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Escherichia coli have evolved adaptive systems to resist strongly acidic habitats in part through the production of 2 biochemically identical isoforms of glutamate decarboxylase (GAD), encoded by the gadA and gadB genes. These genes occur in E. coli and other members of the genospecies (e.g., Shigella spp.) and originated as part of a genomic fitness island acquired early in Escherichia evolution. The present duplicated gad loci are widely spaced on the E. coli chromosome, and the 2 genes are 97% similar in sequence. Comparison of the nucleotide sequences of the gadA and gadB in 16 strains of pathogenic E. coli revealed 3.8% and 5.0% polymorphism in the 2 genes, respectively. Alignment of the homologous genes identified a total of 120 variable sites, including 21 fixed nucleotide differences between the loci within the first 82 codons of the genes. Twenty-three phylogenetically informative sites were polymorphic for the same nucleotides in both genes suggesting recent gene conversions or intergenic recombination. Phylogenetic analysis based on the synonymous substitutions per synonymous site indicated 2 cases in which specific gadA and gadB alleles were more closely related to one another than to other alleles at the corresponding locus. The results indicate that at least 3 gene conversion events have occurred after the gad gene duplication in the evolution of E. coli. Despite multiple gene conversion events, the upstream regulatory regions and the 5' end of each gene remains distinct, suggesting that maintaining functionally different gad genes is important in this acid-resistance mechanism in pathogenic E. coli.

Aims: To compare survival of enterohaemorrhagic Escherichia coli (EHEC) strains of two clonal groups in a simulated gastric environment and to quantify the effect of storage in an acidic food, apple juice, on subsequent survival of EHEC in the simulated gastric environment. Methods and Results: To characterize acid resistance of EHEC under conditions simulating the gastric environment, survival was measured in a model stomach system (MSS) for two clonal groups of EHEC: 14 EHEC 1 strains of serotype O157:H7 and 12 EHEC 2 strains of serotypes O26:H11 and O111:H8. There were significant differences between the two EHEC groups, with the average survival rate of O157 strains in the MSS twice as great as the O26/O111 strains. Strains of the two groups also differed in the quantity of injured cells in MSS and in the transcript levels of the glutamate decarboxylase genes (measured by quantitative PCR) in stationary phase before cultures entered the MSS. Conclusions: The results indicate that E. coli O157:H7 strains have superior ability to survive simulated gastric acidity compared with the non-O157 EHEC. Significance and Impact of the Study: E. coli O157:H7 becomes acid resistant rapidly upon entry into stationary phase, which may underlie the low infectious dose of this pathogen.

SbrE is a ncRNA in Listeria monocytogenes, reported to be up-regulated by the alternative sigma factor σB. Initial quantitative RT-PCR (qRT-PCR) experiments on parent strains and isogenic ΔsigB strains demonstrated σB-dependent expression of SbrE across the four L. monocytogenes lineages and in L. innocua. Microarray and proteomics (MDLC/MS/MS with iTRAQ labeling) experiments with the L. monocytogenes parent strain and an isogenic ΔsbrE strain identified a single gene (lmo0636) and two proteins (Lmo0637 and Lmo2094) that showed lower expression levels in the ΔsbrE strain. qRT-PCR demonstrated an increase in SbrE transcript levels in stationary phase L. monocytogenes and in bacteria exposed to oxidative stress (mean log2 transcript levels 7.68 ± 0.57 and 1.70 ± 0.71 greater than in mid-log phase cells, respectively). However, no significant differences in growth or survival between the parent strain and ΔsbrE strain were confirmed under a variety of environmental stress conditions tested. Our data suggest that σB-dependent transcription of SbrE represents a conserved mechanism that contributes, across Listeria species, to fine-tuning of gene expression under specific environmental conditions that remain to be defined.

Combinations of organic acids are often used in ready-to-eat foods to control the growth of Listeria monocytogenes during refrigerated storage. The purpose of this study was to quantitatively assess synergy between two organic acid growth inhibitors under conditions similar to those present in cold-smoked salmon, and to assess the effect of evolutionary lineage on response to those growth inhibitors. Thirteen strains of L. monocytogenes, representing lineages I and II, were grown at 7 degrees C in broth at pH 6.1 and 4.65% water-phase NaCl, which was supplemented with 2% potassium lactate, 0.14% sodium diacetate, or the combination of both at the same levels. Our data suggest that lineages adapt similarly to these inhibitors, as the only significant growth parameter difference between lineages was a minor effect (+/- 0.16 day, P = 0.0499) on lag phase (lambda). For all strains, lactate significantly extended lambda, from 2.6 +/- 0.4 to 3.8 +/- 0.5 days (P < 0.001), and lowered the maximum growth rate (mu(max)) from 0.54 +/- 0.06 to 0.49 +/- 0.04 log(CFU/ml)/day (P < 0.001), compared with the control. Diacetate was ineffective alone, but in combination with lactate, synergistically increased lambda to 6.6 +/- 1.6 days (P < 0.001) and decreased mu(max) to 0.34 +/- 0.05 log(CFU/ml)/day (P < 0.001). Monte Carlo simulations provided further evidence for synergy between diacetate and lactate by predicting signficantly slower growth to nominal endpoints for the combination of inhibitors. This study shows potassium lactate and sodium diacetate have significant synergistic effects on both lambda and mu(max) of L. monocytogenes at refrigeration temperature in broth, and justifies combining these inhibitors, at effective levels, in food product formulations.