a number of challenges remain in this field. Chief among these is understanding how this pathogen senses acid stress. While it is clear that the stressosome plays a critical role in sensing acid stress and activating the general stress response (Guerreiro et al. ), neither the precise mechanism of sensing nor the nature of the signal that is sensed (it could be proton concentration and/or some secondary effect of reduced pH) has been elucidated. Likewise, although GadR is now known to be a key transcriptional regulator of adaptive acid resistance (Wu et al. ), in this pathogen the post-translational signal transduction mechanisms that activate this regulator remain to be identified. As a member of PCVR family of regulators, it seems likely that interactions with the PTS system will play a part in the regulation of GadR activity (Fig. ). The heterogeneity of acid resistance as a phenotype within the species also presents a significant challenge to food producers and regulators in assessing the risk presented by individual strains and clonal complexes. A better understanding of how genetic variations contribute to acid resistance will be needed to make more informed risk assessments and to develop better control strategies for this pathogen. Larger-scale genome-wide association studies (GWAS) that exploit the availability of large fully sequenced strain collections will play an important role in helping to identify key genetic markers that underpin acid resistance (Lees and Bentley ). While the power of bacterial GWAS is somewhat hampered by the highly clonal population structure of L. monocytogenes (Hingston et al. , Myintzaw et al. ), transposon-directed insertion sequencing might be a suitable complementary method to uncover strain-specific features in acid stress response (Wu et al. ). By carefully harnessing available strain collections and exploiting integrative omics approaches (e.g. transcriptomics, proteomics, and metabolomics), in the future new insights are likely to be uncovered in the sensory, regulatory, and homeostatic mechanisms that combine to influence acid resistance of individual strains (Abram et al. , Toledo-Arana et al. , Maury et al. , Impens et al. , Zhou et al. ). The trade-offs that likely exist between acid resistance and other cell properties may also be better understood using these approaches (Abram et al. ). While more fundamental studies on the mechanistic basis of acid resistance are still needed, there is also a need for additional research on the role of the individual acid resistance systems in persistence and growth in food products where organic acid may be encountered. Understanding which mechanisms contribute to growth and survival in different food products will be essential to improve food safety through a more informed design of food formulations. Ultimately, it may even be possible to design foods that specifically inhibit components of the cells’ acid resistance machinery, thereby limiting the growth and survival of this pathogen in food and in the stomach. For example, maleic acid has been shown to inhibit the GAD system in L. monocytogenes and its presence leads to increased acid sensitivity even in cells growing as biofilm (Paudyal et al. ). Although maleic acid is not permitted as a food additive, it could potentially be used in combination with acidic cleaning agents in food production settings. Manganese was recently shown to be required for the growth of L. monocytogenes at low pH (Wu et al. ). It is known that manganese limitation in cottage cheese, which has an acidic pH, restricts the growth of Listeria (van Gijtenbeek et al. ) and also protects against fungal spoilage in yogurt (Siedler et al. ). Formulating acidic foods with limited manganese could therefore be a potential means of limiting the growth of this pathogen and spoilage organisms. Thus, approaches based on a mechanistic understanding of the underlying stress responses have the potential to limit pathogen growth and extend shelf life, ultimately enhancing food safety and reducing food waste. 

