Conjugative transfer of the Lactococcus lactis sex factor and pRS01 plasmid to Enterococcus faecalis

The low G+C gram-positive bacterium Lactococcus lactis harbours two highly similar conjugative elements: an integrative and conjugative element called sex factor and the pRS01 plasmid. Originally, it was believed that the host range of the sex factor was limited to L. lactis subspecies. Here, it is reported that pTRK28 cointegrates of a spectinomycin-marked L. lactis sex factor and of the pRS01 conjugative plasmid can be transferred from L. lactis to Enterococcus faecalis. These results demonstrate the conjugative transfer of these elements to other bacterial species. Furthermore, it is reported that Ll.LtrB, a mobile group II intron carried by both elements, can invade its recognition site upon pRS01 conjugative transfer to E. faecalis.     

Conjugation mediates transfer of the Ll.LtrB group II intron between different bacterial species

Some self-splicing group II introns (ribozymes) are mobile retroelements. These retroelements, which can insert themselves into cognate intronless alleles or ectopic sites by reverse splicing, are thought to be the evolutionary progenitors of the widely distributed eukaryotic spliceosomal introns. Lateral or horizontal transmission of introns (i.e. between species), although never experimentally demonstrated, is a well-accepted model for intron dispersal and evolution. Horizontal transfer of the ancestral bacterial group II introns may have contributed to the dispersal and wide distribution of spliceosomal introns present in modern eukaryotic genomes. Here, the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis was used as a model system to address the dissemination of introns in the bacterial kingdom. We report the first experimental demonstration of horizontal transfer of a group II intron. We show that the Ll.LtrB group II intron, originally discovered on an L. lactis conjugative plasmid (pRS01) and within a chromosomally located sex factor in L. lactis 712, invades new sites using both retrohoming and retrotransposition pathways after its transfer by conjugation. Ll.LtrB lateral transfer is shown among different L. lactis strains (intraspecies) (retrohoming and retrotransposition) and between L. lactis and Enterococcus faecalis (interspecies) (retrohoming). These results shed light on long-standing questions about intron evolution and propagation, and demonstrate that conjugation is one of the mechanisms by which group II introns are, and probably were, broadly disseminated between widely diverged organisms.     

A conjugation-based system for genetic analysis of group II intron splicing in Lactococcus lactis

The conjugative element pRS01 from Lactococcus lactis encodes the putative relaxase protein LtrB. The ltrB gene is interrupted by the functional group II intron Ll.ltrB. Accurate splicing of the two ltrB exons is required for synthesis of the mRNA encoding the LtrB conjugative relaxase and subsequent plasmid transfer. A conjugation-based genetic assay was developed to identify Ll.ltrB mutations that affect splicing. In this assay a nonsplicing, transfer-defective pRS01 derivative (pM1014) and a shuttle vector carrying the ltrB region, including the Ll.ltrB intron (pCOM9), are used. pCOM9 provides splicing-dependent complementation of the transfer defect of pM1014. Site-directed mutations within Ll.ltrB, either in the catalytic RNA or in the intron-encoded protein gene ltrA, were generated in the context of pCOM9. When these mutants were tested in the conjugation-based assay, significantly reduced mating was observed. Quantitative molecular analysis of in vivo splicing activity confirmed that the observed mating defects resulted from reduced splicing. Once the system was validated for the engineered mutants, random mutagenesis of the intron followed by genetic and molecular screening for splicing defects resulted in identification of point mutations that affect splicing.     

