Unknown,Transcriptomics,Genomics,Proteomics

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The plasmid-borne Type II Secretion System of Escherichia coli O157:H7 isolate Sakai mediates adherence to spinach. Experiment 2: control screen.

ABSTRACT: Food-borne illness arising for Shiga-toxigenic Escherichia coli is often linked to consumption of fruit and vegetables as the bacteria have the ability to interact with plants and use them as alternative or secondary hosts. Attachment of the bacteria to host tissue is one of the first steps in the interaction, and, as with mammalian hosts, has shown to be mediated by a combination of non-specific and specific adhesin-mediated interactions. We took a high-throughput positive-selection approach to investigate adherence mechanisms for E. coli O157:H7 isolate Sakai by inoculating a BAC clone library onto spinach, which was quantified by microarray hybridisation and gene loci enrichment measured using a Bayesian hierarchical model. The screen involved four successive rounds of adherence to spinach roots, resulting in 115 CDS credible candidates, covered by seven contiguous genomic regions. Two candidates regions selected for functional assessment included a chaperone-usher fimbrial gene cluster (loc6) and the type two secretion system (T2SS). The TS22 was found to significantly enhance binding to spinach roots and leaves, demonstrated with a BAC-T2SS clone and by mutagenesis of the secretin protein, EtpD. Both etpD and the inner membrane anchor protein gene etpC were expressed at 18 degree celsius, and expression of etpD was demonstrated for STEC (Sakai) resident in the apoplastic spaces in spinach leaf tissue. Together, these data indicate a novel function for STEC T2SS in adherence to plant tissue. Experiment 2: full replicated control screen. Conditions as for Experiment 1 (E-MTAB-5923), but no spinach root present. Two BAC clones (BAC2B5 & BAC2B24) used for additional positive controls.

INSTRUMENT(S): Agilent G2505B scanner

ORGANISM(S): Escherichia coli

SUBMITTER: Pete Hedley

PROVIDER: E-MTAB-5924 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Food-borne illness arising for Shiga-toxigenic Escherichia coli is often linked to consumption of fruit and vegetables as the bacteria have the ability to interact with plants and use them as alternative or secondary hosts. Attachment of the bacteria to host tissue is one of the first steps in the interaction, and, as with mammalian hosts, has shown to be mediated by a combination of non-specific and specific adhesin-mediated interactions. We took a high-throughput positive-selection approach to investigate adherence mechanisms for E. coli O157:H7 isolate Sakai by inoculating a BAC clone library onto spinach, which was quantified by microarray hybridisation and gene loci enrichment measured using a Bayesian hierarchical model. The screen involved four successive rounds of adherence to spinach roots, resulting in 115 CDS credible candidates, covered by seven contiguous genomic regions. Two candidates regions selected for functional assessment included a chaperone-usher fimbrial gene cluster (loc6) and the type two secretion system (T2SS). The TS22 was found to significantly enhance binding to spinach roots and leaves, demonstrated with a BAC-T2SS clone and by mutagenesis of the secretin protein, EtpD. Both etpD and the inner membrane anchor protein gene etpC were expressed at 18 degree celsius, and expression of etpD was demonstrated for STEC (Sakai) resident in the apoplastic spaces in spinach leaf tissue. Together, these data indicate a novel function for STEC T2SS in adherence to plant tissue. Experiment 1: screening E coli O157:H7 Sakai genes for adherence to spinach roots. A BAC library of Sakai clones in an E coli DH10B background (which has poor root adherence) defined as the 'Input pool', was incubated with spinach roots for 4 rounds of enrichment, defined as the 'Output pool'. Control samples (defined as 'Input control' & 'Output control') were cultures of pV41 vector only. DNA extractions from test pools were labelled with Cy3 throughout. DH10B DNA was used for grid alignment and labelled with Cy5 throughout.

Dataset Information

The plasmid-borne Type II Secretion System of Escherichia coli O157:H7 isolate Sakai mediates adherence to spinach. Experiment 2: control screen.

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