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Burkholderia thailandensis has emerged as a nonpathogenic surrogate for Burkholderia pseudomallei, the causative agent of melioidosis, and an important Gram-negative model bacterium for studying the biosynthesis and regulation of secondary metabolism. We recently reported that subinhibitory concentrations of trimethoprim induce vast changes in both the primary and secondary metabolome of B. thailandensis. In the current work, we show that the folate biosynthetic enzyme FolE2 is permissive under standard growth conditions but essential for B. thailandensis in the presence of subinhibitory doses of trimethoprim. Reasoning that FolE2 may serve as an attractive drug target, we screened for and identified ten inhibitors, including dehydrocostus lactone (DHL), parthenolide, and ß-lapachone, all of which are innocuous individually but form a chemical-synthetic lethal combination with subinhibitory doses of trimethoprim. We show that DHL is a mechanism-based inhibitor of FolE2 and capture the structure of the covalently inhibited enzyme using X-ray crystallography. In vitro, the combination of subinhibitory trimethoprim and DHL is more potent than Bactrim, the current standard of care against melioidosis. Moreover, unlike Bactrim, this combination does not affect the growth of most commensal and beneficial gut bacteria tested, thereby providing a degree of specificity against B. pseudomallei. Our work provides a path for identifying antimicrobial drug targets and for utilizing binary combinations of molecules that form a toxic cocktail based on metabolic idiosyncrasies of specific pathogens. [ABSTRACT FROM AUTHOR]

Bioremediation is considered to be an effective treatment for hydrocarbon removal from polluted soils. However, the effectiveness of this treatment is often limited by the low availability of targeted contaminants. Biosurfactants produced by some microorganisms can increase organic compound solubility and might then overcome this limitation. Two different inocula producers of biosurfactants (Burkholderia thailandensis E264 and SHEMS1 microbial consortium isolated from a hydrocarbon-contaminated soil) were incubated in Bushnell-Haas medium supplemented with hydrocarbons to investigate their biodegradation potential. Experimental results showed their ability to degrade 9.1 and 6.1% of hydrocarbons respectively after 65 days of incubation with an initial total hydrocarbon concentration of 16 g L−1. The biodegradation was more effective for the light and medium fractions (C10 to C36). B. thailandensis and SHEMS1 consortium produced surfactants after 14 days of culture during the stationary phase with hydrocarbons as the sole carbon and energy source. However, biosurfactant production did not appear to directly increase hydrocarbon degradation efficiency. The complexity and recalcitrance of hydrocarbon mixture used in this study appeared to continue to limit its biodegradation even in the presence of biosurfactants. In conclusion, B. thailandensis and SHEMS1 consortium can degrade recalcitrant hydrocarbon compounds and are therefore good candidates for the bioremediation of environments polluted by total hydrocarbons. [ABSTRACT FROM AUTHOR]

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei, an intracellular pathogen with a high mortality rate and significant antibiotic resistance. The high mortality rate and resistance to antibiotics have drawn considerable attention from researchers studying melioidosis. This study evaluated the effects of various concentrations (75, 50, and 25 µg/mL) of promethazine hydrochloride (PTZ), a potent antihistamine, on biofilm formation and lipase activity after 24 h of exposure to B. thailandensis E264. A concentration-dependent decrease in both biofilm biomass and lipase activity was observed. RT-PCR analysis revealed that PTZ treatment not only made the biofilm structure loose but also reduced the expression of btaR1, btaR2, btaR3, and scmR. Single gene knockouts of quorum sensing (QS) receptor proteins (∆btaR1, ∆btaR2, and ∆btaR3) were successfully constructed. Deletion of btaR1 affected biofilm formation in B. thailandensis, while deletion of btaR2 and btaR3 led to reduced lipase activity. Molecular docking and biological performance results demonstrated that PTZ inhibits biofilm formation and lipase activity by suppressing the expression of QS-regulated genes. This study found that repositioning PTZ reduced biofilm formation in B. thailandensis E264, suggesting a potential new approach for combating melioidosis. Key Points: Repurposing promethazine hydrochloride to inhibit biofilms formation. Promethazine hydrochloride suppressed the expression of QS-regulated genes. The deletion of btaR1 affected the biofilm formation of B. thailandensis. [ABSTRACT FROM AUTHOR]

The methylerythritol phosphate (MEP) pathway is a metabolic pathway that produces the isoprenoids isopentyl pyrophosphate and dimethylallyl pyrophosphate. Notably, the MEP pathway is present in bacteria and not in mammals, which makes the enzymes of the MEP pathway attractive targets for discovering new anti‐infective agents due to the reduced chances of off‐target interactions leading to side effects. There are seven enzymes in the MEP pathway, the third of which is IspD. Two crystal structures of Burkholderia thailandensis IspD (BtIspD) were determined: an apo structure and that of a complex with cytidine triphosphate (CTP). Comparison of the CTP‐bound BtIspD structure with the apo structure revealed that CTP binding stabilizes the loop composed of residues 13–19. The apo structure of Mycobacterium paratuberculosis IspD (MpIspD) is also reported. The melting temperatures of MpIspD and BtIspD were evaluated by circular dichroism. The moderate Tm values suggest that a thermal shift assay may be feasible for future inhibitor screening. Finally, the binding affinity of CTP for BtIspD was evaluated by isothermal titration calorimetry. These structural and biophysical data will aid in the discovery of IspD inhibitors. [ABSTRACT FROM AUTHOR]

Introduction: Burkholderia thailandensis is a study model for Burkholderia pseudomallei, a highly virulent pathogen, known to be the causative agent of melioidosis and a potential bioterrorism agent. These two bacteria use an (acylhomoserine lactone) AHL-mediated quorum sensing (QS) system to regulate different behaviors including biofilm formation, secondary metabolite productions, and motility. Methods: Using an enzyme-based quorum quenching (QQ) strategy, with the lactonase SsoPox having the best activity on B. thailandensis AHLs, we evaluated the importance of QS in B. thailandensis by combining proteomic and phenotypic analyses. Results: We demonstrated that QS disruption largely affects overall bacterial behavior including motility, proteolytic activity, and antimicrobial molecule production. We further showed that QQ treatment drastically decreases B. thailandensis bactericidal activity against two bacteria (Chromobacterium violaceum and Staphylococcus aureus), while a spectacular increase in antifungal activity was observed against fungi and yeast (Aspergillus niger, Fusarium graminearum and Saccharomyces cerevisiae). Discussion: This study provides evidence that QS is of prime interest when it comes to understanding the virulence of Burkholderia species and developing alternative treatments. [ABSTRACT FROM AUTHOR]

