Comprehensive genomic analysis of Burkholderia arboris PN-1 reveals its biocontrol potential against Fusarium solani-induced root rot in Panax notoginseng.

Panax notoginseng (Burkill) F.H. Chen, a valuable traditional Chinese medicine, faces significant yield and quality challenges stemming from root rot primarily caused by Fusarium solani. Burkholderia arboris PN-1, isolated from the rhizosphere soil of P. notoginseng, demonstrated a remarkable ability to inhibit the growth of F. solani. This study integrates phenotypic, phylogenetic, and genomic analyses to enhance our understanding of the biocontrol mechanisms employed by B. arboris PN-1. Phenotype analysis reveals that B. arboris PN-1 effectively suppresses P. notoginseng root rot both in vitro and in vivo. The genome of B. arboris PN-1 comprises three circular chromosomes (contig 1: 3,651,544 bp, contig 2: 1,355,460 bp, and contig 3: 3,471,056 bp), with a 66.81% GC content, housing 7,550 protein-coding genes. Notably, no plasmids were detected. Phylogenetic analysis places PN-1 in close relation to B. arboris AU14372, B. arboris LMG24066, and B. arboris MEC_B345. Average nucleotide identity (ANI) values confirm the PN-1 classification as B. arboris. Comparative analysis with seven other B. arboris strains identified 4,628 core genes in B. arboris PN-1. The pan-genome of B. arboris appears open but may approach closure. Whole-genome sequencing revealed 265 carbohydrate-active enzymes and identified 9 gene clusters encoding secondary metabolites. This comprehensive investigation enhances our understanding of B. arboris genomes, paving the way for their potential as effective biocontrol agents against fungal plant pathogens in the future.

Nutrient supplementation by genome-eroded Burkholderia symbionts of scale insects.

Hemipterans are known as hosts to bacterial or fungal symbionts that supplement their unbalanced diet with essential nutrients. Among them, scale insects (Coccomorpha) are characterized by a particularly large diversity of symbiotic systems. Here, using microscopic and genomic approaches, we functionally characterized the symbionts of two scale insects belonging to the Eriococcidae family, Acanthococcus aceris and Gossyparia spuria. These species host Burkholderia bacteria that are localized in the cytoplasm of the fat body cells. Metagenome sequencing revealed very similar and highly reduced genomes (<900KBp) with a low GC content (~38%), making them the smallest and most AT-biased Burkholderia genomes yet sequenced. In their eroded genomes, both symbionts retain biosynthetic pathways for the essential amino acids leucine, isoleucine, valine, threonine, lysine, arginine, histidine, phenylalanine, and precursors for the semi-essential amino acid tyrosine, as well as the cobalamin-dependent methionine synthase MetH. A tryptophan biosynthesis pathway is conserved in the symbiont of G. spuria, but appeared pseudogenized in A. aceris, suggesting differential availability of tryptophan in the two host species' diets. In addition to the pathways for essential amino acid biosynthesis, both symbionts maintain biosynthetic pathways for multiple cofactors, including riboflavin, cobalamin, thiamine, and folate. The localization of Burkholderia symbionts and their genome traits indicate that the symbiosis between Burkholderia and eriococcids is younger than other hemipteran symbioses, but is functionally convergent. Our results add to the emerging picture of dynamic symbiont replacements in sap-sucking Hemiptera and highlight Burkholderia as widespread and versatile intra- and extracellular symbionts of animals, plants, and fungi.

Biocontrol of bacterial seedling rot of rice plants using combination of Cytobacillus firmus JBRS159 and silicon.

