Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

The RpoN Regulon of Escherichia coli O157:H7

ABSTRACT: There is increasing evidence to support a role for sigma factor 54 (RpoN) in the regulation of stress resistance factors and protein secretion systems important to bacterial transmission and pathogenesis. In enterohemorrhagic E. coli O157:H7, acid resistance and type III secretion are essential determinants of gastric passage and colonization. This study thus described the transcriptome of an rpoN null strain of E. coli O157:H7 (EcJR-8) to determine the influence of RpoN on virulence and stress resistance gene regulation, and further explored its contribution to glutamate-dependent acid resistance (GDAR). Inactivation of rpoN resulted in the growth phase-dependent, differential expression of 104 genes. This included type III secretion structural and regulatory genes encoded on the locus of enterocyte effacement (LEE), as well as GDAR genes gadA, gadBC and gadE. Upregulation of gad transcript levels in EcJR-8 during logarithmic growth correlated with increased GDAR and survival in a model stomach. Acid susceptibility was reconstituted in EcJR-8 complemented in trans with wild-type rpoN. Acid resistance in EcJR-8 was dependent on exogenous glutamate, gadE and rpoS, but was independent of hns. Results also suggest that GDAR may be controlled by RpoN at multiple regulatory levels. This study supports the hypothesis that RpoN is an important regulator of virulence and stress resistance factors in E. coli O157:H7, and is the first to examine the mechanism by which it represses GDAR. Hybridizations measured transcriptional differences between an rpoN null and wild-type (WT) strain of E. coli O157:H7 Sakai at logarithmic and transition phase. Image files (TIFF) of hybridized microarray slides were generated using an Axon 4000B scanner (Molecular Devices), and analyzed using GenePix Pro software (Molecular Devices, ver. 6.0). The resulting microarray intensity data was log2-transformed, and normalized using the LOWESS algorithm in MAANOVA ver. 0.98-8 (R ver. 2.2.1).

ORGANISM(S): Escherichia coli O157:H7 str. Sakai

SUBMITTER: James Riordan

PROVIDER: E-GEOD-17467 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Inactivation of alternative sigma factor 54 (RpoN) leads to increased acid resistance, and alters locus of enterocyte effacement (LEE) expression in Escherichia coli O157 : H7.

Riordan James T JT Tietjen Jillian A JA Walsh Coilin W CW Gustafson John E JE Whittam Thomas S TS

Microbiology (Reading, England) 20091126 Pt 3

Alternative sigma factor 54 (RpoN) is an important regulator of stress resistance and virulence genes in many bacterial species. In this study, we report on the gene expression alterations that follow rpoN inactivation in Escherichia coli O157 : H7 strain Sakai (Sakai rpoN : : kan), and the influence of RpoN on the acid resistance phenotype. Microarray gene expression profiling revealed the differential expression of 103 genes in SakairpoN : : kan relative to Sakai. This included the growth-phas ...[more]

PMID: 19942657

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Project description:Escherichia coli O157:H7 has caused serious outbreaks of foodborne illness via transmission in a variety of food vehicles, including unpasteurized apple juice, dried salami, and spinach. To understand how this pathogen responds to the multiple stresses of the food environment, we compared global transcription patterns after exposure to apple juice. Transcriptomes of mid-exponential and stationary phase cells were evaluated after 10 minutes in model apple juice (pH3.5) using microarrays probing 4,886 ORFs. Significant changes in gene expression were determined using R/MAANOVA and the Fs test. A total of 331 ORFs were significantly induced upon exposure of cells to model apple juice and included genes involved in the acid and osmotic stress responses as well as the oxidative stress response and envelope stress. Genes involved in the acid and osmotic stress responses, including asr, osmC, osmB, and osmY were significantly induced in response to model apple juice. Genes involved in the envelope stress response, known to be controlled by CpxR (cpxP, degP, and htpX), were significantly induced 2 to 15 fold upon exposure to apple juice, independent of growth phase. Inactivation of CpxRA resulted in a significant decrease in survival of O157:H7 in model apple juice compared to the isogenic parent strain. Of the 331 ORFs induced in model apple juice, 104 are O157-specific ORFs, including those encoding type three secretion effectors espJ, espB, espM2, espL3 and espZ. By elucidating the response of O157:H7 to acidic foods, we hope to gain insights into how this pathogen is able to survive in food matrices and how exposure to foods affects subsequent transmission and virulence. Keywords: stress The results are based on O157:H7 Sakai exponential and stationary phase cultures grown in MOPS minimal medium and then exposed to model apple juice (pH 3.5, 37C) for 10 minutes. Differences in transcript levels were determined using a mixed model ANOVA in R/MAANOVA which tested for significant differences due to growth phase (exponential or stationary), treatment (MOPS or MAJ) and the interaction of these two factors using the following linear model: array+dye+sample (biological replicate)+ phase+treatment+phase*treatment. We incorporated the dye-swaps among the biological replicates.

Dataset Information

The RpoN Regulon of Escherichia coli O157:H7

Publications

Inactivation of alternative sigma factor 54 (RpoN) leads to increased acid resistance, and alters locus of enterocyte effacement (LEE) expression in Escherichia coli O157 : H7.

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