Rhamnolipids (RLs) are one of the most promising eco-friendly green alternatives to commercially viable fossil fuel-based surfactants. However, the current bioprocess practices cannot meet the required affordability, quantity, and biocompatibility within an industrially relevant framework. To circumvent these issues, our study aims to develop a sustainable biorefinery approach using post-consumption food waste as a second-generation feedstock. In-depth substrate screening revealed that food waste hydrolysate (FWH) was rich in readily assimilable carbohydrates, volatile fatty acids, and amino acids. The fermentative valorization of FWH as a sole carbon and energy source with Burkholderis thailandensis E264 in a bioreactor showed active RLs biosynthesis of up to 0.6–0.8 g/L (34–40 mg/g FWH) in a short duration (72 h). In terms of the kinetic parameters, the FWH-RLs outperformed other supplemented pure/waste streams. Interestingly, the recovered RLs had a long chain length, with Rha-Rha-C12-C14 being the predominant isoform and exhibiting a strong emulsification ability (E24, 54.6%). To the best of our knowledge, this study is the first to prove bioreactor-level RLs production and their abundance in food waste. Moreover, the feasibility of this developed process could propel next-generation biosurfactants, lower waste burdens, and increase the industrial applicability of RLs, thereby significantly contributing to the development of a circular bioeconomy. [ABSTRACT FROM AUTHOR]

In the human body, docosahexaenoic acid (DHA) contained in fish oil is converted to trace amounts of specialized pro-resolving mediators (SPMs) as the principal bioactive metabolites for their pharmacological effects. Protectin Dx (PDX), an SPM, is an important medicinal compound with biological activities such as modulation of endogenous antioxidant systems, inflammation pro-resolving action, and inhibition of influenza virus replication. Although it can be biotechnologically synthesized from DHA, it has not yet been produced quantitatively. Here, we found that 15S-lipoxygenase from Burkholderia thailandensis (BT 15SLOX) converted 10S-hydroxydocosahexaenoic acid (10S-HDHA) to PDX using enzymatic reactions, which was confirmed by LC-MS/MS and NMR analyses. Thus, whole-cell reactions of Escherichia coli cells expressing BT 15SLOX were performed in flasks to produce PDX from lipase-treated DHA-enriched fish oil along with E. coli cells expressing Mus musculus (mouse) 8S-lipoxygenase (MO 8SLOX) that converted DHA to 10S-HDHA. First, 1 mM DHA (DHA-enriched fish oil hydrolysate, DFOH) was obtained from 455 mg/L DHA-enriched fish oil by lipase for 1 h. Second, E. coli cells expressing MO 8SLOX converted 1 mM DHA in DFOH to 0.43 mM 10S-HDHA for 6 h. Finally, E. coli cells expressing BT 15SLOX converted 0.43 mM 10S-HDHA in MO 8SLOX-treated DFOH to 0.30 mM (108 mg/L) PDX for 5 h. Consequently, DHA-enriched fish oil at 455 mg/L was converted to 108 mg/L PDX after a total of 12 h (conversion yield: 24% (w/w); productivity: 4.5 mg/L/h). This study is the first report on the quantitative production of PDX via biotechnological approaches. [ABSTRACT FROM AUTHOR]

The causative agent of melioidosis, Burkholderia pseudomallei, as well as closely related nonpathogenic bacteria of the B. pseudomallei/thailandensis complex of species, are found in soils and water bodies in melioidosis-endemic areas which results in significant biological risks to agricultural workers in these regions. Bacteriophages related to the phiBp-AMP1 phage are the most common known group of B. pseudomallei/thailandensis viruses. The interaction of these bacteriophages with their hosts may be a key factor in the natural control of the B. pseudomallei population, which determines the dynamics of melioidosis infection in humans. In this study, we showed that frequently used herbicides (paraquat, atrazine, 2,4-D, ametrine, and copper dichloride) did not significantly affect the stability and infectivity of the AMP1 phage, while iron(II) salts, which are used as fertilizers, inactivated the phage even at concentrations of about 0.1 mM or lower. Moreover, the concentration of sodium chloride was critical for the infectivity of the AMP1 phage, both against B. thailandensis and against the pathogen B. pseudomallei. When the concentration of this salt in solution was less than 3 g/L, the phages ceased infecting the host cells. Therefore, soil salinity may thus be responsible for the mosaic pattern of B. pseudomallei population dynamics within a number of endemic regions, such as northeastern Thailand. [ABSTRACT FROM AUTHOR]

Background: Burkholderia pseudomallei is the bacterial causative agent of melioidosis, a difficult disease to diagnose clinically with high mortality if not appropriately treated. Definitive diagnosis requires isolation and identification of the organism. With the increased adoption of MALDI-TOF MS for the identification of bacteria, we established a method for rapid identification of B. pseudomallei using the Vitek MS, a system that does not currently have B. pseudomallei in its in-vitro diagnostic database. Results: A routine direct spotting method was employed to create spectra and SuperSpectra. An initial B. pseudomallei SuperSpectrum was created at Shoklo Malaria Research Unit (SMRU) from 17 reference isolates (46 spectra). When tested, this initial SMRU SuperSpectrum was able to identify 98.2 % (54/55) of Asian isolates, but just 46.7 % (35/75) of Australian isolates. Using spectra (430) from different reference and clinical isolates, two additional SMRU SuperSpectra were created. Using the combination of all SMRU SuperSpectra with seven existing SuperSpectra from Townsville, Australia 119 (100 %) Asian isolates and 31 (100 %) Australian isolates were correctly identified. In addition, no misidentifications were obtained when using these 11 SuperSpectra when tested with 34 isolates of other bacteria including the closely related species Burkholderia thailandensis and Burkholderia cepacia. Conclusions: This study has established a method for identification of B. pseudomallei using Vitek MS, and highlights the impact of geographical differences between strains for identification using this technique. [ABSTRACT FROM AUTHOR]

Natural products have been an important source of therapeutic agents and chemical tools. The recent realization that many natural product biosynthetic genes are silent or sparingly expressed during standard laboratory growth has prompted efforts to investigate their regulation and develop methods to induce their expression. Because it is difficult to intuit signals that induce a given biosynthetic locus, we recently implemented a forward chemical-genetic approach to identify such inducers. In the current work, we applied this approach to nine silent biosynthetic loci in the model bacterium Burkholderia thailandensis to systematically screen for elicitors from a library of Food and Drug Administration-approved drugs. We find that β-lactams, fluoroquinolones, antifungals, and, surprisingly, calcimimetics, phenothiazine antipsychotics, and polyaromatic antidepressants are the most effective global inducers of biosynthetic genes. Investigations into the mechanism of stimulation of the silent virulence factor malleicyprol by the β-lactam piperacillin allowed us to elucidate the underlying regulatory circuits. Low-dose piperacillin causes oxidative stress, thereby inducing redox-sensing transcriptional regulators, which activate malR, a pathway-specific positive regulator of the malleicyprol gene cluster. Malleicyprol is thus part of the OxyR and SoxR regulons in B. thailandensis, allowing the bacterium to initiate virulence in response to oxidative stress. Our work catalogs a diverse array of elicitors and a previously unknown regulatory input for secondary metabolism in B. thailandensis. [ABSTRACT FROM AUTHOR]