Specificity determinants of conjugative DNA processing in the Enterococcus faecalis plasmid pCF10 and the Lactococcus lactis plasmid pRS01

The DNA-processing region of the Enterococcus faecalis pheromone-responsive plasmid pCF10 is highly similar to that of the otherwise unrelated plasmid pRS01 from Lactococcus lactis. A transfer-proficient pRS01 derivative was unable to mobilize plasmids containing the pCF10 origin of transfer, oriT. In contrast, pRS01 oriT-containing plasmids could be mobilized by pCF10 at a low frequency. Relaxases PcfG and LtrB were both capable of binding to single-stranded oriT DNAs; LtrB was highly specific for its cognate oriT, whereas PcfG could recognize both pCF10 and pRS01 oriT. However, pcfG was unable to complement an ltrB insertion mutation. Genetic analysis showed that pcfF of pCF10 and ltrF of pRS01 are also essential for plasmid transfer. Purified PcfF and LtrF possess double-stranded DNA binding activities for the inverted repeat within either oriT sequence. PcfG and LtrB were recruited into their cognate F-oriT DNA complex through direct interactions with their cognate accessory protein. PcfG also could interact with LtrF when pCF10 oriT was present. In vivo cross-complementation analysis showed that ltrF partially restored the pCF10DeltapcfF mutant transfer ability when provided in trans, whereas pcfF failed to complement an ltrF mutation. Specificity of conjugative DNA processing in these plasmids involves both DNA-protein and protein-protein interactions.     

Restriction for gene insertion within the Lactococcus lactis Ll.LtrB group II intron

The Ll.LtrB intron, from the low G+C gram-positive bacterium Lactococcus lactis, was the first bacterial group II intron shown to splice and mobilize in vivo. The detailed retrohoming and retrotransposition pathways of Ll.LtrB were studied in both L. lactis and Escherichia coli. This bacterial retroelement has many features that would make it a good gene delivery vector. Here we report that the mobility efficiency of Ll.LtrB expressing LtrA in trans is only slightly affected by the insertion of fragments <100 nucleotides within the loop region of domain IV. In contrast, Ll.LtrB mobility efficiency is drastically decreased by the insertion of foreign sequences >1 kb. We demonstrate that the inhibitory effect caused by the addition of expression cassettes on Ll.LtrB mobility efficiency is not sequence specific, and not due to the expression, or the toxicity, of the cargo genes. Using genetic screens, we demonstrate that in order to maintain intron mobility, the loop region of domain IV, more specifically domain IVb, is by far the best region to insert foreign sequences within Ll.LtrB. Poisoned primer extension and Northern blot analyses reveal that Ll.LtrB constructs harboring cargo sequences splice less efficiently, and show a significant reduction in lariat accumulation in L. lactis. This suggests that cargo-containing Ll.LtrB variants are less stable. These results reveal the potential, yet limitations, of the Ll.LtrB group II intron to be used as a gene delivery vector, and validate the random insertion approach described in this study to create cargo-containing Ll.LtrB variants that are mobile.     

Genetic analysis of regions of the Lactococcus lactis subsp. lactis plasmid pRS01 involved in conjugative transfer.

The genes responsible for conjugative transfer of the 48.4-kb Lactococcus lactis subsp. lactis ML3 plasmid pRS01 were localized by insertional mutagenesis. Integration of the IS946-containing plasmid pTRK28 into pRS01 generated a pool of stable cointegrates, including a number of plasmids altered in conjugative proficiency. Mapping of pTRK28 insertions and phenotypic analysis of cointegrate plasmids identified four distinct regions (Tra1, Tra2, Tra3, and Tra4) involved in pRS01 conjugative transfer. Tra3 corresponds closely to a region previously identified (D. G. Anderson and L. L. McKay, J. Bacteriol. 158:954-962, 1984). Another region (Tra4) was localized within an inversion sequence shown to correlate with a cell aggregation phenotype. Tra1 and Tra2, two previously unidentified regions, were located at a distance of 9 kb from Tra3. When provided in trans, a cloned portion of the Tra3 region complemented Tra3 mutants.     

