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Molecular Cloning of Cecropin B Responsive Endonucleases in Yersinia ruckeri

Marine Biotechnology volume 13, pages 56–65 (2011)Cite this article

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 DD-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 RT-PCR. 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.

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Acknowledgements

We would like to acknowledge Professor Joerg Graf of the Department of Molecular and Cell Biology at the University of Connecticut, Storrs, for his guidance in the construction and screening of the Y. ruckeri genomic library. This work was supported by a grant from United States Department of Agriculture (CONTR #58-1930-0-009) to T.T.C.

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  1. Ulysses W. Sallum

    Present address: Wellman Center for Photomedicine, Massachusetts General Hospital, Derpartment of Dermatology, Harvard Medical School, Bartlett Hall 404C, 40 Blossom St, Boston, MA, 02114, USA

Affiliations

  1. Department of Molecular and Cell Biology, University of Connecticut, 91 N. Eagleville Rd., U-3125, Storrs, CT, 06269-3125, USA

    Ulysses W. Sallum & Thomas T. Chen

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  1. Ulysses W. Sallum
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  2. Thomas T. Chen
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Correspondence to Thomas T. Chen.

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Sallum, U.W., Chen, T.T. Molecular Cloning of Cecropin B Responsive Endonucleases in Yersinia ruckeri . Mar Biotechnol 13, 56–65 (2011). https://doi.org/10.1007/s10126-010-9269-z

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  • DOI: https://doi.org/10.1007/s10126-010-9269-z

Keywords

  • Cecropin B
  • Yersinia ruckeri
  • Extracellular endonuclease
  • Antimicrobial peptide resistance
  • Neutrophil extracellular traps
  • Endonuclease 1
  • ATP dependent endonuclease of the OLD family