Colistin resistance is complex and multifactorial. DbcA is an inner membrane protein belonging to the DedA superfamily required for maintaining extreme colistin resistance of Burkholderia thailandensis. The molecular mechanisms behind this remain unclear. Here, we report that ∆dbcA displays alkaline pH/bicarbonate sensitivity and propose a role of DbcA in extreme colistin resistance of B. thailandensis by maintaining cytoplasmic pH homeostasis. We found that alkaline pH or presence of sodium bicarbonate displays a synergistic effect with colistin against not only extremely colistin resistant species like B. thailandensis and Serratia marcescens, but also a majority of Gram-negative and Gram-positive bacteria tested, suggesting a link between cytoplasmic pH homeostasis and colistin resistance across species. We found that lowering the level of oxygen in the growth media or supplementation of fermentable sugars such as glucose not only alleviated alkaline pH stress, but also increased colistin resistance in most bacteria tested, likely by avoiding cytoplasmic alkalinization. Our observations suggest a previously unreported link between pH, oxygen, and colistin resistance. We propose that maintaining optimal cytoplasmic pH is required for colistin resistance in a majority of bacterial species, consistent with the emerging link between cytoplasmic pH homeostasis and antibiotic resistance. [ABSTRACT FROM AUTHOR]

Melioidosis is a neglected tropical disease with an estimated annual mortality rate of 89,000 in 45 countries across tropical regions. The causative agent is Burkholderia pseudomallei, a gram-negative soil-dwelling bacterium. In Thailand, B. pseudomallei can be found across multiple regions, along with the low-virulence B. thailandensis and the recently discovered B. thailandensis variant (BTCV), which expresses B. pseudomallei-like capsular polysaccharide. Comprehensive studies of human immune responses to B. thailandensis variants and cross-reactivity to B. pseudomallei are not complete. We evaluated human immune responses to B. pseudomallei, B. thailandensis, and BTCV in melioidosis patients and healthy persons in B. pseudomallei-endemic areas using a range of humoral and cellular immune assays. We found immune cross-reactivity to be strong for both humoral and cellular immunity among B. pseudomallei, B. thailandensis, and BTCV. Our findings suggest that environmental exposure to low-virulence strains may build cellular immunity to B. pseudomallei. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Burkholderia thailandensis is a model organism for human pathogens Burkholderia mallei and Burkholderia pseudomallei. The study of B. thailandensis peroxiredoxin is helpful for understanding the survival, pathogenic infection, and antibiotic resistance of its homologous species. Alkyl hydroperoxide reductase subunit C (AhpC) is an important peroxiredoxin involved in oxidative damage defense. Here, we report that Bth AhpC exhibits broad specificity for peroxide substrates, including inorganic and organic peroxides and peroxynitrite. AhpC catalyzes the reduction of oxidants using the N-terminal conserved Cys57 as a peroxidatic Cys and the C-terminal conserved Cys171 and Cys173 as resolving Cys. These three conserved Cys residues play critical roles in the catalytic mechanism. AhpD directly interacts with AhpC as an electron donor, and the conserved Cys residues in active site of AhpD are important for AhpC reduction. AhpC is directly repressed by OxyR as shown by identifying the OxyR binding site in the ahpC promoter with a DNA binding assay. This work sheds light on the function of AhpC in the peroxides and peroxynitrite damage response in B. thailandensis and homologous species. [ABSTRACT FROM AUTHOR]

Intercellular communication and self-recognition are critical for coordinating cooperative and competitive behaviors during sociomicrobiological community development. Contact-dependent growth inhibition (CDI) proteins are polymorphic toxin delivery systems that inhibit the growth of non-self neighboring bacteria that lack the appropriate immunity protein. In Burkholderia thailandensis, CDI system proteins (encoded by bcpAIOB genes) also induce cooperative behaviors among sibling (self) cells, a phenomenon called contact-dependent signaling (CDS). Here we describe a mobile genetic element (MGE) that carries the bcpAIOB genes in B. thailandensis E264. It is a ~210 kb composite transposon with insertion sequence (IS) elements at each end. Although the ISs are most similar to IS2 of Escherichia coli, the transposase-dependent intermediate molecule displays characteristics more similar to those of the IS26 translocatable unit (TU). A reaction requiring only the “left” IS-encoded transposase results in formation of an extrachromosomal circular dsDNA intermediate (“the megacircle”) composed of the left IS and the sequences intervening between the ISs. Insertion of the megacircle into the chromosome occurs next to a pre-existing copy of an IS2-like element, recreating a functional composite transposon. We found that BcpA activity is required for megacircle formation, and in turn, megacircle formation is required for CDS phenotypes. Our data support a model in which the bcpAIOB genes function as both helping and harming greenbeard genes, simultaneously enhancing the fitness of self bacteria that possess the same allele plus tightly linked genes that mediate cooperative behaviors, and killing non-self bacteria that do not possess the same bcpAIOB allele. Mobility of the megacircle between cells could allow bacteria invading a community to be converted to self, and would facilitate propagation of the bcpAIOB genes in the event that the invading strain is capable of overtaking the resident community. [ABSTRACT FROM AUTHOR]

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Since Burkholderia thailandensis is included in the reference spectra of the VITEK MS libraries rather than Burkholderia pseudomallei, B. pseudomallei cannot be correctly identified in the current version of VITEK MS. This study was undertaken to evaluate the utility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with the VITEK MS plus system in the detection of B. pseudomallei and B. thailandensis isolates. For each species, we increased the reference spectra, and then, a SuperSpectrum was created based on the selection of 39 specific masses. In a second step, we validated the SuperSpectra with 106 isolates identified by 16S rRNA gene sequencing. The results showed that there was 100% agreement between the validation strains analyzed by MALDI-TOF MS and those evaluated using 16S rRNA gene sequencing analysis methods. Therefore, MALDI-TOF MS is a promising, rapid, and economical method to monitor the outbreaks and spread of B. pseudomallei and B. thailandensis isolates. [ABSTRACT FROM AUTHOR]

The bacterium Burkholderia thailandensis, a member of the Burkholderia pseudomallei complex, is generally considered nonpathogenic; however, on rare occasions, B. thailandensis infections have been reported. We describe a clinical isolate of B. thailandensis, BtAR2017, recovered from a patient with an infected wound in Arkansas, USA, in 2017. [ABSTRACT FROM AUTHOR]

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 - d35 + 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. [ABSTRACT FROM AUTHOR]