Interaction between Conjugative and Retrotransposable Elements in Horizontal Gene Transfer

Mobile genetic elements either encode their own mobilization machineries or hijack them from other mobile elements. Multiple classes of mobile elements often coexist within genomes and it is unclear whether they have the capacity to functionally interact and even collaborate. We investigate the possibility that molecular machineries of disparate mobile elements may functionally interact, using the example of a retrotransposon, in the form of a mobile group II intron, found on a conjugative plasmid pRS01 in Lactococcus lactis. This intron resides within the pRS01 ltrB gene encoding relaxase, the enzyme required for nicking the transfer origin (oriT) for conjugal transmission of the plasmid into a recipient cell. Here, we show that relaxase stimulates both the frequency and diversity of retrotransposition events using a retromobility indicator gene (RIG), and by developing a high-throughput genomic retrotransposition detection system called RIG-Seq. We demonstrate that LtrB relaxase not only nicks ssDNA of its cognate oriT in a sequence- and strand-specific manner, but also possesses weak off-target activity. Together, the data support a model in which the two different mobile elements, one using an RNA-based mechanism, the other using DNA-based transfer, do functionally interact. Intron splicing facilitates relaxase expression required for conjugation, whereas relaxase introduces spurious nicks in recipient DNA that stimulate both the frequency of intron mobility and the density of events. We hypothesize that this functional interaction between the mobile elements would promote horizontal conjugal gene transfer while stimulating intron dissemination in the donor and recipient cells.     

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

Group 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 endonuclease-dependent pathway. However, in retrotransposition in L. lactis, the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonuclease-independent 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 endonuclease-independent 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.     

Genetic and physical characterization of recombinant plasmids associated with cell aggregation and high-frequency conjugal transfer in Streptococcus lactis ML3.

Restriction mapping was employed to characterize the 104-kilobase (kb) cointegrate lactose plasmids from 15 independent transconjugants derived from Streptococcus lactis ML3 as well as the 55-kb lactose plasmid ( pSK08 ) and a previously uncharacterized 48.4-kb plasmid ( pRS01 ) from S. lactis ML3. The data revealed that the 104-kb plasmids were cointegrates of pSK08 and pRS01 and were structurally distinct. The replicon fusion event occurred within adjacent 13.8- or 7.3-kb PvuII fragments of pSK08 and interrupted apparently random regions of pRS01 . Correlation of the transconjugants' clumping and conjugal transfer capabilities with the interrupted region of pRS01 identified pRS01 regions coding for these properties. In the 104-kb plasmids, the pRS01 region was present in both orientations with respect to the pSK08 region. The replicon fusion occurred in recombination-deficient (Rec-) strains and appeared to introduce a 0.8 to 1.0-kb segment of DNA within the junction fragments. The degeneration of the cointegrate plasmids was monitored by examining the lactose plasmids from nonclumping derivatives of clumping transconjugants. These plasmids displayed either precise or imprecise excision of pRS01 sequences or had dramatically reduced copy numbers. Both alterations occurred by rec-independent mechanisms. Alterations of a transconjugant 's clumping phenotype also occurred by rec-independent inversion of a 4.3-kb KpnI-PvuII fragment within the pRS01 sequences of the cointegrate plasmid.     

Homing of a group II intron from Lactococcus lactis subsp. lactis ML3.

Ll.ltrB is a functional group II intron located within a gene (ltrB) encoding a conjugative relaxase essential for transfer of the lactococcal element pRSO1. In this work, the Ll.ltrB intron was shown to be an independent mobile element capable of inserting into an intronless allele of the ltrB gene. Ll.ltrB was not observed to insert into a deletion derivative of the ltrB gene in which the intron splice site was removed. In contrast, a second vector containing a 271-nucleotide segment of ltrB spanning the Ll.ltrB splice site was shown to be a proficient recipient of intron insertion. Efficient homing was observed in the absence of a functional host homologous recombination system. This work demonstrates that the Ll.ltrB intron is a novel site-specific mobile element in lactococci and that group II intron self-transfer is a mechanism for intron dissemination among bacteria.