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Transposition of an insertion sequence, ISTth7, in the genome of the extreme thermophile Thermus thermophilus HB8.

ABSTRACT: We have identified an active insertion sequence (IS) in the genome of Thermus thermophilus HB8. Transposition was detected as insertional inactivation of a 16S rRNA methyltransferase gene, rsmG, resulting in streptomycin resistance. The IS element, ISTth7, is 1029 bp in length, encodes an imperfect 12 bp inverted repeat, and produces a 9 bp direct repeat of the target sequence. The sequence of a putative transposase encoded by ISTth7 indicates that it is a member of the IS427 group within the IS5 family of ISs. Nine intact copies and several partial copies were identified throughout the chromosome and the megaplasmid pTT27. ISTth7 was also detected in T. thermophilus strain IB-21 and Thermus igniterrae but not Thermus antranikianii, suggesting a widespread occurrence of ISTth7 among Thermus spp.

SUBMITTER: Gregory ST

PROVIDER: S-EPMC3643509 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Transposition of an insertion sequence, ISTth7, in the genome of the extreme thermophile Thermus thermophilus HB8.

Gregory Steven T ST Dahlberg Albert E AE

FEMS microbiology letters 20081028 2

We have identified an active insertion sequence (IS) in the genome of Thermus thermophilus HB8. Transposition was detected as insertional inactivation of a 16S rRNA methyltransferase gene, rsmG, resulting in streptomycin resistance. The IS element, ISTth7, is 1029 bp in length, encodes an imperfect 12 bp inverted repeat, and produces a 9 bp direct repeat of the target sequence. The sequence of a putative transposase encoded by ISTth7 indicates that it is a member of the IS427 group within the IS ...[more]

PMID: 19016874

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Project description:Lysine acetylation in proteins has recently been globally identified in bacteria and eukaryotes. Even though acetylproteins are known to be involved in various cellular processes, its physiological significance has not yet been resolved. Using a proteomics approach in combination with immunoprecipitation, we identified 197 lysine acetylation sites and 4 N-terminal acetylation sites from 128 proteins in Thermus thermophilus HB8, an extremely thermophilic eubacterium. Our analyses revealed that identified acetylproteins are well conserved across all three domains of life and are mainly involved in central metabolism and translation. To further characterize functional significance, we successfully mapped 113 acetylation sites on their 54 authentic and 59 homologous protein structures. The acetylation in the majority of proteins occurs in ordered structures and the sites were situated near the negatively charged glutamic acid residues. In addition, 59 of 103 acetylations were located within considerable distance that can disrupt electrostatic interactions and hydrogen bonding networks on protein surface, demonstrating the physiological significances of the acetylations. Finally, we further summarized 22 critical acetylation sites related to Schiff-base formation, ligand binding, protein-RNA and protein-protein interaction. The structural information of 113 acetylation sites provides new molecular insight into the role of lysine acetylation in the proteins. Data processing, bioinformatics: MS and MS/MS spectral data were processed by DataAnalysis 4.0 software (Bruker Daltonics). The peak lists containing m/z of precursor ions with that of their product ions were generated by the Compound-Auto MS(n) option of the DataAnalysis 4.0 software. Fifty non-deconvoluted peaks over the intensity threshold 150 and charge deconvoluted peaks in each MS/MS spectrum were exported into peak list files. The spectra were searched against our in-house T. thermophilus HB8 database, containing 2,238 protein sequence entries from the complete genome sequence using Mascot search engine (version 2.3; Matrix Science, London, UK). The acetylated peptides were identified using a mass tolerance of ±0.05 Da for precursor and product ions and allowed a maximum of 6 mis-cleavage sites for trypsin. The carbamidomethylation of cysteine was selected as a fixed modification. The oxidation of methionine, deamidation of asparagine and glutamine, acetylation of lysine and acetylation of protein N-terminus were selected as variable modifications. Only peptides in the confidence range of 99% probability (P value < 0.01) in Mascot ion score were assumed to be identified.

Project description:BackgroundPenicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of β-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the thermophilic bacterium Thermus thermophilus (Tth) HB27. Because of the nature of this PAC and its complex maturation that is crucial to reach its functional heterodimeric final conformation, the overexpression of this enzyme in a heterologous mesophilic host was a challenge. Here we describe the purification and characterization of the PAC protein from Tth HB27 overexpressed in Escherichia coli.ResultsFusions to a superfolder green fluorescent protein and differential membrane solubilization assays indicated that the native enzyme remains attached through its amino-terminal end to the outer side of the cytoplasmic membrane of Tth cells. In order to overexpress this PAC in E. coli cells, a variant of the protein devoid of its membrane anchoring segment was constructed. The effect of the co-expression of chaperones and calcium supplementation of the culture medium was investigated. The total production of PAC was enhanced by the presence of DnaK/J and GrpE and even more by trigger factor and GroEL/ES. In addition, 10 mM calcium markedly improved both PAC specific and volumetric activities. Recombinant PAC was affinity-purified and proper maturation of the protein was confirmed by SDS-PAGE and MALDI-TOF analysis of the subunits. The recombinant protein was tested for activity towards several penicillins, cephalosporins and homoserine lactones. Hydrophobic acyl-chain penicillins were preferred over the rest of the substrates. Penicillin K (octanoyl penicillin) was the best substrate, with the highest specificity constant value (16.12 mM-1.seg-1). The optimum pH was aprox. 4 and the optimum temperature was 75 °C. The half-life of the enzyme at this temperature was 9.2 h.ConclusionsThis is the first report concerning the heterologous expression of a pac gene from a thermophilic microorganism in the mesophilic host E. coli. The recombinant protein was identified as a penicillin K-deacylating thermozyme.

Dataset Information

Transposition of an insertion sequence, ISTth7, in the genome of the extreme thermophile Thermus thermophilus HB8.

Publications

Transposition of an insertion sequence, ISTth7, in the genome of the extreme thermophile Thermus thermophilus HB8.

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