Burkholderia glumae causes bacterial panicle blight (BPB) and bacterial seedling rot (BSR) which are difficult to control in rice plants. Seed disinfection using microbes and eco-friendly materials is an efficient alternative practice for managing BPB and BSR. In this study, we applied Cytobacillus firmus JBRS159 (JBRS159) in combination with silicon dioxide (SiO2) nanoparticle or potassium silicate (K2SiO3) solution to control BSR. JBRS159, SiO2 nanoparticle, and K2SiO3 independently suppressed the BSR disease and promoted growths of rice and Arabidopsis. Population of B. glumae in the treated rice seeds was suppressed by the application of JBRS159 via competitions for nutrients and niches. The mixture of JBRS159 and each Si compound (SiO2 nanoparticle or K2SiO3) was complementary for disease-suppressing and growth-promoting activities of individual treatment. The results of this study indicate that mixture of JBRS159 with each Si compound can be harnessed for disease control and growth promotion as efficient alternatives to chemical pesticides and synthetic fertilizers. The efficacy of JBRS159 and Si compounds in the control of BSR and BPB in the field remains to be evaluated.

CysB Is a Key Regulator of the Antifungal Activity of Burkholderia pyrrocinia JK-SH007.

Burkholderia pyrrocinia JK-SH007 can effectively control poplar canker caused by pathogenic fungi. Its antifungal mechanism remains to be explored. Here, we characterized the functional role of CysB in B. pyrrocinia JK-SH007. This protein was shown to be responsible for the synthesis of cysteine and the siderophore ornibactin, as well as the antifungal activity of B. pyrrocinia JK-SH007. We found that deletion of the cysB gene reduced the antifungal activity and production of the siderophore ornibactin in B. pyrrocinia JK-SH007. However, supplementation with cysteine largely restored these two abilities in the mutant. Further global transcriptome analysis demonstrated that the amino acid metabolic pathway was significantly affected and that some sRNAs were significantly upregulated and targeted the iron-sulfur metabolic pathway by TargetRNA2 prediction. Therefore, we suggest that, in B. pyrrocinia JK-SH007, CysB can regulate the expression of genes related to Fe-S clusters in the iron-sulfur metabolic pathway to affect the antifungal activity of B. pyrrocinia JK-SH007. These findings provide new insights into the various biological functions regulated by CysB in B. pyrrocinia JK-SH007 and the relationship between iron-sulfur metabolic pathways and fungal inhibitory substances. Additionally, they lay the foundation for further investigation of the main antagonistic substances of B. pyrrocinia JK-SH007.

Fast degradation of macro alkanes through activating indigenous bacteria using biosurfactants produced by Burkholderia sp.

Soil bacteria that produce biosurfactants can use total petroleum hydrocarbons (TPHs) as a carbon source. This study demonstrated that biosurfactants produced by Burkholderia sp. enhanced the recovery and synergism of soil microbial community, resulting in fast degradation of macro alkanes. Experiments were carried out by applying bio-stimulation after pre-oxidation to investigate the effects of nutrient addition on biosurfactant production, TPH degradation, and microbial community succession in the soil. The results presented that bio-stimulation could produce biosurfactants in high C/N (32.6) and C/H (13.3) conversion after pre-oxidation and increased the total removal rate of TPH (10.59-46.71%). The number of total bacteria had a rapid increase trend (2.94-8.50 Log CFU/g soil). The degradation rates of macro alkanes showed a 4.0-fold (48.07 mg/kg·d-1 versus 186.48 mg/kg·d-1) increase, and the bioremediation time of degrading macro alkanes saved 166 days. Further characterization revealed that the biosurfactants produced by Burkholderia sp. could activate indigenous bacteria to degrade macro alkanes rapidly. A shift in phylum from Actinomycetes to Proteobacteria was observed during bioremediation. The average relative abundance of the microbial community increased from 36.24 to 64.96%, and the predominant genus tended to convert from Allorhizobium (8.57%) to Burkholderia (15.95%) and Bacillus (15.70%). The co-occurrence network and Pearson correlation analysis suggested that the synergism of microbial community was the main reason for the fast degradation of macro alkanes in petroleum-contaminated soils. Overall, this study indicated the potential of the biosurfactants to activate and enhance the recovery of indigenous bacteria after pre-oxidation, which was an effective method to remediate petroleum-contaminated soils.

