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Acta Veterinaria Scandinavica Mar 2015A frame-shift mutation in the flagellum motor gene motB coding for the chemotaxis MotB protein of Burkholderia mallei has been utilized to design a conventional duplex...
BACKGROUND
A frame-shift mutation in the flagellum motor gene motB coding for the chemotaxis MotB protein of Burkholderia mallei has been utilized to design a conventional duplex PCR assay with fluorescent labelled primers.
FINDINGS
Species specificity was tested with a panel of 13 Burkholderia type strains. A total of 41 B. mallei field strains, 36 B. pseudomallei field strains, and 1 B. thailandensis field strain from different geographic regions were tested and correctly identified. Testing of 55 non-Burkholderia bacterial species revealed 100% specificity of the assay. The minimum detection limit was 1 pg DNA or 160 GE for B. mallei and 130 GE for B. pseudomallei, respectively.
CONCLUSIONS
This assay enables the clear distinction between B. mallei and B. pseudomallei/B. thailandensis.
Topics: Bacterial Proteins; Burkholderia mallei; Burkholderia pseudomallei; DNA Primers; Real-Time Polymerase Chain Reaction; Species Specificity
PubMed: 25887130
DOI: 10.1186/s13028-015-0104-4 -
Molecules (Basel, Switzerland) Feb 2023Due to the increase in multidrug-resistant microorganisms, the investigation of novel or more efficient antimicrobial compounds is essential. The World Health... (Review)
Review
Due to the increase in multidrug-resistant microorganisms, the investigation of novel or more efficient antimicrobial compounds is essential. The World Health Organization issued a list of priority multidrug-resistant bacteria whose eradication will require new antibiotics. Among them, , and Enterobacteriaceae are in the "critical" (most urgent) category. As a result, major investigations are ongoing worldwide to discover new antimicrobial compounds. , specifically sensu stricto, is recognized as an antimicrobial-producing group of species. Highly dissimilar compounds are among the molecules produced by this genus, such as those that are unique to a particular strain (like compound CF66I produced by CF-66) or antimicrobials found in a number of species, e.g., phenazines or ornibactins. The compounds produced by include N-containing heterocycles, volatile organic compounds, polyenes, polyynes, siderophores, macrolides, bacteriocins, quinolones, and other not classified antimicrobials. Some of them might be candidates not only for antimicrobials for both bacteria and fungi, but also as anticancer or antitumor agents. Therefore, in this review, the wide range of antimicrobial compounds produced by is explored, focusing especially on those compounds that were tested in vitro for antimicrobial activity. In addition, information was gathered regarding novel compounds discovered by genome-guided approaches.
Topics: Burkholderia; Anti-Infective Agents; Anti-Bacterial Agents; Burkholderia cepacia; Bacteriocins
PubMed: 36838633
DOI: 10.3390/molecules28041646 -
Acta Crystallographica. Section F,... Mar 2023N-Acetyl-(R)-β-phenylalanine acylase is an enzyme that hydrolyzes the amide bond of N-acetyl-(R)-β-phenylalanine to produce enantiopure (R)-β-phenylalanine. In...
Expression, purification and crystallization of N-acetyl-(R)-β-phenylalanine acylases derived from Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 and structure determination of the Burkholderia enzyme.
N-Acetyl-(R)-β-phenylalanine acylase is an enzyme that hydrolyzes the amide bond of N-acetyl-(R)-β-phenylalanine to produce enantiopure (R)-β-phenylalanine. In previous studies, Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 were isolated as (R)-enantiomer-specific N-acetyl-(R)-β-phenylalanine acylase-producing organisms and the properties of the native enzyme from Burkholderia sp. AJ110349 were characterized. In this study, structural analyses were carried out in order to investigate the structure-function relationships of the enzymes derived from both organisms. The recombinant N-acetyl-(R)-β-phenylalanine acylases were crystallized by the hanging-drop vapor-diffusion method under multiple crystallization solution conditions. The crystals of the Burkholderia enzyme belonged to space group P422, with unit-cell parameters a = b = 112.70-112.97, c = 341.50-343.32 Å, and were likely to contain two subunits in the asymmetric unit. The crystal structure was solved by the Se-SAD method, suggesting that two subunits in the asymmetric unit form a dimer. Each subunit was composed of three domains, and they showed structural similarity to the corresponding domains of the large subunit of N,N-dimethylformamidase from Paracoccus sp. strain DMF. The crystals of the Variovorax enzyme grew as twinned crystals and were not suitable for structure determination. Using size-exclusion chromatography with online static light-scattering analysis, the N-acetyl-(R)-β-phenylalanine acylases were clarified to be dimeric in solution.
