Conserved Target for Group II Intron Insertion in Relaxase Genes of Conjugative Elements of Gram-Positive Bacteria

Abstract

The lactococcal group II intron Ll.ltrB interrupts the ltrB relaxase gene within a region that encodes a conserved functional domain. Nucleotides essential for the homing of Ll.ltrB into an intronless version of ltrB are found exclusively at positions required to encode amino acids broadly conserved in a family of relaxase proteins of gram-positive bacteria. Two of these relaxase genes, pcfG from the enterococcal plasmid pCF10 and the ORF4 gene in the streptococcal conjugative transposon Tn5252, were shown to support Ll.ltrB insertion into the conserved motif at precisely the site predicted by sequence homology with ltrB. Insertion occurred through a mechanism indistinguishable from retrohoming. Splicing and retention of conjugative function was demonstrated for pCF10 derivatives containing intron insertions. Ll.ltrB targeting of a conserved motif of a conjugative element suggests a mechanism for group II intron dispersal among bacteria. Additional support for this mechanism comes from sequence analysis of the insertion sites of the E.c.I4 family of bacterial group II introns.Group II introns are self-splicing and mobile RNAs (14) encoded within the genomes of bacteria (27) and in the organelle DNAs of plants, fungi, and protists (3). These RNAs have a conserved secondary structure with six sets of basepairing regions, domains I through VI, arranged radially around a central circle (29). Much of domain IV is looped out of the secondary structure and determines the synthesis of an intron-encoded protein (IEP) with maturase, endonuclease, and reverse transcriptase (RT) activities (28, 33). Maturase assists in splicing, probably by enhancing the formation or stabilization of a catalytic ribozyme structure which mediates splicing (28) via a mechanism very similar to that of eukaryotic spliceosomal introns (38).Group II introns home at high frequency into intronless alleles of the same exon gene and transpose at low frequency into ectopic sites with sequence similarity to the natural site (26). Since group II intron mobility occurs through an RNA intermediate, the terms retrohoming and retrotransposition are used to designate these events (9, 21). During retrohoming, the IEP, which is associated with the spliced-out intron RNA, recognizes nucleotides in the distal 5Ј portion of the DNA target and EBS/␦ ribonucleotides in the intron base pair with IBS/␦Ј nucleotides in the DNA target site. This process is followed by reverse splicing of the intron RNA into target DNA (20,28,34,40). The IEP then interacts with the 3Ј portion of the DNA target, and the IEP endonuclease nicks the antisense strand of recipient DNA a few base pairs downstream of the insertion site (20,28). Using the 3Ј end of the nicked target DNA as a primer, IEP RT reverse transcribes the intron RNA via a mechanism reminiscent of non-long terminal repeat retrotransposon target DNA-primed reverse transcription (14, 28). In retrohoming by yeast mitochondrial group II introns aI1 and aI2, completion of the insertion event generally occurs by double-strand...

Genetic characterization of the conjugative DNA processing system of enterococcal plasmid pCF10

“…Four plasmid-free E. faecalis strains derived from OG1 were also used in this study. OG1ES is resistant to erythromycin and streptomycin (Staddon et al, 2004). Because of the high degree of natural resistance of other OG1 strains to streptomycin and their exquisite sensitivity to erythromycin, erythromycin alone was typically used to select for OG1ES in mating assays.…”

“…Plasmids used in pcfG functional analysis-Plasmid pCF10::Ll.ltrBΔORF-Kan consists of a splicing-deficient Ll.ltrB derivative inserted into the Ll.ltrB target site in pcfG. This plasmid was constructed by introducing pLEIItd + KR" ΔORF (Staddon et al, 2004) and pCOMΔA13.16 (Klein et al, 2004) into OG1RF/(pCF10) by electroporation. PLEIItd + KR" ΔORF encodes for the splicing-deficient Ll.ltrBΔORF-Kan intron (Staddon et al, 2004).…”

“…An interesting feature of ltrB is that the wild-type allele is interrupted by the self-splicing, mobile group II intron Ll.ltrB, the first fully functional group II intron identified in bacteria (Mills et al, 1996). We recently showed that Ll.ltrB efficiently targets DNA of a conserved relaxase domain in pcfG for insertion by a retro-transposition mechanism (Staddon et al, 2004). L1.1trB splices out of pcfG mRNA, such that the host bacterium retains conjugation proficiency.…”

Pheromone-inducible conjugation in Enterococcus faecalis: A model for the evolution of biological complexity?

“…They actually show more relatedness to putative conjugative elements from pathogenic streptococci than to the other enterococal pheromone plasmids (Hirt et al, 2005), suggesting that they evolved from a distinct source. Finally at the distal end of transfer genes, we have identified the genes encoding DNA processing machinery, including the conjugative relaxase gene pcfG, and the functional origin of transfer sequence (Staddon et al, 2004). This latter group of genes is most closely related to the pRS01 conjugative element of Lactococcus lactis, also studied in our laboratory (Staddon et al, 2004); other pheromone plasmids such as pAD1 use completely different DNA processing genes.…”

“…Finally at the distal end of transfer genes, we have identified the genes encoding DNA processing machinery, including the conjugative relaxase gene pcfG, and the functional origin of transfer sequence (Staddon et al, 2004). This latter group of genes is most closely related to the pRS01 conjugative element of Lactococcus lactis, also studied in our laboratory (Staddon et al, 2004); other pheromone plasmids such as pAD1 use completely different DNA processing genes. Thus even though the entire set of genes required for efficient transfer of pCF10 is coordinately regulated by the cCF10 pheromone, at least three distinct modules were used to assemble the system.…”