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

Abstract

Colonization of the cystic fibrosis lung byPseudomonas aeruginosais 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 theP. aeruginosachromosome. This paper reports the nucleotide sequence of a previously uncharacterized gene,algK,which lies between thealg44andalgEgenes of the operon. DNA sequencing data foralgKpredicted 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 ofalgKusing either T7 ortacpromoter expression systems, andin vivolabelling studies with [³⁵S]methionine, indicated thatalgKencodes a polypeptide of approximately 53 kDa which is processed to a mature protein of approximately 50 kDa when expressed inEscherichia coliorP. aeruginosa,in agreement with the nucleotide sequence analysis. Results from an AlgK-β-lactamase fusion survey support this interpretation and also provide evidence that mature AlgK is entirely periplasmic and is probably membrane-anchored.