Topics: Burkholderia; Crystallization; Crystallography, X-Ray; Phenylalanine
PubMed: 36862095
DOI: 10.1107/S2053230X23000730 -
Journal of Bacteriology Jun 2020The nonpathogenic soil saprophyte is a member of the group, which also comprises the closely related human pathogens and responsible for the melioidosis and glanders...
The nonpathogenic soil saprophyte is a member of the group, which also comprises the closely related human pathogens and responsible for the melioidosis and glanders diseases, respectively. ScmR, a recently identified LysR-type transcriptional regulator in , acts as a global transcriptional regulator throughout the stationary phase and modulates the production of a wide range of secondary metabolites, including -acyl-l-homoserine lactones and 4-hydroxy-3-methyl-2-alkylquinolines and virulence in the nematode worm host model, as well as several quorum sensing (QS)-dependent phenotypes. We have investigated the role of ScmR in strain E264 during the exponential phase. We used RNA sequencing transcriptomic analyses to identify the ScmR regulon, which was compared to the QS-controlled regulon, showing a considerable overlap between the ScmR-regulated genes and those controlled by QS. We characterized several genes modulated by ScmR using quantitative reverse transcription-PCR or mini-CTX- transcriptional reporters, including the oxalate biosynthetic gene required for pH homeostasis, the orphan LuxR-type transcriptional regulator BtaR5-encoding gene, and the ( secretion apparatus) type III secretion system genes essential for both and pathogenicity, as well as the gene itself. We confirmed that the transcription of is under QS control, presumably ensuring fine-tuned modulation of gene expression. Finally, we demonstrated that ScmR influences virulence using the fruit fly model host We conclude that ScmR represents a central component of QS regulatory network. Coordination of the expression of genes associated with bacterial virulence and environmental adaptation is often dependent on quorum sensing (QS). The QS circuitry of the nonpathogenic bacterium , widely used as a model system for the study of the human pathogen , is complex. We found that the LysR-type transcriptional regulator, ScmR, which is highly conserved and involved in the control of virulence/survival factors in the genus, is a global regulator mediating gene expression through the multiple QS systems coexisting in , as well as QS independently. We conclude that ScmR represents a key QS modulatory network element, ensuring tight regulation of the transcription of QS-controlled genes, particularly those required for acclimatization to the environment.
Topics: Acids; Acyl-Butyrolactones; Animals; Bacterial Proteins; Burkholderia; Burkholderia Infections; Caenorhabditis elegans; Drosophila melanogaster; Gene Expression Regulation, Bacterial; Genes, Regulator; Homeostasis; Humans; Hydrogen-Ion Concentration; Male; Quorum Sensing; Virulence
PubMed: 32312745
DOI: 10.1128/JB.00776-19 -
Journal of Natural Products Mar 2020Bactobolin is a hybrid natural product with potent cytotoxic activity. Its production from was reported as part of a collaboration between the Greenberg and Clardy... (Review)
Review
Bactobolin is a hybrid natural product with potent cytotoxic activity. Its production from was reported as part of a collaboration between the Greenberg and Clardy laboratories in 2010. The collaboration sparked a series of studies leading to the discovery of new analogues and associated structure-activity relationships, the identification of the bactobolin biosynthetic gene cluster and assembly of its unusual amino acid building block, the molecular target of and resistance to the antibiotic, and finally an X-ray crystal structure of the ribosome-bactobolin complex. Herein, we review the collaborations that led to our current understanding of the chemistry and biology of bactobolin.
