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

The pathways of pectin and galacturonate catabolism in Erwinia chrysan themi 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 E scherichia coli kdgK mutation, using an RP4-derivative plasmid. One of the kdgK R-prime plasmids harbored a DNA insert of about 80 kb and ca rried the uxuA and uxuB genes involved in glucuronate catabolism and t he celY gene coding for an E. chrysanthemi cellulase. The kdgK and cel Y genes were precisely located on this plasmid, and their respective t ranscriptional directions were determined. The nucleotide sequence of the kdgK region indicated that the kdgK reading frame is 981 bases lon g, corresponding to a protein of 329 amino acids with a molecular mass of 36,377 Da. Analysis of the deduced primary amino acid sequence sho wed that this enzyme is a new member of the PfkB family of carbohydrat e kinases. Expression of kdgK is controlled by a negative regulatory g ene, kdgR, which represses all the steps of pectin degradation. Near t he putative promoter of the kdgK gene, we identified a putative KdgR-b inding 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 th e intracellular inducer concentration, appears to be a key enzyme in i nduction of the whole catabolic pathway.