SummaryPseudomonas 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 one (mono-rhamnolipid) or two (di-rhamnolipid) L-rhamnose molecules. Rhamnosyltransferase 1 (RhlAB) catalyses the synthesis of mono-rhamnolipid from dTDP-L-rhamnose and b-hydroxydecanoyl-b-hydroxydecanoate, whereas di-rhamnolipid is produced from mono-rhamnolipid and dTDP-L-rhamnose. We report here the molecular characterization of rhlC, a gene encoding the rhamno-production in P. aeruginosa. RhlC is a protein consisting of 325 amino acids with a molecular mass of 35.9 kDa. It contains consensus motifs that are found in other glycosyltransferases involved 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 contrast, a null rhlA mutant (PAO1-rhlA) was incapable of producing both mono-and di-rhamnolipid. Complementation of mutant RTII-2 with plasmid pRTII-26 containing rhlC restored the level of di-rhamnolipid production in the recombinant to a level similar to that of the wild-type strain PAO1. The rhlC gene was located in an operon with an upstream gene (PA1131) of unknown function. A s 54 -type promoter for the PA1131±rhlC operon was identified, and 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 genetic 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 responsible for the biosynthesis of di-rhamnolipid.
Pseudomonas aeruginosa produces exoproducts correlated with its pathogenicity. One of these virulence-associated traits is the surfactant rhamnolipid. The production of alginate and lipopolysaccharide (LPS) are also of importance for P. aeruginosa virulence. The product of the algC gene (which is involved in alginate production through its phosphomannomutase activity and in LPS synthesis through its phosphoglucomutase activity) participates in rhamnolipid production, presumably catalyzing the first step in the deoxy-thymidine-diphospho-L-rhamnose (dTDP-L-rhamnose) pathway, the conversion of glucose-6-phosphate to glucose-1-phosphate. Other structural alg genes, encoded in the alg operon, are not involved in rhamnolipid nor LPS production. These results show that the AlgC protein plays a central role in the production of the three P. aeruginosa virulence-associated saccharides: alginate, LPS and rhamnolipid. ß
Pseudomonas aeruginosa produces the biosurfactants rhamnolipids and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs). In this study, we report the production of one family of rhamnolipids, specifically the monorhamnolipids, and of HAAs in a recombinant Escherichia coli strain expressing P. aeruginosa rhlAB operon. We found that the availability in E. coli of dTDP-L: -rhamnose, a substrate of RhlB, restricts the production of monorhamnolipids in E. coli. We present evidence showing that HAAs and the fatty acid dimer moiety of rhamnolipids are the product of RhlA enzymatic activity. Furthermore, we found that in the recombinant E. coli, these compounds have the same chain length of the fatty acid dimer moiety as those produced by P. aeruginosa. These data suggest that it is RhlAB specificity, and not the hydroxyfatty acid relative abundance in the bacterium, that determines the profile of the fatty acid moiety of rhamnolipids and HAAs. The rhamnolipids level produced in recombinant E. coli expressing rhlAB is lower than the P. aeruginosa level and much higher than those reported by others in E. coli, showing that this metabolic engineering strategy lead to an increased rhamnolipids production in this heterologous host.
The gene coding for inulosucrase in Leuconostoc citreum CW28, islA, was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme catalyzed inulin synthesis from sucrose like the wild-type enzyme. Inulosucrase presents an unusual structure: its N-terminal region is similar to the variable region of glucosyltransferases, its catalytic domain is similar to fructosyltransferases from various microorganisms, and its C-terminal domain presents similarity to the glucan binding domain from alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355. From sequence comparison, it was found that this fructosyltransferase is a natural chimeric enzyme resulting from the substitution of the catalytic domain of alternansucrase by a fructosyltransferase. Two different forms of the islA gene truncated in the C-terminal glucan binding domain were successfully expressed in E. coli and retained their ability to synthesize inulin but lost thermal stability. This is the first report of an inulosucrase bearing structural features of both glucosyltransferases and fructosyltransferases.
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