Burkholderia thailandensis is often used as a model for more virulent members of this genus of proteobacteria that are highly antibiotic‐resistant and are potential agents of biological warfare that are infective by inhalation. As part of ongoing efforts to identify potential targets for the development of rational therapeutics, the structures of enzymes that are absent in humans, including that of chorismate mutase from B. thailandensis, have been determined by the Seattle Structural Genomics Center for Infectious Disease. The high‐resolution structure of chorismate mutase from B. thailandensis was determined in the monoclinic space group P21 with three homodimers per asymmetric unit. The overall structure of each protomer has the prototypical AroQγ topology and shares conserved binding‐cavity residues with other chorismate mutases, including those with which it has no appreciable sequence identity. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei is the causative agent of melioidosis and regarded as a bioterrorism threat. It can adapt to the nutrient-limited environment as the bacteria can survive in triple distilled water for 16 years. Moreover, B. pseudomallei exhibits intrinsic resistance to diverse groups of antibiotics in particular while growing in biofilms. Recently, nutrient-limited condition influenced both biofilm formation and ceftazidime (CAZ) tolerance of B. pseudomallei were found. However, there is no information about how nutrient-limitation together with antibiotics used in melioidosis treatment affects the structure of the biofilm produced by B. pseudomallei. Moreover, no comparative study to investigate the biofilm architectures of B. pseudomallei and the related B. thailandensis under different nutrient concentrations has been reported. Therefore, this study aims to provide new information on the effects of four antibiotics used in melioidosis treatment, viz. ceftazidime (CAZ), imipenem (IMI), meropenem (MEM) and doxycycline (DOX) on biofilm architecture of B. pseudomallei and B. thailandensis with different nutrient concentrations under static and flow conditions using confocal laser scanning microscopy. Impact of nutritional stress on drug susceptibility of B. pseudomallei and B. thailandensis grown planktonically or as biofilm was also evaluated. The findings of this study indicate that nutrient-limited environment enhanced survival of B. pseudomallei in biofilm after exposure to the tested antibiotics. The shedding planktonic B. pseudomallei and B. thailandensis were also found to have increased CAZ tolerance in nutrient-limited environment. However, killing activities of MEM and IMI were stronger than CAZ and DOX on B. pseudomallei and B. thailandensis both in planktonic cells and in 2-day old biofilm. In addition, MEM and IMI were able to inhibit B. pseudomallei and B. thailandensis biofilm formation to a larger extend compared to CAZ and DOX. Differences in biofilm architecture were observed for biofilms grown under static and flow conditions. Under static conditions, biofilms grown in full strength modified Vogel and Bonner’s medium (MVBM) showed honeycomb-like architecture while a knitted-like structure was observed under limited nutrient condition (0.1×MVBM). Under flow conditions, biofilms grown in MVBM showed a multilayer structure while merely dispersed bacteria were found when grown in 0.1×MVBM. Altogether, this study provides more insight on the effect of four antibiotics against B. pseudomallei and B. thailandensis in biofilm under different nutrient and flow conditions. Since biofilm formation is believed to be involved in disease relapse, MEM and IMI may be better therapeutic options than CAZ for melioidosis treatment. [ABSTRACT FROM AUTHOR]

Objectives: Although meropenem is widely used to treat Burkholderia infections, the response of Burkholderia pathogens to this antibiotic is largely unexplored.Methods: Burkholderia thailandensis, a model for Burkholderia spp., particularly Burkholderia mallei and Burkholderia pseudomallei, was challenged with a lethal level of meropenem and survivors were isolated. The genomes of two of the isolates were analysed to identify mutated genes and these genes were then specifically examined in more isolates to profile mutation diversity. Mutants were characterized to investigate the biological basis underlying survival against meropenem.Results: One of two genes associated with tRNA metabolism [metG or trmD, encoding methionyl-tRNA synthetase or tRNA (guanine-N1)-methyltransferase, respectively] was found to be mutated in the two survivors. A single nucleotide substitution and a frameshift mutation were found in metG and trmD, respectively. Five different substitution mutations affecting methionine- or tRNA-binding sites were found in metG during further screening. The mutants exhibited slowed growth and increased tolerance not only to meropenem but also various other antibiotics. This tolerance required intact RelA, a key stringent response.Conclusions: Specific mutations affecting the tRNA pool, particularly those in metG, play a pivotal role in the B. thailandensis response to meropenem challenge. This mechanism of antibiotic tolerance is important because it can reduce the effectiveness of meropenem and thereby facilitate chronic infection by Burkholderia pathogens. In addition, specific mutations found in MetG will prove useful in the effort to develop new drugs to completely inhibit this essential enzyme, while preventing stringent-response-mediated antibiotic tolerance in pathogens. [ABSTRACT FROM AUTHOR]

Background: Burkholderia pseudomallei is an environmental Gram-negative bacillus and the cause of melioidosis. B. thailandensis, some strains of which express a B. pseudomallei-like capsular polysaccharide (BTCV), is also commonly found in the environment in Southeast Asia but is considered non-pathogenic. The aim of the study was to determine the distribution of B. thailandensis and its capsular variant in Thailand and investigate whether its presence is associated with a serological response to B. pseudomallei. Methodology/principal findings: We evaluated the presence of B. pseudomallei and B. thailandensis in 61 rice fields in Northeast (n = 21), East (n = 19) and Central (n = 21) Thailand. We found BTCV in rice fields in East and Central but not Northeast Thailand. Fourteen fields were culture positive for B. pseudomallei alone, 8 for B. thailandensis alone, 11 for both B. pseudomallei and B. thailandensis, 6 for both B. thailandensis and BTCV, and 5 for B. pseudomallei, B. thailandensis and BTCV. Serological testing using the indirect hemagglutination assay (IHA) of 96 farmers who worked in the study fields demonstrated that farmers who worked in B. pseudomallei-positive fields had higher IHA titers than those who worked in B. pseudomallei-negative fields (median 1:40 [range: <1:10–1:640] vs. <1:10 [range: <1:10–1:320], p = 0.002). In a multivariable ordered logistic regression model, IHA titers were significantly associated with the presence of B. pseudomallei (aOR = 3.7; 95% CI 1.8–7.8, p = 0.001) but were not associated with presence of B. thailandensis (p = 0.32) or BTCV (p = 0.32). One sequence type (696) was identified for the 27 BTCV isolates tested. Conclusions/significance: This is the first report of BTCV in Thailand. The presence of B. pseudomallei and B. thailandensis in the same field was not uncommon. Our findings suggest that IHA positivity of healthy rice farmers in Thailand is associated with the presence of B. pseudomallei in rice fields rather than B. thailandensis or BTCV. [ABSTRACT FROM AUTHOR]

