Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: February 1, 2003
Publication Date: May 18, 2003
Citation: Uhlich, G.A., Wonderling, L.D., Luchansky, J.B. 2003. Phenotypic analyses of the putative crp/fnr family of transcriptional regulators in a serotype 4b strain of listeria monocytogenes. American Society for Microbiology. Abstract #K-125, p. 381. Technical Abstract: Whole genome sequence analyses indicated that Listeria monocytogenes strain F2365 (serotype 4b, food isolate, 1985 Mexican-style cheese outbreak) contained 15 potential members of the Crp/Fnr family of transcriptional regulatory proteins. Primers were designed to amplify the open-reading-frames and flanking DNA of all 15 members from strain F2365. Amplified products were cloned into a T/A vector, subjected to in vitro transpositional mutagenesis (Tn7-based, SpR), transferred to the integration vector pCON1, and recombined back into the parent 4b strain. Integrations were verified by PCR using opposing transposon and nested chromosomal primers. Stable mutants were produced for 13 of the 15 family members. The parent strain and all 13 mutants were screened for differences in carbon, nitrogen, sulfur, and phosphorus metabolism using a commercial, colorimetric, assay system. The wild-type and 2 of the mutants were identical in metabolizing 29 of 190 carbon substrates. The remaining mutants either failed to utilize one of the carbon substrates or utilized as many as three additional carbon substrates. The wild-type F2365 utilized 24 of 95 nitrogen substrates. None of the mutants were completely identical to F2365 and the differences among mutants did not allow grouping of members based on nitrogen utilization. The wild-type F2365 metabolized 10 of 95 phosphorus and sulfur substrates; 6 of the mutants had only slight variations in utilization when compared to the wild-type. However, 4 mutants showed distinct variations in utilization of purine and pyrimidine 5'-monophosphates while another mutant showed enhanced metabolism of purine and pyrimidine 3'-monophosphates or 2',3'-cyclic monophosphates. These results indicate that each Crp/Fnr mutant has a substrate utilization pattern unique from the wild-type strain and from each other. These differing patterns suggest a unique regulatory specificity for each mutant and indicate that future screening of these mutants for phenotypes important in pathogenicity and persistence in food will have to be done on an individual basis.