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Thermococcus kodakarensis genetics: TK1827-encoded beta-glycosidase, new positive-selection protocol, and targeted and repetitive deletion technology.

ABSTRACT: Inactivation of TK1761, the reporter gene established for Thermococcus kodakarensis, revealed the presence of a second beta-glycosidase that we have identified as the product of TK1827. This enzyme (pTK1827) has been purified and shown to hydrolyze glucopyranoside but not mannopyranoside, have optimal activity at 95 degrees C and from pH 8 to 9.5, and have a functional half-life of approximately 7 min at 100 degrees C. To generate a strain with both TK1761 and TK1827 deleted, a new selection/counterselection protocol has been developed, and the levels of beta-glycosidase activity in T. kodakarensis strains with TK1761 and/or TK1827 deleted and with these genes expressed from heterologous promoters are described. Genetic tools and strains have been developed that extend the use of this selection/counterselection procedure to delete any nonessential gene from the T. kodakarensis chromosome. Using this technology, TK0149 was deleted to obtain an agmatine auxotroph that grows on nutrient-rich medium only when agmatine is added. Transformants can therefore be selected rapidly, and replicating plasmids can be maintained in this strain growing in rich medium by complementation of the TK0149 deletion.

SUBMITTER: Santangelo TJ

PROVIDER: S-EPMC2820962 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Thermococcus kodakarensis genetics: TK1827-encoded beta-glycosidase, new positive-selection protocol, and targeted and repetitive deletion technology.

Santangelo Thomas J TJ Cubonová L'ubomíra L Reeve John N JN

Applied and environmental microbiology 20091218 4

Inactivation of TK1761, the reporter gene established for Thermococcus kodakarensis, revealed the presence of a second beta-glycosidase that we have identified as the product of TK1827. This enzyme (pTK1827) has been purified and shown to hydrolyze glucopyranoside but not mannopyranoside, have optimal activity at 95 degrees C and from pH 8 to 9.5, and have a functional half-life of approximately 7 min at 100 degrees C. To generate a strain with both TK1761 and TK1827 deleted, a new selection/cou ...[more]

PMID: 20023088

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Project description:Prokaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic Archaeon, Thermococcus kodakarensis, under different physiological conditions.Sequencing cDNA libraries, generated from RNA isolated from cells under different growth and metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements. The primary transcription start sites (TSS) upstream of 1,254 annotated genes, plus 644 primary TSS and their promoters within genes, are identified. Most mRNAs have a 5'-untranslated region (5'-UTR) 10 to 50 nt long (median = 16 nt), but ~20% have 5'-UTRs from 50 to 300 nt long and ~14% are leaderless. Approximately 50% of mRNAs contain a consensus ribosome binding sequence. The results identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA, and confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 predicted snoRNAs. Two putative riboswitch RNAs were present in growing but not in stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in T. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. Many previously unanticipated small RNAs have been identified, some with relative low GC contents (? 50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile.The results identify >2,700 TSS, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. The T.?kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the transcriptomes established also contain many non-coding RNAs and predict extensive RNA-based regulation in this model Archaeon.

Dataset Information

Thermococcus kodakarensis genetics: TK1827-encoded beta-glycosidase, new positive-selection protocol, and targeted and repetitive deletion technology.

Publications

Thermococcus kodakarensis genetics: TK1827-encoded beta-glycosidase, new positive-selection protocol, and targeted and repetitive deletion technology.

Similar Datasets

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