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Establishment of a gene expression system in Ochrobactrum anthropi.

ABSTRACT: Genetic studies of Ochrobactrum anthropi are hindered by the lack of a suitable gene expression system. We constructed a set of vectors containing several promoters and a His tag fusion in the N terminus to facilitate protein detection and purification. The new vectors should significantly enhance the genetic manipulation and characterization of O. anthropi.

SUBMITTER: Seleem MN

PROVIDER: S-EPMC1610320 | biostudies-literature | 2006 Oct

REPOSITORIES: biostudies-literature

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Establishment of a gene expression system in Ochrobactrum anthropi.

Seleem Mohamed N MN Ali Mohammed M Boyle Stephen M SM Mukhopadhyay Biswarup B Witonsky Sharon G SG Schurig Gerhardt G GG Sriranganathan Nammalwar N

Applied and environmental microbiology 20061001 10

Genetic studies of Ochrobactrum anthropi are hindered by the lack of a suitable gene expression system. We constructed a set of vectors containing several promoters and a His tag fusion in the N terminus to facilitate protein detection and purification. The new vectors should significantly enhance the genetic manipulation and characterization of O. anthropi. ...[more]

PMID: 17021239

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Clinical characteristics of Ochrobactrum anthropi bacteremia.

Project description:The clinical picture of Ochrobactrum anthropi infection is not well described because the infection is rare in humans and identification of the pathogen is difficult. We present a case of O. anthropi bacteremia that was initially misidentified as Ralstonia paucula and later identified by 16S rRNA sequencing and recA analysis.

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Purification, characterization, and gene cloning of purine nucleosidase from Ochrobactrum anthropi.

Project description:A bacterium, Ochrobactrum anthropi, produced a large amount of a nucleosidase when cultivated with purine nucleosides. The nucleosidase was purified to homogeneity. The enzyme has a molecular weight of about 170,000 and consists of four identical subunits. It specifically catalyzes the irreversible N-riboside hydrolysis of purine nucleosides, the K(m) values being 11.8 to 56.3 microM. The optimal activity temperature and pH were 50 degrees C and pH 4.5 to 6.5, respectively. Pyrimidine nucleosides, purine and pyrimidine nucleotides, NAD, NADP, and nicotinamide mononucleotide are not hydrolyzed by the enzyme. The purine nucleoside hydrolyzing activity of the enzyme was inhibited (mixed inhibition) by pyrimidine nucleosides, with K(i) and K(i)' values of 0.455 to 11.2 microM. Metal ion chelators inhibited activity, and the addition of Zn(2+) or Co(2+) restored activity. A 1.5-kb DNA fragment, which contains the open reading frame encoding the nucleosidase, was cloned, sequenced, and expressed in Escherichia coli. The deduced 363-amino-acid sequence including a 22-residue leader peptide is in agreement with the enzyme molecular mass and the amino acid sequences of NH(2)-terminal and internal peptides, and the enzyme is homologous to known nucleosidases from protozoan parasites. The amino acid residues forming the catalytic site and involved in binding with metal ions are well conserved in these nucleosidases.

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Draft Genome Sequence of Organophosphate-Degrading Ochrobactrum anthropi FRAF13.

Project description:ITALIC! Ochrobactrum anthropiFRAF13 was isolated from farmland soil in Jersey Village, Texas. FRAF13 is a bacterial microorganism with broad antibiotic resistance that possesses a number of metal-dependent ?-lactam enzymes with secondary phosphotriesterase activity that can initiate the breakdown of organophosphate compounds.

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Molecular cloning, expression and site-directed mutagenesis of glutathione S-transferase from Ochrobactrum anthropi.

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Complete Genome Assembly of Reference Strain Ochrobactrum anthropi ATCC 49687.

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Project description:Ochrobactrum anthropi, formerly known as CDC group Vd, is an oxidase-producing, gram-negative, obligately aerobic, non-lactose-fermenting bacillus of low virulence that occasionally causes human infections. It is highly resistant to all beta-lactams except imipenem. A clinical isolate, SLO74, and six reference strains were tested. MICs of penicillins, aztreonam, and most cephalosporins tested, including cefotaxime and ceftazidime, were >128 microg/ml and of cefepime were 64 to >128 microg/ml. Clavulanic acid was ineffective and tazobactam had a weak effect in association with piperacillin. Two genes, ampR and ampC, were cloned by inserting restriction fragments of genomic DNA from the clinical strain O. anthropi SLO74 into pBK-CMV to give the recombinant plasmid pBK-OA1. The pattern of resistance to beta-lactams of this clone was similar to that of the parental strain, except for its resistance to cefepime (MIC, 0.5 ,micro/ml). The deduced amino acid sequence of the AmpC beta-lactamase (pI, 8.9) was only 41 to 52% identical to the sequence of other chromosomally encoded and plasmid-encoded class C beta-lactamases. The kinetic properties of this beta-lactamase were typical for this class of beta-lactamases. Upstream from the ampC gene, the ampR gene encodes a protein with a sequence that is 46 to 62% identical to those of other AmpR proteins and with an amino-terminal DNA-binding domain typical of transcriptional activators of the Lys-R family. The deduced amino acid sequences of the ampC genes of the six reference strains were 96 to 99% identical to the sequence of the clinical strain. The beta-lactamase characterized from strain SLO74 was named OCH-1 (gene, bla(OCH-I)).

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?-Transaminase from Ochrobactrum anthropi is devoid of substrate and product inhibitions.

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A semi-quantitative GeLC-MS analysis of temporal proteome expression in the emerging nosocomial pathogen Ochrobactrum anthropi.

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