Rosalina L. J. Goverde

The psychrotrophic bacterium Yersinia enterocolitica is characterized by temperature‐dependent adaptations. To investigate Y. enterocolitica genes involved in cold adaptation, a mutant restricted in its ability to grow at 5°C was isolated from a transposon mutant library. The transposon insertion site in this psychrotrophy‐defective (PD) mutant mapped 16 bp upstream of an open reading frame whose predicted amino acid sequence showed 93% similarity with the Escherichia coli exoribonuclease polynucleotide phosphorylase (PNPase), encoded by pnp. Expression of this gene was blocked in the PD mutant. However, the introduction of a second copy of pnp, including 0.33 kbp sequences upstream of its coding region, into the chromosome of the PD mutant restored pnp expression as well as the ability to grow at 5°C. Furthermore, the expression of pnp appeared to be temperature dependent: in the parental Y. enterocolitica strain, the levels of both pnp mRNA and PNPase were 1.6‐fold higher at 5°C compared with 30°C. A similarly enhanced level of PNPase at 5°C was observed in the merodiploid recombinant strain, which indicates that the 0.33 kbp region upstream of pnp harboured a cold‐inducible promoter. A putative cold shock promoter motif (ATTGG) was observed in this region.

The effect of temperature on the growth rate, protein pattern and fatty acid composition of Yersinia enterocolitica strain W22703 pYV ⁺ , its plasmidless isogenic derivative W22703 pYV ^‐ and four recent field isolates was examined. The growth rate was clearly influenced by presence or absence of the virulence plasmid: pYV ^‐ strains grew consistently faster than pYV ⁺ strains. This difference in growth rate was high at 30–35°C, moderate at 1–10°C and 25°C, but hardly significant at 15–20°C. Increasing the growth temperature above 25°C resulted in the induction of the 220 kDa virulence plasmid‐encoded Yop1 protein. In the 1–20°C range no obvious temperature‐ or plasmid‐related differences in protein patterns could be detected. The fatty acid composition showed a clear temperature‐dependent change: with all strains the degree of saturation was low at 1°C and gradually increased with raising temperatures. All strains had similar fatty acid patterns, except one of the field isolates which showed aberrant C16 : 1 and cyclic fatty acid contents in the 5–25°C and 15°C ranges respectively. With strain W22703, the presence or absence of the virulence plasmid did not significantly alter the fatty acid pattern.

A non‐radioactive colony hybridization method was developed for the rapid detection of Yersinia enterocolitica in primary isolates and for differentiation between pathogenic and non‐pathogenic strains. The method is based on, respectively, the presence of the inv ‐locus in all Yersinia spp. and the presence of the ail ‐gene in pathogenic Y. enterocolitica only. Hybridization results with ail ‐probes of 132 strains of Y. enterocolitica were in good agreement with pathogenicity phenotypes as indicated by a tissue culture invasion (TCI) assay and by serotyping. All TCI ⁺ strains and only two TCI ^‐ strains were positive by hybridization with ail. Hybridization results with inv ‐ or ail ‐probes of 150 primary isolates of human, animal or slaughterhouse origin were compared with those of conventional methods to detect and identify Y. enterocolitica. All samples that were positive for Yersinia spp. by cultivation (four of 66) or were positive for pathogenic Y. enterocolitica by cultivation and serotyping (six of 84) were also positive by hybridization with, respectively, the inv ‐ or ail ‐probe. In three slaughterhouse swab samples, in which Yersinia spp. were not detected by cultivation (2%), strong positive hybridization signals were obtained with the inv ‐ and/or ail ‐probe. Four other swab samples which were negative by cultivation produced weak positive signals by hybridization with inv ‐ and/or ail ‐ probes. These results indicate that the method can be used for (1) the identification of pathogenic Y. enterocolitica isolates and (2) the detection of Yersinia spp. in primary isolates of naturally contaminated samples.