Identification and characterization of a chloroacetate-inducible glycolate operon in Burkholderia caribensis MBA4

Abstract

It has been confirmed that the ability of Burkholderia caribensis MBA4 in metabolizing halogenated organic compounds is due to a haloacid operon containing a dehalogenase Deh4a and a haloacid transporterDeh4p. Haloacetates are transported across the cell membrane with the assistance of Deh4p and degraded by Deh4a to form glycolate. Previous studies showed that the transcription of this haloacid operon is negatively regulated. Furthermore, the binding of putative regulators to the promoter of this operon is affected by monochloroacetate. Previous studies mainly focused on the dehalogenation mechanism of monochloroacetate where glycolate is the metabolite. Glycolate is subsequently catabolized by enzymes of the central metabolic pathways. In Escherichia coli the transcription of a glycolate operon, encoding for glycolate oxidase and malate synthase G, is induced by glycolate. In MBA4 a glycolate operon was identified and which is inducible by glycolate but much more by monochloroacetate. In Escherichia coli the glycolate operon is regulated by GlcC. Similar gene is also identified in MBA4. The aim of this study is to characterize this chloroacetate-inducible glycolate operon. Reverse-transcription PCR analysis showed that the glycolate operon in MBA4 composed of glcDEFGB genes (K788_0009107, K788_0000598, etc.). The regulator gene glcC (K788_0000597) is located immediately upstream on the opposite strand of the glycolate operon. Gene disruption analysis confirmed the activator role of this GlcC. Sequencing and bioinformatic analyses have identified the presence of two putative GlcC binding sites between glcC and glcDEFGB. A distal site, located at the beginning of glcC, and a proximal site, located upstream of glcDEFGB. The functional roles of these sites were evaluated with 5’-deletion assays and RegPrecise V3.3. Electrophoretic mobility shift assays implied the binding of GlcC to these two sites. When glycolate was the growth substrate, the distal site was occupied. The proximal site was bound when monochloroacetate was used. Confirmation of the specific binding of GlcC was conducted with cell extracts of glcC‾ mutant and with cell extracts of E. coli BL21(DE3) pLysS producing GlcC. Further analysis showed that monochloroacetate is required for the switching of binding of GlcC from the distal to the proximal site. This also implied that the binding specificity of this GlcC of MBA4 to the proximal site is chloroacetate-dependent. In this study a chloroacetate-inducible glc operon has been identified and characterized in Burkholderia caribensis MBA4. The presence of this operon enables the efficient metabolism of haloacetate to glycolate then to metabolite that can be used by the central metabolic pathways. This implied that MBA4 is a bacterium well adapted for the degradation of haloacid.