The fish pathogen Aliivibrio salmonicida LFI1238 can degrade and metabolize chitin despite major gene loss in the chitinolytic pathway

The fish pathogen Aliivibrio (Vibrio) salmonicida LFI1238 is thought to be incapable of utilizing chitin as a nutrient source since approximately half of the genes representing the chitinolytic pathway are disrupted by insertion sequences. In the present study, we combined a broad set of analytical methods to investigate this hypothesis. Cultivation studies revealed that Al. salmonicida grew efficiently on N -acetylglucosamine (GlcNAc) and chitobiose ((GlcNAc) 2 ), the primary soluble products resulting from enzymatic chitin hydrolysis. The bacterium was also able to grow on chitin particles, albeit at a lower rate compared to the soluble substrates. The genome of the bacterium contains five disrupted chitinase genes (pseudogenes) and three intact genes encoding a glycoside hydrolase family 18 (GH18) chitinase and two auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenases (LPMOs). Biochemical characterization showed that the chitinase and LPMOs were able to depolymerize both α- and β-chitin to (GlcNAc) 2 and oxidized chitooligosaccharides, respectively. Notably, the chitinase displayed up to 50-fold lower activity compared to other well-studied chitinases. Deletion of the genes encoding the intact chitinolytic enzymes showed that the chitinase was important for growth on β-chitin, whereas the LPMO gene-deletion variants only showed minor growth defects on this substrate. Finally, proteomic analysis of Al. salmonicida LFI1238 growth on β-chitin showed expression of all three chitinolytic enzymes, and intriguingly also three of the disrupted chitinases. In conclusion, our results show that Al. salmonicida LFI1238 can utilize chitin as a nutrient source and that the GH18 chitinase and the two LPMOs are needed for this ability. IMPORTANCE The ability to utilize chitin as a source of nutrients is important for the survival and spread of marine microbial pathogens in the environment. One such pathogen is Aliivibrio (Vibrio) salmonicida , the causative agent of cold water vibriosis. Due to extensive gene decay, many key enzymes in the chitinolytic pathway have been disrupted, putatively rendering this bacterium incapable of chitin degradation and utilization. In the present study we demonstrate that Al. salmonicida can degrade and metabolize chitin, the most abundant biopolymer in the ocean. Our findings shed new light on the environmental adaption of this fish pathogen.

The fish pathogen Aliivibrio salmonicida LFI1238 can degrade and metabolize chitin despite major gene loss in the chitinolytic pathway

The fish pathogen Aliivibrio (Vibrio) salmonicida LFI1238 is thought to be incapable of utilizing chitin as a nutrient source since approximately half of the genes representing the chitinolytic pathway are disrupted by insertion sequences. In the present study, we combined a broad set of analytical methods to investigate this hypothesis. Cultivation studies revealed that Al. salmonicida grew efficiently on N-acetylglucosamine (GlcNAc) and chitobiose ((GlcNAc)2), the primary soluble products resulting from enzymatic chitin hydrolysis. The bacterium was also able to grow on chitin particles, albeit at a lower rate compared to the soluble substrates. The genome of the bacterium contains five disrupted chitinase genes (pseudogenes) and three intact genes encoding a glycoside hydrolase family 18 (GH18) chitinase and two auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenases (LPMOs). Biochemical characterization showed that the chitinase and LPMOs were able to depolymerize both a- and b-chitin to (GlcNAc)2 and oxidized chitooligosaccharides, respectively. Notably, the chitinase displayed up to 50-fold lower activity compared to other well-studied chitinases. Deletion of the genes encoding the intact chitinolytic enzymes showed that the chitinase was important for growth on β-chitin, whereas the LPMO gene-deletion variants only showed minor growth defects on this substrate. Finally, proteomic analysis of Al. salmonicida LFI1238 growth on β-chitin showed expression of all three chitinolytic enzymes, and intriguingly also three of the disrupted chitinases. In conclusion, our results show that Al. salmonicida LFI1238 can utilize chitin as a nutrient source and that the GH18 chitinase and the two LPMOs are needed for this ability.

LitR of Vibrio salmonicida Is a Salinity-Sensitive Quorum-Sensing Regulator of Phenotypes Involved in Host Interactions and Virulence

ABSTRACTVibrio(Aliivibrio)salmonicidais the causal agent of cold-water vibriosis, a fatal bacterial septicemia primarily of farmed salmonid fish. The molecular mechanisms of invasion, colonization, and growth ofV. salmonicidain the host are still largely unknown, and few virulence factors have been identified. Quorum sensing (QS) is a cell-to-cell communication system known to regulate virulence and other activities in several bacterial species. The genome ofV. salmonicidaLFI1238 encodes products presumably involved in several QS systems. In this study, the gene encoding LitR, a homolog of the master regulator of QS inV. fischeri, was deleted. Compared to the parental strain, thelitRmutant showed increased motility, adhesion, cell-to-cell aggregation, and biofilm formation. Furthermore, thelitRmutant produced less cryptic bioluminescence, whereas production of acylhomoserine lactones was unaffected. Our results also indicate a salinity-sensitive regulation of LitR. Finally, reduced mortality was observed in Atlantic salmon infected with thelitRmutant, implying that the fish were more susceptible to infection with the wild type than with the mutant strain. We hypothesize that LitR inhibits biofilm formation and favors planktonic growth, with the latter being more adapted for pathogenesis in the fish host.