Topics: Benzopyrans; Biological Products; Burkholderia; Molecular Structure; Multigene Family; Ribosomes; Structure-Activity Relationship
PubMed: 32105069
DOI: 10.1021/acs.jnatprod.9b01237 -
Journal of Global Antimicrobial... Mar 2020Members of the Burkholderia cepacia complex (Bcc) have been isolated from various environmental and clinical samples and reportedly pose a threat to human health. Here... (Comparative Study)
Comparative Study
Draft genome sequence reveals co-occurrence of multiple antimicrobial resistance and plant probiotic traits in rice root endophytic strain Burkholderia sp. LS-044 affiliated to Burkholderia cepacia complex.
OBJECTIVES
Members of the Burkholderia cepacia complex (Bcc) have been isolated from various environmental and clinical samples and reportedly pose a threat to human health. Here we examine the draft genome sequence of Burkholderia sp. LS-044, an antibiotic-resistant endophytic strain affiliated to the Bcc (ST895) inhabiting rice (Oryza sativa ssp. japonica cv. Tainung 71) root.
METHODS
Antimicrobial susceptibility of LS-044 was evaluated comparatively with other Burkholderia sp. (CC-Al74 and CC-3XP9) using commercial ATB PSE 5 test strips. The genome of LS-044 was sequenced using an Illumina MiSeq platform. Plant probiotic and antimicrobial resistance genes were screened by Rapid Annotation using Subsystem Technology (RAST), CARD 2017, NCBI and/or UniProt.
RESULTS
Plant-associated members of Bcc (LS-044 and CC-Al74) exhibited greater resistance to the majority of antibiotics tested. The draft genome sequence of LS-044 contained 8.78 Mbp in 62 contigs having a G + C content of 66.5%, 8868 coding sequences and 75 RNAs. The genome harboured genes coding for LysR-type β-lactamase transcription regulator, classes A, C and D β-lactamases, several metal-dependent β-lactamases, antibiotic efflux proteins, and proteins conferring resistance to colistin, streptothricin, colicin and fluoroquinolones. Similarly, it also possessed genes for copper homeostasis, copper-cobalt-zinc-cadmium-chromium resistance and reduction of mercury. Genes involved in flagellar motility, hydrolysis of murein and chitin, production of siderophore and auxin, and metabolism of aromatic compounds were also found.
CONCLUSION
Genome sequence data revealed an interlinked occurrence of plant probiotic traits and antimicrobial resistance in the rice root endophyte LS-044.
Topics: Base Composition; Burkholderia; Drug Resistance, Multiple, Bacterial; Genome Size; Genome, Bacterial; High-Throughput Nucleotide Sequencing; Humans; Molecular Sequence Annotation; Oryza; Plant Roots; Probiotics; Whole Genome Sequencing
PubMed: 31809939
DOI: 10.1016/j.jgar.2019.11.017 -
Communications Biology Mar 2022Burkholderia pseudomallei lethal factor 1 (BLF1) exhibits site-specific glutamine deamidase activity against the eukaryotic RNA helicase, eIF4A, thereby blocking...
Burkholderia pseudomallei lethal factor 1 (BLF1) exhibits site-specific glutamine deamidase activity against the eukaryotic RNA helicase, eIF4A, thereby blocking mammalian protein synthesis. The structure of a complex between BLF1 C94S and human eIF4A shows that the toxin binds in the cleft between the two RecA-like eIF4A domains forming interactions with residues from both and with the scissile amide of the target glutamine, Gln339, adjacent to the toxin active site. The RecA-like domains adopt a radically twisted orientation compared to other eIF4A structures and the nature and position of conserved residues suggests this may represent a conformation associated with RNA binding. Comparison of the catalytic site of BLF1 with other deamidases and cysteine proteases reveals that they fall into two classes, related by pseudosymmetry, that present either the re or si faces of the target amide/peptide to the nucleophilic sulfur, highlighting constraints in the convergent evolution of their Cys-His active sites.
