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The Lactococcus lactis Ll .LtrB group II intron retrohomes by reverse splicing into one strand of a double -stranded DNA target site , while the intron -encoded protein cleaves the opposite strand and uses it as a primer for reverse transcription of the inserted intron RNA . The protein and intron RNA function in a ribonucleoprotein particle , with much of the DNA target sequence recognized by base pairing of the intron RNA . Consequently , Ll .LtrB introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA . Here , I show that an Escherichia coli gene disruption library obtained using randomly inserted Ll .LtrB introns contains most viable E . coli gene disruptions . Further , each inserted intron is targeted to a specific site by its unique base -pairing regions , and in most cases , could be recovered by PCR and used unmodified to obtain the desired single disruptant . I also demonstrate that Ll .LtrB introns can be used for efficient gene targeting in a variety of Gram -negative and positive bacteria , including E . coli , Pseudomonas aeruginosa , Agrobacterium tumefaciens , Bacillus subtilis , and Staphylococcus aureus . Ll .LtrB introns expressed from a broad -host -range vector or an E . coli -S . aureus shuttle vector yielded targeted disruptions in a variety of test genes in these organisms at frequencies of 1 -100 % without selection . By using an Ll .LtrB intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing , I disrupted the essential gene hsa in S . aureus . Because the splicing of the Ll .LtrB intron by the intron -encoded protein is temperature -sensitive , this method yields a conditional hsa disruptant that grows at 32oC , but not at 43oC . Finally , I developed high -throughput screens to identify E . coli genes that affect either the splicing or retrohoming of the Ll .LtrB intron . By using these screens , I identified fourteen mutants in a variety of genes that have decreased intron retrohoming efficiencies and additional mutants that have increased intron retrohoming efficiencies , in some cases apparently resulting from increased stability of the intron RNA . |
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