Microbially produced rhamnolipids have significant commercial potential; however, the main bacterial producer, Pseudomonas aeruginosa, is an opportunistic human pathogen, which limits biotechnological exploitation. The non-pathogenic species Burkholderia thailandensis produces rhamnolipids; however, yield is relatively low. The aim of this study was to determine whether rhamnolipid production could be increased in Burkholderia thailandensis through mutation of genes responsible for the synthesis of the storage material polyhydroxyalkanoate (PHA), thereby increasing cellular resources for the production of rhamnolipids. Potential PHA target genes were identified in B. thailandensis through comparison with known function genes in Pseudomonas aeruginosa. Multiple knockout strains for the phbA, phbB and phbC genes were obtained and their growth characteristics and rhamnolipid and PHA production determined. The wild-type strain and an rhamnolipid (RL)-deficient strain were used as controls. Three knockout strains (Δ phbA1, Δ phbB1 and Δ phbC1) with the best enhancement of rhamnolipid production were selected for detailed study. Δ phbB1 produced the highest level of purified RL (3.78 g l) compared to the wild-type strain (1.28 g l). In Δ phbB1, the proportion of mono-rhamnolipid was also increased compared to the wild-type strain. The production of PHA was reduced by at least 80% in all three phb mutant strains, although never completely eliminated. These results suggest that, in contrast to Pseudomonas aeruginosa, knockout of the PHA synthesis pathway in Burkholderia thailandensis could be used to increase rhamnolipid production. The evidence of residual PHA production in the phb mutant strains suggests B. thailandensis possesses a secondary unelucidated PHA synthesis pathway. [ABSTRACT FROM AUTHOR]

Following escape into the cytoplasm of host cells, Burkholderia pseudomallei and the related species Burkholderia thailandensis employ the type VI secretion system 5 (T6SS-5) to induce plasma membrane fusion with an adjacent host cell. This process leads to the formation of multinucleated giant cells and facilitates bacterial access to an uninfected host cell in a direct manner. Despite its importance in virulence, the mechanism of the T6SS-5 and the role of host cell factors in cell-cell fusion remain elusive. To date, the T6SS-5 is the only system of bacterial origin known to induce host-cell fusion. To gain insight into the nature of T6SS-5-stimulated membrane fusion, we investigated the contribution of cholesterol and proteins exposed on the host cell surface, which were shown to be critically involved in virus-mediated giant cell formation. In particular, we analyzed the effect of host cell surface protein and cholesterol depletion on the formation of multinucleated giant cells induced by B. thailandensis. Acute protease treatment of RAW264.7 macrophages during infection with B. thailandensis followed by agarose overlay assays revealed a strong reduction in the number of cell-cell fusions compared with EDTA treated cells. Similarly, proteolytic treatment of specifically infected donor cells or uninfected recipient cells significantly decreased multinucleated giant cell formation. Furthermore, modulating host cell cholesterol content by acute cholesterol depletion from cellular membranes by methyl- β-cyclodextrin treatment or exogenous addition of cholesterol impaired the ability of B. thailandensis to induce cell-cell fusions. The requirement of physiological cholesterol levels suggests that the membrane organization or mechanical properties of the lipid bilayer influence the fusion process. Altogether, our data suggest that membrane fusion induced by B. pseudomallei and B. thailandensis involves a complex interplay between the T6SS-5 and the host cell. [ABSTRACT FROM AUTHOR]

Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-!, in Burkholderia infection and characterized PKC-η/MARCKS signaling as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-a and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-a, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis, we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. Identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective host-targeted therapies against infectious disease caused by intracellular pathogens. [ABSTRACT FROM AUTHOR]

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) upor down-regulated in a scmR deletion mutant (ΔscmR). Metabolically, the ΔscmR 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 ΔscmR 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. [ABSTRACT FROM AUTHOR]

Significant impact of Bp-AMP1-related bacteriophages on the population dynamics of Burkholderia pseudomallei in agroecosystems of the regions endemic for melioidosis has been repeatedly hypothesised in recent years. Temperature-dependent life cycle of these viruses may be responsible for modulation of the natural phage-mediated biocontrol, and thus it may contribute to the complexity of the seasonal dynamics of melioidosis observed. Refined data on Bp-AMP1 phage genome organization are a prerequisite for the ongoing research on infection physiology of this virus. In the present work, location of the physical ends of the virion-encapsidated phage DNA was determined, 389-bp long terminal repeats were revealed, and the annotation of the phage Bp-AMP1 genome sequence was revised. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in the orchestration of immune responses. S1P levels within the mammalian host are tightly regulated, in part through the activity of S1P lyase (S1PL) which catalyses its irreversible degradation. Herein, we describe the identification and characterization of secreted S1PL orthologues encoded by the facultative intracellular bacteria Burkholderia pseudomallei and Burkholderia thailandensis. These bacterial orthologues exhibited S1PL enzymatic activity, functionally complemented an S1PL-deficient yeast strain and conferred resistance to the antimicrobial sphingolipid D- erythro-sphingosine. We report that secretion of these bacterial S1PLs is pH-dependent, and is observed during intracellular infection. S1PL-deficient mutants displayed impaired intracellular replication in murine macrophages (associated with an inability to evade the maturing phagosome) and were significantly attenuated in murine and larval infection models. Furthermore, treatment of Burkholderia-infected macrophages with either S1P or a selective agonist of S1P receptor 1 enhanced bacterial colocalisation with LAMP-1 and reduced their intracellular survival. In summary, our studies confirm bacterial-encoded S1PL as a critical virulence determinant of B. pseudomallei and B. thailandensis, further highlighting the pivotal role of S1P in host-pathogen interactions. In addition, our data suggest that S1P pathway modulators have potential for the treatment of intracellular infection. [ABSTRACT FROM AUTHOR]

Melioidosis caused by gram negative bacteria, B. pseudomallei, is a fatal disease in the tropical and sub-tropical regions. However, sporadic cases have been reported in elsewhere. Early detection is imperative to reduce the mortality rate. Serological tests have being substantially developed using recombinant proteins as specific targeted antigens to melioidosis antibodies. In the present study, we focus on a truncated flagellin fragment (FLAG300) and outer membrane protein A (OmpABT) of B. thailandensis E264 as potential antigens for developing indirect ELISA to improve the serodiagnosis of melioidosis. Recombinant proteins were overexpressed and purified by immobilized metal affinity chromatography with denaturing conditions. The sensitivity and specificity of individual test were calculated within culture-confirmed melioidosis sera (n = 42) and non-melioidosis serum samples (n = 241) using the cut-off point at average of absorbance plus 2 standard deviations. The results demonstrated that a FLAG 300 based indirect ELISA showed 90.48 % sensitivity and 87.14 % specificity and an OmpABT based this assay revealed sensitivity of 80.95 % and specificity of 89.21 %. Their use in a double-antigen ELISA resulted in improve specificity (92.95 %) and still high degree of sensitivity (85.71 %). These data suggest a facile method for serodiagnosis of melioidosis by the use of antigens from a non-pathogenic strain. [ABSTRACT FROM AUTHOR]

