Project description:Bacterial acyl-homoserine lactones upregulated an uncharacterized gene cluster (bta) in Burkholderia thailandensis E264 to produce an uncharacterized polar antibiotic. The antibiotic is identified as a mixture of four bactobolins. Annotation of the bta cluster allows us to propose a biosynthetic scheme for bactobolin and reveals unusual enzymatic reactions for further study.
Project description:Three new bicyclic depsipeptides, which are related to the previously reported thailandepsins A (1), B (2) and C (3), were discovered from the culture broth of Burkholderia thailandensis E264 when supplemented with amino acid precursors, and were subsequently named as thailandepsins D (4), E (5) and F (6), respectively. Enzyme assays showed that 1-6 are potent histone deacetylase (HDAC) inhibitors, particularly toward HDAC1 which represents class I human HDACs.
Project description:Rhamnolipid production was monitored for a period of 216 h using different substrates in Pseudomonas aeruginosa PAO1 and Burkholderia thailandensis E264 which showed comparable crude yields attained by both after 216 h. The crude yield for P. aeruginosa, however, was significantly higher at the early stages of fermentation (72 or 144 h). Additionally, P. aeruginosa produced rhamnolipid with odd and even carbon chain lipid moieties using odd carbon chain fatty acid substrates (up to 45.97 and 67.57%, respectively). In contrast, B. thailandensis produced rhamnolipid with predominantly even carbon chain lipid moieties (up to 99.26). These results indicate the use of the fatty acid synthesis (FAS II) pathway as the main source of lipid precursors in rhamnolipid biosynthesis by B. thailandensis. Isotope tracing using 0.25% stearic acid - d 35 +?1% glycerol as carbon substrate showed a single pattern of deuterium incorporation: with predominantly less than 15 deuterium atoms incorporated into a single Di-C14-C14 rhamnolipid molecule. This further indicates that the FAS II pathway is the main source of the lipid precursor in rhamnolipid biosynthesis by B. thailandensis. The pathogenicity of these strains was also assessed, and results showed that B. thailandensis is significantly less pathogenic than P. aeruginosa with an LC50 at 24 h >?2500, approximately three logs higher than P. aeruginosa using the Galleria mellonella larva model.
Project description:Natural product gene clusters are often tightly regulated, resulting in gene cluster silencing in laboratory fermentation studies. The systematic overexpression of transcription factors (TFs) associated with biosynthetic gene clusters found in the genome of Burkholderia thailandensis E264 identified a set of TFs that, when overexpressed, alter the secondary metabolome of this bacterium. The isolation and characterization of burkholdacs A and B, two new acyldepsitripeptide histone deacetylase inhibitors produced by B. thailandensis overexpressing the TF bhcM, is reported.
Project description:Burkholderia thailandensis E264 is a potential non-pathogenic substitute for producing rhamnolipid biosurfactant, replacing the pathogenic Pseudomonas aeruginosa. However, it has low rhamnolipid production and longer fermentation time. We have earlier suggested that media supplementation with exogenous quorum sensing (QS) molecules could lead to early onset of biosynthesis and increased rhamnolipid yield. Here, we assessed the effect of single, double or triple mutations in the various QS systems of B. thailandensis on rhamnolipid production, with the view to see which system(s) have the most impact on rhamnolipid yield and subsequently use the QS molecule to potentially increase yield in the wild-type B. thailandensis. The triple mutant strain had a rhamnolipid yield of 4.46 ± 0.345 g/l at 240 h of fermentation which was significantly higher than that of the wild type (0.94 ± 0.06 g/l), an unexpected outcome. To gain more insight as to how this might occur, we studied substrate metabolism and energy storage in the form of polyhydroxyalkanoate (PHA) by both the triple mutant and the wild type. We observed increased glycerol metabolism and reduced PHA production in the triple mutant compared with the wild type. Glycerol concentration at 240 h and maximum PHA productivity (g/gDCB) were 8.76 g/l or 16.19 g/l and 21.80% or 31.4% in either the triple mutant or the wild type respectively. Complementation of the triple-mutant cultures with exogenous QS molecules restored rhamnolipid production to similar levels as the wild type. QS therefore is a potential target for increased rhamnolipid production in B. thailandensis.
