Mathiesen, G. | bioRxiv Science

bioRxiv2021

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 - and {beta}-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 {beta}-chitin, whereas the LPMO gene-deletion variants only showed minor growth defects on this substrate. Finally, proteomic analysis of Al. salmonicida LFI1238 growth on {beta}-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. IMPORTANCEThe 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.

biochemistry↗

bioRxiv2018

Over 2000-fold increased production of the leaderless bacteriocin garvicin KS by genetic engineering and optimization of culture conditions

The leaderless bacteriocin Garvicin KS (GarKS) is a potent antimicrobial, being active against a wide range of important pathogens. GarKS production by the native producer Lactococcus garvieae KS1546 was however relatively low (80 BU/ml) under standard laboratory growth conditions (batch culture in GM17 at 30{degrees}C). To improve the production of GarKS, we systematically evaluated the impact of different media and media components on bacteriocin production. Based on the outcomes a new medium formulation was made to greatly improve bacteriocin production. The new medium composed of pasteurized milk and tryptone (PM-T), increased GarKS production about 60-fold compared to that achieved in GM17. GarKS production was increased further 4-fold (i.e., to 20,000 BU/ml) by increasing gene dose of the bacteriocin gene cluster (gak) in the native producer. Finally, a combination of the newly composed medium (PM-T), an increased gene dose and a cultivation at a constant pH 6 and a 50-60% dissolved oxygen level in growth medium, gave rise to a GarKS production of 164,000 BU/ml. This high production, which is about 2000-fold higher compared to that initially achieved in GM17, corresponds to a GarKS production of 1.2 g/L. To our knowledge, this is one of the highest bacteriocin production reported hitherto.\n\nImportanceLow bacteriocin production is a well-known bottle-neck in developing bacteriocins into large-scaled and useful applications. The present study shows different approaches that significantly improve bacteriocin production. This is an important research field to better exploit the antimicrobial potential of bacteriocins, especially with regard to the decreasing effect of antibiotics in infection treatments due to the global emergence of antibiotic resistance.

microbiology↗