Publications

2006
Mohr S, Matsuura M, Perlman PS, Lambowitz AM. A DEAD-box protein alone promotes group II intron splicing and reverse splicing by acting as an RNA chaperone. Proc Natl Acad Sci U S A. 103 (10) :3569-74.Abstract
Group II intron RNAs self-splice in vitro but only at high salt and/or Mg2+ concentrations and have been thought to require proteins to stabilize their active structure for efficient splicing in vivo. Here, we show that a DEAD-box protein, CYT-19, can by itself promote the splicing and reverse splicing of the yeast aI5gamma and bI1 group II introns under near-physiological conditions by acting as an ATP-dependent RNA chaperone, whose continued presence is not required after RNA folding. Our results suggest that the folding of some group II introns may be limited by kinetic traps and that their active structures, once formed, do not require proteins or high Mg2+ concentrations for structural stabilization. Thus, during evolution, group II introns could have spliced and transposed by reverse splicing by using ubiquitous RNA chaperones before acquiring more specific protein partners to promote their splicing and mobility. More generally, our results provide additional evidence for the widespread role of RNA chaperones in folding cellular RNAs.
Zagorski N. Profile of Alan M. Lambowitz. Proc Natl Acad Sci U S A. 103 (6) :1669-71.
Yao J, Zhong J, Fang Y, Geisinger E, Novick RP, Lambowitz AM. Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing. RNA. 12 (7) :1271-81.Abstract
We show that a targetron based on the Lactococcus lactis Ll.LtrB group II intron can be used for efficient chromosomal gene disruption in the human pathogen Staphylococcus aureus. Targetrons expressed from derivatives of vector pCN37, which uses a cadmium-inducible promoter, or pCN39, a derivative of pCN37 with a temperature-sensitive replicon, gave site-specific disruptants of the hsa and seb genes in 37%-100% of plated colonies without selection. To disrupt hsa, an essential gene, we used a group II intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, enabling the production of functional HSa protein. We show that because splicing of the Ll.LtrB intron by the intron-encoded protein is temperature-sensitive, this method yields a conditional hsa disruptant that grows at 32 degrees C but not 43 degrees C. The temperature sensitivity of the splicing reaction suggests a general means of obtaining one-step conditional disruptions in any organism. In nature, temperature sensitivity of group II intron splicing could limit the temperature range of an organism containing a group II intron inserted in an essential gene.
2005
Blocker FJH, Mohr G, Conlan LH, Qi L, Belfort M, Lambowitz AM. Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase. RNA. 11 (1) :14-28.Abstract
Group II intron-encoded proteins (IEPs) have both reverse transcriptase (RT) activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active RNA structure. The LtrA protein encoded by the Lactococcus lactis Ll.LtrB group II intron contains an N-terminal RT domain, with conserved sequence motifs RT1 to 7 found in the fingers and palm of retroviral RTs; domain X, associated with maturase activity; and C-terminal DNA-binding and DNA endonuclease domains. Here, partial proteolysis of LtrA with trypsin and Arg-C shows major cleavage sites in RT1, and between the RT and X domains. Group II intron and related non-LTR retroelement RTs contain an N-terminal extension and several insertions relative to retroviral RTs, some with conserved features implying functional importance. Sequence alignments, secondary-structure predictions, and hydrophobicity profiles suggest that domain X is related structurally to the thumb of retroviral RTs. Three-dimensional models of LtrA constructed by "threading" the aligned sequence on X-ray crystal structures of HIV-1 RT (1) account for the proteolytic cleavage sites; (2) suggest a template-primer binding track analogous to that of HIV-1 RT; and (3) show that conserved regions in splicing-competent LtrA variants include regions of the RT and X (thumb) domains in and around the template-primer binding track, distal regions of the fingers, and patches on the protein's back surface. These regions potentially comprise an extended RNA-binding surface that interacts with different regions of the intron for RNA splicing and reverse transcription.