CRISPR detection in metagenome-assembled genomes (MAGs) of coal mine.

Bacterial and archaeal CRISPR-Cas systems provide adaptive immune protection against foreign mobile genetic elements. When viruses infect bacteria, a small portion of the viral DNA is inserted into the bacterial DNA in a specific pattern to produce segments known as CRISPR arrays. Metagenome assembled genomes (MAGs) were used in our study to identify the CRISPR sequence for determining the interacted phage. Metagenomic data from a coal mine was used to perform a computational study. From raw reads, 206151 contigs were assembled. Then contigs were clustered into 150 Metagenome assembled genomes from which 78 non-redundant MAGs were selected. Using the CHECKM standard, seven MAGs were found to have >80 completeness and <20 contaminations. Those MAGs were analyzed for the presence of CRISPR elements. Out of seven MAGs, four MAGs have the CRISPR elements and are searched against the VIROblast database. CRISPR arrays have 4, 1, 3, and 7 spacer sequences in the MAGs of Burkholderia, Acinetobacter, Oxalobacteraceae, and Burkholderia multivorans respectively. The uncultured Caudovirales phage genomic regions were present in the genomes of Burkholderia, Oxalobacteriaceae, and Burkholderia multivorans. This study follows the unconventional metagenomics workflow to provide a better understanding of bacteria and phage interactions.

Burkholderia semiarida sp. nov. and Burkholderia sola sp. nov., two novel B. cepacia complex species causing onion sour skin.

Two putative novel Burkholderia cenocepacia lineages found in the semi-arid region of north-east Brazil causing onion sour skin were studied using genomic approaches to determine their taxonomic position. Four strains belonging to one novel lineage (CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171) and one strain (CCRMBC51) belonging to another novel lineage had their whole genome sequenced to carry out taxogenomic analyses. The phylogenomic tree built using the type (strain) genome server (TYGS) clustered the strains CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171 into the same clade, while grouped the strain CCRMBC51 separately. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) analysis showed values above 99.21 % and 93.2 %, respectively, among the strains CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171, while ANI and dDDH values between these strains and the strain CCRMBC51 were below 94.49 % and 56.6 %, respectively. All these strains showed ANI and dDDH values below 94.78 % and 58.8 % concerning type strains of the B. cepacia complex (Bcc) species. The phylogenetic maximum likelihood tree constructed based on the multilocus sequence analysis of core genes (cMLSA) clustered the strains CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171 and the strain CCRMBC51 in two exclusive clades, which did not cluster with any known species of the Bcc. Therefore, combined data from TYGS, ANI, dDDH, and cMLSA demonstrated that the strains represent two novel species of the Bcc, which we classified as Burkholderia semiarida sp. nov. and Burkholderia sola sp. nov., and proposed the strains CCRMBC74T (=IBSBF 3371 T = CBAS 905 T) and CCRMBC51T (=IBSBF3370T = CBAS 904 T) as type strains, respectively.

Transformation mechanism of methylphosphonate to methane by Burkholderia sp: Insight from multi-labeled water isotope probing and transcriptomic.

