scholarly journals Conjugative Transfer of the Lactococcus lactis Chromosomal Sex Factor Promotes Dissemination of the Ll.LtrB Group II Intron

2005 ◽  
Vol 187 (3) ◽  
pp. 930-939 ◽  
Author(s):  
Kamila Belhocine ◽  
Karen K. Yam ◽  
Benoit Cousineau
Keyword(s):  
Lactococcus Lactis ◽  
High Efficiency ◽  
Group Ii Intron ◽  
Conjugative Plasmid ◽  
Conjugative Transfer ◽  
Bacterial Group ◽  
Positive Bacterium ◽  
Group Ii ◽  
Integrative And Conjugative Element

ABSTRACT 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.

DNA Target Site Requirements for Homing in Vivo of a Bacterial Group II Intron Encoding a Protein Lacking the DNA Endonuclease Domain

2003 ◽  
Vol 326 (2) ◽  
pp. 413-423 ◽  
Author(s):  
José I Jiménez-Zurdo ◽  
Fernando M Garcı́a-Rodrı́guez ◽  
Antonio Barrientos-Durán ◽  
Nicolás Toro
Keyword(s):  
Group Ii Intron ◽  
Target Site ◽  
Bacterial Group ◽  
Dna Endonuclease ◽  
Endonuclease Domain ◽  
Group Ii

scholarly journals Quantitative Analysis of Group II Intron Expression and Splicing in Lactococcus lactis

2005 ◽  
Vol 71 (5) ◽  
pp. 2576-2586 ◽  
Author(s):  
Yuqing Chen ◽  
Joanna R. Klein ◽  
Larry L. McKay ◽  
Gary M. Dunny
Keyword(s):  
Lactococcus Lactis ◽  
Mrna Level ◽  
Group Ii Intron ◽  
Mrna Levels ◽  
Growth Phases ◽  
Physiological Conditions ◽  
Reverse Transcription Pcr ◽  
Group Ii ◽  
Atp Binding Protein

ABSTRACT The group II intron Ll.ltrB is found within the ltrB relaxase gene of the conjugative element pRS01 in Lactococcus lactis. Precise splicing of the intron is essential for pRS01 transfer. The transcription regulation and in vivo splicing activity of Ll.ltrB have not been investigated thoroughly in L. lactis in the natural pRS01 context. We developed absolute quantitative real-time reverse transcription-PCR assays to quantify RNA levels of the 5′ exon (ltrBE1) and the spliced relaxase (ltrB) and intron-encoded protein (ltrA) genes, as well as Ll.ltrB splicing activity under different physiological conditions. The mRNA levels for the ATP-binding protein OppD were assayed for comparison to the ltrB transcripts. The oppD mRNA ranged from 10- to 10,000-fold higher than ltrB region genes. ltrBE1 expression was growth-phase dependent. The mRNA level of ltrA was almost constant during all growth phases and in all media tested. Ll.ltrB in vivo splicing activity ranged from (6.5 ± 2.1)% to (22.1 ± 8.0)%. Acid challenge significantly decreased both ltrB region mRNA levels and intron splicing activity. The presence of recipient cells, different mating environments, and temperature stress had no significant effects on expression and splicing. Western blotting showed that the level of LtrB protein expressed from an intronless ltrB gene was much higher (about 20-fold) than the level of protein expressed from an intron-containing construct. Interestingly, LtrB protein showed a tendency to function in cis on its oriT target. The low level of ltrB transcript and relatively inefficient splicing of the intron may limit Ll.ltrB mobility and dissemination in nature.

scholarly journals A Conjugation-Based System for Genetic Analysis of Group II Intron Splicing in Lactococcus lactis

2004 ◽  
Vol 186 (7) ◽  
pp. 1991-1998 ◽  
Author(s):  
Joanna R. Klein ◽  
Yuqing Chen ◽  
Dawn A. Manias ◽  
Jin Zhuo ◽  
Liang Zhou ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Shuttle Vector ◽  
Point Mutations ◽  
Random Mutagenesis ◽  
Group Ii Intron ◽  
Catalytic Rna ◽  
Splicing Defects ◽  
Group Ii ◽  
Genetic Assay