Burkholderia thailandensis E264 is a rhamnolipid (RL)-producing gram-negative bacterium first isolated from the soils and stagnant waters of central and north-eastern Thailand. Growth of B. thailandensis E264 under two different incubation temperatures (25 and 30 °C) resulted in a significantly higher dry cell biomass production at 30 °C (7.71 g/l) than at 25 °C (4.75 g/l) after 264 h; however, incubation at the lower temperature resulted in consistently higher concentration of RL production throughout the growth period. After 264 h, the concentration of crude RL extract for the 25 °C culture was 2.79 g/l compared to 1.99 g/l for the 30 °C culture. Overall RL production concentration after 264 h was 0.258 g/g dry cell biomass (DCB) for the 30 °C culture compared to 0.587 g/g DCB for the 25 °C culture. Real-time PCR (qPCR) was also used to analyse expression of the RL biosynthesis genes throughout the incubation period at 25 °C showing that the expression of the rhlA, rhlB and rhlC genes is continuous. During the log and early stationary phases of growth, expression levels remain low and are increased upon entry to the late stationary phase. B. thailandensis E264 produces mostly di-RLs and the Di-RL C14-C14 in most abundance (41.88 %). Fermentations were also carried out in small-scale bioreactors (4 l working volume) under controlled conditions, and results showed that RL production was maintained. Our findings show that B. thailandensis E264 has excellent potential for industrial scale RL production. [ABSTRACT FROM AUTHOR]

In prokaryotes and eukaryotes, cell-cell communication and recognition of self are critical to coordinate multicellular functions. Although kin and kind discrimination are increasingly appreciated to shape naturally occurringmicrobe populations, the underlying mechanisms that govern these interbacterial interactions are insufficiently understood. Here, we identify a mechanism of interbacterial signal transduction that is mediated by contact-dependent growth inhibition (CDI) system proteins. CDI systems have been characterized by their ability to deliver a polymorphic protein toxin into the cytoplasm of a neighboring bacterium, resulting in growth inhibition or death unless the recipient bacterium produces a corresponding immunity protein. Using the model organism Burkholderia thailandensis, we show that delivery of a catalytically active CDI system toxin to immune (self) bacteria results in gene expression and phenotypic changes within the recipient cells. Termed contact-dependent signaling (CDS), this response promotes biofilm formation and other community-associated behaviors. Engineered strains that are isogenic with B. thailandensis, except the DNA region encoding the toxin and immunity proteins, did not display CDS, whereas a strain of Burkholderia dolosa producing a nearly identical toxin-immunity pair induced signaling in B. thailandensis. Our data indicate that bcpAIOB loci confer dual benefits; they direct antagonism toward non-self bacteria and promote cooperation between self bacteria, with self being defined by the bcpAIOB allele and not by genealogic relatedness. [ABSTRACT FROM AUTHOR]

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The use of in vitro models to understand the interaction of bacteria with host cells is well established. In vitro bacterial infection models are often used to quantify intracellular bacterial load by lysing cell populations and subsequently enumerating the bacteria. Modern established techniques employ the use of fluorescence technologies such as flow cytometry, fluorescent microscopy, and/or confocal microscopy. However, these techniques often lack either the quantification of large data sets (microscopy) or use of gross fluorescence signal which lacks the visual confirmation that can provide additional confidence in data sets. Multispectral imaging flow cytometry (MIFC) is a novel emerging field of technology. This technology captures a bright field and fluorescence image of cells in a flow using a charged coupled device camera. It allows the analysis of tens of thousands of single cell images, making it an extremely powerful technology. Here MIFC was used as an alternative method of analyzing intracellular bacterial infection using Burkholderia thailandensis E555 as a model organism. It has been demonstrated that the data produced using traditional enumeration is comparable to data analyzed using MIFC. It has also been shown that by using MIFC it is possible to generate other data on the dynamics of the infection model rather than viable counts alone. It has been demonstrated that it is possible to inhibit the uptake of bacteria into mammalian cells and identify differences between treated and untreated cell populations. The authors believe this to be the first use of MIFC to analyze a Burkholderia bacterial species during intracellular infection. © 2016 Crown copyright. Published by Wiley Periodicals Inc. on behalf of ISAC. [ABSTRACT FROM AUTHOR]

Background: Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bmp) are Gram-negative facultative intracellular pathogens, which are the causative agents of melioidosis and glanders, respectively. Depending on the route of exposure, aerosol or transcutaneous, infection by Bp or Bm can result in an extensive range of disease - from acute to chronic, relapsing illness to fatal septicemia. Both diseases are associated with difficult diagnosis and high fatality rates. About ninety five percent of patients succumb to untreated septicemic infections and the fatality rate is 50 % even when standard antibiotic treatments are administered. Results: The goal of this study is to profile murine macrophage-mediated phenotypic and molecular responses that are characteristic to a collection of Bp, Bm, Burkholderia thailandensis (Bt) and Burkholderia oklahomensis (Bo) strains obtained from humans, animals, environment and geographically diverse locations. Burkholderia spp. (N = 21) were able to invade and replicate in macrophages, albeit to varying degrees. All Bp (N = 9) and four Bm strains were able to induce actin polymerization on the bacterial surface following infection. Several Bp and Bm strains showed reduced ability to induce multinucleated giant cell (MNGC) formation, while Bo and Bp 776 were unable to induce this phenotype. Measurement of host cytokine responses revealed a statistically significant Bm mediated IL-6 and IL- 10 production compared to Bp strains. Hierarchical clustering of transcriptional data from 84 mouse cytokines, chemokines and their corresponding receptors identified 29 host genes as indicators of differential responses between the Burkholderia spp. Further validation confirmed Bm mediated Il-1b, Il-10, Tnfrsf1b and Il-36a mRNA expressions were significantly higher when compared to Bp and Bt. Conclusions: These results characterize the phenotypic and immunological differences in the host innate response to pathogenic and avirulent Burkholderia strains and provide insight into the phenotypic alterations and molecular targets underlying host-Burkholderia interactions. [ABSTRACT FROM AUTHOR]

Linoleate 13-lipoxygenase from Burkholderia thailandensis was expressed in Escherichia coli for the production of 13-hydroxyoctadecadienoic acid (13-HODE), an antiseptic emulsifier. Linoleate 13-lipoxygenase in cells had higher thermal stability than the purified enzyme. To increase 13-HODE production, recombinant cells were permeabilized by solvents, detergents, salts, and other chemicals. The enzymatic activity in cells was the highest for permeabilized cells treated with 0.5 M NaCl among the permeabilizers tested. The optimal reaction conditions for the production of 13-HODE from linoleic acid by permeabilized cells treated with 0.5 M NaCl were at pH 7.5, 25 °C, 20 g/l linoleic acid, 15 g/l cells, 0.15 mM Cu, and 6 % (v/v) methanol in a 100-ml baffled flask containing a 5-ml working volume with agitation at 200 rpm. Under these conditions, permeabilized cells produced 15.8 g/l 13-HODE after 30 min with a conversion yield of 79 % (w/w) and a productivity of 31.6 g/l/h. The conversion yield and productivity of permeabilized cells for 13-HODE production were higher than those of purified and crude enzymes as well as nonpermeabilized cells. Therefore, permeabilized cells were efficient biocatalysts for 13-HODE production. To the best of our knowledge, this is the first report of the production of 13-HODE using cells. [ABSTRACT FROM AUTHOR]

