As we have previously reported for P aeruginosa [14],

As we have previously reported for P. aeruginosa [14], LCZ696 manufacturer within each isomeric pair, the rhamnolipid congener with the shortest chain adjacent to the sugar moiety is more abundant. To verify whether the rhamnolipids produced by B. thailandensis share this characteristic, they were subjected to an enzymatic hydrolysis of their rhamnose groups with naringinase [30] to produce the corresponding HAAs. The same stoichiometrical preference was confirmed. Figure 2 Congener analysis of rhamnolipids from B. thailandensis. A) Mass spectra of the fragmented m/z 587, 615 and 643 pseudomolecular ions of congeners Rha-C12-C14, Rha-C14-C12, Rha-C14-C14, Rha-C14-C16 and Rha-C16-C14.

B) Schematic representation of observed fragmentation patterns of a monorhamnolipid. click here C) Daughter ions generated by fragmentation of the specified congeners. With these results in hand, we investigated the potential of the highly genetically related species B. pseudomallei to produce a range of rhamnolipids other than the previously described Rha-Rha-C14-C14. Figure 3 shows the production of the most abundant rhamnolipids by this pathogen. The same long-chain bearing congeners found in B. thailandensis were also discovered in B. pseudomallei, including the dominant Rha-Rha-C14-C14.

Figure 3 Production of the most abundant dirhamnolipids in a B. pseudomallei 1026b culture. Bacteria were grown in NB supplemented with 4% glycerol as carbon source. Rhamnolipids were quantified by LC/MS. Critical Micelle Concentration (CMC) and surface tension assays To investigate the potential of the long-chain rhamnolipids produced by Burkholderia species for lowering surface tension, the critical micelle concentration of this mixture of rhamnolipid congeners was established (Figure 4). At the CMC of about 250 mg/L, the surface tension is lowered to 43 mN/m. Figure 4 Surface tension and CMC value. Surface tension of the total mixture of rhamnolipids and HAAs extracted from a B. thailandensis E264 culture. Each data point shows the mean of triplicate measurements. Error

bars represent the Standard Deviation (SD). Oxalosuccinic acid Both rhlA alleles are functional and necessary for maximal rhamnolipid production The contribution to rhamnolipid production of the two identical rhl gene clusters found on the B. thailandensis genome was tested by creating single ΔrhlA mutants for each allele, as well as a double ΔrhlA mutant. These three mutants were then investigated for their ability to produce rhamnolipids (Figure 5). Five sets of replicates confirmed that the B. thailandensis ΔrhlA1 mutant produces more rhamnolipids than the ΔrhlA2 mutant. The rhamnolipids produced by each of these mutants are composed of the same congeners in the same proportions as the wild type strain and only a quantitative difference is observed.

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