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Carbon and nitrogen substrate utilization by archival Salmonella typhimurium LT2 cells.

ABSTRACT: BACKGROUND:A collection of over 20,000 Salmonella typhimurium LT2 mutants, sealed for four decades in agar stabs, is a unique resource for study of genetic and evolutionary changes. Previously, we reported extensive diversity among descendants including diversity in RpoS and catalase synthesis, diversity in genome size, protein content, and reversion from auxotrophy to prototrophy. RESULTS:Extensive and variable losses and a few gains of catabolic functions were observed by this standardized method. Thus, 95 catabolic reactions were scored in each of three plates in wells containing specific carbon and nitrogen substrates. CONCLUSION:While the phenotype microarray did not reveal a distinct pattern of mutation among the archival isolates, the data did confirm that various isolates have used multiple strategies to survive in the archival environment. Data from the MacConkey plates verified the changes in carbohydrate metabolism observed in the Biolog system.

SUBMITTER: Tracy BS

PROVIDER: S-EPMC126257 | biostudies-literature | 2002 Sep

REPOSITORIES: biostudies-literature

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Carbon and nitrogen substrate utilization by archival Salmonella typhimurium LT2 cells.

Tracy Breca S BS Edwards Kelly K KK Eisenstark Abraham A

BMC evolutionary biology 20020908

<h4>Background</h4>A collection of over 20,000 Salmonella typhimurium LT2 mutants, sealed for four decades in agar stabs, is a unique resource for study of genetic and evolutionary changes. Previously, we reported extensive diversity among descendants including diversity in RpoS and catalase synthesis, diversity in genome size, protein content, and reversion from auxotrophy to prototrophy.<h4>Results</h4>Extensive and variable losses and a few gains of catabolic functions were observed by this s ...[more]

PMID: 12217081

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Project description:Emergence of genetic variants with increased resistance/tolerance to natural antimicrobials, such as essential oils, has been previously evidenced; however, it is unknown whether mutagenesis follows a general or a specific pattern. For this purpose, we carried out four adaptive laboratory evolutions (ALE) in parallel of Salmonella enterica Typhimurium with carvacrol. After 10 evolution steps, we selected and characterized one colony from each lineage (SeCarA, SeCarB, SeCarC, and SeCarD). Phenotypic characterization of the four evolved strains revealed enhanced survival to lethal treatments; two of them (SeCarA and SeCarB) showed an increase of minimum inhibitory concentration of carvacrol and a better growth fitness in the presence of carvacrol compared to wild-type strain. Whole genome sequencing revealed 10 mutations, of which four (rrsH, sseG, wbaV, and flhA) were present in more than one strain, whereas six (nirC, fliH, lon, rob, upstream yfhP, and upstream argR) were unique to individual strains. Single-mutation genetic constructs in SeWT confirmed lon and rob as responsible for the increased resistance to carvacrol as well as to antibiotics (ampicillin, ciprofloxacin, chloramphenicol, nalidixic acid, rifampicin, tetracycline, and trimethoprim). wbaV played an important role in increased tolerance against carvacrol and chloramphenicol, and flhA in cross-tolerance to heat treatments. As a conclusion, no common phenotypical or genotypical pattern was observed in the isolated resistant variants of Salmonella Typhimurium emerged under carvacrol stress. Furthermore, the demonstration of cross-resistance against heat and antibiotics exhibited by resistant variants raises concerns regarding food safety. KEY POINTS: • Stable resistant variants of Salmonella Typhimurium emerged under carvacrol stress • No common pattern of mutagenesis after cyclic exposures to carvacrol was observed • Resistant variants to carvacrol showed cross-resistance to heat and to antibiotics.

Project description:Fosmidomycin is a time-dependent nanomolar inhibitor of methylerythritol phosphate (MEP) synthase, which is the enzyme that catalyzes the first committed step in the MEP pathway to isoprenoids. Importantly, fosmidomycin is one of only a few MEP pathway-specific inhibitors that exhibits antimicrobial activity. Most inhibitors identified to date only exhibit activity against isolated pathway enzymes. The MEP pathway is the sole route to isoprenoids in many bacteria, yet has no human homologs. The development of inhibitors of this pathway holds promise as novel antimicrobial agents. Similarly, analyses of the bacterial response toward MEP pathway inhibitors provides valuable information toward the understanding of how emergent resistance may ultimately develop to this class of antibiotics. We have examined the transcriptional response of Salmonella enterica serovar typhimurium LT2 to sub-inhibitory concentrations of fosmidomycin via cDNA microarray and RT-PCR. Within the regulated genes identified by microarray were a number of genes encoding enzymes associated with the mediation of reactive oxygen species (ROS). Regulation of a panel of genes implicated in the response of cells to oxidative stress (including genes for catalases, superoxide dismutases, and alkylhydrogen peroxide reductases) was investigated and mild upregulation in some members was observed as a function of fosmidomycin exposure over time. The extent of regulation of these genes was similar to that observed for comparable exposures to kanamycin, but differed significantly from tetracycline. Furthermore, S. typhimurium exposed to sub-inhibitory concentrations of fosmidomycin displayed an increased sensitivity to exogenous H2O2 relative to either untreated controls or kanamycin-treated cells. Our results suggest that endogenous oxidative stress is one consequence of exposures to fosmidomycin, likely through the temporal depletion of intracellular isoprenoids themselves, rather than other mechanisms that have been proposed to facilitate ROS accumulation in bacteria (e.g. cell death processes or the ability of the antibiotic to redox cycle).

Dataset Information

Carbon and nitrogen substrate utilization by archival Salmonella typhimurium LT2 cells.

Publications

Carbon and nitrogen substrate utilization by archival Salmonella typhimurium LT2 cells.

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