A conjugation-based system for genetic analysis of group II intron splicing in Lactococcus lactis.
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.
Klein
Joanna R
JR
Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Chen
Yuqing
Y
Manias
Dawn A
DA
Zhuo
Jin
J
Zhou
Liang
L
Peebles
Craig L
CL
Dunny
Gary M
GM
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003227:Conjugation, Genetic; D004251:DNA Transposable Elements / Q000235:genetics*; D007438:Introns / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D012326:RNA Splicing
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; C099786:LtrB protein, Lactococcus lactis