Topics: Amides; Animals; Burkholderia; Eukaryotic Initiation Factor-4A; Glutamine; Humans; Mammals; Protein Biosynthesis
PubMed: 35347220
DOI: 10.1038/s42003-022-03186-2 -
PloS One 2020Bacterial efflux pumps are an important pathogenicity trait because they extrude a variety of xenobiotics. Our laboratory previously identified in silico Burkholderia...
Burkholderia collagen-like protein 8, Bucl8, is a unique outer membrane component of a putative tetrapartite efflux pump in Burkholderia pseudomallei and Burkholderia mallei.
Bacterial efflux pumps are an important pathogenicity trait because they extrude a variety of xenobiotics. Our laboratory previously identified in silico Burkholderia collagen-like protein 8 (Bucl8) in the hazardous pathogens Burkholderia pseudomallei and Burkholderia mallei. We hypothesize that Bucl8, which contains two predicted tandem outer membrane efflux pump domains, is a component of a putative efflux pump. Unique to Bucl8, as compared to other outer membrane proteins, is the presence of an extended extracellular region containing a collagen-like (CL) domain and a non-collagenous C-terminus (Ct). Molecular modeling and circular dichroism spectroscopy with a recombinant protein, corresponding to this extracellular CL-Ct portion of Bucl8, demonstrated that it adopts a collagen triple helix, whereas functional assays screening for Bucl8 ligands identified binding to fibrinogen. Bioinformatic analysis of the bucl8 gene locus revealed it resembles a classical efflux-pump operon. The bucl8 gene is co-localized with downstream fusCDE genes encoding fusaric acid (FA) resistance, and with an upstream gene, designated as fusR, encoding a LysR-type transcriptional regulator. Using reverse transcriptase (RT)-qPCR, we defined the boundaries and transcriptional organization of the fusR-bucl8-fusCDE operon. We found exogenous FA induced bucl8 transcription over 80-fold in B. pseudomallei, while deletion of the entire bucl8 locus decreased the minimum inhibitory concentration of FA 4-fold in its isogenic mutant. We furthermore showed that the putative Bucl8-associated pump expressed in the heterologous Escherichia coli host confers FA resistance. On the contrary, the Bucl8-associated pump did not confer resistance to a panel of clinically-relevant antimicrobials in Burkholderia and E. coli. We finally demonstrated that deletion of the bucl8-locus drastically affects the growth of the mutant in L-broth. We determined that Bucl8 is a component of a novel tetrapartite efflux pump, which confers FA resistance, fibrinogen binding, and optimal growth.
Topics: Bacterial Outer Membrane Proteins; Burkholderia; Burkholderia mallei; Burkholderia pseudomallei; Collagen; Drug Resistance, Multiple, Bacterial; Escherichia coli; Escherichia coli Proteins; Genes, Bacterial; Membrane Transport Proteins; Operon; Transcription Factors
PubMed: 33227031
DOI: 10.1371/journal.pone.0242593 -
BMC Bioinformatics Sep 2016Burkholderia mallei and B. pseudomallei are the causative agents of glanders and melioidosis, respectively, diseases with high morbidity and mortality rates. B. mallei...
BACKGROUND
Burkholderia mallei and B. pseudomallei are the causative agents of glanders and melioidosis, respectively, diseases with high morbidity and mortality rates. B. mallei and B. pseudomallei are closely related genetically; B. mallei evolved from an ancestral strain of B. pseudomallei by genome reduction and adaptation to an obligate intracellular lifestyle. Although these two bacteria cause different diseases, they share multiple virulence factors, including bacterial secretion systems, which represent key components of bacterial pathogenicity. Despite recent progress, the secretion system proteins for B. mallei and B. pseudomallei, their pathogenic mechanisms of action, and host factors are not well characterized.
