A novel gene, algK, from the alginate biosynthesis cluster of Pseudomonas aeruginosa

Colonization of the cystic fibrosis lung by Pseudomonas aeruginosa is greatly facilitated by the production of an exopolysaccharide called alginate. Many of the enzymes involved in alginate biosynthesis are clustered in an operon at 34 min on the P. aeruginosa chromosome. This paper reports the nucleotide sequence of a previously uncharacterized gene, algK, which lies between the alg44 and algE genes of the operon. DNA sequencing data for algK predicted a protein product of approximately 52.5 kDa which contains a putative 27 amino acid N-terminal signal sequence and a consensus cleavage and lipid attachment site for signal peptidase II. Expression of algK using either T7 or tac promoter expression systems, in vivo labelling studies with [35S]methionine, indicated that algK encodes a polypeptide of approximately 53 kDa which is processed to a mature protein of approximately 50 kDa when expressed in Escherichia coli or P. aeruginosa, in agreement with the nucleotide sequence analysis. Results from an algK-beta-lactamase fusion survey support this interpretation and also provide evidence that mature AlgK is entirely periplasmic and is probably membrane-anchored.