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A conjugation-based system for genetic analysis of group II intron splicing in Lactococcus lactis
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Klein JR Chen Y Manias DA Zhuo J Zhou L Peebles CL Dunny GM 《Journal of bacteriology》2004,186(7):1991-1998
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. 相似文献
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Conjugative transfer of the Lactococcus lactis chromosomal sex factor promotes dissemination of the Ll.LtrB group II intron
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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. 相似文献
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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. 相似文献
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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. 相似文献
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Group II introns are mobile genetic elements that can be redirected to invade specific genes. Here we describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome of Lactococcus lactis IL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, the tra gene found in IS904 (tra904), tra981, and tra983, of which 9, 10, and 14 copies, respectively, were present in IL1403-UCD. Intron invasion of tra904, tra981, and tra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the respective tra alleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into the tra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade the L. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in different tra904 alleles were obtained. In general, increasing the chromosomal copy number of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same number of Ll.ltrB::GFP copies within different sets of tra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was observed from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addition, the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements. 相似文献
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Frazier CL San Filippo J Lambowitz AM Mills DA 《Applied and environmental microbiology》2003,69(2):1121-1128
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. 相似文献
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Conserved target for group II intron insertion in relaxase genes of conjugative elements of gram-positive bacteria
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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. 相似文献
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The Lactococcus lactis group II intron (Ll.ltrB) retrohomes into the ltrB gene at high efficiency. To date, the critical DNA bases recognized in vivo by the Ll.ltrB ribonucleoprotein (RNP) have been exclusively elucidated in Escherichia coli. However, recent evidence indicates host-dependant differences in Ll.ltrB mobility, raising the possibility of limitations of the current model for RNP-homing site recognition in the native L. lactis host. In this work, intron retargeting experiments in L. lactis have demonstrated that adherence to specific target site critical bases is not sufficient to predict success or failure of chromosomal invasion, as in E. coli. Accordingly, a quantitative real-time PCR (QPCR) assay was developed to test target site nucleotides previously demonstrated as critical for homing in E. coli, for relevance in its native host. This two-plasmid QPCR homing assay is highly sensitive and, unlike previous E. coli-based assays, resolves differential homing efficiencies in the absence of selection. As in E. coli, deviation from wild type at target site positions -23, -21, -20, -19, and +5 resulted in lower homing efficiencies in L. lactis. Furthermore, the same trends are observed when assaying select variants in Enterococcus faecalis. Our results suggest that these target site positions are critical in both E. coli and L. lactis. 相似文献
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Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection
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Courtney L. Frazier Joseph San Filippo Alan M. Lambowitz David A. Mills 《Applied microbiology》2003,69(2):1121-1128
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. 相似文献
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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. 相似文献
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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. 相似文献