ABSTRACT 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.

scholarly journals Exon sequence requirements for excision in vivo of the bacterial group II intron RmInt1

2011 ◽  
Vol 12 (1) ◽  
Author(s):  
Antonio Barrientos-Durán ◽  
Isabel Chillón ◽  
Francisco Martínez-Abarca ◽  
Nicolás Toro
Keyword(s):  
Group Ii Intron ◽  
Bacterial Group ◽  
Exon Sequence ◽  
Group Ii ◽  
Sequence Requirements

scholarly journals Methylation of rRNA as a host defense against rampant group II intron retrotransposition

Mobile DNA ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Justin M. Waldern ◽  
Dorie Smith ◽  
Carol Lyn Piazza ◽  
E. Jake Bailey ◽  
Nicholas J. Schiraldi ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Fold Increase ◽  
Group Ii Intron ◽  
In Vivo Experiments ◽  
Cellular Factors ◽  
Group Ii ◽  
Self Splicing ◽  
Native Host

Abstract Background Group II introns are mobile retroelements, capable of invading new sites in DNA. They are self-splicing ribozymes that complex with an intron-encoded protein to form a ribonucleoprotein that targets DNA after splicing. These molecules can invade DNA site-specifically, through a process known as retrohoming, or can invade ectopic sites through retrotransposition. Retrotransposition, in particular, can be strongly influenced by both environmental and cellular factors. Results To investigate host factors that influence retrotransposition, we performed random insertional mutagenesis using the ISS1 transposon to generate a library of over 1000 mutants in Lactococcus lactis, the native host of the Ll.LtrB group II intron. By screening this library, we identified 92 mutants with increased retrotransposition frequencies (RTP-ups). We found that mutations in amino acid transport and metabolism tended to have increased retrotransposition frequencies. We further explored a subset of these RTP-up mutants, the most striking of which is a mutant in the ribosomal RNA methyltransferase rlmH, which exhibited a reproducible 20-fold increase in retrotransposition frequency. In vitro and in vivo experiments revealed that ribosomes in the rlmH mutant were defective in the m3Ψ modification and exhibited reduced binding to the intron RNA. Conclusions Taken together, our results reinforce the importance of the native host organism in regulating group II intron retrotransposition. In particular, the evidence from the rlmH mutant suggests a role for ribosome modification in limiting rampant retrotransposition.

scholarly journals Group II intron domain 5 facilitates a trans-splicing reaction.

1988 ◽  
Vol 8 (6) ◽  
pp. 2361-2366 ◽  
Author(s):  
K A Jarrell ◽  
R C Dietrich ◽  
P S Perlman
Keyword(s):  
Mitochondrial Dna ◽  
Group Ii Intron ◽  
Intron Sequence ◽  
Trans Splicing ◽  
Group Ii ◽  
Self Splicing ◽  
Yeast Mitochondrial Dna ◽  
Domain 4

A self-splicing group II intron of yeast mitochondrial DNA (aI5g) was divided within intron domain 4 to yield two RNAs that trans-spliced in vitro with associated trans-branching of excised intron fragments. Reformation of the domain 4 secondary structure was not necessary for the trans reaction, since domain 4 sequences were shown to be dispensable. Instead, the trans reaction depended on a previously unpredicted interaction between intron domain 5, the most highly conserved region of group II introns, and another region of the RNA. Domain 5 was shown to be essential for cleavage at the 5' splice site. It stimulated that cleavage when supplied as a trans-acting RNA containing only 42 nucleotides of intron sequence. The relevance of our findings to in vivo trans-splicing mechanisms is discussed.

Splicing of a group II intron in a functional transfer gene of Lactococcus lactis

1996 ◽  
Vol 21 (1) ◽  
pp. 45-53 ◽  
Author(s):  
Claire Shearman ◽  
Jean‐Jacques Godon ◽  
Michael Gasson
Keyword(s):  
Lactococcus Lactis ◽  
Group Ii Intron ◽  
Transfer Gene ◽  
Group Ii
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