Del Campo M, Recinos C, Yanez G, Pomerantz SC, Guymon R, Crain PF, McCloskey JA, Ofengand J. Number, position, and significance of the pseudouridines in the large subunit ribosomal RNA of Haloarcula marismortui and Deinococcus radiodurans. RNA. 11 (2) :210-9.Abstract
The number and position of the pseudouridines of Haloarcula marismortui and Deinococcus radiodurans large subunit RNA have been determined by a combination of total nucleoside analysis by HPLC-mass spectrometry and pseudouridine sequencing by the reverse transcriptase method and by LC/MS/MS. Three pseudouridines were found in H. marismortui, located at positions 1956, 1958, and 2621 corresponding to Escherichia coli positions 1915, 1917, and 2586, respectively. The three pseudouridines are all in locations found in other organisms. Previous reports of a larger number of pseudouridines in this organism were incorrect. Three pseudouridines and one 3-methyl pseudouridine (m3Psi) were found in D. radiodurans 23S RNA at positions 1894, 1898 (m3Psi), 1900, and 2584, the m3Psi site being determined by a novel application of mass spectrometry. These positions correspond to E. coli positions 1911, 1915, 1917, and 2605, which are also pseudouridines in E. coli (1915 is m3Psi). The pseudouridines in the helix 69 loop, residues 1911, 1915, and 1917, are in positions highly conserved among all phyla. Pseudouridine 2584 in D. radiodurans is conserved in eubacteria and a chloroplast but is not found in archaea or eukaryotes, whereas pseudouridine 2621 in H. marismortui is more conserved in eukaryotes and is not found in eubacteria. All the pseudoridines are near, but not exactly at, nucleotides directly involved in various aspects of ribosome function. In addition, two D. radiodurans Psi synthases responsible for the four Psi were identified.
Huang H-R, Rowe CE, Mohr S, Jiang Y, Lambowitz AM, Perlman PS. The splicing of yeast mitochondrial group I and group II introns requires a DEAD-box protein with RNA chaperone function. Proc Natl Acad Sci U S A. 102 (1) :163-8.Abstract
Group I and II introns self-splice in vitro, but require proteins for efficient splicing in vivo, to stabilize the catalytically active RNA structure. Recent studies showed that the splicing of some Neurospora crassa mitochondrial group I introns additionally requires a DEAD-box protein, CYT-19, which acts as an RNA chaperone to resolve nonnative structures formed during RNA folding. Here we show that, in Saccharomyces cerevisiae mitochondria, a related DEAD-box protein, Mss116p, is required for the efficient splicing of all group I and II introns, some RNA end-processing reactions, and translation of a subset of mRNAs, and that all these defects can be partially or completely suppressed by the expression of CYT-19. Results for the aI2 group II intron indicate that Mss116p is needed after binding the intron-encoded maturase, likely for the disruption of stable but inactive RNA structures. Our results suggest that both group I and II introns are prone to kinetic traps in RNA folding in vivo and that the splicing of both types of introns may require DEAD-box proteins that function as RNA chaperones.
Paukstelis PJ, Coon R, Madabusi L, Nowakowski J, Monzingo A, Robertus J, Lambowitz AM. A tyrosyl-tRNA synthetase adapted to function in group I intron splicing by acquiring a new RNA binding surface. Mol Cell. 17 (3) :417-28.Abstract
We determined a 1.95 A X-ray crystal structure of a C-terminally truncated Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) that functions in splicing group I introns. CYT-18's nucleotide binding fold and intermediate alpha-helical domains superimpose on those of bacterial TyrRSs, except for an N-terminal extension and two small insertions not found in nonsplicing bacterial enzymes. These additions surround the cyt-18-1 mutation site and are sites of suppressor mutations that restore splicing, but not synthetase activity. Highly constrained models based on directed hydroxyl radical cleavage assays show that the group I intron binds at a site formed in part by the three additions on the nucleotide binding fold surface opposite that which binds tRNATyr. Our results show how essential proteins can progressively evolve new functions.
Zhao J, Lambowitz AM. A bacterial group II intron-encoded reverse transcriptase localizes to cellular poles. Proc Natl Acad Sci U S A. 102 (45) :16133-40.Abstract
The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase (LtrA protein) that binds the intron RNA to promote RNA splicing and intron mobility. Here, we used LtrA-GFP fusions and immunofluorescence microscopy to show that LtrA localizes to cellular poles in Escherichia coli and Lactococcus lactis. This polar localization occurs with or without coexpression of Ll.LtrB intron RNA, is observed over a wide range of cellular growth rates and expression levels, and is independent of replication origin function. The same localization pattern was found for three nonoverlapping LtrA subsegments, possibly reflecting dependence on common redundant signals and/or protein physical properties. When coexpressed in E. coli, LtrA interferes with the polar localization of the Shigella IcsA protein, which mediates polarized actin tail assembly, suggesting competition for a common localization determinant. The polar localization of LtrA could account for the preferential insertion of the Ll.LtrB intron in the origin and terminus regions of the E. coli chromosome, may facilitate access to exposed DNA in these regions, and could potentially link group II intron mobility to the host DNA replication and/or cell division machinery.
