MOLECULAR CHARACTERIZATION OF THE ERWINIA-CHRYSANTHEMI KDGK GENE INVOLVED IN PECTIN DEGRADATION

The pathways of pectin and galacturonate catabolism in Erwinia chrysanthemi converge to form a common intermediate, 2-keto-3-deoxygluconate (KDG), which is phosphorylated by KDG kinase encoded by the kdgK gene. We cloned the kdgK gene of E. chrysanthemi 3937 by complementing an Escherichia coli kdgK mutation, using an RP4-derivative plasmid. One of the kdgK R-prime plasmids harbored a DNA insert of about 80 kb and carried the uxuA and uxuB genes involved in glucuronate catabolism and the celY gene coding for an E. chrysanthemi cellulase. The kdgK and celY genes were precisely located on this plasmid, and their respective transcriptional directions were determined. The nucleotide sequence of the kdgK region indicated that the kdgK reading frame is 981 bases long, corresponding to a protein of 329 amino acids with a molecular mass of 36,377 Da. Analysis of the deduced primary amino acid sequence showed that this enzyme is a new member of the PfkB family of carbohydrate kinases. Expression of kdgK is controlled by a negative regulatory gene, kdgR, which represses all the steps of pectin degradation. Near the putative promoter of the kdgK gene, we identified a putative KdgR-binding site and demonstrated that the KdgR protein specifically binds in vitro to this DNA region. The KdgR-KDG couple directly mediates the phenomenon of repression or induction. The KDG kinase, by limiting the intracellular inducer concentration, appears to be a key enzyme in induction of the whole catabolic pathway.