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Genomic and proteomic characterization of the large Myoviridae bacteriophage ?TMA of the extreme thermophile Thermus thermophilus.

ABSTRACT: A lytic phage, designated as ?TMA, was isolated from a Japanese hot spring using Thermus thermophilus HB27 as an indicator strain. Electron microscopic examination showed that ?TMA had an icosahedral head and a contractile tail. The circular double-stranded DNA sequence of ?TMA was 151,483 bp in length, and its organization was essentially same as that of ?YS40 except that the ?TMA genome contained genes for a pair of transposase and resolvase, and a gene for a serine to asparagine substituted ortholog of the protein involved in the initiation of the ?YS40 genomic DNA synthesis. The different host specificities of ?TMA and ?YS40 could be explained by the sequence differences in the C-terminal regions of their distal tail fiber proteins. The ?pilA knockout strains of T. thermophilus showed simultaneous loss of sensitivity to their cognate phages, pilus structure, twitching motility and competence for natural transformation, thus suggesting that the phage infection required the intact host pili. Pulsed-field gel electrophoresis analysis of the ?TMA and ?YS40 genomes revealed that the length of their DNA exceeded 200 kb, indicating that the terminal redundancy is more than 30% of the closed circular form. Proteomic analysis of the ?TMA virion using a combination of N-terminal sequencing and mass spectrometric analysis of peptide fragments suggested that the maturation of several proteins involved in the phage assembly process was mediated by a trypsin-like protease. The gene order of the phage structural proteins was also discussed.

SUBMITTER: Tamakoshi M

PROVIDER: S-EPMC3225780 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Genomic and proteomic characterization of the large Myoviridae bacteriophage ϕTMA of the extreme thermophile Thermus thermophilus.

Tamakoshi Masatada M Murakami Aya A Sugisawa Motoki M Tsuneizumi Kenji K Takeda Shigeki S Saheki Toshihiko T Izumi Takashi T Akiba Toshihiko T Mitsuoka Kaoru K Toh Hidehiro H Yamashita Atsushi A Arisaka Fumio F Hattori Masahira M Oshima Tairo T Yamagishi Akihiko A

Bacteriophage 20110501 3

A lytic phage, designated as ϕTMA, was isolated from a Japanese hot spring using Thermus thermophilus HB27 as an indicator strain. Electron microscopic examination showed that ϕTMA had an icosahedral head and a contractile tail. The circular double-stranded DNA sequence of ϕTMA was 151,483 bp in length, and its organization was essentially same as that of ϕYS40 except that the ϕTMA genome contained genes for a pair of transposase and resolvase, and a gene for a serine to asparagine substituted o ...[more]

PMID: 22164349

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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

Genomic and proteomic characterization of the large Myoviridae bacteriophage ?TMA of the extreme thermophile Thermus thermophilus.

Publications

Genomic and proteomic characterization of the large Myoviridae bacteriophage ϕTMA of the extreme thermophile Thermus thermophilus.

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