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Article: pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

TitlepZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis
Authors
Issue Date2014
PublisherBioMed Central Ltd. The Journal's web site is located at http://www.biomedcentral.com/bmcmicrobiol/
Citation
B M C Microbiology, 2014, v. 14 n. 1, p. article no. 68 How to Cite?
AbstractBackground The ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications. Results A quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli - Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified. Conclusions We show that a shuttle vector-based protein affinity 'pull-down' approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.
Persistent Identifierhttp://hdl.handle.net/10722/195904
ISSN
2015 Impact Factor: 2.581
2015 SCImago Journal Rankings: 1.391
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorSo, LYen_US
dc.contributor.authorChen, Wen_US
dc.contributor.authorLacap-Bugler, DCen_US
dc.contributor.authorSeemann, Men_US
dc.contributor.authorWatt, RMen_US
dc.date.accessioned2014-03-21T02:17:17Z-
dc.date.available2014-03-21T02:17:17Z-
dc.date.issued2014en_US
dc.identifier.citationB M C Microbiology, 2014, v. 14 n. 1, p. article no. 68en_US
dc.identifier.issn1471-2180-
dc.identifier.urihttp://hdl.handle.net/10722/195904-
dc.description.abstractBackground The ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications. Results A quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli - Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified. Conclusions We show that a shuttle vector-based protein affinity 'pull-down' approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.en_US
dc.languageengen_US
dc.publisherBioMed Central Ltd. The Journal's web site is located at http://www.biomedcentral.com/bmcmicrobiol/en_US
dc.relation.ispartofB M C Microbiologyen_US
dc.rightsCreative Commons: Attribution 3.0 Hong Kong Licenseen_US
dc.titlepZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilisen_US
dc.typeArticleen_US
dc.identifier.emailChen, W: chenwy@hku.hken_US
dc.identifier.emailLacap-Bugler, DC: dclacap@hkusua.hku.hken_US
dc.identifier.emailWatt, RM: rmwatt@hku.hken_US
dc.identifier.authorityChen, W=rp01487en_US
dc.identifier.authorityWatt, RM=rp00043en_US
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.1186/1471-2180-14-68en_US
dc.identifier.pmid24629064-
dc.identifier.hkuros228327en_US
dc.identifier.volume14en_US
dc.identifier.issue1-
dc.identifier.spagearticle no. 68en_US
dc.identifier.epagearticle no. 68en_US
dc.identifier.isiWOS:000335407600001-
dc.publisher.placeUnited Kingdom-

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