Bacterial gene targeting using group II intron L1.LtrB splicing and retrohoming

Yao, Jun, 1974-
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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.