Methylphosphonate (MPn), has been identified as a likely source of methane in aerobic ocean and may be responsible for the "ocean methane paradox", that is oversaturation of dissolved methane in oxic sea waters. However, the mechanism underlying the cleavage of C-P bonds during microbial degradation is not well understood. Using multi-labeled water isotope probing (MLWIP) and transcriptome analysis, we investigated the phosphate oxygen isotope systematics and mechanisms of microbial-mediated degradation of MPn in this study. In the aerobic culture containing MPn as the only phosphorus source, there was a significant release of inorganic phosphate (149.4 µmol/L) and free methane (268.3 mg/L). The oxygen isotopic composition of inorganic phosphorus (δ18OP) of accumulated released phosphate was 4.50‰, 23.96‰, and 40.88‰, respectively, in the corresponding 18O-labeled waters of -10.3‰, 9.9‰, and 30.6‰, and the slope obtained in plots of δ18OP versus the oxygen isotopic composition of water (δ18OW) was 0.89. Consequently, 89% of the oxygen atoms (Os) in phosphate (PO4) were exchanged with 18O-labeled waters in the medium, while the rest were exchanged with intracellular metabolic water. It has been confirmed that the C-P bond cleavage of MPn occurs in the cell with both ambient and metabolic water participation. Moreover, phn gene clusters play significant roles to cleave the C-P bond of MPn for Burkholderia sp. HQL1813, in which phnJ, phnM and phnI genes are significantly up-regulated during MPn decomposition to methane. In conclusion, the aerobic biotransformation of MPn to free methane by Burkholderia sp. HQL1813 has been elucidated, providing new insights into the mechanism that bio-cleaves C-P bonds to produce methane aerobically in aqueous environments for representative phosphonates.

Cost-effective rhamnolipid production by Burkholderia thailandensis E264 using agro-industrial residues.

The agro-industrial by-products corn steep liquor (CSL) and olive mill wastewater (OMW) were evaluated as low-cost substrates for rhamnolipid production by Burkholderia thailandensis E264. In a culture medium containing CSL (7.5% (v/v)) as sole substrate, B. thailandensis E264 produced 175 mg rhamnolipid/L, which is about 1.3 times the amount produced in the standard medium, which contains glycerol, peptone, and meat extract. When the CSL medium was supplemented with OMW (10% (v/v)), rhamnolipid production further increased up to 253 mg/L in flasks and 269 mg/L in a bioreactor. Rhamnolipids produced in CSL + OMW medium reduced the surface tension up to 27.1 mN/m, with a critical micelle concentration of 51 mg/L, better than the values obtained with the standard medium (28.9 mN/m and 58 mg/L, respectively). However, rhamnolipids produced in CSL + OMW medium displayed a weak emulsifying activity when compared to those produced in the other media. Whereas di-rhamnolipid congeners represented between 90 and 95% of rhamnolipids produced by B. thailandensis E264 in CSL and the standard medium, the relative abundance of mono-rhamnolipids increased up to 55% in the culture medium containing OMW. The difference in the rhamnolipid congeners produced in each medium explains their different surface-active properties. To the best of our knowledge, this is the first report of rhamnolipid production by B. thailandensis using a culture medium containing agro-industrial by-products as sole ingredients. Furthermore, rhamnolipids produced in the different media recovered around 60% of crude oil from contaminated sand, demonstrating its potential application in the petroleum industry and bioremediation. KEY POINTS: • B. thailandensis produced RL using agro-industrial by-products as sole substrates • Purified RL displayed excellent surface activity (minimum surface tension 27mN/m) • Crude RL (cell-free supernatant) recovered 60% of crude oil from contaminated sand.

