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.     

Conjugative transfer of the Lactococcus lactis chromosomal sex factor promotes dissemination of the Ll.LtrB group II intron

The Ll.LtrB group II 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. This retroelement interrupts the relaxase gene (ltrB) of three L. lactis conjugative elements: plasmids pRS01 and pAH90 and the chromosomal sex factor. Conjugative transfer of a plasmid harboring a segment of the pRS01 conjugative plasmid including the Ll.LtrB intron allows dissemination of Ll.LtrB among L. lactis strains and lateral transfer of this retroelement from L. lactis to Enterococcus faecalis. Here we report the dissemination of the Ll.LtrB group II intron among L. lactis strains following conjugative transfer of the native chromosomally embedded L. lactis sex factor. We demonstrated that Ll.LtrB dissemination is highly variable and often more efficient from this integrative and conjugative element than from an engineered conjugative plasmid. Cotransfer among L. lactis strains of both Ll.LtrB-containing elements, the conjugative plasmid and the sex factor, was detected and shown to be synergistic. Moreover, following their concurrent transfer, both mobilizable elements supported the spread of their respective copies of the Ll.LtrB intron. Our findings explain the unusually high efficiency of Ll.LtrB mobility observed following conjugation of intron-containing plasmids.     

Genetic manipulation of Lactococcus lactis by using targeted group II introns: generation of stable insertions without selection

Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci.     

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.     

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 characterization of the conjugative DNA processing system of enterococcal plasmid pCF10

Conjugation is a major contributor to lateral gene transfer in bacteria, and pheromone-inducible conjugation systems in Enterococcus faecalis play an important role in the dissemination of antibiotic resistance and virulence in enterococci and related bacteria. We have genetically dissected the determinants of DNA processing of the enterococcal conjugative plasmid pCF10. Insertional inactivation of a predicted relaxase gene pcfG, via insertion of a splicing-deficient group II intron, severely reduced pCF10 transfer. Restoration of intron splicing ability by genetic complementation restored conjugation. The pCF10 origin of transfer (oriT) was localized to a 40-nucleotide sequence within a non-coding region with sequence similarity to origins of transfer of several other plasmids in gram positive bacteria. Deletion of the oriT reduced pCF10 transfer by more than five orders of magnitude without affecting pCF10-dependent mobilization of co-resident oriT-containing plasmids. Although the host range for pCF10 replication is limited to enterococci, we found that the pCF10 conjugation system promotes mobilization of oriT-containing plasmids to multiple bacterial genera. Therefore, this transfer system may have applications for gene delivery to a variety of poorly-transformed bacteria.     

Overexpression of DEAD box protein pMSS116 promotes ATP-dependent splicing of a yeast group II intron in vitro.

The group II intron bI1, the first intron of the mitochondrial cytochrome b gene in yeast is self-splicing in vitro. Genetic evidence suggests that trans-acting factors are required for in vivo splicing of this intron. In accordance with these findings, we present in vitro data showing that splicing of bI1 under physiological conditions depends upon the presence of proteins of a mitochondrial lysate. ATP is an essential component is this reaction. Overexpression of the nuclear-encoded DEAD box protein pMSS-116 results in a marked increase in the ATP-dependent splicing activity of the extract, suggesting that pMSS116 may play an important role in splicing of bI1.     

Recruitment of a peptidyl-tRNA hydrolase as a facilitator of group II intron splicing in chloroplasts

Group II introns are catalytic RNAs that have been proposed to be the evolutionary precursors to the spliceosome. Most group II introns require accessory factors to splice efficiently in vivo, but few such factors have been identified. We have cloned the maize nuclear gene crs2, which is required for the splicing of nine group II introns in chloroplasts. CRS2 is related to peptidyl-tRNA hydrolase enzymes. However, CRS2 expression failed to rescue an Escherichia coli pth(ts) mutant and CRS2 lacks several conserved amino acids that are important for the activity of the E.coli enzyme, indicating that it may lack peptidyl-tRNA hydrolase activity. CRS2 is localized to the chloroplast stroma, where it is found in a large salt-stable complex that contains RNA. CRS2 co-sediments with group II intron RNA during centrifugation of stroma through sucrose gradients, suggesting that CRS2 facilitates splicing via direct interaction with intron RNA. Sequence comparisons indicate how evolutionary tinkering may have allowed an enzyme that interacts with peptidyl-tRNAs to acquire a function in group II intron splicing.