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Applied and Environmental Microbiology, December 2006, p. 7785-7792, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01564-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of Pyruvate Carboxylase Genes in Pseudomonas aeruginosa PAO1 and Development of a P. aeruginosa-Based Overexpression System for {alpha}4- and {alpha}4ß4-Type Pyruvate Carboxylases{triangledown}

Huafang Lai,1,{dagger} Jessica L. Kraszewski,1,2 Endang Purwantini,1,4 and Biswarup Mukhopadhyay1,2,3*

Virginia Bioinformatics Institute,1 Departments of Biochemistry,2 Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia,3 Department of Pharmacy, Institute Teknolgi Bandung, Bandung, Indonesia4

Received 6 July 2006/ Accepted 12 September 2006

Pyruvate carboxylase (PYC) is an ecologically, medically, and industrially important enzyme. It is widespread in all three domains of life, the archaea, bacteria, and eukarya. PYC catalyzes ATP-dependent carboxylation of pyruvate to oxaloacetate. Detailed structure-function studies of this enzyme have been hampered due to the unavailability of a facile recombinant overexpression system. Except for the {alpha}4 enzyme from a thermophilic Bacillus species, Escherichia coli has been unsuitable for overexpression of PYCs. We show that a Pseudomonas aeruginosa strain carrying the T7 polymerase gene can serve as a host for the overexpression of Mycobacterium smegmatis {alpha}4 PYC and Pseudomonas aeruginosa {alpha}4ß4 PYC under the control of the T7 promoter from a broad-host-range conjugative plasmid. Overexpression occurred both in aerobic (LB medium) and nitrate-respiring anaerobic (LB medium plus glucose and nitrate) cultures. The latter system presented a simpler option because it involved room temperature cultures in stationary screw-cap bottles. We also developed a P. aeruginosa {Delta}pyc strain that allowed the expression of recombinant PYCs in the absence of the native enzyme. Since P. aeruginosa can be transformed genetically and lysed for cell extract preparation rather easily, our system will facilitate site-directed mutagenesis, kinetics, X-ray crystallographic, and nuclear magnetic resonance-based structure-function analysis of PYCs. During this work we also determined that, contrary to a previous report (C. K. Stover et al., Nature 406:959-964, 2000), the open reading frame (ORF) PA1400 does not encode a PYC in P. aeruginosa. The {alpha}4ß4 PYC of this organism was encoded by the ORFs PA5436 and PA5435.

* Corresponding author. Mailing address: Virginia Bioinformatics Institute, Bioinformatics I, Virginia Polytechnic Institute and State University, Washington Street, MC 0477, Blacksburg, VA 24061. Phone: (540) 231-8015. Fax: (540) 231-2606. E-mail: biswarup{at}vt.edu.

{triangledown} Published ahead of print on 22 September 2006.

{dagger} Present address: Stem Cell Research Program, T603 Waisman Center, University of Wisconsin, 1500 Highland Ave., Madison, WI 53705.

Applied and Environmental Microbiology, December 2006, p. 7785-7792, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01564-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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