Housekeeping recA gene interrupted by group II intron in the thermophilic Geobacillus kaustophilus

Chee, Gab-Joo; Takami, Hideto

doi:10.1016/j.gene.2005.08.017

Cited by 19 publications

(22 citation statements)

References 40 publications

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“…Moreover, sporadic insertion of group II introns in nature could rapidly change the fitness of organisms to grow at different temperatures. Nevertheless, at least some bacterial group II introns can splice at elevated temperatures, possibly reflecting adaptation of the intron to the host after insertion (Chee and Takami 2005). aureus RN4220 using targetron Hsa-24s expressed from pNL9163 and pNL9164, respectively.…”

Section: Discussionmentioning

confidence: 99%

Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing

Yao¹,

Zhong²,

Fang³

et al. 2006

RNA

105

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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°C but not 43°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.

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Section: Discussionmentioning

confidence: 99%

Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing

Yao¹,

Zhong²,

Fang³

et al. 2006

RNA

105

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“…GsI-IIB is a subgroup IIB2 intron that is inserted in the G. stearothermophilus recA gene and is related to previously described RT-encoding group II introns in the recA genes of Geobacillus kaustophilus (Chee and Takami 2005) and Bacillus caldolyticus (Ng et al 2007). GsI-IIC is a group IIC intron found in multiple copies in the G. stearothermophilus genome (Moretz and Lampson 2010).…”

Section: Recombinant Plasmidsmentioning

confidence: 97%

“…Group II intron RTs from thermophiles are expected to combine these useful properties with thermostability. Thus far, however, only one mobile group II intron RT, the LtrA protein encoded by the Ll.LtrB intron, has been expressed in bacteria and purified with high yield and activity (Saldanha et al 1999), while other group II intron RTs, including those from thermophiles, are poorly expressed and largely insoluble in the absence of bound RNAs (Vellore et al 2004;Chee and Takami 2005;Ng et al 2007). A further challenge for biotechnological development is that group II introns RTs often have mutations that decrease or abolish RT activity, reflecting that they are under selective pressure to suppress intron mobility, which is deleterious to their hosts (Mohr et al 2010).…”

Section: Introductionmentioning

confidence: 99%

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing

Mohr

Ghanem

Smith

et al. 2013

RNA

196

301

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Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3 ′ ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

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“…13) However, no intein or group II introns, much less IVS harboring a HEG, were found in flagellin genes, unlike the previously reported bacterial group I introns in proteincoding regions. 4,5,[13][14][15][16] Group I intron-like sequences were also found in locations immediately after flagellin genes in A. flavithermus and Exiguobacterium sibiricum, although E. sibiricum has no intron in the flagellin gene ( Fig. 2A).…”

mentioning

confidence: 93%

A Group I Self-Splicing Intron in the Flagellin Gene of the Thermophilic BacteriumGeobacillus stearothermophilus

Hayakawa

Ishizuka

2009

Bioscience, Biotechnology, and Biochemistry

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Housekeeping recA gene interrupted by group II intron in the thermophilic Geobacillus kaustophilus

Cited by 19 publications

References 40 publications

Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing

Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing

A Group I Self-Splicing Intron in the Flagellin Gene of the Thermophilic BacteriumGeobacillus stearothermophilus

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