Project description:The bacterial SOS response is a well-characterized regulatory network encoded by most prokaryotic bacterial species and is involved in DNA repair. In addition to nucleic acid repair, the SOS response is involved in pathogenicity, stress-induced mutagenesis, and the emergence and dissemination of antibiotic resistance. Using high-throughput sequencing technology (SOLiD RNA-Seq), we analyzed the Burkholderia thailandensis global SOS response to the fluoroquinolone antibiotic, ciprofloxacin (CIP), and the DNA-damaging chemical, mitomycin C (MMC). We demonstrate that a B. thailandensis recA mutant (RU0643) is ∼4-fold more sensitive to CIP in contrast to the parental strain B. thailandensis DW503. Our RNA-Seq results show that CIP and MMC treatment (P < 0.01) resulted in the differential expression of 344 genes in B. thailandensis and 210 genes in RU0643. Several genes associated with the SOS response were induced and include lexA, uvrA, dnaE, dinB, recX, and recA. At the genome-wide level, we found an overall decrease in gene expression, especially for genes involved in amino acid and carbohydrate transport and metabolism, following both CIP and MMC exposure. Interestingly, we observed the upregulation of several genes involved in bacterial motility and enhanced transcription of a B. thailandensis genomic island encoding a Siphoviridae bacteriophage designated E264. Using B. thailandensis plaque assays and PCR with B. mallei ATCC 23344 as the host, we demonstrate that CIP and MMC exposure in B. thailandensis DW503 induces the transcription and translation of viable bacteriophage in a RecA-dependent manner. This is the first report of the SOS response in Burkholderia spp. to DNA-damaging agents. We have identified both common and unique adaptive responses of B. thailandensis to chemical stress and DNA damage.
Project description:<h4>Background</h4>Burkholderia pseudomallei is a human pathogen causing severe infections in tropical and subtropical regions and is classified as a bio-threat agent. B. thailandensis strain E264 has been proposed as less pathogenic surrogate for understanding the interactions of B. pseudomallei with host cells.<h4>Results</h4>We show that, unlike B. thailandensis strain E264, the pattern of growth of B. thailandensis strain E555 in macrophages is similar to that of B. pseudomallei. We have genome sequenced B. thailandensis strain E555 and using the annotated sequence identified genes and proteins up-regulated during infection. Changes in gene expression identified more of the known B. pseudomallei virulence factors than changes in protein levels and used together we identified 16% of the currently known B. pseudomallei virulence factors. These findings demonstrate the utility of B. thailandensis strain E555 to study virulence of B. pseudomallei.<h4>Conclusions</h4>A weakness of studies using B. thailandensis as a surrogate for B. pseudomallei is that the strains used replicate at a slower rate in infected cells. We show that the pattern of growth of B. thailandensis strain E555 in macrophages closely mirrors that of B. pseudomallei. Using this infection model we have shown that virulence factors of B. pseudomallei can be identified as genes or proteins whose expression is elevated on the infection of macrophages. This finding confirms the utility of B. thailandensis strain E555 as a surrogate for B. pseudomallei and this strain should be used for future studies on virulence mechanisms.
Project description:Bacteria produce a diverse array of secondary metabolites that have been invaluable in the clinic and in research. These metabolites are synthesized by dedicated biosynthetic gene clusters (BGCs), which assemble architecturally complex molecules from simple building blocks. The majority of BGCs in a given bacterium are not expressed under normal laboratory growth conditions, and our understanding of how they are silenced is in its infancy. Here, we have addressed this question in the Gram-negative model bacterium Burkholderia thailandensis E264 using genetic, transcriptomic, metabolomic, and chemical approaches. We report that a previously unknown, quorum-sensing-controlled LysR-type transcriptional regulator, which we name ScmR (for secondary metabolite regulator), serves as a global gatekeeper of secondary metabolism and a repressor of numerous BGCs. Transcriptionally, we find that 13 of the 20 BGCs in B. thailandensis are significantly (threefold or more) up- or down-regulated in a scmR deletion mutant (?scmR) Metabolically, the ?scmR strain displays a hyperactive phenotype relative to wild type and overproduces a number of compound families by 18- to 210-fold, including the silent virulence factor malleilactone. Accordingly, the ?scmR mutant is hypervirulent both in vitro and in a Caenorhabditis elegans model in vivo. Aside from secondary metabolism, ScmR also represses biofilm formation and transcriptionally activates ATP synthesis and stress response. Collectively, our data suggest that ScmR is a pleiotropic regulator of secondary metabolism, virulence, biofilm formation, and other stationary phase processes. A model for how the interplay of ScmR with pathway-specific transcriptional regulators coordinately silences virulence factor production is proposed.