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      The transcriptome of Escherichia coli O157: H7 reveals a role for oxidative stress resistance in its survival from predation by Tetrahymena

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          ABSTRACT

          Pathogenic E. coli remains undigested upon phagocytosis by Tetrahymena and is egested from the ciliate as viable cells in its fecal pellets. Factors that are involved in the survival of Shiga toxin-producing E. coli serovar O157: H7 (EcO157) from digestion by Tetrahymena were identified by microarray analysis of its transcriptome in the protozoan phagosome. Numerous genes belonging to anaerobic metabolism and various stress responses were upregulated significantly ≥ 2-fold in EcO157 cells in the food vacuoles compared with in planktonic cells that remained uningested by the protist. Among these were the oxidative stress response genes, ahpF and katG. Fluorescence microscopy and staining with CellROX® Orange confirmed the presence of reactive oxygen species in food vacuoles containing EcO157 cells. Frequency distribution analysis of the percentage of EcO157 viable cells in Tetrahymena fecal pellets revealed that the ΔahpCF and ΔahpCFΔkatG mutants were less fit than the wild type strain and ΔkatG mutant after passage through the protist. Given the broad use of oxidants as sanitizers in the food industry, our observation of the oxidative stress response in EcO157 during its interaction with Tetrahymena emphasizes the importance of furthering our knowledge of the physiology of this human pathogen in environments relevant to its ecology and to food safety.

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          Most cited references62

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          One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.

          We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.
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            Alkyl hydroperoxide reductase is the primary scavenger of endogenous hydrogen peroxide in Escherichia coli.

            Hydrogen peroxide is generated during aerobic metabolism and is capable of damaging critical biomolecules. However, mutants of Escherichia coli that are devoid of catalase typically exhibit no adverse phenotypes during growth in aerobic media. We discovered that catalase mutants retain the ability to rapidly scavenge H(2)O(2) whether it is formed internally or provided exogenously. Analysis of candidate genes revealed that the residual activity is due to alkyl hydroperoxide reductase (Ahp). Mutants that lack both Ahp and catalase could not scavenge H(2)O(2). These mutants excreted substantial amounts of H(2)O(2), and they grew poorly in air. Ahp is kinetically a more efficient scavenger of trace H(2)O(2) than is catalase and therefore is likely to be the primary scavenger of endogenous H(2)O(2). Accordingly, mutants that lack Ahp accumulated sufficient hydrogen peroxide to induce the OxyR regulon, whereas the OxyR regulon remained off in catalase mutants. Catalase still has an important role in wild-type cells, because the activity of Ahp is saturated at a low (10(-5) M) concentration of H(2)O(2). In contrast, catalase has a high K(m), and it therefore becomes the predominant scavenger when H(2)O(2) concentrations are high. This arrangement is reasonable because the cell cannot provide enough NADH for Ahp to rapidly degrade large amounts of H(2)O(2). In sum, E. coli does indeed generate substantial H(2)O(2), but damage is averted by the scavenging activity of Ahp.
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              Off the hook--how bacteria survive protozoan grazing.

              Bacterial growth and survival in numerous environments are constrained by the action of bacteria-consuming protozoa. Recent findings suggest that bacterial adaptations against protozoan predation might have a significant role in bacterial persistence and diversification. We argue that selective predation has given rise to diverse routes of bacterial defense, including adaptive mechanisms in bacterial biofilms, and has promoted major transitions in bacterial evolution, such as multicellularity and pathogenesis. We propose that studying predation-driven adaptations will provide an exciting frontier for microbial ecology and evolution at the interface of prokaryotes and eukaryotes.
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                Author and article information

                Contributors
                (View ORCID Profile)
                Journal
                FEMS Microbiology Ecology
                Oxford University Press (OUP)
                0168-6496
                1574-6941
                March 01 2020
                March 2020
                February 03 2020
                March 01 2020
                March 2020
                : 96
                : 3
                Affiliations
                [1 ]Produce Safety and Microbiology Research Unit, Western Regional Research Center, Agricultural Research Service, US Department of Agriculture, Albany, CA, USA
                Article
                10.1093/femsec/fiaa014
                32009174
                cf1ba404-f202-4d3d-8e83-c4d04e8556c3
                © 2020
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