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Genetics and Molecular Biology |
Max von Pettenkofer Institut für Medizinische Mikrobiologie und Hygiene, Ludwig Maximilians Universität, Pettenkoferstraße 9a, 80336 München, Germany1
Author for correspondence: J. Heesemann. Tel: +49 89 5160 5200. Fax: +49 89 5160 5202. e-mail: heesemann{at}m3401.mpk.med.uni-muenchen.de
The gene encoding periplasmic 2',3'-cyclic phosphodiesterase in Yersinia enterocolitica O:8 (designated cpdB), was cloned and expressed in Escherichia coli. This enzyme enables Y. enterocolitica to grow on 2',3'-cAMP as a sole source of carbon and energy. Sequencing and analysis of a 3 kb EcoRI fragment containing the cpdB gene revealed an open reading frame of 1179 bp, corresponding to a protein with a molecular mass of 71 kDa. The first 25 amino acid residues show features of a typical prokaryotic signal sequence. The predicted molecular mass of the mature peptide is therefore in agreement with the molecular mass estimated by SDS gel electrophoresis (68 kDa). The putative cpdB promoter region contains two possible -10 and -35 regions. Furthermore, the 5' untranslated region contains sequences with significant homology to the cyclic AMP–cyclic AMP receptor protein binding site and the 
28 consensus. This region is interrupted by an enterobacterial repetitive intergenic consensus (ERIC) sequence. Deletion of the ERIC element from the cpdB promoter region had no effect on cpdB expression. In the 3' untranslated region, a possible rho-independent transcriptional terminator was identified. The deduced amino acid sequence of the Y. enterocolitica CpdB protein shows 76% identity with CpdB of Salmonella typhimurium and E. coli. CpdB of Y. enterocolitica is exported to the periplasmic space. An isogenic Y. enterocolitica cpdB mutant strain, constructed by allelic exchange, was no longer able to grow on 2',3'-cAMP as sole source of carbon and energy. The CpdB mutant showed no significant change in virulence in an oral and intravenous mouse infection model.
Keywords: cpdB gene, 2',3'-cAMP, ERIC, cAMP–CRP-binding site
Abbreviations: cAMP–CRP, cyclic AMP–cyclic AMP receptor protein; ERIC sequence, enterobacterial repetitive intergenic consensus sequence; NPPC, p-nitrophenyl phosphorylcholine; PNPP, p-nitrophenyl phosphate
The GenBank accession number for the cpdB gene fragment of Yersinia enterocolitica O:8 strain WA-314 reported in this paper is X85742.
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