Yao J, Zhong J, Lambowitz AM. Gene targeting using randomly inserted group II introns (targetrons) recovered from an Escherichia coli gene disruption library. Nucleic Acids Res. 33 (10) :3351-62.Abstract
The Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse-splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it to prime reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base-pairing of the intron RNA. Consequently, group II introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, we show that an Escherichia coli gene disruption library obtained using such randomly inserting Ll.LtrB introns contains most viable E.coli gene disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. Additionally, we identified a subset of introns that insert at sites lacking T+5, a nucleotide residue critical for second-strand cleavage. All such introns tested individually gave the desired specific disruption, some by switching to an alternate retrohoming mechanism targeting single-stranded DNA and using a nascent lagging DNA strand to prime reverse transcription.
Smith D, Zhong J, Matsuura M, Lambowitz AM, Belfort M. Recruitment of host functions suggests a repair pathway for late steps in group II intron retrohoming. Genes Dev. 19 (20) :2477-87.Abstract
Retrohoming of group II introns occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Host repair enzymes are predicted to complete this process. Here, we screened a battery of Escherichia coli mutants defective in host functions that are potentially involved in retrohoming of the Lactococcus lactis Ll.LtrB intron. We found strong (greater than threefold) effects for several enzymes, including nucleases directed against RNA and DNA, replicative and repair polymerases, and DNA ligase. A model including the presumptive roles of these enzymes in resection of DNA, degradation of the intron RNA template, traversion of RNA-DNA junctions, and second-strand DNA synthesis is described. The completion of retrohoming is viewed as a DNA repair process, with features that may be shared by other non-LTR retroelements.
Jones JP, Kierlin MN, Coon RG, Perutka J, Lambowitz AM, Sullenger BA. Retargeting mobile group II introns to repair mutant genes. Mol Ther. 11 (5) :687-94.Abstract
Retroposable elements such as retroviral and lentiviral vectors have been employed for many gene therapy applications. Unfortunately, such gene transfer vectors integrate genes into many different DNA sequences and unintended integration of the vector near a growth-promoting gene can engender pathological consequences. For example, retroviral vector-mediated gene transfer induced leukemia in 2 of 11 children treated for severe combined immunodeficiency, raising significant safety issues for gene transfer strategies that cannot be targeted to specific sequences. Here, we examine the use of a mobile retroposable genetic element that can be targeted to introduce therapeutic sequences site specifically into mutant genes. The data demonstrate that the mobile group II intron from Lactococcus lactis can be targeted to insert into and repair mutant lacZ (approved gene symbol GLB1) and beta-globin (approved gene symbol HBB) genes with high efficiency and fidelity in model systems in bacteria. These results suggest that these mobile genetic elements represent a novel class of agents for performing targeted genetic repair.
Coros CJ, Landthaler M, Piazza CL, Beauregard A, Esposito D, Perutka J, Lambowitz AM, Belfort M. Retrotransposition strategies of the Lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment. Mol Microbiol. 56 (2) :509-24.Abstract
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.
