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postgraduate thesis: Cloning and characterization of an IclR protein of Burkholderia sp. MBA4

TitleCloning and characterization of an IclR protein of Burkholderia sp. MBA4
Authors
Advisors
Advisor(s):Tsang, JSH
Issue Date2012
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Xu, X. [徐信一]. (2012). Cloning and characterization of an IclR protein of Burkholderia sp. MBA4. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4784950
AbstractBurkholderia 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.
DegreeDoctor of Philosophy
SubjectRalstonia solanacearum.
Molecular genetics.
Dept/ProgramBiological Sciences

 

DC FieldValueLanguage
dc.contributor.advisorTsang, JSH-
dc.contributor.authorXu, Xinyi-
dc.contributor.author徐信一-
dc.date.issued2012-
dc.identifier.citationXu, X. [徐信一]. (2012). Cloning and characterization of an IclR protein of Burkholderia sp. MBA4. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4784950-
dc.description.abstractBurkholderia 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.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.source.urihttp://hub.hku.hk/bib/B47849502-
dc.subject.lcshRalstonia solanacearum.-
dc.subject.lcshMolecular genetics.-
dc.titleCloning and characterization of an IclR protein of Burkholderia sp. MBA4-
dc.typePG_Thesis-
dc.identifier.hkulb4784950-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiological Sciences-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b4784950-
dc.date.hkucongregation2012-

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