Project description:The Listeria monocytogenes genome contains more than 20 genes that encode cell surface-associated proteins termed internalins, which are characterized by the presence of multiple leucine-rich repeats. Subsets of internalin genes have been reported previously as transcriptionally regulated by the stress responsive sigma factor B (inlA, inlB, inlC2 and inlD) and the pleiotropic transcriptional activator PrfA (inlA, inlB, inlC). To investigate contributions of σB and PrfA to internalin gene regulation, we designed a sub-genomic microarray containing two probes for each of the 24 internalin-like genes present in the L. monocytogenes 10403S genome. Competitive microarray hybridization was performed on RNA extracted from (i) the 10403S parent strain and an isogenic ΔsigB strain; (ii) 10403S and an isogenic ΔprfA strain; (iii) a 10403S derivative that expresses the constitutively active PrfA* (G155S) and the ΔprfA strain; and (iv) 10403S and an isogenic ΔsigBΔprfA strain. σB- and PrfA-dependent transcription of selected genes was further confirmed by quantitative reverse transcriptase PCR. Statistical analyses of microarray data demonstrated that use of two probes on a microarray for a given target gene yielded more reliable transcriptional profiling information than use of a single probe. The suitability of the PrfA* strain for examining PrfA-dependent gene expression was established quantitatively by the observation that the plcA and prfA transcript levels generated by the PrfA* strain were similar to those obtained from intracellular L. monocytogenes and significantly higher than those in a wildtype strain grown in BHI broth. Among the 24 internalin-like genes examined, 4 and 6 were positively regulated by PrfA and σB, respectively, including inlA and inlB, which were positively regulated by both PrfA and σB. In summary, our findings clearly establish broad roles for both PrfA and σB in regulating L. monocytogenes internalin gene expression Keywords: Listeria monocytogenes, Internalins, sigB, PrfA, Microarrays

Project description:The Listeria monocytogenes genome contains more than 20 genes that encode cell surface-associated proteins termed internalins, which are characterized by the presence of multiple leucine-rich repeats. Subsets of internalin genes have been reported previously as transcriptionally regulated by the stress responsive sigma factor B (inlA, inlB, inlC2 and inlD) and the pleiotropic transcriptional activator PrfA (inlA, inlB, inlC). To investigate contributions of Ï?B and PrfA to internalin gene regulation, we designed a sub-genomic microarray containing two probes for each of the 24 internalin-like genes present in the L. monocytogenes 10403S genome. Competitive microarray hybridization was performed on RNA extracted from (i) the 10403S parent strain and an isogenic Î?sigB strain; (ii) 10403S and an isogenic Î?prfA strain; (iii) a 10403S derivative that expresses the constitutively active PrfA* (G155S) and the Î?prfA strain; and (iv) 10403S and an isogenic Î?sigBÎ?prfA strain. Ï?B- and PrfA-dependent transcription of selected genes was further confirmed by quantitative reverse transcriptase PCR. Statistical analyses of microarray data demonstrated that use of two probes on a microarray for a given target gene yielded more reliable transcriptional profiling information than use of a single probe. The suitability of the PrfA* strain for examining PrfA-dependent gene expression was established quantitatively by the observation that the plcA and prfA transcript levels generated by the PrfA* strain were similar to those obtained from intracellular L. monocytogenes and significantly higher than those in a wildtype strain grown in BHI broth. Among the 24 internalin-like genes examined, 4 and 6 were positively regulated by PrfA and Ï?B, respectively, including inlA and inlB, which were positively regulated by both PrfA and Ï?B. In summary, our findings clearly establish broad roles for both PrfA and Ï?B in regulating L. monocytogenes internalin gene expression Keywords: Listeria monocytogenes, Internalins, sigB, PrfA, Microarrays For all experiments, each strain was grown in BHI at 37oC with shaking (200 rpm) to OD600=0.4, then diluted 1:100 into fresh BHI and grown to OD600=0.4. Cells were then exposed to conditions designed to induce PrfA or sB activity (0.2% charcoal or 0.3 M NaCl, respectively). Specifically, to collect RNA for identification of PrfA-dependent genes, 0.2% charcoal (â??BHI-charcoalâ??) was added to early-log phase (OD600=0.4) 10403S and DprfA cells, which were subsequently incubated with shaking for 120 min at 37Â°C. To collect RNA for identification of PrfA-and sB-dependent genes, early-log phase 10403S and DprfADsigB cells were incubated with shaking for 120 min at 37Â°C in BHI with 0.2% charcoal. NaCl was added (0.3 M final concentration) for the final 10 min (â??BHI-charcoal+NaClâ??). To collect RNA for identification of sB-dependent genes, early-log phase 10403S and DsigB cells were incubated for 120 min in BHI with NaCl added (0.3 M final concentration) for the final 10 min (â??BHI+NaClâ??). To collect RNA for identification of PrfA- dependent genes in the prfA* strain, early-log phase prfA* and DprfA cells were subsequently incubated for 120 min in BHI. After these exposures, RNA was stabilized by the addition of two volumes of RNAprotectâ?¢ (Qiagen, CA, USA). Bacterial cells were then harvested by centrifugation and stored at â??80oC for no longer than 24 h before RNA isolation. For each growth condition described above, RNA isolations were performed on three different days to provide independent biological replicates. Each set of RNA samples was used to perform competitive hybridization microarray experiments.

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Temperature-dependent expression of internalin and internalin-like genes

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