Gram-positive bacteria possess a myriad of acid resistance systems that can help them to overcome the challenge posed by different acidic environments. In this review the most common mechanisms are described: i.e., the use of proton pumps, the protection or repair of macromolecules, cell membrane changes, production of alkali, induction of pathways by transcriptional regulators, alteration of metabolism, and the role of cell density and cell signaling. We also discuss the reponses of Listeria monocytogenes, Rhodococcus, Mycobacterium, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, oral streptococci, and lactic acid bacteria to acidic environments and outline ways in which this knowledge has been or may be used to either aid or prevent bacterial survival in low-pH environments.

Surviving the Acid Test: Responses of Gram-Positive Bacteria to Low pH

Preservative agents in foods. Mode of action and microbial resistance mechanisms.

The sigma(S) (RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli and related bacteria. While rapidly growing cells contain very little sigma(S), exposure to many different stress conditions results in rapid and strong sigma(S) induction. Consequently, transcription of numerous sigma(S)-dependent genes is activated, many of which encode gene products with stress-protective functions. Multiple signal integration in the control of the cellular sigma(S) level is achieved by rpoS transcriptional and translational control as well as by regulated sigma(S) proteolysis, with various stress conditions differentially affecting these levels of sigma(S) control. Thus, a reduced growth rate results in increased rpoS transcription whereas high osmolarity, low temperature, acidic pH, and some late-log-phase signals stimulate the translation of already present rpoS mRNA. In addition, carbon starvation, high osmolarity, acidic pH, and high temperature result in stabilization of sigma(S), which, under nonstress conditions, is degraded with a half-life of one to several minutes. Important cis-regulatory determinants as well as trans-acting regulatory factors involved at all levels of sigma(S) regulation have been identified. rpoS translation is controlled by several proteins (Hfq and HU) and small regulatory RNAs that probably affect the secondary structure of rpoS mRNA. For sigma(S) proteolysis, the response regulator RssB is essential. RssB is a specific direct sigma(S) recognition factor, whose affinity for sigma(S) is modulated by phosphorylation of its receiver domain. RssB delivers sigma(S) to the ClpXP protease, where sigma(S) is unfolded and completely degraded. This review summarizes our current knowledge about the molecular functions and interactions of these components and tries to establish a framework for further research on the mode of multiple signal input into this complex regulatory system.

Signal transduction and regulatory mechanisms involved in control of the sigma(S) (RpoS) subunit of RNA polymerase.

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl2. Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.

https://www.thermoscientific.com/content/dam/tfs/LPG/LCD/LCD%20Documents/Peer%20Reviewed%20Papers%20OR%20Third-Party%20Papers/Microplate%20Instrumentation/Microplate%20Readers/D00570~.pdf

Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane

Perspectives on the use of organic acids and short chain fatty acids as antimicrobials

The enteric microorganisms Salmonella typhimurium, Escherichia coli and Shigella flexneri prefer to grow in neutral pH environments. They nevertheless experience dramatic pH fluctuations in nature and during pathogenesis. In response to environmental encounters with acid, these organisms have evolved complex, inducible acid survival strategies. Regulatory features include an alternative sigma factor (σS), 2-component signal transduction systems (PhoP/Q; MviA/?) and the major iron regulatory protein Fur. Specific survival mechanisms include emergency pH homeostasis by inducible amino acid decarboxylases and probable roles for DNA repair, chaparonins, membrane biogenesis as well as others that remain poorly defined. Continued study of acid survival in these organisms will provide general insights regarding stress management and will have a direct impact on our understanding of pathogenesis.

Acid stress responses in enterobacteria

Salmonella typhimurium encounters a variety of acid stress situations during growth in host and nonhost environments. The organism can survive potentially lethal acid conditions (pH <4) if it is first able to adapt to mild or more moderate acid levels. The molecular events that occur during this adaptive process are collectively referred to as the acid tolerance response and vary depending on whether the cells are in log- or stationary-phase growth. The acid tolerance response of logarithmically growing cells includes the participation of an alternate sigma factor, sigmaS (RpoS), commonly associated with stationary-phase physiology. Of 51 acid shock proteins (ASPs) induced during shifts to pH 4.4, 8 are clearly dependent on sigmaS for production (I. S. Lee, J. Lin, H. K. Hall, B. Bearson, and J. W. Foster, Mol. Microbiol. 17:155-167, 1995). The acid shock induction of these proteins appears to be the result of an acid shock-induced increase in the level of sigmaS itself. We have discovered that one component of a potential signal transduction system responsible for inducing rpoS expression is the product of the mouse virulence gene mviA+. MviA exhibits extensive homology to the regulatory components of certain two-component signal transduction systems (W. H. Benjamin, Jr., and P. D. Hall, abstr. B-67, p. 38, in Abstracts of the 93rd General Meeting of the American Society for Microbiology 1993, 1993). Mutations in mviA (mviA::Km) caused the overproduction of sigmaS and sigmaS-dependent ASPs in logarithmically growing cells, as well as increases in tolerances to acid, heat, osmolarity and oxidative stresses and significant decreases in growth rate and colony size. Mutations in rpoS suppressed the mviA::Km-associated defects in growth rate, colony size, ASP production, and stress tolerance, suggesting that the effects of MviA on cell physiology occur via its control of sigmaS levels. Western blot (immunoblot) analyses of sigmaS produced from natural or arabinose-regulated promoters revealed that acid shock and MviA posttranscriptionally regulate sigmaS levels. Turnover experiments suggest that MviA regulates the stability of sigmaS protein rather than the translation of rpoS message. We propose a model in which MviA or its unknown signal transduction partner senses some consequence of acid shock, and probably other stresses, and signals the release of sigmaS from proteolysis. The increased concentration of sigmaS drives the elevated expression of the sigmaS-dependent ASPs, resulting in an increase in stress tolerance. The avirulent nature of mviA insertion mutants, therefore, appears to result from inappropriate sigmaS-dependent gene expression during pathogenesis.

