Gene Targeting in Gram-Negative Bacteria by Use of a Mobile Group II Intron (“Targetron”) Expressed from a Broad-Host-Range Vector

Abstract

Mobile group II introns ("targetrons") can be programmed for insertion into virtually any desired DNA target with high frequency and specificity. Here, we show that targetrons expressed via an m-toluic acidinducible promoter from a broad-host-range vector containing an RK2 minireplicon can be used for efficient gene targeting in a variety of gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. Targetrons expressed from donor plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies of 1 to 58% of screened colonies in the E. coli lacZ, P. aeruginosa pqsA and pqsH, and A. tumefaciens aopB and chvI genes. The development of this broad-host-range system for targetron expression should facilitate gene targeting in many bacteria.Mobile group II introns ("targetrons") have been used for targeted gene disruption and site-specific DNA insertion in diverse gram-negative and gram-positive bacteria, including Escherichia coli (21, 30), Shigella flexneri (21), Salmonella enterica serovar Typhimurium (21), Lactococcus lactis (11), Clostridium perfringens (3), and Staphylococcus aureus (45). Group II introns are useful for gene targeting because they can be programmed for insertion into virtually any desired DNA target with high frequency and specificity (22,30). This ability derives from their unique DNA integration mechanism, called retrohoming, in which the intron RNA is inserted via reverse splicing directly into a DNA target site and is then reverse transcribed by the intron-encoded protein (IEP) (reviewed in reference 23). Retrohoming is mediated by a ribonucleoprotein (RNP) particle that is formed during RNA splicing and contains the IEP and excised intron lariat RNA. RNPs initiate mobility by using both the IEP and base pairing of the intron RNA to recognize DNA target sequences, with the latter contributing most of the target specificity (10,16,17,41). Consequently, it is possible to retarget group II introns for insertion into preselected sites simply by modifying the base-pairing sequences in the intron RNA (16,22,28,30).DNA target site interactions for the L. lactis Ll.LtrB group II intron used in the present work are shown in Fig. 1A (16, 28, 30, 41). The DNA target site positions recognized by base pairing of the intron RNA span positions Ϫ12 to ϩ2 from the intron insertion site and are denoted intron-binding sites 1 and 2 (IBS1 and IBS2) in the 5Ј exon (E1) and ␦Ј in the 3Ј exon (E2). The complementary intron RNA sequences are located in two different RNA stem loops and are denoted exon-binding sites 1 and 2 (EBS1 and EBS2) and ␦ sequences (sequences adjacent to EBS1). The IEP (LtrA protein) recognizes additional positions in the distal 5Ј exon and 3Ј exon regions of the DNA target site, interactions that are important for local DNA melting and bottom-strand cleavage for generating the primer for reverse transcription of the inserted intron RNA (key positions recognized by the IEP are underlined in Fig. 1A) (22, 23). ...

EcI5, a group IIB intron with high retrohoming frequency: DNA target site recognition and use in gene targeting

“…Analysis of the E. coli K12 genome for potential EcI5 targeting sites using the computer algorithm developed here revealed 14,938 such sites on either DNA strand with log-odds scores >8.2, an average of one highly ranked target site per 621 nucleotide residues. In the E. coli lacZ gene (z3 kb), the Ll.LtrB intron has five potential targeting sites with log-odds scores >8.2, of which two have been validated (targeting efficiencies >10%; Yao and Lambowitz 2007;Zhao RNA, Vol. 15, No.…”

“…with several Ll.LtrB targetrons in different bacteria and appeared to be as efficient as pACD3 for expressing Ll.LtrB targetron LacZ635s in E. coli HMS174(DE3) (Yao and Lambowitz 2007). However, additional tests comparing other Ll.LtrB LacZ targetrons in E. coli HMS174(DE3) showed that some targetrons function as efficiently when expressed from pBL1 as from pACD3, while others function less efficiently (J.…”

“…We also tested whether EcI5 could be expressed from the broad-host range vector pBL1, which can be used in diverse Gram-negative bacteria without introducing a gene encoding phage T7 RNA polymerase (Yao and Lambowitz 2007). This plasmid expresses targetrons by using an m-toluic acid-inducible promoter (P m ) recognized by the host RNA polymerase.…”

“…Donor plasmid pBL1 is a derivative of the broad-host-range vector pJB866 (Blatny et al 1997), which uses an m-toluic acidinducible promoter P m to express a cassette consisting of the Ll.LtrB-DORF intron with flanking exons followed by the IEP (Yao and Lambowitz 2007). A parallel construct expressing EcI5 targetron LacZ1806s was constructed by PCR amplifying the EcI5-DORF intron, flanking exon sequences, and EcI5 IEP from pACD3-EcI5A containing the retargeted intron (see below), using primers EcI5 59 and EcI5 39 (Supplemental Table 2), then digesting the PCR product with HindIII and XhoI and cloning it between the corresponding sites of pJB866.…”

“…This feature made it possible to develop Ll.LtrB into a highly efficient bacterial gene targeting vector (''targetron''), which has programmable target specificity. The Ll.LtrB targetron has been used in diverse Gram-negative and Gram-positive bacteria (Karberg et al 2001;Frazier et al 2003;Perutka et al 2004;Chen et al 2005Chen et al , 2007Yao et al 2006;Heap et al 2007;Shao et al 2007;Yao and Lambowitz 2007;Malhotra and Srivastava 2008;Rodriguez et al 2008;Sayeed et al 2008), and has the potential to be used similarly for gene targeting in eukaryotes Mastroianni et al 2008). The ability to retarget the Ll.LtrB intron and use it as a gene targeting vector reflects a combination of beneficial properties, including that active intron RNA and IEP can be expressed readily from a donor plasmid, that IEP interactions with the DNA target site are relatively few and flexible, that the base-pairing interactions between the intron RNA and DNA target sequence can be modified, and that these base-pairing interactions are sufficiently long to confer high specificity.…”