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Journal of Bacteriology, April 2004, p. 1991-1998, Vol. 186, No. 7
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.7.1991-1998.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A Conjugation-Based System for Genetic Analysis of Group II Intron Splicing in Lactococcus lactis

Joanna R. Klein,1 Yuqing Chen,1 Dawn A. Manias,1 Jin Zhuo,2 Liang Zhou,1 Craig L. Peebles,2 and Gary M. Dunny1*

Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455,1 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 152602

Received 7 August 2003/ Accepted 5 December 2003

The conjugative element pRS01 from Lactococcus lactis encodes the putative relaxase protein LtrB. The ltrB gene is interrupted by the functional group II intron Ll.ltrB. Accurate splicing of the two ltrB exons is required for synthesis of the mRNA encoding the LtrB conjugative relaxase and subsequent plasmid transfer. A conjugation-based genetic assay was developed to identify Ll.ltrB mutations that affect splicing. In this assay a nonsplicing, transfer-defective pRS01 derivative (pM1014) and a shuttle vector carrying the ltrB region, including the Ll.ltrB intron (pCOM9), are used. pCOM9 provides splicing-dependent complementation of the transfer defect of pM1014. Site-directed mutations within Ll.ltrB, either in the catalytic RNA or in the intron-encoded protein gene ltrA, were generated in the context of pCOM9. When these mutants were tested in the conjugation-based assay, significantly reduced mating was observed. Quantitative molecular analysis of in vivo splicing activity confirmed that the observed mating defects resulted from reduced splicing. Once the system was validated for the engineered mutants, random mutagenesis of the intron followed by genetic and molecular screening for splicing defects resulted in identification of point mutations that affect splicing.

* Corresponding author. Mailing address: Department of Microbiology, University of Minnesota Medical School, 1460 Mayo Bldg./MMC 196, 420 Delaware St., SE, Minneapolis, MN 55455-0312. Phone: (612) 625-9930. Fax: (612) 626-0623. E-mail: gary-d{at}biosci.cbs.umn.edu.

Journal of Bacteriology, April 2004, p. 1991-1998, Vol. 186, No. 7
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.7.1991-1998.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hu, X., Van der Auwera, G., Timmery, S., Zhu, L., Mahillon, J. (2009). Distribution, Diversity, and Potential Mobility of Extrachromosomal Elements Related to the Bacillus anthracis pXO1 and pXO2 Virulence Plasmids. Appl. Environ. Microbiol. 75: 3016-3028 [Abstract] [Full Text]  
  • Belhocine, K., Mak, A. B., Cousineau, B. (2007). Trans-splicing of the Ll.LtrB group II intron in Lactococcus lactis. Nucleic Acids Res 35: 2257-2268 [Abstract] [Full Text]  
  • Chen, Y., Klein, J. R., McKay, L. L., Dunny, G. M. (2005). Quantitative Analysis of Group II Intron Expression and Splicing in Lactococcus lactis. Appl. Environ. Microbiol. 71: 2576-2586 [Abstract] [Full Text]  
  • Belhocine, K., Yam, K. K., Cousineau, B. (2005). Conjugative Transfer of the Lactococcus lactis Chromosomal Sex Factor Promotes Dissemination of the Ll.LtrB Group II Intron. J. Bacteriol. 187: 930-939 [Abstract] [Full Text]  


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