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.     

Group II intron–ribosome association protects intron RNA from degradation

The influence of the cellular environment on the structures and properties of catalytic RNAs is not well understood, despite great interest in ribozyme function. Here we report on ribosome association of group II introns, which are ribozymes that are important because of their putative ancestry to spliceosomal introns and retrotransposons, their retromobility via an RNA intermediate, and their application as gene delivery agents. We show that group II intron RNA, in complex with the intron-encoded protein from the native Lactoccocus lactis host, associates strongly with ribosomes in vivo. Ribosomes have little effect on intron ribozyme activities; rather, the association with host ribosomes protects the intron RNA against degradation by RNase E, an enzyme previously shown to be a silencer of retromobility in Escherichia coli. The ribosome interacts strongly with the intron, exerting protective effects in vivo and in vitro, as demonstrated by genetic and biochemical experiments. These results are consistent with the ribosome influencing the integrity of catalytic RNAs in bacteria in the face of degradative nucleases that regulate intron mobility.     

Bipolar localization of the group II intron Ll.LtrB is maintained in Escherichia coli deficient in nucleoid condensation, chromosome partitioning and DNA replication

Group II introns are mobile genetic elements that invade their cognate intron-minus alleles via an RNA intermediate, in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. In Escherichia coli, retrotransposition of the lactococcal group II intron, Ll.LtrB, occurs preferentially within the Ori and Ter macrodomains of the E. coli chromosome. These macrodomains migrate towards the poles of the cell, where the intron-encoded protein, LtrA, localizes. Here we investigate whether alteration of nucleoid condensation, chromosome partitioning and replication affect retrotransposition frequencies, as well as bipolar localization of the Ll.LtrB intron integration and LtrA distribution in E. coli. We thus examined these properties in the absence of the nucleoid-associated proteins H-NS, StpA and MukB, in variants of partitioning functions including the centromere-like sequence migS and the actin homologue MreB, as well as in the replication mutants DeltaoriC, seqA, tus and topoIV (ts). Although there were some dramatic fluctuations in retrotransposition levels in these hosts, bipolar localization of integration events was maintained. LtrA was consistently found in nucleoid-free regions, with its localization to the cellular poles being largely preserved in these hosts. Together, these results suggest that bipolar localization of group II intron retrotransposition results from the residence of the intron-encoded protein at the poles of the cell.     

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.     

Use of RmInt1, a group IIB intron lacking the intron-encoded protein endonuclease domain, in gene targeting

The group IIA intron Ll.LtrB from Lactococcus lactis and the group IIB intron EcI5 from Escherichia coli have intron-encoded proteins (IEP) with a DNA-binding domain (D) and an endonuclease domain (En). Both have been successfully retargeted to invade target DNAs other than their wild-type target sites. RmInt1, a subclass IIB3/D intron with an IEP lacking D and En domains, is highly active in retrohoming in its host, Sinorhizobium meliloti. We found that RmInt1 was also mobile in E. coli and that retrohoming in this heterologous host depended on temperature, being more efficient at 28°C than at 37°C. Furthermore, we programmed RmInt1 to recognize target sites other than its wild-type site. These retargeted introns efficiently and specifically retrohome into a recipient plasmid target site or a target site present as a single copy in the chromosome, generating a mutation in the targeted gene. Our results extend the range of group II introns available for gene targeting.     

Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes

Mobile group II introns are site-specific retroelements that use a novel mobility mechanism in which the excised intron RNA inserts directly into a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Because the DNA target site is recognized primarily by base-pairing of the intron RNA with only a small number of positions recognized by the protein, it has been possible to develop group II introns into a new type of gene targeting vector ("targetron"), which can be reprogrammed to insert into desired DNA targets simply by modifying the intron RNA. Here, we used databases of retargeted Lactococcus lactis Ll.LtrB group II introns and a compilation of nucleotide frequencies at active target sites to develop an algorithm that predicts optimal Ll.LtrB intron-insertion sites and designs primers for modifying the intron to insert into those sites. In a test of the algorithm, we designed one or two targetrons to disrupt each of 28 Escherichia coli genes encoding DExH/D-box and DNA helicase-related proteins and tested for the desired disruptants by PCR screening of 100 colonies. In 21 cases, we obtained disruptions at frequencies of 1-80% without selection, and in six other cases, where disruptants were not identified in the initial PCR screen, we readily obtained specific disruptions by using the same targetrons with a retrotransposition-activated selectable marker. Only one DExH/D-box protein gene, secA, which was known to be essential, did not give viable disruptants. The apparent dispensability of DExH/D-box proteins in E.coli contrasts with the situation in yeast, where the majority of such proteins are essential. The methods developed here should permit the rapid and efficient disruption of any bacterial gene, the computational analysis provides new insight into group II intron target site recognition, and the set of E.coli DExH/D-box protein and DNA helicase disruptants should be useful for analyzing the function of these proteins.     

RraA rescues Escherichia coli cells over-producing RNase E from growth arrest by modulating the ribonucleolytic activity

RraA is an evolutionary conserved protein inhibitor of RNase E, which catalyzes the initial step in the decay and processing of numerous RNAs in Escherichia coli and forms the core component of the degradosome, a large protein complex involved in RNA metabolism. Here, we report that co-expression of RraA reduces the ribonucleolytic activity in cells over-producing RNase E and consequently rescues these cells from growth arrest. These findings suggest that inability of cells over-producing RNase E to normally grow results from increased cellular ribonucleolytic activity and RraA is able to effectively modulate the catalytic activity of RNase E in vivo.     

Coincident Hfq binding and RNase E cleavage sites on mRNA and small regulatory RNAs

The Escherichia coli RNA chaperone Hfq was discovered originally as an accessory factor of the phage Qbeta replicase. More recent work suggested a role of Hfq in cellular physiology through its interaction with ompA mRNA and small RNAs (sRNAs), some of which are involved in translational regulation. Despite their stability under certain conditions, E. coli sRNAs contain putative RNase E recognition sites, that is, A/U-rich sequences and adjacent stem-loop structures. We show herein that an RNase E cleavage site coincides with the Hfq-binding site in the 5'-untranslated region of E. coli ompA mRNA as well as with that in the sRNA, DsrA. Likewise, Hfq protects RyhB RNA from in vitro cleavage by RNase E. These in vitro data are supported by the increased abundance of DsrA and RyhB sRNAs in an RNase E mutant strain as well as by their decreased stability in a hfq(-) strain. It is commonly believed that the RNA chaperone Hfq facilitates or promotes the interaction between sRNAs and their mRNA targets. This study reveals another role for Hfq, that is, protection of sRNAs from endonucleolytic attack.     

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.