2004
Cui X, Matsuura M, Wang Q, Ma H, Lambowitz AM. A group II intron-encoded maturase functions preferentially in cis and requires both the reverse transcriptase and X domains to promote RNA splicing. J Mol Biol. 340 (2) :211-31.Abstract
Mobile group II introns encode proteins with both reverse transcriptase activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active structure of the intron RNA. Previous studies with the Lactococcus lactis Ll.LtrB intron suggested a model in which the intron-encoded protein binds first to a high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of its own coding region, and then makes additional contacts with conserved catalytic core regions to stabilize the active RNA structure. Here, we developed an Escherichia coli genetic assay that links the splicing of the Ll.LtrB intron to the expression of green fluorescent protein and used it to study the in vivo splicing of wild-type and mutant introns and to delineate regions of the maturase required for splicing. Our results show that the maturase functions most efficiently when expressed in cis from the same transcript as the intron RNA. In agreement with previous in vitro assays, we find that the high-affinity binding site in DIVa is required for efficient splicing of the Ll.LtrB intron in vivo, but in the absence of DIVa, 6-10% residual splicing occurs by the direct binding of the maturase to the catalytic core. Critical regions of the maturase were identified by statistically analyzing ratios of missense to silent mutations in functional LtrA variants isolated from a library generated by mutagenic PCR ("unigenic evolution"). This analysis shows that both the reverse transcriptase domain and domain X, which likely corresponds to the reverse transcriptase thumb, are required for RNA splicing, while the C-terminal DNA-binding and DNA endonuclease domains are not required. Within the reverse transcriptase domain, the most critical regions for maturase activity include parts of the fingers and palm that function in template and primer binding in HIV-1 reverse transcriptase, but the integrity of the reverse transcriptase active site is not required. Biochemical analysis of LtrA mutants indicates that the N terminus of the reverse transcriptase domain is required for high-affinity binding of the intron RNA, possibly via direct interaction with DIVa, while parts of domain X interact with conserved regions of the catalytic core. Our results support the hypothesis that the intron-encoded protein adapted to function in splicing by using, at least in part, interactions used initially to recognize the intron RNA as a template for reverse transcription.
Watanabe K, Lambowitz AM. High-affinity binding site for a group II intron-encoded reverse transcriptase/maturase within a stem-loop structure in the intron RNA. RNA. 10 (9) :1433-43.Abstract
Mobile group II introns encode proteins that have reverse transcriptase and maturase activities and bind specifically to the intron RNA to promote both RNA splicing and intron mobility. Previous studies with the Lactococcus lactis Ll.LtrB intron showed that the intron-encoded protein (LtrA) has a high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure containing the translation initiation region of the LtrA open reading frame, and that this binding site consists of a small stem-loop emanating from a purine-rich internal loop. The binding of LtrA to DIVa is important for translational regulation, RNA splicing, and intron mobility. Here, we show by in vitro selection that part of the purine-rich internal loop can be closed by base pairing, enabling the LtrA binding site to be represented as an extended stem-loop structure with a bulged A (A556) required for tight binding of LtrA. The deletion or pairing of A556 has relatively little effect on maturase-promoted RNA splicing, but significantly inhibits intron mobility. The wild-type DIVa structure has a second bulged A (A553), which is selected against in tightly binding variants. As expected from the selection, the deletion or pairing of A553 results in tighter binding of LtrA, but surprisingly, also inhibits intron mobility. These findings suggest that the binding of LtrA to DIVa is delicately balanced, so that either too weak or too tight binding can be deleterious. The nature of the maturase/DIVa interaction and its role in translational regulation are reminiscent of the coat protein/RNA hairpin interactions of single-stranded RNA phages.
Lambowitz AM, Zimmerly S. Mobile group II introns. Annu Rev Genet. 38 :1-35.Abstract
Mobile group II introns, found in bacterial and organellar genomes, are both catalytic RNAs and retrotransposable elements. They use an extraordinary mobility mechanism in which the excised intron RNA reverse splices directly into a DNA target site and is then reverse transcribed by the intron-encoded protein. After DNA insertion, the introns remove themselves by protein-assisted, autocatalytic RNA splicing, thereby minimizing host damage. Here we discuss the experimental basis for our current understanding of group II intron mobility mechanisms, beginning with genetic observations in yeast mitochondria, and culminating with a detailed understanding of molecular mechanisms shared by organellar and bacterial group II introns. We also discuss recently discovered links between group II intron mobility and DNA replication, new insights into group II intron evolution arising from bacterial genome sequencing, and the evolutionary relationship between group II introns and both eukaryotic spliceosomal introns and non-LTR-retrotransposons. Finally, we describe the development of mobile group II introns into gene-targeting vectors, "targetrons," which have programmable target specificity.
