Genetic manipulation of Lactococcus lactis by using targeted group II introns: generation of stable insertions without selection.
Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci.
Frazier
Courtney L
CL
Department of Viticulture and Enology, University of California at Davis, Davis, California 95616-8749, USA.
San Filippo
Joseph
J
Lambowitz
Alan M
AM
Mills
David A
DA
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000235:genetics; D003227:Conjugation, Genetic; D004251:DNA Transposable Elements; D005818:Genetic Engineering / Q000379:methods*; D007438:Introns / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D012326:RNA Splicing; D011995:Recombination, Genetic; D013753:Tetracycline Resistance / Q000235:genetics
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; C099786:LtrB protein, Lactococcus lactis; D002262:Carboxy-Lyases; C041240:malate carboxy-lyase