The Heat Sensitive Factor (HSF) of Yersinia Ruckeri is Produced by an Alkyl Sulphatase Involved in Sodium Dodecyl Sulphate (SDS) Degradation but Not in Virulence

Background: The heat sensitive factor (HSF) of the fish pathogen Yersinia ruckeri was previously identified as an unusual band on SDS-PAGE. According to this, Y. ruckeri strains were classified in HSF+ and HSF - in terms of the presence/absence of the factor. Experiments carried out by injection challenge with HSF + strains caused high mortalities in rainbow trout. In contrast, HSF - strains did not cause mortality. In conclusion, HSF appeared to be a relevant virulence factor in Y. ruckeri.

Results: We report here the identification and study of the gene coding for the enzyme involved in the production of HSF. Culture medium containing SDS and Coomassie brilliant blue dye was used to screen a mini-Tn5 Km2 mutant library of Y. ruckeri 150. Blue colonies lacking a surrounding creamy deposit, a phenotype described in former studies as HSF - , were identified. DNA sequence analysis of a selected mutant revealed that this had a transposon interruption in a chromosome-located gene which codes for a heat sensitive alkyl sulphatase of 78.7 kDa (YraS; Yersinia ruckeri alkyl sulphatase) which is able to degrade SDS to 1-dodecanol. As it was expected, the introduction of the yraS gene into an HSF - strain turned this into HSF + . Surprisingly, although the protein allows Y. ruckeri to degrade SDS, the bacterium could not use this compound as the sole carbon source. Moreover, the yraS mutant showed a similar level of SDS resistance to the parental strain. It was the interruption of the acrA gene which made Y. ruckeri sensitive to this compound. LD50 experiments showed a similar virulence of the yraS mutant and parental strain.

Conclusions: The HSF of Y. ruckeri is the product of the alkyl sulphatase YraS, able to degrade SDS to 1-dodecanol. This degradation is not linked to the utilization of SDS as a carbon source and surprisingly, the enzyme is not involved in bacterial virulence or in the high SDS resistance displayed by the bacterium. This role is played by the AcrAB-TolC system.