Burkholderia thailandensis is a Gram-negative soil bacterium used as a model organism for B. pseudomallei, the causative agent of melioidosis and an organism classified category B priority pathogen and a Tier 1 select agent for its potential use as a biological weapon. Burkholderia species are reportedly “highly resistant” to antimicrobial agents, including cyclic peptide antibiotics, due to multiple resistance systems, a hypothesis we decided to test using antimicrobial (host defense) peptides. In this study, a number of cationic antimicrobial peptides (CAMPs) were tested in vitro against B. thailandensis for both antimicrobial activity and inhibition of biofilm formation. Here, we report that the Chinese cobra (Naja atra) cathelicidin NA-CATH was significantly antimicrobial against B. thailandensis. Additional cathelicidins, including the human cathelicidin LL-37, a sheep cathelicidin SMAP-29, and some smaller ATRA peptide derivatives of NA-CATH were also effective. The D-enantiomer of one small peptide (ATRA-1A) was found to be antimicrobial as well, with EC50 in the range of the L-enantiomer. Our results also demonstrate that human alpha-defensins (HNP-1 & -2) and a short beta-defensin-derived peptide (Peptide 4 of hBD-3) were not bactericidal against B. thailandensis. We also found that the cathelicidin peptides, including LL-37, NA-CATH, and SMAP-29, possessed significant ability to prevent biofilm formation of B. thailandensis. Additionally, we show that LL-37 and its D-enantiomer D-LL-37 can disperse pre-formed biofilms. These results demonstrate that although B. thailandensis is highly resistant to many antibiotics, cyclic peptide antibiotics such as polymyxin B, and defensing peptides, some antimicrobial peptides including the elapid snake cathelicidin NA-CATH exert significant antimicrobial and antibiofilm activity towards B. thailandensis. [ABSTRACT FROM AUTHOR]

A recombinant putative lipoxygenase from Burkholderia thailandensis with a specific activity of 26.4 U mg was purified using HisTrap affinity chromatography. The native enzyme was a 75-kDa dimer with a molecular mass of 150 kDa. The enzyme activity and catalytic efficiency ( k/ K) were the highest for linoleic acid ( k of 93.7 s and K of 41.5 μM), followed by arachidonic acid, α-linolenic acid, and γ-linolenic acid. The enzyme was identified as an omega-6 linoleate lipoxygenase (or a linoleate 13 S-lipoxygenase) based on genetic and HPLC analyses as well as substrate specificity. The reaction conditions for the enzymatic production of 13-hydroxy-9,11( Z, E)-octadecadienoic acid (13-HODE) were optimal at pH 7.5, 25 °C, 20 g l linoleic acid, 2.5 g l enzyme, 0.1 mM Cu, and 6 % ( v/ v) methanol. Under these conditions, linoleate 13-lipoxygenase from B. thailandensis produced 20.8 g l 13-HODE (70.2 mM) from 20 g l linoleic acid (71.3 mM) for 120 min, with a molar conversion yield of 98.5 % and productivity of 10.4 g l h. The molar conversion yield and productivity of 13-HODE obtained using B. thailandensis lipoxygenase were 151 and 158 % higher, respectively, than those obtained using commercial soybean lipoxygenase under the optimum conditions for each enzyme at the same concentrations of substrate and enzyme. [ABSTRACT FROM AUTHOR]

Background: Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis, an often fatal disease in tropical countries. Burkholderia thailandensis is a non-virulent but closely related species. Both species are soil saprophytes but are almost never isolated together. Results: We identified two mechanisms by which B. pseudomallei affects the growth of B. thailandensis. First, we found that six different isolates of B. pseudomallei inhibited the growth of B. thailandensis on LB agar plates. Second, our results indicated that 55% of isolated strains of B. pseudomallei produced a secreted compound that inhibited the motility but not the viability of B. thailandensis. Analysis showed that the active compound was a pH-sensitive and heat-labile compound, likely a protein, which may affect flagella processing or facilitate their degradation. Analysis of bacterial sequence types (STs) demonstrated an association between this and motility inhibition. The active compound was produced from B. pseudomallei during the stationary growth phase. Conclusion: Taken together, our results indicate that B. pseudomallei inhibits both the growth and motility of its close relative B. thailandensis. The latter phenomenon appears to occur via a previously unreported mechanism involving flagellar processing or degradation. [ABSTRACT FROM AUTHOR]

Burkholderia thailandensis infection in humans is uncommon. We describe a case of B. thailandensis infection in a person in China, a location heretofore unknown for B. thailandensis. We identified the specific virulence factors of B. thailandensis, which may indicate a transition to a new virulent form. [ABSTRACT FROM AUTHOR]

There is an increasing awareness of the multiple ways that bacteriophages (phages) influence bacterial evolution, population dynamics, physiology, and pathogenicity. By studying a novel group of phages infecting a soil borne pathogen, we revealed a paradigm shifting observation that the phages switch their lifestyle according to temperature. We sampled soil from an endemic area of the serious tropical pathogen Burkholderia pseudomallei, and established that podoviruses infecting the pathogen are frequently present in soil, and many of them are naturally occurring variants of a common virus type. Experiments on one phage in the related model B. thailandensis demonstrated that temperature defines the outcome of phage-bacteria interactions. At higher temperatures (37°C), the phage predominantly goes through a lytic cycle, but at lower temperatures (25°C), the phage remains temperate. This is the first report of a naturally occurring phage that follows a lytic or temperate lifestyle according to temperature. These observations fundamentally alter the accepted views on the abundance, population biology and virulence of B. pseudomallei. Furthermore, when taken together with previous studies, our findings suggest that the phenomenon of temperature dependency in phages is widespread. Such phages are likely to have a profound effect on bacterial biology, and on our ability to culture and correctly enumerate viable bacteria. [ABSTRACT FROM AUTHOR]

LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265-284 and lactoferricin17-30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead. [ABSTRACT FROM AUTHOR]

