Characterization of a universal stress protein UspA1171 of Burkholderia caribensis MBA4

Abstract

Burkholderia caribensis MBA4 is able to utilize haloacetic acids as growth substrates. A key enzyme, dehalogenase Deh4a, that enables bioremediation of halogenated organic compounds, was isolated from MBA4. A haloacid operon, which consists the structural genes for Deh4a and a transporter, Deh4p, was identified and characterized. So far, the transcription of this operon was found to regulate negatively. However, the mechanism has not been fully understood. Recently, a protein designated as TetR6620, encoded by gene K788_006620, has been found to bind to DNA fragment containing 100-bp of upstream non-coding region of the haloacid operon. Previous study showed that cell extracts prepared from glycolate-grown MBA4 produced two retardation complexes, A & B. When gene K788_006620 was disrupted in a mutant, Ins6620, the formation of the retardation complex A was abolished. Moreover, when purified recombinant TetR6620 with an N-terminal histidine-tag was used in similar bandshift assay, at least two retardation complexes were detected. The mobilities of these complexes are different from that of complexes A & B identified previously. When recombinant TetR6620 was supplemented to cell extracts of glycolate-grown Ins6620, the formation of complex A was restored. These suggest that while TetR6620 is able to bind to the upstream non-coding region of the haloacid operon, it probably interacted with another protein to perform its binding activity. By means of pull-down experiment, a universal stress protein UspA1171, encode by gene K788_001171, was envisaged to interact with TetR6620.

In this study, the role of UspA1171 was characterized. A K788_001171 disruptant was constructed. Plasmid pKNOCK-Cm was used as a backbone and a derivative containing part of K788_001171 was used to disrupt gene K788_001171 in MBA4. If UspA1171 is a repressor for the haloacid operon, then a disruptant will have a relieved expression of Deh4a in glycolate-grown cells. Quantitative RT-PCR will be used to determine the expression levels of deh4a.

The results showed that the slow migrating retardation complex A, identified in wildtype MBA4, was not found in glycolate-grown Ins6620. The gene K788_001171 has been amplified from the genome of MBA4 and cloned into an E. coli expression vector pET-14b. The plasmid was constructed and used to express UspA1171 in E. coli BL21(DE3). The recombinant histidine-tagged UspA1171 was over-expressed, purified and used in immobilized-metal affinity chromatography (IMAC) to pull down proteins that might interact with it. In addition, UspA1171 was used in BS3 crosslinking experiments with TetR6620. Western blot analysis was used to verify their interaction. The IMAC results showed that a 24 kDa protein was successfully pulled down from glycolate-grown Ins1171 by UspA1171 conjugated Ni-NTA agarose column. Previous results of chemical crosslinking showed that TetR6620 can form dimer but there is no evidence of UspA1171 capable of forming dimer. Nonetheless, with the presence of UspA1171 and BS3 crosslinker, there might be interaction between TetR6620 and UspA1171. For the future work, purified histidine-tagged UspA1171 will be supplemented to cell extracts of K788_001171 disruptant to see whether the formation of complex A will be recovered. Furthermore, the purified recombinant UspA1171 will be mixed with TetR6620 to see whether retardation complex A can be formed binding to the 100 bp of upstream non-coding region of the haloacid operon.