Retrotransposition strategies of the Lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment

Abstract

SummaryGroup II introns are mobile retroelements that invade their cognate intron-minus gene in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Previous studies of the Lactococcus lactis intron Ll.LtrB indicated that in its native host, as in Escherichia coli , retrohoming occurs by the intron RNA reverse splicing into double-stranded DNA (dsDNA) through an endonucleasedependent pathway. However, in retrotransposition in L. lactis , the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonucleaseindependent manner. This work describes the retrotransposition of the Ll.LtrB intron in E. coli , using a retrotransposition indicator gene previously employed in our L. lactis studies. Unlike in L. lactis , in E. coli , Ll.LtrB retrotransposed frequently into dsDNA, and the process was dependent on the endonuclease activity of the intron-encoded protein.Further, the endonuclease-dependent insertions preferentially occurred around the origin and terminus of chromosomal DNA replication. Insertions in E. coli can also occur through an endonucleaseindependent pathway, and, as in L. lactis , such events have a more random integration pattern. Together these findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. Additionally, growth conditions affect the insertion pattern. We propose a model in which DNA replication, compactness of the nucleoid and chromosomal localization influence target site preference.

Insertion of group II intron retroelements after intrinsic transcriptional terminators

“…The most probable source is ssDNA at the DNA replication fork because this ssDNA is the substrate for other group II introns that lack endonuclease domains (20,21,32). In this pathway, the intron reverse splices into either ssDNA or dsDNA at the replication fork and then uses nascent DNA of the leading or lagging strands to prime reverse transcription of the intron.…”

Atomic Force Microscopy Reveals DNA Bending during Group II Intron Ribonucleoprotein Particle Integration into Double-Stranded DNA

“…The first set consists of 66 retrohoming sites in the E. coli genome used by an Ll.LtrB-ΔORF intron having randomized EBS2, EBS1, and δ sequences (19); the 66 sequences were culled from 88 total sequences by excluding sites lacking T+5, which may have been utilized via En-independent retrohoming pathways. The second set consists of 33 retrotransposition sites in the E. coli genome used by the wild-type Ll.LtrB intron, again culled from a larger set by excluding sites lacking T+5 (12). For each test set, the average bendability was calculated for positions −30 to +10 from the intron-insertion site, using a six-nucleotide sliding window with a shift increment of one, and compared with that in control sets consisting of the same number of 45-bp sequences selected randomly from the E. coli genome.…”

“…The algorithm was used to calculate an average bendability profile for two sets of Ll.LtrB insertions sites in the E. coli genome, one consisting of 66 retrohoming sites used by an Ll.LtrB-ΔORF intron having randomized target-site recognition sequences (19), and the other consisting of 33 retrotransposition sites used by the wild-type Ll.LtrB intron (12). Both test sets were culled for larger sets of sites by excluding those lacking T+5, which may have been utilized via Enindependent pathways that do not require the same IEP interactions with the 3' exon (see Introduction; (11,12)). The average bendability profiles for the tests sets were then compared with those for 100 different control sets, each consisting of the same number of randomly selected 45-bp E. coli genomic sequences (a total of 6600 and 3300 control sequences for the retrohoming and retrotransposition sites, respectively; see Experimental Procedures).…”

“…While polymers with a ratio "t" of contour length to persistence length of about two can only probe bend angles up to 90° with significant probability, more flexible polymers that have reduced persistence lengths probe larger bend angles. Given that the persistence length of DNA is generally 40−45 nm (38) or about half the length of our DNA, the distribution of bend angles in the −30/+15 DNA show the predicted behavior for t = 2, while the wild-type DNA shows the behavior expected for t > 2 or much reduced stiffness.Both the retrohoming and retrotransposition sites contain only a small number of nucleotide residues whose bases are recognized directly by the IEP, and with the exception of T+5, these are only moderately conserved in different sites (e.g., T-23, 41%; G-21, 79%; and A-20, 61% in the 66 retrohoming sites, and T-23, 15%; G-21, 48%; and A-20, 33% in the retrotransposition sites (12,19)). The region of the retrohoming sites recognized by base pairing of the intron RNA also has variable sequences, due to the method by which the sites were selected (i.e., with an Ll.LtrB intron having randomized EBS2, EBS1, and δ sequences).…”

Recurrent insertion of 5′-terminal nucleotides and loss of the branchpoint motif in lineages of group II introns inserted in mitochondrial preribosomal RNAs

“…As already emphasized (see Results), all 10 introns with a 59 insert actually lack the entire subdomain that the EBS2 segment is normally part of (Michel et al 1989;Dai et al 2003). The EBS2-IBS2 pairing is known to be important for insertion of group II introns by reverse-splicing into double-stranded DNA, presumably because it helps stabilize interactions between the intron and its target relative to DNA:DNA base-pairing, but it does not appear to be required for transposition into single-stranded nucleic acids (Coros et al 2005). Still, that this interaction should persist not only in introns that have lost the coding sequence for a reverse transcriptase, but in several of those that encode a LAGLIDADG homing endonuclease ( Table 1), implies that the EBS2-IBS2 pairing has some significant function in splicing as well.…”