The degradation of hydrocarbons plays an important role in the eco-balancing of petroleum products, pesticides and other toxic products in the environment. The degradation of hydrocarbons by microbes such as Geobacillus thermodenitrificans, Burkhulderia, Gordonia sp. and Acinetobacter sp. has been studied intensively in the literature. The present study focused on the in silico protein engineering of alkane monooxygenase (ladA)-a protein involved in the alkane degradation pathway. We demonstrated the improvement in substrate binding energy with engineered ladA in Burkholderia thailandensis MSMB121. We identified an ortholog of ladA monooxygenase found in B. thailandensis MSMB121, and showed it to be an enzyme involved in an alkane degradation pathway studied extensively in Geobacillus thermodenitrificans. Homology modeling of the three-dimensional structure of ladA was performed with a crystal structure (protein databank ID: 3B9N) as a template in MODELLER 9v11, and further validated using PROCHECK, VERIFY-3D and WHATIF tools. Specific amino acids were substituted in the region corresponding to amino acids 305-370 of ladA protein, resulting in an enhancement of binding energy in different alkane chain molecules as compared to wild protein structures in the docking experiments. The substrate binding energy with the protein was calculated using Vina (Implemented in VEGAZZ). Molecular dynamics simulations were performed to study the dynamics of different alkane chain molecules inside the binding pockets of wild and mutated ladA. Here, we hypothesize an improvement in binding energies and accessibility of substrates towards engineered ladA enzyme, which could be further facilitated for wet laboratory-based experiments for validation of the alkane degradation pathway in this organism. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Transposon-directed insertion site sequencing was used to identify genes required by Burkholderia thailandensis to survive in plant/soil microcosms. A total of 1,153 genetic loci fulfilled the criteria as being likely to encode survival characteristics. Of these, 203 (17.6 %) were associated with uptake and transport systems; 463 loci (40.1 %) coded for enzymatic properties, 99 of these (21.4 %) had reduction/oxidation functions; 117 (10.1 %) were gene regulation or sensory loci; 61 (5.3 %) encoded structural proteins found in the cell envelope or with enzymatic activities related to it, distinct from these, 46 (4.0 %) were involved in chemotaxis and flagellum, or pilus synthesis; 39 (3.4 %) were transposase enzymes or were bacteriophage-derived; and 30 (2.6 %) were involved in the production of antibiotics or siderophores. Two hundred and twenty genes (19.1 %) encoded hypothetical proteins or those of unknown function. Given the importance of motility and pilus formation in microcosm persistence the nature of the colonization of the rhizosphere was examined by confocal microscopy. Wild type B. thailandensis expressing red fluorescent protein was inoculated into microcosms. Even though the roots had been washed, the bacteria were still present but they were motile with no attachment having taken place, perhaps being retained in a biofilm. [ABSTRACT FROM AUTHOR]

Background Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with downstream target mRNAs to attenuate translation of mRNA into protein at the post-transcriptional level. In response to specific environmental changes, sRNAs can modulate the expression levels of target genes, thus enabling adaptation of cellular physiology. Results We profiled sRNA expression in the Gram-negative bacteria Burkholderia thailandensis cultured under 54 distinct growth conditions using a Burkholderia-specific microarray that contains probe sets to all intergenic regions greater than 90 bases. We identified 38 novel sRNAs and performed experimental validation on five sRNAs that play a role in adaptation of Burkholderia to cell stressors. In particular, the trans-encoded BTH_s1 and s39 exhibited differential expression profiles dependent on growth phase and cell stimuli, such as antibiotics and serum. Furthermore, knockdown of the highly-expressed BTH_s39 by antisense transcripts reduced B. thailandensis cell growth and attenuated host immune response upon infection, indicating that BTH_s39 functions in bacterial metabolism and adaptation to the host. In addition, expression of cis-encoded BTH_s13 and s19 found in the 5' untranslated regions of their cognate genes correlated with tight regulation of gene transcript levels. This sRNA-mediated downregulation of gene expression may be a conserved mechanism of post-transcriptional gene dosage control. Conclusions These studies provide a broad analysis of differential Burkholderia sRNA expression profiles and illustrate the complexity of bacterial gene regulation in response to different environmental stress conditions. [ABSTRACT FROM AUTHOR]

Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors. [ABSTRACT FROM AUTHOR]

Aims To develop a simple gas chromatography-mass spectrometry ( GC- MS) method for the detection and differentiation of Burkholderia pseudomallei and Burkholderia mallei from each other, Burkholderia thailandensis and several members of the Burkholderia cepacia complex. Methods and Results Biomarkers were generated by one-step thermochemolysis ( TCM) and analysed using a GC- MS system. Fragments of poly-3-hydroxybutyrate- co-hydroxyvalerate [poly(3 HBA- co-3 HVA)] produced by TCM were useful biomarkers. Several cellular fatty acid methyl esters were important in differentiating the various Burkholderia species. A statistical discrimination algorithm was constructed using a combination of biomarkers. The identities of four B. pseudomallei strains, four B. mallei strains and one strain of each near neighbour were confirmed in a statistically designed test using the algorithm. The detection limit for this method was found to be approximately 4000 cells. Conclusions The method is fast, accurate and easy to use. The algorithm is robust against different growth conditions (medium and temperature). Significance and Impact of the Study This assay may prove beneficial in a clinical diagnostic setting, where the rapid identification of B. pseudomallei is essential to effective treatment. This method could also be easily employed after a biological attack to confirm the presence of either B. pseudomallei or B. mallei. [ABSTRACT FROM AUTHOR]

Die langgesuchte Struktur von Malleobactin, einem Virulenzfaktor von Pathogenen der Familie Burkholderia mallei, wurde durch genetische und chemische Analysen aufgeklärt. Der neue nitrosubstituierte Siderophor ist von einem ungewöhnlichen ungeschützten Hydroxylamin abgeleitet, das spontane Oxidation erfährt, wie in In ‐ vitro ‐ Assays und durch Nachweis von Analoga mit Hydroxylamin ‐ , Nitroso ‐ und Azoxid ‐ Gruppen gezeigt wurde. [ABSTRACT FROM AUTHOR]

The elusive structure of malleobactin, a virulence factor of pathogens belonging to the Burkholderia mallei family, was finally unveiled by genetic and chemical analyses. The novel nitro‐substituted siderophore is derived from an unusual, unprotected hydroxylamine, which undergoes spontaneous oxidation, as shown by in vitro assays and detection of analogues featuring hydroxylamino, nitroso, and azoxide groups. [ABSTRACT FROM AUTHOR]

5′-Methylthioadenosine/ S-adenosylhomocysteine nucleosidase ( MTAN) plays crucial roles in the production of autoinducers and methionine metabolism. Putative genes encoding MTAN and Ado Hcyase from Burkholderia thailandensis were cloned and characterized. The Km values of MTAN for 5′-methylthioadenosine ( MTA) and S-adenosylhomocysteine ( SAH) were 19 and 58 μM, respectively. The catalytic efficiency of MTAN for SAH was only 0.004% of the value for MTA, indicating an almost complete substrate preference of MTAN for MTA. The results of autoinducer-2 assay of B. thailandensis and recombinants indicated that LuxS enzyme activity was lacking in Burkholderia species. Instead, Ado Hcyase hydrolysed SAH directly to homocysteine and adenosine in the activated methyl cycle. Meanwhile, the Km value of Ado Hcyase for SAH was determined to be 40 μM. Sequence analysis revealed that MTAN had much higher diversity than Ado Hcyase, which likely contributes to its substrate preference for MTA. Furthermore, the phylogenetic tree of MTAN sequences revealed that LuxS+ bacteria could be discriminated from LuxS− bacteria. These results suggested that the substrate preference of MTAN for MTA and SAH degradation pathway evolved with the bacterial-activated methyl cycle. [ABSTRACT FROM AUTHOR]