Burkholderia thailandensis harbors two identical rhl gene clusters responsible for the biosynthesis of rhamnolipids

Danielle Dubeau 1 Eric Dziel 1 Donald E Woods 0 Franois Lpine 1 0 Department of Microbiology and Infectious Diseases, Faculty of Medicine, University of Calgary Health Sciences Centre , Calgary, Alberta, T2N 4N1 , Canada 1 INRS-Institut Armand Frappier , Laval, Quebec, H7V 1B7 , Canada Background: Rhamnolipids are surface active molecules composed of rhamnose and hydroxydecanoic acid. These biosurfactants are produced mainly by Pseudomonas aeruginosa and have been thoroughly investigated since their early discovery. Recently, they have attracted renewed attention because of their involvement in various multicellular behaviors. Despite this high interest, only very few studies have focused on the production of rhamnolipids by Burkholderia species. Results: Orthologs of rhlA, rhlB and rhlC, which are responsible for the biosynthesis of rhamnolipids in P. aeruginosa, have been found in the non-infectious Burkholderia thailandensis, as well as in the genetically similar important pathogen B. pseudomallei. In contrast to P. aeruginosa, both Burkholderia species contain these three genes necessary for rhamnolipid production within a single gene cluster. Furthermore, two identical, paralogous copies of this gene cluster are found on the second chromosome of these bacteria. Both Burkholderia spp. produce rhamnolipids containing 3-hydroxy fatty acid moieties with longer side chains than those described for P. aeruginosa. Additionally, the rhamnolipids produced by B. thailandensis contain a much larger proportion of dirhamnolipids versus monorhamnolipids when compared to P. aeruginosa. The rhamnolipids produced by B. thailandensis reduce the surface tension of water to 42 mN/m while displaying a critical micelle concentration value of 225 mg/L. Separate mutations in both rhlA alleles, which are responsible for the synthesis of the rhamnolipid precursor 3-(3-hydroxyalkanoyloxy)alkanoic acid, prove that both copies of the rhl gene cluster are functional, but one contributes more to the total production than the other. Finally, a double rhlA mutant that is completely devoid of rhamnolipid production is incapable of swarming motility, showing that both gene clusters contribute to this phenotype. Conclusions: Collectively, these results add another Burkholderia species to the list of bacteria able to produce rhamnolipids and this, by the means of two identical functional gene clusters. Our results also demonstrate the very impressive tensio-active properties these long-chain rhamnolipids possess in comparison to the well-studied short-chain ones from P. aeruginosa. - Background Rhamnolipids are surface-active compounds that have been extensively studied since their early identification in Pseudomonas aeruginosa cultures in the late 1940s [1]. However, it was only in the mid 1960s that the structure of a rhamnolipid molecule was first reported [2]. Due to their excellent tensioactive properties, low toxicity and high biodegradability, these biosurfactants are promising candidates for a variety of industrial applications as well as bioremediation processes [3,4]. Furthermore, rhamnolipids have recently received renewed attention because of their involvement in P. aeruginosa multicellular behavior, such as biofilm development and swarming motility [57]. Rhamnolipids are also considered virulence factors as they interfere with the normal functioning of the tracheal ciliary system and are found in sputa of cystic fibrosis (CF) patients infected by P. aeruginosa [8-10]. Moreover, rhamnolipids inhibit the phagocytic response of macrophages and are known as the heat-stable extracellular hemolysin produced by P. aeruginosa [11,12]. These amphiphilic molecules are usually produced by P. aeruginosa as a complex mixture of congeners composed of one or two molecules of L-rhamnose coupled to a 3hydroxyalkanoic acid dimer, the most abundant being Lrhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rha-C10-C10) and L-rhamnosyl-L-rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rha-Rha-C10-C10) [1315]. The biosynthetic pathway of rhamnolipids has been the subject of many studies that have demonstrated the implication of three crucially important genes, rhlA, rhlB and rhlC. The first enzyme, RhlA, is responsible for the interception of two molecules of -hydroxydecanoyl-ACP, an intermediate in the de novo fatty acid biosynthesis cycle, to produce 3-hydroxyalkanoic acid dimers, known as 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) [16,17]. The second reaction, implicating the membranebound RhlB rhamnosyltransferase, uses dTDP-L-rhamnose to add the first rhamnose moiety to an HAA molecule, thus forming a monorhamnolipid (L-rhamnosyl-3hydroxyalkanoyl-3-hydroxyalkanoate). Finally, an additional rhamnosyltransferase, RhlC, couples a second rhamnose molecule to a monorhamnolipid by the means of another dTDP-L-rhamnose, producing the final dirhamnolipid (L-rhamnosyl-L-rhamnosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate) [18,19]. Previously assigned to the Pseudomonas genus, Burkholderia spp. are attracting increasing interest because of their involvement in human infections. Burkholderia is best known for its pathogenic members like B. pseudomallei, the causative agent of melioidosis, as well as the opportunistic pathogens belonging to the B. cepacia complex [20,21]. Two studies have reported evidence of the production of a single dirhamnolipid by B. pseudomallei as well as by another member of the same genus, B. plantarii [22,23]. Here, we investigate the production of rhamnolipids by B. thailandensis, a non-infectious Burkholderia species closely related to B. pseudomallei [24], and by B. pseudomallei itself. In contrast to the mandated B. pseudomallei guidelines, an advantage to studying B. thailandensis is that it does not require biosafety level 3 conditions, and there is no restriction on the use of antibiotic-resistance markers for its genetic manipulation. In addition, numerous studies have shown to what extreme level these two Burkholderia species are closely related from a genetic point of view and that B. thailandensis can serve as a surrogate for studying many different traits, including physiological characteristics as well as pathogenic factors in regards to B. pseudomallei [25,26]. Results Presence of rhlABC homologs in B. thailandensis and B. pseudomallei Following a nucleotide and protein similarity search using algorithms blastn and blastp with standard parameters http://blast.ncbi.nlm.nih.gov/Blast.cgi, respectively, in sequenced B. thailandensis and B. pseudomallei genome sequences, close orthologs of the P. aeruginosa rhamnolipid-biosynthesis genes rhlA, rhlB and rhlC were found in all associated strains as gene clusters. Interestingly, both species possess two 100% identical rhl gene clusters on their second chromosome (Figure 1). A search in the partially sequenced genome of B. pseudomallei 1026b (Genomes OnLine Database; http://www.genomeson line.org), the strain used in this study, i (...truncated)