Cloning and characterization of an IclR protein of Burkholderia sp. MBA4

Abstract

Burkholderia sp. MBA4 was identified from soil for its ability to grow on

monobromoacetic acid. A dehalogenase, Deh4a, confers a dehalogenation

function in MBA4. A permease gene, deh4p, forms a haloacid operon with deh4a.

Deh4a has been well characterized but the regulatory mechanism of the haloacid

operon was unknown. Electrophoretic mobility shift assay shows that at least one

regulatory protein exists and binds with the promoter of deh4a. A DNA-affinity

column purified several DNA-binding proteins and two proteins were identified

by tandem mass spectroscopy as putative transcriptional regulators of B.

xenovorans LB400. One of these proteins was named IclR1 and subjected to

further analysis in this study. Here I report the cloning of the iclR1 gene and the

functional study of this protein.

The iclR1 gene was cloned by means of chromosome walking. The iclR1 gene has

837 bases and encodes 278 amino acids. The putative protein is classified as a

member of the IclR family. Recombinant IclR1 was produced in E. coli and

purified by Ni-NTA column. The experimental size of IclR1 is 27.5 kD and a

dimer of 52.3 kD can be identified in vitro with cross-linking reagent. Purified

IclR1 failed to bind the deh4a promoter, and mutants with a disrupted iclR1 gene

or over-expressing IclR1 has no effect on the deh4a expression. It is likely that

IclR1 is not a regulator of the haloacid operon. EMSA shows that IclR1 binds to

its own promoter which contains a palindrome sequence and a pair of inverted

repeats upstream of the start codon. The transcription start site of iclR1 was

determined to be a G 110 bp upstream of the start codon by 5’ RACE. The iclR1

promoter region was ligated with a lacZ reporter gene, and transformed into wild

type MBA4, a disruptant and an over-producer mutant. ONPG assay shows that

the expression of the reporter is induced by NaCl. The transcript level of iclR1 is

also higher in NaCl-containing medium. Over-expression of IclR1 inhibits the

expression of the reporter, indicating that IclR1 is a self-regulated repressor. The

growth of an iclR1 disruptant is more sensitive to salt. These results suggest that

IclR1 is beneficial for the survival of the cell in NaCl stress, but excessive IclR1

prevent the responding system from overworking. Since MBA4 is very sensitive

to NaCl, understanding the NaCl-related physiology of MBA4 is important. The

gene(s) under direct control of IclR1 is unknown and the specific function of

IclR1 awaits further study.