Chen X, Mohr G, Lambowitz AM. The Neurospora crassa CYT-18 protein C-terminal RNA-binding domain helps stabilize interdomain tertiary interactions in group I introns. RNA. 10 (4) :634-44.Abstract
The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by stabilizing the catalytically active RNA structure. To accomplish this, CYT-18 recognizes conserved structural features of group I intron RNAs using regions of the N-terminal nucleotide-binding fold, intermediate alpha-helical, and C-terminal RNA-binding domains that also function in binding tRNA(Tyr). Curiously, whereas the splicing of the N. crassa mitochondrial large subunit rRNA intron is completely dependent on CYT-18's C-terminal RNA-binding domain, all other group I introns tested thus far are spliced efficiently by a truncated protein lacking this domain. To investigate the function of the C-terminal domain, we used an Escherichia coli genetic assay to isolate mutants of the Saccharomyces cerevisiae mitochondrial large subunit rRNA and phage T4 td introns that can be spliced in vivo by the wild-type CYT-18 protein, but not by the C-terminally truncated protein. Mutations that result in dependence on CYT-18's C-terminal domain include those disrupting two long-range GNRA tetraloop/receptor interactions: L2-P8, which helps position the P1 helix containing the 5'-splice site, and L9-P5, which helps establish the correct relative orientation of the P4-P6 and P3-P9 domains of the group I intron catalytic core. Our results indicate that different structural mutations in group I intron RNAs can result in dependence on different regions of CYT-18 for RNA splicing.
Perutka J, Wang W, Goerlitz D, Lambowitz AM. Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes. J Mol Biol. 336 (2) :421-39.Abstract
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.
2003
Noah JW, Lambowitz AM. Effects of maturase binding and Mg2+ concentration on group II intron RNA folding investigated by UV cross-linking. Biochemistry. 42 (43) :12466-80.Abstract
The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase/maturase (LtrA protein) that promotes RNA splicing by stabilizing the catalytically active RNA structure. Here, we mapped 17 UV cross-links induced in both wild-type Ll.LtrB RNA and Ll.LtrB-Delta2486 RNA, which has a branch-point deletion that prevents splicing, and we used these cross-links to follow tertiary structure formation under different conditions in the presence or absence of the LtrA protein. Twelve of the cross-links are long-range, with six near known tertiary interaction sites in the active RNA structure. In a reaction medium containing 0.5 M NH(4)Cl, eight of the 17 cross-links were detected in the absence of Mg(2+) or the presence of EDTA, and all were detected at 5 mM Mg(2+), where efficient splicing requires the LtrA protein. The frequencies of all but four cross-links increased with increasing Mg(2+) concentrations, becoming maximal between 4 and 50 mM Mg(2+), where the intron is self-splicing. These findings suggest that a high Mg(2+) concentration induces self-splicing by globally stabilizing tertiary structure, including key tertiary interactions that are required for catalytic activity. Significantly, the binding of the maturase under protein-dependent splicing conditions (0.5 M NH(4)Cl and 5 mM Mg(2+)) increased the frequency of only nine cross-links, seven of which are long-range, suggesting that, in contrast to a high Mg(2+) concentration, LtrA promotes splicing by stabilizing critical tertiary structure interactions, while leaving other regions of the intron relatively flexible. This difference may contribute to the high rate of protein-dependent splicing, relative to the rate of self-splicing. The propensity of the intron RNA to form tertiary structure even at relatively low Mg(2+) concentrations raises the possibility that the maturase functions at least in part by tertiary structure capture. Finally, an abundant central wheel cross-link, present in >50% of the molecules at 5 mM Mg(2+), suggests models in which group II intron domains I and II are either coaxially stacked or aligned in parallel, bringing the 5'-splice site together with the 3'-splice site and catalytic core elements at JII/III. This and other cross-links provide new constraints for three-dimensional structural modeling of the group II intron catalytic core.
Frazier CL, San Filippo J, Lambowitz AM, Mills DA. Genetic manipulation of Lactococcus lactis by using targeted group II introns: generation of stable insertions without selection. Appl Environ Microbiol. 69 (2) :1121-8.Abstract
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.
Zhong J, Lambowitz AM. Group II intron mobility using nascent strands at DNA replication forks to prime reverse transcription. EMBO J. 22 (17) :4555-65.Abstract
The Lactococcus lactis Ll.LtrB group II intron uses a major retrohoming mechanism in which the excised intron RNA reverse splices into one strand of a DNA target site, while the intron-encoded protein uses a C-terminal DNA endonuclease domain to cleave the opposite strand and then uses the cleaved 3' end as a primer for reverse transcription of the inserted intron RNA. Here, experiments with mutant introns and target sites indicate that Ll.LtrB can retrohome without second-strand cleavage by using a nascent strand at a DNA replication fork as the primer for reverse transcription. This mechanism connecting intron mobility to target DNA replication may be used by group II intron species that encode proteins lacking the C-terminal DNA endonuclease domain and for group II intron retrotransposition to ectopic sites.

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