Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria

Karberg, Michael; Guo, Haixun; Zhong, Jin; Coon, Robert; Perůtka, Jiřı́; Lambowitz, Alan M.

doi:10.1038/nbt1201-1162

Cited by 196 publications

(239 citation statements)

References 25 publications

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“…To do so, we used a TargeTron-based group II intron (Karberg et al, 2001) that was retargeted for integration into dltD at nucleotide 353 of the coding sequence (see Methods). The resulting strain, MC120, was then tested for the MICs of nisin, polymyxin B, gallidermin and vancomycin.…”

Section: Effects Of the Disruption Of Dltd On Camp Resistancementioning

confidence: 99%

The dlt operon confers resistance to cationic antimicrobial peptides in Clostridium difficile

2011

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The dlt operon in Gram-positive bacteria encodes proteins that are necessary for the addition of D-alanine to teichoic acids of the cell wall. The addition of D-alanine to the cell wall results in a net positive charge on the bacterial cell surface and, as a consequence, can decrease the effectiveness of antimicrobials, such as cationic antimicrobial peptides (CAMPs). Although the roles of the dlt genes have been studied for some Gram-positive organisms, the arrangement of these genes in Clostridium difficile and the life cycle of the bacterium in the host are markedly different from those of other pathogens. In the current work, we determined the contribution of the putative C. difficile dlt operon to CAMP resistance. Our data indicate that the dlt operon is necessary for full resistance of C. difficile to nisin, gallidermin, polymyxin B and vancomycin. We propose that the D-alanylation of teichoic acids provides protection against antimicrobial peptides that may be essential for growth of C. difficile in the host. INTRODUCTIONClostridium difficile causes a potentially fatal intestinal disease that is increasing in incidence and severity (Dubberke et al., 2010). Infection is often chronic and difficult to eradicate (O'Brien et al., 2007). C. difficile is transmitted as dormant endospores that are highly resistant to killing by heat, antibiotics, oxidizing agents, UV radiation and many other treatments that are usually effective for killing bacteria (Vonberg et al., 2008;Setlow, 1995). C. difficile spores germinate in the intestine to produce vegetatively growing cells capable of producing toxins A and B, which have been implicated in disease progression and severity (Bartlett et al., 1977;McDonald et al., 2005).Upon germination, actively growing C. difficile must contend with the onslaught of host defences present in the intestinal environment. The host produces a number of innate immune factors, such as cationic antimicrobial peptides (CAMPs) and bacteriolytic enzymes that protect the intestine from invading bacterial pathogens (Müller et al., 2005;Gudmundsson & Agerberth, 1999). Some of these CAMPs are products of host cells and others are produced by the intestinal microflora. These host effectors are attracted to the overall negative charge of bacterial cells and, as a consequence, bacterial resistance to these effectors can be achieved by charge modification of cell wall and membrane components (Nizet, 2006;Peschel, 2002). In many Gram-positive bacteria, this charge modification occurs by D-alanyl esterification of teichoic acid in the cell wall, which results in an increased positive charge on the cell (Neuhaus & Baddiley, 2003). The addition of D-alanine esters to teichoic acids is typically mediated by the products of the dlt operon, which encodes four proteins: DltA, a D-alanine : D-alanyl carrier protein ligase; DltB, a D-alanyl transfer protein; DltC, the D-alanyl carrier protein; and DltD, a D-alanine esterase. All four proteins are required for successful addition of D-alanine to the cell wall (Ne...

