Cloning and functional characterization of the Pseudomonas aeruginosa rhlCgene that encodes rhamnosyltransferase 2, an enzyme responsible for di-rhamnolipid biosynthesis

Pseudomonas aeruginosa is an opportunistic pathogen capable of producing a wide variety of virulence factors, including extracellular rhamnolipids and lipopolysaccharide. Rhamnolipids are tenso-active glycolipids containing o ne (mono-rhamnolipid) or two (di-rhamnolipid) l-rhamnose molecules. Rhamnos yltransferase 1 (RhlAB) catalyses the synthesis of mono-rhamnolipid from dT DP-L-rhamnose and beta -hydroxydecanoyl-beta -hydroxydecanoate, whereas di- rhamnolipid is produced from mono-rhamnolipid and dTDP-L-rhamnose. We repor t here the molecular characterization of rhlC, a gene encoding the rhamnosy ltransferase involved in di-rhamnolipid (L-rhamnose-L-rhamnose-beta -hydrox ydecanoyl-beta -hydroxydecanoate) production in P. aeruginosa. RhlC is a pr otein consisting of 325 amino acids with a molecular mass of 35.9 kDa. It c ontains consensus motifs that are found in other glycosyltransferases invol ved in the transfer of l-rhamnose to nascent polymer chains. To verify the biological function of RhlC, a chromosomal mutant, RTII-2, was generated by insertional mutagenesis and allelic replacement. This mutant was unable to produce di-rhamnolipid, whereas mono-rhamnolipid was unaffected. In contra st, a null rhlA mutant (PAO1-rhlA) was incapable of producing both mono- an d di-rhamnolipid. Complementation of mutant RTII-2 with plasmid pRTII-26 co ntaining rhlC restored the level of di-rhamnolipid production in the recomb inant to a level similar to that of the wild-type strain PAO1. The rhlC gen e was located in an operon with an upstream gene (PA1131) of unknown functi on. A sigma (54)-type promoter for the PA1131-rhlC operon was identified, a nd a single transcriptional start site was mapped. Expression of the PA1131 -rhlC operon was dependent on the P. aeruginosa rhl quorum-sensing system, and a well-conserved lux box was identified in the promoter region. The gen etic regulation of rhlC by RpoN and RhlR was in agreement with the observed increasing RhlC rhamnosyltransferase activity during the stationary phase of growth. This is the first report of a rhamnosyltransferase gene responsi ble for the biosynthesis of di-rhamnolipid.