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. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/174508 |
| Date | January 2012 |
| Creators | Xu, Xinyi, 徐信一 |
| Contributors | Tsang, JSH |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B47849502 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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