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Section: Effects Of the Disruption Of Dltd On Camp Resistancementioning

confidence: 99%

The dlt operon confers resistance to cationic antimicrobial peptides in Clostridium difficile

2011

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“…12 This subsequently led to the formulation of an algorithm whereby the changes necessary to redirect the Ll.LtrB-derived introns to a gene of interest could be reliably predicted. 10,13 A further pivotal finding was the demonstration that ltrA (encoding the IEP) need not be located within the intron, but could be provided in trans. This allows ltrA to be positioned on the backbone of the group II intron delivery plasmid.…”

Section: Clostron Technologymentioning

confidence: 99%

“…Subsequent phenotypic screening of the cell population within these positive colonies, based on the presence or absence of halos around colonies following serial defined the mechanisms by which bacterial introns move from one location to another and thereafter exploited the system to direct the insertion of the element to any intended target. [9][10][11] Group II introns are catalytic RNAs that excise themselves from RNA transcripts via a lariat intermediate and insert themselves into a new distal target site. Bacterial group II introns tend to reside outside of structural genes (i.e., are exon-less) and are closely associated with mobile elements.…”

Section: Clostron Technologymentioning

confidence: 99%

ClosTron-Mediated Engineering of Clostridium

Kuehne

Heap

Cooksley

et al. 2011

Methods in Molecular Biology

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“…We determined the retrohoming efficiency of the Ll.LtrB intron in E. coli mutants with targetron-mediated disruptions of the corA and mgtA genes (27) by using a plasmid-based assay (28) (Fig. 2A).…”

Section: E Colimentioning

confidence: 99%

Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core

Truong

Sidote

Russell

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Mobile group II introns are bacterial retrotransposons thought to be evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of a catalytically active intron RNA ("ribozyme") and an intron-encoded reverse transcriptase, which function together to promote RNA splicing and intron mobility via reverse splicing of the intron RNA into new DNA sites ("retrohoming"). Although group II introns are active in bacteria, their natural hosts, they function inefficiently in eukaryotes, where lower free Mg 2+ concentrations decrease their ribozyme activity and constitute a natural barrier to group II intron proliferation within nuclear genomes. Here, we show that retrohoming of the Ll.LtrB group II intron is strongly inhibited in an Escherichia coli mutant lacking the Mg 2+ transporter MgtA, and we use this system to select mutations in catalytic core domain V (DV) that partially rescue retrohoming at low Mg 2+ concentrations. We thus identified mutations in the distal stem of DV that increase retrohoming efficiency in the MgtA mutant up to 22-fold. Biochemical assays of splicing and reverse splicing indicate that the mutations increase the fraction of intron RNA that folds into an active conformation at low Mg 2+ concentrations, and terbium-cleavage assays suggest that this increase is due to enhanced Mg 2+ binding to the distal stem of DV. Our findings indicate that DV is involved in a critical Mg 2+ -dependent RNA folding step in group II introns and demonstrate the feasibility of selecting intron variants that function more efficiently at low Mg 2+ concentrations, with implications for evolution and potential applications in gene targeting. directed evolution | Mg 2+ transport | RNA structure M obile group II introns are retrotransposons that are found in prokaryotes and the mitochondrial and chloroplast DNAs of some eukaryotes and are thought to be evolutionary ancestors of spliceosomal introns, the spliceosome, retrotransposons, and retroviruses in higher organisms (1). They consist of two components-an autocatalytic intron RNA ("ribozyme") and an intron-encoded protein (IEP) with reverse transcriptase activity-that function together in a ribonucleoprotein (RNP) complex to promote RNA splicing and site-specific integration of the intron into new DNA sites in a process called retrohoming (1). Like spliceosomal introns, group II introns splice via two sequential transesterification reactions that yield an excised intron lariat RNA (2). For group II introns, the splicing reactions are catalyzed by the intron RNA with the assistance of the IEP, which binds specifically to the intron RNA and stabilizes the catalytically active RNA structure. The IEP then remains bound to the excised intron lariat RNA in an RNP that promotes retrohoming via reverse splicing of the intron RNA directly into DNA sites followed by reverse transcription by the IEP. The resulting intron cDNA is integrated into the genome by host enzymes (3, 4). The ribozyme-based, site-specific DNA integration mechanism used by g...

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Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria

Cited by 196 publications

References 25 publications

The dlt operon confers resistance to cationic antimicrobial peptides in Clostridium difficile

The dlt operon confers resistance to cationic antimicrobial peptides in Clostridium difficile

ClosTron-Mediated Engineering of Clostridium

Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core

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