Conor P. O'Byrne

Role of sigma(B) in heat, ethanol, acid, and oxidative stress resistance and during carbon starvation in Listeria monocytogenes.

ABSTRACT To determine the contribution of sigma B (ςB) to survival of stationary-phase Listeria monocytogenescells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolarsigB null mutant strain after exposure to heat (50°C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45°C, 5% ethanol, or pH 4.5). The ΔsigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the ΔsigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the ΔsigB strain. These results suggest the existence in L. monocytogenes of both a ςB-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. ςB contributed to resistance to both oxidative stress and carbon starvation inL. monocytogenes. The ΔsigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the ΔsigB strain lost viability more rapidly than the parent strain. ςB contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that ςB plays a role in protecting L. monocytogenes against environmental adversities.

Acid tolerance in Listeria monocytogenes : the adaptive acid tolerance response (ATR) and growth-phase-dependent acid resistance

Listeria monocytogenes acquired increased acid tolerance during exponential growth upon exposure to sublethal acid stress, a response designated the acid tolerance response (ATR). Maximal acid resistance was seen when the organism was exposed to pH 5.0 for 1 h prior to challenge at pH 3.0, although intermediate levels of protection were afforded by exposure to pH values ranging from 4.0 to 6.0. A 60 min adaptive period was required for the development of maximal acid tolerance; during this period the level of acid tolerance increased gradually. Full expression of the ATR required de novo protein synthesis; chloramphenicol, a protein synthesis inhibitor, prevented full induction of acid tolerance. Analysis of protein expression during the adaptive period by two-dimensional gel electrophoresis revealed a change in the expression of at least 23 proteins compared to the non-adapted culture. Eleven proteins showed induced expression while 12 were repressed, implying that the ATR is a complex response involving a modulation in the expression of a large number of genes. In addition to the exponential phase ATR, L.monocytogenes also developed increased acid resistance upon entry into the stationary phase; this response appeared to be independent of the pH-dependent ATR seen during exponential growth.

Inhibition of Escherichia coli growth by acetic acid: a problem with methionine biosynthesis and homocysteine toxicity

The mechanism by which methionine relieves the growth inhibition of Escherichia coli K-12 that is caused by organic weak acid food preservatives was investigated. In the presence of 8 mM acetate the specific growth rate of E. coli Frag1 (in MacIlvaines minimal medium pH 6.0) is reduced by 50%. Addition of methionine restores growth to 80% of that observed in untreated controls. Similar relief was seen with cultures treated with either benzoate or propionate. Mutants with an elevated intracellular methionine pool were almost completely resistant to the inhibitory effects of acetate, suggesting that the methionine pool becomes limiting for growth in acetate-treated cells. Measurement of the intracellular concentrations of pathway intermediates revealed that the homocysteine pool is increased dramatically in acetate-treated cells, suggesting that acetate inhibits a biosynthetic step downstream from this intermediate. Supplementation of the medium with homocysteine inhibits the growth of E. coli cells. Acetate inhibition of growth arises from the depletion of the intracellular methionine pool with the concomitant accumulation of the toxic intermediate homocysteine and this augments the effect of lowering cytoplasmic pH.

Role of Listeria monocytogenes σB in Survival of Lethal Acidic Conditions and in the Acquired Acid Tolerance Response

ABSTRACT The food-borne pathogen Listeria monocytogenes can acquire enhanced resistance to lethal acid conditions through multiple mechanisms. We investigated contributions of the stress-responsive alternative sigma factor, σB, which is encoded by sigB, to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in L. monocytogenes. At various points throughout growth, we compared the relative survival of L. monocytogenes wild-type and ΔsigB strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation. Under these conditions, survival of the ΔsigB strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase. Survival of both ΔsigB and wild-type L. monocytogenes strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the L. monocytogenes growth phase-dependent AR mechanism is σB independent. σB-mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth. Loss of a functional σB reduced the survival of L. monocytogenes at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that L. monocytogenes protection against organic and inorganic acid may be mediated through different mechanisms. σB does not appear to contribute to pHi homeostasis through regulation of net proton movement across the cell membrane or by regulation of pHi buffering by the GAD system under the conditions examined in this study. In summary, a functional σB protein is necessary for full resistance of L. monocytogenes to lethal acid treatments.

Weak organic acids: a panoply of effects on bacteria.

Weak organic acids have been used for centuries to preserve foods, but only recently has the possible mechanism for bacterial growth inhibition been investigated. Although the lowering of internal pH was favored as the cause of growth inhibition, the emphasis has shifted to the anion and its specificity. There are a number of applications of weak organic acids to foods and in the food industry be they pre-or postharvest, However, there is concern that the ability of foodborne pathogens to adapt to these acids may allow longer survival in these commodities and also to better survive transit through the gastric acid barrier of the stomach. Genomic and proteomic approaches have been applied to the identification of genes and proteins that may allow prokaryotes to cope with organic acid stress. These technologies in combination with genetic approaches may provide better identification of genes essential for survival to organic acids. These acids may have other roles: they can induce phenotypic antibiotic resistance, and the high concentrations of these acids in the colon may signal a relationship to diet, colonic microflora, and human health.

