Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Identification of genes regulated by gadE in E. coli O157:H7

ABSTRACT: Integrating laterally acquired virulence genes into the backbone regulatory network is important for the pathogenesis of Escherichia coli O157:H7, which has captured many virulence genes through horizontal transfer during evolution. GadE is an essential transcriptional activator of glutamate decarboxylase (GAD) system, the most efficient acid resistance mechanism in E. coli. The full contribution of GadE to the acid resistance and virulence of pathogenic E. coli O157:H7 remains largely unknown. We inactivated gadE in E. coli O157:H7 Sakai and compared global transcription profiles with that of wild type in exponential and stationary phases of growth using microarrays containing 6088 ORFs from three E. coli genomes. gadE inactivation significantly altered the expression of 60 genes independent of growth phase and 122 genes in a growth phase-dependent manner. Inactivation of gadE markedly down-regulated the expression of gadA, gadB, gadC and many acid fitness island genes in a growth phase-dependent manner. Nineteen genes encoded on the locus of enterocyte effacement (LEE), including ler, showed a significant increase in expression upon gadE inactivation. Altogether, our data indicate that GadE is critical for acid resistance of E. coli O157:H7 and plays an important role in virulence by down-regulating expression of LEE. The results are based on O157:H7 Sakai wild type and gadE mutant exponential and stationary phase cultures grown in MOPS minimal medium. 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), strain (wild type or mutant) and the interaction of these two factors using the following linear model: array+dye+sample (biological replicate)+ phase+strain+phase*strain. We incorporated the dye-swaps among the biological replicates.

ORGANISM(S): Escherichia coli

SUBMITTER: Sivapriya Kailasan Vanaja

PROVIDER: E-GEOD-13132 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Characterization of the Escherichia coli O157:H7 Sakai GadE regulon.

Kailasan Vanaja Sivapriya S Bergholz Teresa M TM Whittam Thomas S TS

Journal of bacteriology 20081229 6

Integrating laterally acquired virulence genes into the backbone regulatory network is important for the pathogenesis of Escherichia coli O157:H7, which has captured many virulence genes through horizontal transfer during evolution. GadE is an essential transcriptional activator of the glutamate decarboxylase (GAD) system, the most efficient acid resistance (AR) mechanism in E. coli. The full contribution of GadE to the AR and virulence of E. coli O157:H7 remains largely unknown. We inactivated ...[more]

PMID: 19114477

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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.

Project description: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).

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Project description:A total of 192 genes were found to be differentially expressed; 38 genes of them were up regulated and 154 genes were down regulated. Most notably the functional categories that were affected include carbohydrate metabolism and cell envelop realted genes ( 56, 29.1% of the total),Virulence genes (12 genes 6.25% of the total significantly differentiated genes), and aminoacid transport genes (7 genes, 3.6 %). Among the virulence-related genes, the most notable ones were those belong to the production of capsule, the M protein, exotoxin, NAD glycohydrolase and Streptolysin S. Some of the capsule related genes were down regulated by more than 64 fold. Additionally 11 differentially expressed lipid metabolism related genes and four aminoacid transport and metabolism-related genes were upregulated. These results indicate that the C-terminal CHAP domain of CdhA (group A Streptococcal Cell division controlling and Chain-forming cell wall hydrolase) plays an important role in the regulation of virulence. Since these mutant lack cell wall hydrolase activity but are not defective in cell division or growth, we believe that CdhA isa multifunctional protein with N-terminal region controlling cell division and c-terminal region responsible for regulating bacterial virulence. S. pyogenes strain SF370 (wild-type) was procured from ATCC (ATCC 700294). M1-CdhA55(-) mutant strain was obtained using pFW5 vector containing spectinomycin resistance marker (aad9). The mutant expressing truncated CdhA that lack C-terminal 55 residues constituting catalytic site of the CHAP domain is not defective in cell division and growth, but lacks antiphagocytic property and attenuated for virulence. Both strains (M1-Wild-type and M1-CdhA55(-) were grown in Todd-Hewitt broth to their late log phase. Bacteria were harvested by centrifugation and washed twice with sterile PBS. Total RNA was isolated from these washed streptococci using Qiagen RNeasy Mini kit. For synthesis and labeling of cDNA, 20 ug of total RNA, 1.5 ug of random hexamer, 0.5mM dNTPs (except that 0.2mM of dTTP was replaced by the same amount of amino-allyl dUTP to incorporate dUTP into first-strand cDNA) were used in each reverse transcriptase reaction (Superscript II Reverse Transcriptase, Invitrogen). Purified cDNA preparations from the wild-type and M1-CdhA55(-) mutant strains were labeled with either Alexafluor-555 or Alexafluor-647 depending on the experimental design ( i.e. dye swap experiment). Differentially labeled probes were then combined and purified. Using three independently isolated RNA preparations (biological replicates), a total of 7 experiments (incorporating dye swaps) were performed. Accordingly 7 hybridization measurements for this mutant were performed. Thus Exp-1 and 2 (GSM389993, GSM389994) are the technical replicates of the biological sample-1, Exp-3 and 4 (GSM389995, GSM390054) are technical replicates of biological sample-2 and Exp-5,-6, and -7 (GSM390055,GSM390056, and GSM390057) are technical replicates of the biological sample-3. Signals of the bound reagents on the microarray spots in terms of relative fluorescence values were measured and quantified by a laser scanner (GenePix 4100 ) at 10um/pixel resolution. The resulting images were processed using Gene Pix Pro software (version 4.0, Axon instruments). The raw data were obtained in the form of GenPix *.gpr output files. The web application CARMAweb (Comprehensive R based microarray analysis web service) was used for the normalization and analysis of microarray data. All raw data (in the form of *. GPR files) were uploaded to the web application in the data directory. Using appropriate navigation tree, background correction from the foreground signal was applied and within microarray normalization was achieved using the Lowess method. Genes flagged as bad spots by the scanning software were excluded from the analysis and all flagged spots were given a weight of zero. The normalized data were then subjected to fold-change analysis and t-statistics using Bioconductor multtest package. CARMAweb allows to set Log2 cut-off values for both the M (regulation) and the A (average expression) values. Differentially expressed genes (Log2 values of Mutant647-red vs. wild-type555-green or Log2 values Mutant-555(green) vs Wild-type 647-red) were defined based on cut-off value >1 Log2< i.e. all genes that show a two or more-fold up- or down-regulation. BioConductors Multtest package provided suitable method to adjust P values according to multiple hypothesis testing problems.

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Dataset Information

Identification of genes regulated by gadE in E. coli O157:H7

Publications

Characterization of the Escherichia coli O157:H7 Sakai GadE regulon.

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