RESULTS
We previously developed a manually curated database, DBSecSys, of bacterial secretion system proteins for B. mallei. Here, we report an expansion of the database with corresponding information about B. pseudomallei. DBSecSys 2.0 contains comprehensive literature-based and computationally derived information about B. mallei ATCC 23344 and literature-based and computationally derived information about B. pseudomallei K96243. The database contains updated information for 163 B. mallei proteins from the previous database and 61 additional B. mallei proteins, and new information for 281 B. pseudomallei proteins associated with 5 secretion systems, their 1,633 human- and murine-interacting targets, and 2,400 host-B. mallei interactions and 2,286 host-B. pseudomallei interactions. The database also includes information about 13 pathogenic mechanisms of action for B. mallei and B. pseudomallei secretion system proteins inferred from the available literature or computationally. Additionally, DBSecSys 2.0 provides details about 82 virulence attenuation experiments for 52 B. mallei secretion system proteins and 98 virulence attenuation experiments for 61 B. pseudomallei secretion system proteins. We updated the Web interface and data access layer to speed-up users' search of detailed information for orthologous proteins related to secretion systems of the two pathogens.
CONCLUSIONS
The updates of DBSecSys 2.0 provide unique capabilities to access comprehensive information about secretion systems of B. mallei and B. pseudomallei. They enable studies and comparisons of corresponding proteins of these two closely related pathogens and their host-interacting partners. The database is available at http://dbsecsys.bhsai.org .
Topics: Animals; Bacterial Proteins; Bacterial Secretion Systems; Burkholderia mallei; Burkholderia pseudomallei; Databases, Protein; Humans; Mice; Virulence Factors
PubMed: 27650316
DOI: 10.1186/s12859-016-1242-z -
Applied and Environmental Microbiology Aug 2021A diverse genetic toolkit is critical for understanding bacterial physiology and genotype-phenotype relationships. Inducible promoter systems are an integral part of...
A diverse genetic toolkit is critical for understanding bacterial physiology and genotype-phenotype relationships. Inducible promoter systems are an integral part of this toolkit. In and related species, the l-rhamnose-inducible promoter is among the first choices due to its tight control and the lack of viable alternatives. To improve upon its maximum activity and dynamic range, we explored the effect of promoter system modifications in Burkholderia cenocepacia with a LacZ-based reporter. By combining the bacteriophage T7 gene stem-loop and engineered transcription factor-binding sites, we obtained a rhamnose-inducible system with a 6.5-fold and 3.0-fold increases in maximum activity and dynamic range, respectively, compared to the native promoter. We then added the modified promoter system to pSCrhaB2 and pSC201, common genetic tools used for plasmid-based and chromosome-based gene expression, respectively, in , creating pSCrhaB2plus and pSC201plus. We demonstrated the utility of pSCrhaB2plus for gene expression in B. thailandensis, , and and used pSC201plus to control highly expressed essential genes from the chromosome of B. cenocepacia. The utility of the modified system was demonstrated as we recovered viable mutants to control , , and , whereas the unmodified promoter was unable to control . The modified expression system allowed control of an essential gene depletion phenotype at lower levels of l-rhamnose, the inducer. pSCRhaB2plus and pSC201plus are expected to be valuable additions to the genetic toolkit for and related species. Species of are dually recognized as being of attractive biotechnological potential but also opportunistic pathogens for immunocompromised individuals. Understanding the genotype-phenotype relationship is critical for synthetic biology approaches in to disentangle pathogenic from beneficial traits. A diverse genetic toolkit, including inducible promoters, is the foundation for these investigations. Thus, we sought to improve on the commonly used rhamnose-inducible promoter system. Our modifications resulted in both higher levels of heterologous protein expression and broader control over highly expressed essential genes in B. cenocepacia. The significance of our work is in expanding the genetic toolkit to enable more comprehensive studies into and related bacteria.
Topics: Burkholderia; Gene Expression Regulation, Bacterial; Mutation; Promoter Regions, Genetic; Rhamnose; beta-Galactosidase
PubMed: 34190606
DOI: 10.1128/AEM.00647-21