Acid shock induction of RpoS is mediated by the mouse virulence gene mviA of Salmonella typhimurium.

Salmonella typhimurium encounters a variety of acid stress situations during pathogenesis and in the natural environment. These include the extreme low pH encountered in the stomach and a less acidic intestinal environment containing large amounts of organic weak acids (volatile fatty acids). The acid tolerance response (ATR) is a complex defence system that can minimize the lethal effects of extreme low pH (pH 3). The data presented illustrate that the ATR can also defend against weak acids such as butyric, acetic or propionic acids. Although an acid shock of pH 4.4 induced the ATR, growth in subinhibitory concentrations of weak acids did not. Various mutations shown to affect tolerance to extreme acid conditions (pH 3) were tested for their effects on tolerance to weak acids. An rpoS mutant lacking the alternative sigma factor sS failed to protect cells against weak acids as well as extreme acid pH. The fur (ferric uptake regulator) and atp (Mg2+-dependent ATPase) mutants defective in extreme acid tolerance showed no defects in their tolerance to weak acids. Curiously, the atbR mutant that exhibits increased tolerance to extreme acid pH proved sensitive to weak acids. Several insertions that rendered cells sensitive to organic acids were isolated, all of which proved to be linked to the rpoS locus.

/pdf/the-acid-tolerance-response-of-salmonella-typhimurium-2l90537qmp.pdf

The acid tolerance response of Salmonella typhimurium provides protection against organic acids

The starvation-stress response (SSR) of Salmonella typhimurium includes gene products necessary for starvation avoidance, starvation survival and virulence for this bacterium. Numerous genetic loci induced during carbon-source starvation and required for the long-term-starvation survival of this bacterium have been identified. The SSR not only protects the cell against the adverse effects of long-term starvation but also provides cross-resistance to other environmental stresses, e.g. thermal challenge (55 °C) or acid-pH challenge (pH 2·8). One carbon-starvation-inducible lac fusion, designated stiA was previously reported to be a σS-dependent SSR locus that is phosphate-starvation, nitrogen-starvation and H2O2 inducible, positively regulated by (p)ppGpp in a relA-dependent manner, and negatively regulated by cAMP:cAMP receptor protein complex and OxyR. We have discovered through sequence analysis and subsequent biochemical analysis that the stiA::lac fusion, and a similarly regulated lac fusion designated sti-99, lie at separate sites within the first gene (narZ) of an operon encoding a cryptic nitrate reductase (narZYWV) of unknown physiological function. In this study, it was demonstrated that narZ was negatively regulated by the global regulator Fnr during anaerobiosis. Interestingly, narZ(YWV) was required for carbon-starvation-inducible thermotolerance and acid tolerance. In addition, narZ expression was induced ∼20-fold intracellularly in Madin-Darby canine kidney epithelial cells and ∼16-fold in intracellular salts medium, which is believed to mimic the intracellular milieu. Also, a narZ1 knock-out mutation increased the LD50 ∼10-fold for S. typhimurium SL1344 delivered orally in the mouse virulence model. Thus, the previously believed cryptic and constitutive narZYWV operon is in fact highly regulated by a complex network of environmental-stress signals and global regulatory functions, indicating a central role in the physiology of starved and stressed cells.

The rpoS-dependent starvation stress response locus stiA encodes a nitrate reductase (narZYWV) required for carbon-starvation-inducible thermotolerance and acid tolerance in Salmonella typhimurium

Since the stomach is a first line of defense for the host against ingested microorganisms, an ex vivo swine stomach contents (SSC) assay was developed to search for genes important for Salmonella enterica serovar Typhimurium survival in the hostile gastric environment. Initial characterization of the SSC assay (pH 3.87) using previously identified, acid-sensitive serovar Typhimurium mutants revealed a 10-fold decrease in survival for a phoP mutant following 20 min of challenge and no survival for mutants of rpoS or fur. To identify additional genes, a signature-tagged mutagenesis bank was constructed and screened in the SSC assay. Nineteen mutants were identified and individually analyzed in the SSC and acid tolerance response assays; 13 mutants exhibited a 10-fold or greater sensitivity in the SSC assay compared to the wild-type strain, but only 3 mutants displayed a 10-fold or greater decrease in survival following pH 3.0 acidic challenge. Further examination determined that the lethal effects of the SSC are pH dependent but that low pH is not the sole killing mechanism(s). Gas chromatography analysis of the SSC revealed lactic acid levels of 126 mM. Upon investigating the effects of lactic acid on serovar Typhimurium survival in a synthetic gastric fluid, not only was a concentration- and time-dependent lethal effect observed, but the phoP, rpoS, fur, and pnp genes were identified as involved in protection against lactic acid exposure. These studies indicate a role in gastric survival for several serovar Typhimurium genes and imply that the stomach environment is defined by more than low pH.

/pdf/identification-of-salmonella-enterica-serovar-typhimurium-1l6uj0cyri.pdf

Shawn M. D. Bearson

Papers

Acid stress responses in enterobacteria

Acid shock induction of RpoS is mediated by the mouse virulence gene mviA of Salmonella typhimurium.

The acid tolerance response of Salmonella typhimurium provides protection against organic acids

The rpoS-dependent starvation stress response locus stiA encodes a nitrate reductase (narZYWV) required for carbon-starvation-inducible thermotolerance and acid tolerance in Salmonella typhimurium

Identification of Salmonella enterica Serovar Typhimurium Genes Important for Survival in the Swine Gastric Environment