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postgraduate thesis: Identification and characterization of a conserved haloacids transporter gene in the Burkholderia genus

TitleIdentification and characterization of a conserved haloacids transporter gene in the Burkholderia genus
Authors
Advisors
Advisor(s):Tsang, JSH
Issue Date2012
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Su, X. [苏现斌]. (2012). Identification and characterization of a conserved haloacids transporter gene in the Burkholderia genus. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4961785
AbstractBacterial degradation is an important way to detoxify environmental pollutants haloacids, and the key enzyme involved is dehalogenase. In contrast to the well characterized dehalogenases, haloacids transporters that mediate uptake of haloacids are poorly understood. The deh4p gene in a haloacids-degrading bacterium Burkholderia species MBA4 is the first reported haloacids transporter gene. It is located downstream of the dehalogenase gene deh4a and the two forms a haloacids operon. The role of Deh4p as a haloacids transporter was confirmed by heterologous expression. It was later found that a mutant of MBA4 without functional Deh4p was still able to grow in monochloroacetic acid (MCA), and Deh4p seems to be not the only haloacids transporter in MBA4. This study aimed at gaining a deeper understanding of the haloacids transport process in MBA4, and establishing the role of a newly identified gene dehp2 as a conserved haloacids transporter gene in the Burkholderia genus. Disruption of deh4p in MBA4 caused a 32% decrease in MCA uptake rate, confirming the role of Deh4p as a haloacids transporter, but not the only one. A gene showing homology to deh4p in MBA4 was identified and named dehp2. The role of Dehp2 as a second haloacids transporter in MBA4 was confirmed by both gene disruption and heterologous expression. Like deh4p, the expression of dehp2 is also MCA-inducible. A double mutant with both deh4p and dehp2 disrupted only retained 36% MCA uptake rate, further confirming the roles of Deh4p and Dehp2 as haloacids transporters. Dehp2 and Deh4p were also shown to be the two major haloacids transporters. Both Dehp2 and Deh4p are specific toward acetate and its halogenated derivatives, but Dehp2 has a broader spectrum of substrates than Deh4p. Deh4p was found to be a better MCA transporter than Dehp2, and also has a higher affinity for MCA. The effects of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) and pH on MCA uptake supported the symport of proton(s) and the inclusion of both Dehp2 and Deh4p in the metabolites:H+ symporter family. Orthologs of dehp2 are widely found in the Burkholderia genus, and phylogenetic analysis showed that they were conserved in the genus. The roles of dehp2 orthologs in haloacids transport in three non-pathogenic Burkholderia species were studied. The three species were engineered to gain the ability to utilize MCA as the sole carbon source, and were shown to have MCA-inducible MCA uptake activities. Expressions of the dehp2 orthologs in them are MCA-inducible, strongly suggesting their relationship with MCA uptake. Fusion analysis with lacZ as a reporter gene confirmed the presence of MCA-inducible promoter activity in the upstream non-coding region of dehp2, and the results of electrophoretic mobility shift assay (EMSA) suggested a positive regulation of dehp2. This study established Dehp2 as a second haloacids transporter in MBA4, and also confirmed dehp2 as a conserved haloacids transporter gene in the Burkholderia genus. The presence of at least two haloacids transporters in MBA4 demonstrated the complexity of this process, and future work should figure out the transport mechanisms.
DegreeDoctor of Philosophy
SubjectBacterial genetics.
Ralstonia solanacearum.
Organohalogen compounds.
Dept/ProgramBiological Sciences
Persistent Identifierhttp://hdl.handle.net/10722/180965

 

DC FieldValueLanguage
dc.contributor.advisorTsang, JSH-
dc.contributor.authorSu, Xianbin.-
dc.contributor.author苏现斌.-
dc.date.accessioned2013-02-07T06:21:38Z-
dc.date.available2013-02-07T06:21:38Z-
dc.date.issued2012-
dc.identifier.citationSu, X. [苏现斌]. (2012). Identification and characterization of a conserved haloacids transporter gene in the Burkholderia genus. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4961785-
dc.identifier.urihttp://hdl.handle.net/10722/180965-
dc.description.abstractBacterial degradation is an important way to detoxify environmental pollutants haloacids, and the key enzyme involved is dehalogenase. In contrast to the well characterized dehalogenases, haloacids transporters that mediate uptake of haloacids are poorly understood. The deh4p gene in a haloacids-degrading bacterium Burkholderia species MBA4 is the first reported haloacids transporter gene. It is located downstream of the dehalogenase gene deh4a and the two forms a haloacids operon. The role of Deh4p as a haloacids transporter was confirmed by heterologous expression. It was later found that a mutant of MBA4 without functional Deh4p was still able to grow in monochloroacetic acid (MCA), and Deh4p seems to be not the only haloacids transporter in MBA4. This study aimed at gaining a deeper understanding of the haloacids transport process in MBA4, and establishing the role of a newly identified gene dehp2 as a conserved haloacids transporter gene in the Burkholderia genus. Disruption of deh4p in MBA4 caused a 32% decrease in MCA uptake rate, confirming the role of Deh4p as a haloacids transporter, but not the only one. A gene showing homology to deh4p in MBA4 was identified and named dehp2. The role of Dehp2 as a second haloacids transporter in MBA4 was confirmed by both gene disruption and heterologous expression. Like deh4p, the expression of dehp2 is also MCA-inducible. A double mutant with both deh4p and dehp2 disrupted only retained 36% MCA uptake rate, further confirming the roles of Deh4p and Dehp2 as haloacids transporters. Dehp2 and Deh4p were also shown to be the two major haloacids transporters. Both Dehp2 and Deh4p are specific toward acetate and its halogenated derivatives, but Dehp2 has a broader spectrum of substrates than Deh4p. Deh4p was found to be a better MCA transporter than Dehp2, and also has a higher affinity for MCA. The effects of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) and pH on MCA uptake supported the symport of proton(s) and the inclusion of both Dehp2 and Deh4p in the metabolites:H+ symporter family. Orthologs of dehp2 are widely found in the Burkholderia genus, and phylogenetic analysis showed that they were conserved in the genus. The roles of dehp2 orthologs in haloacids transport in three non-pathogenic Burkholderia species were studied. The three species were engineered to gain the ability to utilize MCA as the sole carbon source, and were shown to have MCA-inducible MCA uptake activities. Expressions of the dehp2 orthologs in them are MCA-inducible, strongly suggesting their relationship with MCA uptake. Fusion analysis with lacZ as a reporter gene confirmed the presence of MCA-inducible promoter activity in the upstream non-coding region of dehp2, and the results of electrophoretic mobility shift assay (EMSA) suggested a positive regulation of dehp2. This study established Dehp2 as a second haloacids transporter in MBA4, and also confirmed dehp2 as a conserved haloacids transporter gene in the Burkholderia genus. The presence of at least two haloacids transporters in MBA4 demonstrated the complexity of this process, and future work should figure out the transport mechanisms.-
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/B49617850-
dc.subject.lcshBacterial genetics.-
dc.subject.lcshRalstonia solanacearum.-
dc.subject.lcshOrganohalogen compounds.-
dc.titleIdentification and characterization of a conserved haloacids transporter gene in the Burkholderia genus-
dc.typePG_Thesis-
dc.identifier.hkulb4961785-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiological Sciences-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b4961785-
dc.date.hkucongregation2013-

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