dltA gene mutation in the teichoic acids alanylation system of Lactococcus garvieae results in diminished proliferation in its natural host.

The dltABCD cluster is involved in the d-alanylation of teichoic acids in gram positive bacteria. In order to determine the role of this alanylation in the physiology and virulence of Lactococcus garvieae, a previously isolated dltA Delta Tn917 signature tagged mutagenesis (STM) clone was analyzed. RT-PCR results revealed that dltABCD genes form an operon. No major differences could be established between the parental and mutant strains with respect to growth rate, autolytic properties, and susceptibility to acid conditions, lysozyme treatment, anionic detergents, or oxidant agents. However, the dltA mutant was more susceptible to nisin than the parental strain, with minimum inhibitory concentration (MIC) values of 8 and 16 microg/ml, respectively. Less proliferation of the mutant was observed in in vivo competence index experiments (CI=0.08). Furthermore, the mutant strain had a 50% lethal dose (LD(50)) 3-fold that of the parental strain. These results, together with the fact that the dltA Delta Tn917 mutant was isolated as a STM clone, reveal that the dltA locus of Lactococcus garvieae is required for full growth and pathogenesis on rainbow trout.

A chromosomally located traHIJKCLMN operon encoding a putative type IV secretion system is involved in the virulence of Yersinia ruckeri.

Nucleotide sequence analysis of the region surrounding the pIVET8 insertion site in Yersinia ruckeri 150RiviXII, previously selected by in vivo expression technology (IVET), revealed the presence of eight genes (traHIJKCLMN [hereafter referred to collectively as the tra operon or tra cluster]), which are similar both in sequence and organization to the tra operon cluster found in the virulence-related plasmid pADAP from Serratia entomophila. Interestingly, the tra cluster of Y. ruckeri is chromosomally encoded, and no similar tra cluster has been identified yet in the genomic analysis of human pathogenic yersiniae. A traI insertional mutant was obtained by homologous recombination. Coinfection experiments with the mutant and the parental strain, as well as 50% lethal dose determinations, indicate that this operon is involved in the virulence of this bacterium. All of these results suggest the implication of the tra cluster in a virulence-related type IV secretion/transfer system. Reverse transcriptase PCR studies showed that this cluster is transcribed as an operon from a putative promoter located upstream of traH and that the mutation of traI had a polar effect. A traI::lacZY transcriptional fusion displayed higher expression levels at 18 degrees C, the temperature of occurrence of the disease, and under nutrient-limiting conditions. PCR detection analysis indicated that the tra cluster is present in 15 Y. ruckeri strains from different origins and with different plasmid profiles. The results obtained in the present study support the conclusion, already suggested by different authors, that Y. ruckeri is a very homogeneous species that is quite different from the other members of the genus Yersinia.