Location: Cool and Cold Water Aquaculture ResearchTitle: Molecular cloning of cecropin B responsive endonucleases in Yersinia ruckeri Author
Submitted to: Marine Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/19/2010
Publication Date: 3/30/2010
Publication URL: http://handle.nal.usda.gov/10113/55682
Citation: Sullum, U.W., Chen, T.T. 2010. Molecular cloning of cecropin B responsive endonucleases in Yersinia ruckeri. Marine Biotechnology. DOI: 10.1007/s10126-010-9269-z. Interpretive Summary: Outbreaks of fish diseases are a major cause of economic losses in aquaculture. Although application of antibiotics, chemicals and vaccines to control fish diseases has been somewhat effective, these remedies are expensive and frequently resulted in severe environmental contamination. We had previously introduced antimicrobial genes into fish and the resulting transgenic fish exhibited resistance to bacterial and viral pathogens. However, it had not been shown that exposure of pathogens to antimicrobial peptides will render them resistant to the peptide. We also had demonstrated that exposure of fish bacterial pathogens to cecropin B, one of the known antimicrobial peptides, resulted in resistance developed via physiological adaptation; but resistance characteristics disappeared when the selection pressure was removed. To understand the cause of this physiological adaption induced by cecropin B, we initiated studies to identify genes in bacterial pathogens that might be regulated by cecropin B, using the fish pathogen Yersinia ruckeri as an experimental organism. We report that cecropin B induces the expression of an endonuclease gene which may be responsible for this resistant characteristic. These results will help aquaculture researches to develop alternative approaches to prevent fish from being infected by bacterial pathogens.
Technical Abstract: We have previously demonstrated that Yersinia ruckeri resists cecropin B in an inducible manner. In this study, we sought to identify the molecular changes responsible for the inducible cecropin B resistance of Y. ruckeri. Differences in gene expression associated with the inducible resistance were investigated. Cultures of Y. ruckeri were exposed to a sublethal concentration of cecropin B and resultant changes in the messenger RNA population of the bacteria were assayed using the differential display reverse transcription polymerase chain reaction (DD-RT-PCR). A single band was consistently increased in intensity in all repeats of the experiment. The band was excised, cloned, sequenced, and used to screen a Y. ruckeri genomic DNA library. The DD-RT-PCR fragment shared 100% identity to the cDNA sequence of an ATP-dependent endonuclease of the overcome lysogenization defect (OLD) family of Y. ruckeri 29473. The genomic clone that was recovered was not identical to the D-RT-PCR clone, but harbored a gene for a secreted endonuclease 1 (nucM) homologue. It was determined that transcription of the gene was upregulated following exposure to cecropin B via RTPCR. Furthermore, an increase in the nuclease activity of culture supernatants of Y. ruckeri following exposure to cecropin B was demonstrated. These findings demonstrate that cecropin B exposure increases the expression of at least two endonucleases in Y. ruckeri. The production and secretion of an endonuclease by Y. ruckeri in response to an antimicrobial peptide indicates the involvement of both intracellular and extracellular DNA in the toxic effects of cecropin B.