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Molecular characterization of several genes involved in gluconate transport and phosphorylation, and induction of the gluconate peripheral pathway in Escherichia coli
Abstract
In this work, gntT, gntU, gntK, gntR and gntV have been cloned from the Kohara library by genetic complementation of appropriate E.coli mutants. The gntR, K, U region (77.0 minutes) has been sequenced and shows the gene order to be gntU-gntK-gntR. The deduced amino acid sequence analysis reveals that GntR contains a helix turn helix motif typical of a negative regulator, GntK shows homology to GntV but not to any other known sugar kinase, and GntU strongly resembles GntP (gluconate permease) of Bacillus subtilis. GntR prevents transcription from gluconate inducible promoters. The expression of gntR is constitutive. The gntK and gntU structural genes overlap, and Northern hybridization indicates that gntK and gntU are co-transcribed from a gluconate-inducible promoter which is subject to partial catabolite repression by glucose. Interestingly, gntK and gntU are only expressed in Log phase when induced by gluconate, but not in stationary phase. In contrast, the gntV is transcribed from a promoter which is subject to catabolite repression and occurs only in stationary phase cultures. The gntV promoter region contains a CAP binding site and a gear box, a stationary-phase induced promoter. The gntT gene is monocistronic, coding for a gluconate transport protein with a molecular weight of 48.07 kDa. Northern blot analysis indicate that gntT is transcribed from a single gluconate inducible promoter which is not subject to catabolite repression. It is apparently only active in log-phase cultures, as shown by the complete degradation of the transcript in stationary phase. Three putative gluconate transporter genes, gnt96P, gnt97P, gnt98P (96.8 minutes, 97.4 minutes, 98 minutes respectively), have been identified as a result of Dr. F. Blattner's E.coli genomic sequence project. Northern blot analysis shows that gnt96P is co-transcribed with two upstream unidentified open reading frames from a promoter which is active only in the stationary phase and is inhibited by catabolite repression. The transcripts of gnt98P is apparently from a single promoter which is subject to catabolite repression by glucose and gluconate. Uptake experiments of $\rm\sp{14}C$-gluconate showed that both Gnt96P and Gnt98P are high affinity gluconate transporters with an apparent Km of 60 uM and 20 uM, respectively.
Subject Area
Molecular biology|Microbiology|Genetics
Recommended Citation
Tong, Suxiang, "Molecular characterization of several genes involved in gluconate transport and phosphorylation, and induction of the gluconate peripheral pathway in Escherichia coli" (1995). ETD collection for University of Nebraska-Lincoln. AAI9538657.
https://digitalcommons.unl.edu/dissertations/AAI9538657