Banat, Ibrahim

Marchant, Roger

Twigg, Matthew

English

Rhamnolipids (RL) were first shown to be produced by the opportunistic pathogen Pseudomonas aeruginosa. RL production is achieved via a biosynthesis pathway comprising three separate enzymes; RhlA, responsible for the synthesis of the fatty acid dimer precursor moieties (HAA); RhlB, a rhamnosyltransferase enzyme which conjugates HAA to dTDP-L-rhamnose to form mono-RL and RhlC, a second rhamnosyltransferase enzyme that adds a second rhamnose to form di-RL. Within P. aeruginosa the genes encoding the first two enzymes, rhlA and rhlB, are present in a single operon alongside genes encoding an AHL-mediated quorum sensing system rhlR and rhlI. The gene encoding the last enzyme, rhlC, is located approx. 1 Mb downstream of this operon. Since the discovery of RL synthesis by P. aeruginosa other bacterial species have also been shown to synthesise RLs, notably Burkholderia thailandensis and Burkholderia pseudomallei. Interestingly the RL biosynthesis genes within Burkholderia only show 40% similarity to those of P. aeruginosa, are located together in a single operon and are duplicated within the genome to give two functional copies of each gene. Recently we have shown RL synthesis in two marine bacteria; Pseudomonas sp. MCTG214(3b1) and Marinobacter sp. MCTG107b. In the Pseudomonas sp. we identified rhlA and rhlB homologues with 99% similarity to those of P. aeruginosa, however we have yet to identify any rhlC homologue. This presents a paradox as we have demonstrated di-RL synthesis by this strain. RL synthesis within the Marinobacter sp. presents an even greater paradox as we have yet to identify any RL synthase homologs. Here we explain the molecular differences in RL synthase homologues, the effects that these differences may create and try to explain the apparent lack of synthesis genes in the two marine strains. We also discus the methods used to investigate the molecular mechanisms of RL synthesis

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1 Zhu, K. & Rock, C.O. (2008). J. Bacteriol. 190 (9): 3147 4 Soberón-Chávez, G., Lépine, F. & Déziel, E. (2005). App Micro Biotech 68 (6): 718 7 Winsor, G.L. Khaira, B. Van Rossum, T. et al (2008). Bioinformatics 24 (23): 28032 Rahim, R., Ochsner, U.A., Olvera, C. et al. (2001). Mol Microbiol 40 (3): 708 5 Funston, S. Tsaous, K. Rudden, M. et al. (2016). App Micro Biotech 100 (18): 7945 8 Twigg, M.S., Tripathi,. Zompra, A. et al. (2018). App Micro Biotech 102 (19): 85373 Ochsner U.A., Fiechter, A. & Reiser, J. (1994). J Biol Chem 269 (31): 19787 6 Winsor, G.L., Griffiths, E.J. Lo, R. et al. (2016). Nucleic Acids Res 44 (D1): D646Pseudomonas aeruginosa PAO1Pseudomonas sp. MCTG214(3b1)rhlI rhlR rhlB rhlArhlC2.67 Mbp3’ 5’rhlB rhlA3’ 5’RhlC1rhlB1rhlA1Burkholderia thailandensis & B. pseudomalleiRhlC2rhlB2rhlA23’3’5’5’The Riddle of Rhamnolipid Molecular BiosynthesisMatthew S. Twigg*, Roger Marchant & Ibrahim M. Banat.School of Biomedical Sciences, Ulster University, Coleraine, Northern Ireland BT52 1SA, UK.Rhamnolipids (RLs) are the best characterised glycolipid biosurfactants. Rhamnolipid biosynthesis is actioned by three enzymes which were first discovered in the classic RL producing bacterium Pseudomonas aeruginosa, fig 1.RhlA – produces fatty acid dimers by intercepting intermediates in the de novo fatty acid synthesis pathway (1). RhlB – Conjugates a single rhamnose moiety onto the fatty acid dimers synthesised by RhlA forming  mono-RLs (2)RhlC – Using mono-RL as a substrate conjugates a second rhamnose moiety forming di-RLs (3)   The enzymes involved with RL biosynthesis are encoded by three genes; rhlA,rhlB and rhlC. Orthologues of these gene have been identified in a number ofspecies of bacteria belonging to the Pseudomonas & Burkholderia genera (4,5).The arrangement of these genes can however be dramatically differentdepending on species, fig 2.We recently identified a marine bacterium phylogenetically similar toPseudomonas mendocina with the ability to synthesise RLs. Using primersbased on the sequences of other RL producing Pseudomonas strains weamplified DNA fragments matching the size of rhlA and rhlB, fig 3 (8).DNA sequencing showed these genes to be 99% homologous to rhlA andrhlB of P. aeruginosa and genomic sequencing showed these genes to belocated together in the chromosome, fig 2 (8). Interestingly our PCRscreening did not identify any rhlC ortholog in this strain presenting aparadox on how this strain synthesises the di-RLs observed in chemicalanalysis of culture extracts (8). Based on our genetic analysis we postulatethat this strain may have obtained its RL synthesis genes via horizontalgene transfer and possesses an as yet unidentified gene responsible fordi-RL synthesis.Fig. 2 The genomic arrangement of RL biosynthesis genes in four RL producing speciesbelonging to the Pseudomonas and Burkholderia genera. Diagram generated based on dataprovided in Pseudomonas and Burkholderia genome databases (6,7).*Corresponding Author – m.twigg@ulster.ac.ukWe have also identified RL production in another marine bacterial strainbelonging to the genus Marinobacter. PCR screening using primers for boththe Pseudomonas and Burkholderia rhl genes have not yielded any amplificationproducts. We therefore predict that RL synthesis is being carried out either bya novel biochemical pathway or, more likely, by RL synthases structurallydifferent from those already elucidated. Therefore we have developed and areemploying a strategy for the detection of novel RL synthases and the genesencoding them, fig 4.The first difference between the RL synthesis genes in Burkholderia speciescompared to that in P. aeruginosa is that Burkholderia possesses two identicaland functional copies of each gene which are located separately on thechromosome, fig 2 (6,7).A second key difference is the level of sequence homology of these twogenera. At both the genetic and amino acid levels the two genera only shareapprox. 40% homology. This sequence homology rises significantly whenyou compare RL producing species belonging to the same genus i.e B.thailandensis and B. pseudomallei.These differences in both genomic arrangement and sequence homologymay indicate early divergence in RL biosynthesis evolution and could alsoaccount for the differences in RL congener profiles observed in these twogenera.Fig. 3 PCR amplification using primers for rhlA (A) and rhlB (B) and genomic DNAextracted from Pseudomonas sp. MCTG214(3b1) and P. aeruginosa PAO1 resulted inamplicons of matching size. Subsequent DNA sequencing showed the MCTG214(3b1)amplicons to posses 99% identity to rhlA and rhlB of P. aeruginosa (8).Fatty Acid De NovoPathwayRhlAHAARhlCMono-rhamnolipidDi-rhamnolipidD-Glucose-5-PdTDP-L-RhamnoseD-Glucose-6-PRmlA-DAlgCRhlBFig. 1 Diagram depicting the biosynthesis of both monoand di-RLs by enzymes RhlA, RhlB and RhlC (1-3)Fig. 4 Diagram illustrating a rigorous experimentalpipeline for the detection of novel RL synthasegenes/ proteins.

The Riddle of Rhamnolipid Molecular Biosynthesis

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The Riddle of Rhamnolipid Molecular Biosynthesis

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