Pathogenic Yersinia species carry a novel, cold-inducible major cold shockprotein tandem gene duplication producing both bicistronic and monocistronic mRNA

Inverse PCR was used to amplify major cold shock protein (MCSP) gene famili es from a diverse range of bacteria, including the psychrotolerant Yersinia enterocolitica, which was found to have two almost identical MCSP coding r egions (cspA1 and cspA2) located approximately 300 bp apart. This tandem ge ne duplication was also found in Y. pestis, Y. pseudotuberculosis, and Y. r uckeri but not in other bacteria. Analysis of the transcriptional regulatio n of this MCSP gene in Y. enterocolitica, performed by using both reverse t ranscriptase-PCR and Northern blot assays, showed there to be two cold-indu cible mRNA templates arising from this locus: a monocistronic template of a pproximately 450 bp (cspA1) and a bicistronic template of approximately 900 bp (cspA1/A2). The former may be due to a secondary structure between cspA 1 and cspA2 causing either 3' degradation protection of cspA1 or, more prob ably, partial termination after cspA1. Primer extension experiments identif ied a putative transcriptional start site (+1) which is flanked by a cold-b ox motif and promoter elements (-10 and -35) similar to those found in Esch erichia coli cold-inducible MCSP genes. At 30 degrees C, the level of both mRNA molecules was negligible; however, upon a temperature downshift to 10 degrees C, transcription of the bicistronic mRNA was both substantial (300- fold increase) and immediate, with transcription of the monocistronic mRNA being approximately 10-fold less (30-fold increase) and significantly slowe r. The ratio of bicistronic to monocistronic mRNA changed with time after c old shock and was higher when cells were shocked to a lower temperature. Hi gh-resolution, two-dimensional protein gel electrophoresis showed that synt hesis of the corresponding proteins, both CspA1 and CspA2, was apparent aft er only 10 min of cold shock from 30 degrees C to 10 degrees C. The data de monstrate an extraordinary capacity of the psychrotolerant Y. enterocolitic a to produce major cold shock proteins upon cold shock.