Dataset Information

Engineering of tellurite-resistant genetic tools for single-copy chromosomal analysis of Burkholderia spp. and characterization of the Burkholderia thailandensis betBA operon.

ABSTRACT: There are few appropriate single-copy genetic tools for most Burkholderia species, and the high level of antibiotic resistance in this genus further complicates the development of genetic tools. In addition, the utilization of resistance genes for clinically important antibiotics is prohibited for the bioterrorism agents Burkholderia pseudomallei and Burkholderia mallei, necessitating the development of additional nonantibiotic-based genetic tools. Three single-copy systems devoid of antibiotic selection based on two nonantibiotic selectable markers, tellurite resistance (Tel(r)) and Escherichia coli aspartate-semialdehyde dehydrogenase (asd(Ec)), were developed to facilitate genetic manipulation in Burkholderia species. These systems include one mariner transposon, a mini-Tn7-derived site-specific transposon, and six FRT reporter fusion vectors based on the lacZ, gfp, and luxCDABE reporter genes. Initially, we showed that the random mariner transposon pBT20-Deltabla-Tel(r)-FRT efficiently transposed within Burkholderia cenocepacia, Burkholderia thailandensis, B. pseudomallei, and B. mallei. We then utilized the mini-Tn7-Tel(r)-based transposon vector (mini-Tn7-Tel(r)-betBA) and a transposase-containing helper plasmid (pTNS3-asd(Ec)) to complement the B. thailandensis DeltabetBA mutation. Next, one of the FRT-lacZ fusion vectors (pFRT1-lacZ-Tel(r)) was integrated by Flp (encoded on a helper plasmid, pCD13SK-Flp-oriT-asd(Ec)) to construct the B. thailandensis DeltabetBA::FRT-lacZ-Tel(r) reporter fusion strain. The betBA operon was shown to be induced in the presence of choline and under osmotic stress conditions by performing beta-galactosidase assays on the B. thailandensis DeltabetBA::FRT-lacZ-Tel(r) fusion strain. Finally, we engineered B. thailandensis DeltabetBA::FRT-gfp-Tel(r) and DeltabetBA::FRT-lux-Tel(r) fusion strains by utilizing fusion vectors pFRT1-gfp-Tel(r) and pFRT1-lux-Tel(r), respectively. The induction of the betBA operon by choline and osmotic stress was confirmed by performing fluorescent microscopy and bioluminescent imaging analyses.

SUBMITTER: Kang Y

PROVIDER: S-EPMC2698364 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Engineering of tellurite-resistant genetic tools for single-copy chromosomal analysis of Burkholderia spp. and characterization of the Burkholderia thailandensis betBA operon.

Kang Yun Y Norris Michael H MH Barrett Ashley R AR Wilcox Bruce A BA Hoang Tung T TT

Applied and environmental microbiology 20090417 12

There are few appropriate single-copy genetic tools for most Burkholderia species, and the high level of antibiotic resistance in this genus further complicates the development of genetic tools. In addition, the utilization of resistance genes for clinically important antibiotics is prohibited for the bioterrorism agents Burkholderia pseudomallei and Burkholderia mallei, necessitating the development of additional nonantibiotic-based genetic tools. Three single-copy systems devoid of antibiotic ...[more]

PMID: 19376905

Similar Datasets

Project description:Tellurite containing compounds are in use for industrial processes and increasing delivery into the environment generates specific pollution that may well result in contamination and subsequent potential adverse effects on public health. It was the aim of the current study to reveal mechanism of toxicity in tellurite-sensitive and tellurite-resistant E. coli at the protein level. In this work an approach using gel-based mass spectrometrical analysis to identify a differential protein profile related to tellurite toxicity was used and the mechanism of ter operon-mediated tellurite resistance was addressed. E. coli BL21 was genetically manipulated for tellurite-resistance by the introduction of the resistance-conferring ter genes on the pLK18 plasmid. Potassium tellurite was added to cultures in order to obtain a final 3.9 micromolar concentration. Proteins from tellurite-sensitive and tellurite-resistant E. coli were run on 2-D gel electrophoresis, spots of interest were picked, in-gel digested and subsequently analysed by nano-LC-MS/MS (ion trap). In addition, Western blotting and measurement of enzymatic activity were performed to verify the expression of certain candidate proteins. Following exposure to tellurite, in contrast to tellurite-resistant bacteria, sensitive cells exhibited increased levels of antioxidant enzymes superoxide dismutases, catalase and oxidoreductase YqhD. Cysteine desulfurase, known to be related to tellurite toxicity as well as proteins involved in protein folding: GroEL, DnaK and EF-Tu were upregulated in sensitive cells. In resistant bacteria, several isoforms of four essential Ter proteins were observed and following tellurite treatment the abovementioned protein levels did not show any significant proteome changes as compared to the sensitive control. The absence of general defense mechanisms against tellurite toxicity in resistant bacteria thus provides further evidence that the four proteins of the ter operon function by a specific mode of action in the mechanism of tellurite resistance probably involving protein cascades from antioxidant and protein folding pathways.

Dataset Information

Engineering of tellurite-resistant genetic tools for single-copy chromosomal analysis of Burkholderia spp. and characterization of the Burkholderia thailandensis betBA operon.

Publications

Engineering of tellurite-resistant genetic tools for single-copy chromosomal analysis of Burkholderia spp. and characterization of the Burkholderia thailandensis betBA operon.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets