Molecular characterization of piezotolerant and stress-resistant mutants of Staphylococcus aureus.

AIMS: In the previous work, following a pressure treatment with wild-type Staphylococcus aureus, we obtained piezotolerant isolates showing altered phenotypic characteristics. This work focuses on understanding the genetic background of their altered phenotype. METHODS AND RESULTS: AK23, a representative piezotolerant isolate was subjected to DNA microarrays, corroborated by PCR product sequencing and revealed 10-gene deletion. All other piezotolerant isolates possessed the mutation encompassing the region from SAR0665 to SAR0674 genes (9351 bp) which was most likely the result of recombination between two homologous loci (ATTGCGGGTG) present in both genes. RNA microarray transcriptomic analysis showed that due to partial deletion of the low-affinity phosphate transporter pitA, the high-affinity PhoU-PstABCS operon was upregulated in AK23 which could be the reason for piezotolerance. Furthermore, AK23 showed low levels of the virulence gene regulator rnaIII resulting in the downregulation of several agr system genes explaining the impaired virulence characteristics of the mutant. CONCLUSIONS: Naturally occurring mutations can result in piezotolerance which can be of a concern for high hydrostatic pressure-treated foods. SIGNIFICANCE AND IMPACT OF THE STUDY: A locus has been identified in piezotolerant S. aureus mutants providing insight into possible mechanisms associated with phenotypic characteristics of S. aureus. Further work should study each individual gene of the locus.

Role of glutamate metabolism in bacterial responses towards acid and other stresses.

Glutamate plays a central role in a wide range of metabolic processes in bacterial cells. This review focuses on the involvement of glutamate in bacterial stress responses. In particular, it reviews the role of glutamate metabolism in response against acid stress and other stresses. The glutamate decarboxylase (GAD) system has been implicated in acid tolerance in several bacterial genera. This system facilitates intracellular pH homoeostasis by consuming protons in a decarboxylation reaction that produces γ-aminobutyrate (GABA) from glutamate. An antiporter system is usually present to couple the uptake of glutamate to the efflux of GABA. Recent insights into the functioning of this system will be discussed. Finally, the intracellular fate of GABA will also be discussed. Many bacteria are capable of metabolizing GABA to succinate via the GABA shunt pathway. The role and regulation of this pathway will be addressed in the review.

Assessing the microbial oxidative stress mechanism of ozone treatment through the responses of Escherichia coli mutants.

AIMS: To investigate the effect of the oxidative stress of ozone on the microbial inactivation, cell membrane integrity and permeability and morphology changes of Escherichia coli. METHODS AND RESULTS: Escherichia coli BW 25113 and its isogenic mutants in soxR, soxS, oxyR, rpoS and dnaK genes were treated with ozone at a concentration of 6 µg ml⁻¹ for a period up to 240 s. A significant effect of ozone exposure on microbial inactivation was observed. After ozonation, minor effects on the cell membrane integrity and permeability were observed, while scanning electron microscopy analysis showed slightly altered cell surface structure. CONCLUSIONS: The results of this study suggest that cell lysis was not the major mechanism of microbial inactivation. The deletion of oxidative stress-related genes resulted in increased susceptibility of E. coli cells to ozone treatment, implying that they play an important role for protection against the radicals produced by ozone. However, DnaK that has previously been shown to protect against oxidative stress did not protect against ozone treatment in this study. Furthermore, RpoS was important for the survival against ozone. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides important information about the role of oxidative stress in the responses of E. coli during ozonation.

A modified rapid enzymatic microtiter plate assay for the quantification of intracellular γ-aminobutyric acid and succinate semialdehyde in bacterial cells.

The GABase assay is widely used to rapidly and accurately quantify levels of extracellular γ-aminobutyric acid (GABA). Here we demonstrate a modification of this assay that enables quantification of intracellular GABA in bacterial cells. Cells are lysed by boiling and ethanolamine-O-sulphate, a GABA transaminase inhibitor is used to distinguish between GABA and succinate semialdehyde.

Identification of components of the sigma B regulon in Listeria monocytogenes that contribute to acid and salt tolerance.

Sigma B (sigma(B)) 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 DeltasigB 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 DeltasigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that sigma(B) 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 DeltasigB 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 sigma(B) 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 DeltasigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of sigma(B). It also demonstrated clear roles for sigma(B) in both osmotic and low-pH stress tolerance and identified specific components of the sigma(B) regulon that contribute to the responses observed.

Effects of repeated cycles of acid challenge and growth on the phenotype and virulence of Salmonella enterica.

AIMS: The aim of the study was to investigate how stresses like low pH, which may be encountered in farms or food preparation premises, shape populations of Salmonella enterica by the selection of stress-resistant variants. METHODS AND RESULTS: Stationary-phase cultures of S. enterica serovar Enteritidis and serovar Typhimurium (one strain of each) were exposed to pH 2.5 for up to 4 h, followed by growth at pH 7 for 48 h. This process was repeated 15 times in two separate experiments, which increased the acid resistance of the three out of four populations we obtained, by three- to fourfold. Sustainable variants derived from the populations showed changes in colony morphology, expression of SEF17 fimbriae, growth, increased heat resistance and reduced virulence. CONCLUSIONS: The study demonstrates that low pH environments can select for populations of S. enterica with persistent phenotypic changes such as increased acid resistance and occasionally increased SEF17 expression and lower virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: There is a common belief that increased acid resistance coincides with increased virulence. This study demonstrates for the first time that increased acid resistance often impairs virulence and affects the general phenotype of S. enterica.

Proteomic analyses of a Listeria monocytogenes mutant lacking sigmaB identify new components of the sigmaB regulon and highlight a role for sigmaB in the utilization of glycerol.

In Listeria monocytogenes the alternative sigma factor sigmaB plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the sigmaB 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 delta sigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was sigmaB dependent; 17 of these proteins were found to require the presence of sigmaB for full expression, while 21 were expressed at a higher level in the delta sigB 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 sigmaB in the general stress response and highlighted a probable role for sigmaB in metabolism, especially in the utilization of alternative carbon sources. Proteomic and physiological data revealed the involvement of sigmaB in glycerol metabolism. Five newly identified members of the sigmaB regulon were shown to be under direct regulation of sigmaB using reverse transcription-PCR (RT-PCR), while random amplification of cDNA ends-PCR was used to map four sigmaB-dependent promoters upstream from lmo0796, lmo1830, lmo2391, and lmo2695. Using RT-PCR analysis of known and newly identified sigmaB-dependent genes, as well as proteomic analyses, sigmaB was shown to play a major role in the stationary phase of growth in complex media.