Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0221-7) contains supplementary material, which is available to authorized users.
Highlights
The heat sensitive factor (HSF) of the fish pathogen Yersinia ruckeri was previously identified as an unusual band on SDS-PAGE
Creamy white Y. ruckeri colony phenotype (HSF+) growing in the presence of SDS is caused by an alkyl sulphatase (YraS) A mini-Tn5 Km2 Y. ruckeri transposon library was screened for colonies lacking the creamy white deposit around the colonies on TSA medium containing SDS and Coomassie brilliant blue (Figure 1)
The product of the interrupted gene, a protein of 708 amino acids, shows a high degree of identity with other proteins defined as alkyl sulphatases or SDS hydrolases from different bacteria such as Microcystis aeruginosa (71%) (CCI04676), Thiocystis violascens (63%) (YP_006413521), Vibrio cholerae (52%) (EGS71352) and Pseudomonas aeruginosa (51%) [11]
Summary
The heat sensitive factor (HSF) of the fish pathogen Yersinia ruckeri was previously identified as an unusual band on SDS-PAGE. Yersinia ruckeri is a Gram-negative bacterium alternating between planktonic and host interaction states. It is the causative agent of enteric redmouth disease (ERM) affecting mainly salmonids in fish farms, which leads to important economic losses in aquaculture worldwide. These authors concluded that HSF appeared to be a relevant virulence factor in Y. ruckeri. In order to be able to distinguish HSF+ and HSF− strains routinely, Furones et al [10] developed a differential culture medium containing SDS and the Coomassie brilliant blue dye. The colonies of the HSF+ strains appeared as white, due to a creamy deposit around the colony, whereas those of HSF− strains did not form this deposit and became deeply blue
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