Xanthobacter flavus employs a single triosephosphate isomerase for heterotrophic and autotrophic metabolism

Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands Author for correspondence: Wim G. Meijer. Tel: +31 50 3632150. Fax: +31 50 3632154. e-mail: w.g.meijer@biol.rug.nl ABSTRACT The expression of the cbb and gap-pgk operons of Xanthobacter flavus encoding enzymes of the Calvin cycle is regulated by the transcriptional regulator CbbR. In order to identify other genes involved in the regulation of these operons, a mutant was isolated with a lowered activity of a fusion between the promoter of the cbb operon and the reporter gene lacZ. This mutant was unable to grow autotrophically and had a reduced growth rate of medium supplemented with gluconate or succinate. The regulation of the gap-pgk operon in the mutant was indistinguishable from the wild-type strain, but induction of the cbb operon upon transition to autotrophic growth conditions was delayed. Complementation of the mutant with a genomic library of X. flavus resulted in the isolation of a 1.1 kb Apa l fragment which restored autotrophic growth of the mutant. One open reading frame (ORF) was present on the Apa l fragment, which could encode a protein highly similar to triosephosphate isomerase proteins from other bacteria. Cell extracts of the mutant grown under glycolytic or gluconeogenic conditions had severely reduced triosephosphate isomerase activities. The ORF was therefore identified as tpi, encoding triosephosphate isomerase. The tpi gene is not linked to the previously identified operons encoding Calvin cycle enzymes and therefore represents a third transcriptional unit required for autotrophic metabolism. Keywords: RuBisCO, triosephosphate isomerase, autotrophy, regulation, Xanthobacter flavus