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Burkholderia pseudomallei is the causative agent of the lethal disease melioidosis. This bacterium infects animals and humans and is increasingly resistant to multiple antibiotics. Recently, genes associated with survival of the bacterium in the infected host have been identified. One of these genes, bpsl0741, is annotated as a hypothetical protein of 185 amino acids. Here, recombinant BPSL0741 (rBPSL0741) protein was expressed, purified, verified by mass spectrometry, crystallized and analyzed by X‐ray diffraction. rBPSL0741 was crystallized by vapor diffusion using a reservoir solution consisting of 0.2 M ammonium acetate, 0.1 M sodium acetate trihydrate pH 4.6, 30% PEG 4000. The crystals diffracted to 2.1 Å resolution using an in‐house X‐ray diffractometer and belonged to an orthorhombic space group, with unit‐cell parameters a = 62.92, b = 64.57, c = 89.16 Å. The Matthews coefficient (VM) was calculated to be 2.18 Å3 Da−1, suggesting the presence of two molecules per asymmetric unit and an estimated solvent content of 43.5%. The crystal was deemed to be suitable for further structural studies, which are currently ongoing. [ABSTRACT FROM AUTHOR]

Introduction: Burkholderia pseudomallei, a soil-dwelling microbe that infects humans and animals is the cause of the fatal disease melioidosis. The molecular mechanisms that underlie B. pseudomallei's versatility to survive within a broad range of environments are still not well defined. Methods: We used the genome-wide screening tool TraDIS (Transposon Directed Insertion-site Sequencing) to identify B. pseudomallei essential genes. Transposon-flanking regions were sequenced and gene essentiality was assessed based on the frequency of transposon insertions within each gene. Transposon mutants were grown in LB and M9 minimal medium to determine conditionally essential genes required for growth under laboratory conditions. The Caenorhabditis elegans infection model was used to assess genes associated with in vivo B. pseudomallei survival. Transposon mutants were fed to the worms, recovered from worm intestines, and sequenced. Two selected mutants were constructed and evaluated for the bacteria's ability to survive and proliferate in the nematode intestinal lumen. Results: Approximately 500,000 transposon-insertion mutants of B. pseudomallei strain R15 were generated. A total of 848,811 unique transposon insertion sites were identified in the B. pseudomallei R15 genome and 492 genes carrying low insertion frequencies were predicted to be essential. A total of 96 genes specifically required to support growth under nutrient-depleted conditions were identified. Genes most likely to be involved in B. pseudomallei survival and adaptation in the C. elegans intestinal lumen, were identified. When compared to wild type B. pseudomallei, a Tn5 mutant of bpsl2988 exhibited reduced survival in the worm intestine, was attenuated in C. elegans killing and showed decreased colonization in the organs of infected mice. Discussion: The B. pseudomallei conditional essential proteins should provide further insights into the bacteria's niche adaptation, pathogenesis, and virulence. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters mediate bacteria uptake or export of a variety of solutes across biological membranes. Bacterial uptake of the monosaccharides is important as a source of carbohydrate building blocks that contribute to the bacteria's major structure. Burkholderia pseudomallei is the etiological agent of melioidosis and within its genome, 33 genes related to monosaccharide ABC transporters have been predicted. The presence of these transporters is believed to assist in bacterial survival and adaptation in various environments. Despite a large number of genes in the genome, most of these systems have yet to be characterized, including the bpss0140-bpss0142 operon. Here, we predicted the 3D structure of each protein encoded by bpss0140-0142 and identified the specifically associated monosaccharides. In silico analyses of the structures demonstrated that BPSS0140 is a sugar-binding protein, BPSS0141 is a transmembrane permease and BPSS0142 is an ATPase. Through protein structure modeling and protein-ligand docking, several specific monosaccharide sugars were found to interact with the BPSS0140-BPSS0142 ABC transporter. To validate the in silico prediction, knock-out mutants for each of the genes were constructed. A growth profile between wild-type and mutants in an M9 medium supplemented with glucose, fructose, ribose, and galactose as predicted from the protein-ligand docking was then performed. The growth of mutants decreased significantly compared to the wild-type bacteria when grown in M9 supplemented with fructose as the sole carbon source indicating that this transporter is potentially the main fructose transporter in B. pseudomallei. [ABSTRACT FROM AUTHOR]

Abstract Background The sRNAs of bacterial pathogens are known to be involved in various cellular roles including environmental adaptation as well as regulation of virulence and pathogenicity. It is expected that sRNAs may also have similar functions for Burkholderia pseudomallei, a soil bacterium that can adapt to diverse environmental conditions, which causes the disease melioidosis and is also able to infect a wide variety of hosts. Results By integrating several proven sRNA prediction programs into a computational pipeline, available Burkholderia spp. genomes were screened to identify sRNA gene candidates. Orthologous sRNA candidates were then identified via comparative analysis. From the total prediction, 21 candidates were found to have Rfam homologs. RT-PCR and sequencing of candidate sRNA genes of unknown functions revealed six putative sRNAs which were highly conserved in Burkholderia spp. and two that were unique to B. pseudomallei present in a normal culture conditions transcriptome. The validated sRNAs include potential cis-acting elements associated with the modulation of methionine metabolism and one B. pseudomallei-specific sRNA that is expected to bind to the Hfq protein. Conclusions The use of the pipeline developed in this study and subsequent comparative analysis have successfully aided in the discovery and shortlisting of sRNA gene candidates for validation. This integrated approach identified 29 B. pseudomallei sRNA genes - of which 21 have Rfam homologs and 8 are novel.