Characterization of chondroitin AC lyase in Flavobacterium columnare
Flavobacterium columnare is a Gram-negative, rod shaped bacterium and is the causative agent of columnaris disease (CD) in many types of freshwater fish. This disease is a significant problem in the aquaculture industry, particularly in the production of channel catfish (Ictularis punctatus). CD is the second largest cause of economic loss in the catfish industry, following closely behind enteric septicemia (Edwardsiella ictaluri). F. columnare possesses a number of putative virulence factors; however, due to the limited number of genetic tools available to manipulate this organism, most of these designations are speculative. ^ Despite this, it is agreed by most that the chondroitin AC lyase must play a role in virulence. Chondroitinases are extracellular enzymes that hydrolyze chondroitin, the molecule responsible for the rigidity of connective tissues. It has been hypothesized that dissolution of chondroitin from connective tissues allows for easier dissemination of the organism throughout its host. In this study, we report the first successful attempt to create an insertional mutation in this gene. We also report the first successful attempt to complement a mutated gene in F. columnare. ^ Mutation of the cslA gene shows a 98% decrease in chondroitinase activity. However, complementation of the cslA mutation only resulted in an activity of 10% in comparison to wild type. Although a significant difference compared to the cslA mutant (p<0.05), the complement failed to restore the wild type phenotype. In an effort to explain this finding, we identified five genes downstream of cslA, arranged in an operon, as confirmed by RT-PCR analysis of wild type total RNA. RT-PCR analysis of cslA mutant revealed a lack of transcription from the downstream genes in the csl operon, demonstrating a polar effect due to the insertion mutation. ^ SDS-PAGE analysis of supernatant proteins revealed two distinct bands that are present in the wild type but absent in the mutant. Although neither band corresponds to the predicted 85kDa size of CslA, we predict that CslA appears as the 67kDa band and undergoes multiple processing steps prior to full activation of the enzyme. Analysis of the complement strain supernatant proteins reveals a 67kDa band at 10% density of the wild type. Restoration of the second distinct band did not occur. Analysis of periplasmic and cytoplasmic fractions did not reveal differences in the protein profiles of the three strains. ^ Finally, the data show that chondroitinase activity was not induced by the addition of chondroitin to liquid culture. However, the addition of chondroitin to an F. columnare culture resulted in the aggregation of previously planktonic cells. This phenomenon may have several implications in the study of this organism, including some possible insight into the regulation of its virulence factors. Further research will be required in order to fully elucidate the role of cslA and chondroitin in the virulence of this very important pathogen of fish.^
Michael J Pereira,
"Characterization of chondroitin AC lyase in Flavobacterium columnare"
Dissertations and Master's Theses (Campus Access).