Identification of Components of the Sigma B Regulon in Listeria monocytogenes That Contribute to Acid and Salt Tolerance

ABSTRACT Sigma B (σB) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the ΔsigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The ΔsigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σB contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the ΔsigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σB in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the ΔsigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σB. It also demonstrated clear roles for σB in both osmotic and low-pH stress tolerance and identified specific components of the σB regulon that contribute to the responses observed.

Identification and characterization of an ATP binding cassette L-carnitine transporter in Listeria monocytogenes

ABSTRACT We identified an operon in Listeria monocytogenes EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from Bacillus subtilis, which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated opuC, consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated proteins presumed to form the permease (OpuCB and OpuCD). The operon is preceded by a potential SigB-dependent promoter. An opuC-defective mutant was generated by the insertional inactivation of theopuCA gene. The mutant was impaired for growth at high osmolarity in brain heart infusion broth and failed to grow in a defined medium. Supplementation of the defined medium with peptone restored the growth of the mutant in this medium. The mutant was found to accumulate the compatible solutes glycine betaine and choline to same extent as the parent strain but was defective in the uptake ofl-carnitine. We conclude that the opuC operon in L. monocytogenes encodes an ABC compatible solute transporter which is capable of transporting l-carnitine and which plays an important role in osmoregulation in this pathogen.

Sensing and adapting to acid stress

Bacteria and archaea occupy a considerable diversity of niches that vary with respect to the physical conditions. Survival and colonisation requires the capacity to sense, and adapt to, environmental change. In this short review we consider the issues of adaptation to acidic conditions, in particular the mechanisms that might be employed by different bacteria to respond to the specific challenges of their niche. We lay particular emphasis on the protection of the cytoplasm during alterations of the cytoplasmic pH and, in the Gram negative bacteria, on recent work that suggests that protection of the periplasm is critical for survival of exposure to extreme acid. Finally, we discuss potential mechanisms by which pH might be sensed and consider the insights gained from proteins that sense and respond specifically to changes in pH.

Role of σB in Regulating the Compatible Solute Uptake Systems of Listeria monocytogenes: Osmotic Induction of opuC Is σB Dependent

ABSTRACT The regulation of the compatible solute transport systems in Listeria monocytogenes by the stress-inducible sigma factor σB was investigated. Using wild-type strain 10403S and an otherwise isogenic strain carrying an in-frame deletion in sigB, we have examined the role of σB in regulating the ability of cells to utilize betaine and carnitine during growth under conditions of hyperosmotic stress. Cells lacking σB were defective for the utilization of carnitine but retained the ability to utilize betaine as an osmoprotectant. When compatible solute transport studies were performed, the initial rates of uptake of both betaine and carnitine were found to be reduced in the sigB mutant; carnitine transport was almost abolished, whereas betaine transport was reduced to approximately 50% of that of the parent strain. Analysis of the cytoplasmic pools of compatible solutes during balanced growth revealed that both carnitine and betaine steady-state pools were reduced in the sigB mutant. Transcriptional reporter fusions to the opuC (which encodes an ABC carnitine transporter) and betL (which encodes an a secondary betaine transporter) operons were generated by using a promoterless copy of the gus gene from Escherichia coli. Measurement of β-glucuronidase activities directed by opuC-gus and betL-gus revealed that transcription of opuC is largely σB dependent, consistent with the existence of a potential σB consensus promoter motif upstream from opuCA. The transcription of betL was found to be sigB independent. Reverse transcriptase PCR experiments confirmed these data and indicated that the transcription of all three known compatible solute uptake systems (opuC, betL, and gbu), as well as a gene that is predicted to encode a compatible solute transporter subunit (lmo1421) is induced in response to elevated osmolarity. The osmotic induction of opuCA and lmo1421 was found to be strongly σB dependent. Together these observations suggest that σB plays a major role in the regulation of carnitine utilization by L. monocytogenes but is not essential for betaine utilization by this pathogen.

Proteomic Analyses of a Listeria monocytogenes Mutant Lacking σB Identify New Components of the σB Regulon and Highlight a Role for σB in the Utilization of Glycerol

ABSTRACT In Listeria monocytogenes the alternative sigma factor σB plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the σB regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic ΔsigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was σB dependent; 17 of these proteins were found to require the presence of σB for full expression, while 21 were expressed at a higher level in the ΔsigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for σB in the general stress response and highlighted a probable role for σB in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of σB in glycerol metabolism. Five newly identified members of the σB regulon were shown to be under direct regulation of σB using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four σB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified σB-dependent genes, as well as proteomic analyses, σB was shown to play a major role in the stationary phase of growth in complex media.