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In order to solve the problem that soil soluble phosphorus content in most cultivated land in China is insufficient and the plant growth is inhibited, a phosphate solubilizing microorganism (PB) was screened and identified, and its phosphate solubilizing performance was optimized. The results showed that the PB strain was belonged to Burkholderia stabilis. It had the ability of nitrogen fixation and indole-3-acetic acid (IAA) secretion, as well as a certain inhibitory effect on Escherichia coli. It could maintain high activity and phosphorus solubilizing ability at pH 8.0-10.0, indicating good alkali resistance. The results of phosphorus dissolving performance optimization showed that the phosphate solubilizing capacity of strain PB reached the best at 30℃, pH 7.0, 180 r·min-1, using glucose as carbon source, ammonium sulfate as nitrogen source, tricalcium phosphate as phosphorus source and adding 50 µmol·L-1 lysine. The amount of dissolved phosphorus was 569.33 mg·L-1, which was 1.9 times of that before optimization. The strain mainly secreted citric acid, malonic acid, and glucuronic acid during metabolism. After adding lysine, the type of organic acids secreted by the strain did not change, but the content increased significantly. Results from pot experiments showed that the application of PB bacterial fertilizer could significantly improve the growth and physiological indicators of garlic seedlings, and that the promotion effect was more obvious after adding lysine. Compared with the control, the height of seedling was increased by 18.6%, seedling diameter was increased by 16.7%, aboveground fresh and dry weight were increased by 22.1% and 15.7%, and belowground fresh and dry weight were increased by 22.0% and 28.7%, respectively in PB with lysine treatment. Soil available phosphorus content was 2.1 and 2.3 times of the control in PB and PB+lysine treatments, indicating that PB could improve soil available phosphate content. Adding lysine could strengthen such function.

Isolation and Characterization of Phosphate Solubilizing Bacteria from Paddy Field Soils in Japan.

Phosphorus (P) is abundant in soil and is essential for plant growth and development; however, it is easily rendered insoluble in complexes of different types of phosphates, which may lead to P deficiency. Therefore, increases in the amount of P released from phosphate minerals using microbial inoculants is an important aspect of agriculture. The present study used inorganic phosphate solubilizing bacteria (iPSB) in paddy field soils to develop microbial inoculants. Soils planted with rice were collected from different regions of Japan. Soil P was sequentially fractionated using the Hedley method. iPSB were isolated using selective media supplemented with tricalcium phosphate (Ca-P), aluminum phosphate (Al-P), or iron phosphate (Fe-P). Representative isolates were selected based on the P solubilization index and soil sampling site. Identification was performed using 16S rRNA and rpoB gene sequencing. Effectiveness was screened based on rice cultivar Koshihikari growth supplemented with Ca-P, Al-P, or Fe-P as the sole P source. Despite the relatively homogenous soil pH of paddy field sources, three sets of iPSB were isolated, suggesting the influence of fertilizer management and soil types. Most isolates were categorized as ß-Proteobacteria (43%). To the best of our knowledge, this is the first study to describe the genera Pleomorphomonas, Rhodanobacter, and Trinickia as iPSB. Acidovorax sp. JC5, Pseudomonas sp. JC11, Burkholderia sp. JA6 and JA10, Sphingomonas sp. JA11, Mycolicibacterium sp. JF5, and Variovorax sp. JF6 promoted plant growth in rice supplemented with an insoluble P source. The iPSBs obtained may be developed as microbial inoculants for various soil types with different P fixation capacities.

cDNA Transcriptome of Arabidopsis Reveals Various Defense Priming Induced by a Broad-Spectrum Biocontrol Agent Burkholderia sp. SSG.

Burkholderia sp. SSG is a potent biological control agent. Even though its survival on the leaf surface declined rapidly, SSG provided extended, moderate plant protection from a broad spectrum of pathogens. This study used Arabidopsis Col-0 and its mutants, eds16-1, npr1-1, and pad4-1 as model plants and compared treated plants with non-treated controls to elucidate whether SSG triggers plant defense priming. Only eds16-1 leaves with SSG became purplish, suggesting the involvement of salicylic acid (SA) in SSG-induced priming. cDNA sequencing of Col-0 plants and differential gene expression analysis identified 120 and 119 differentially expressed genes (DEGs) at 6- and 24-h post-treatment (hpt) with SSG, respectively. Most of these DEGs encoded responses to biotic and abiotic stimuli or stresses; four DEGs had more than two isoforms. A total of 23 DEGs were shared at 6 and 24 hpt, showing four regulation patterns. Functional categorization of these shared DEGs, and 44 very significantly upregulated DEGs revealed that SSG triggered various defense priming mechanisms, including responses to phosphate or iron deficiency, modulation of defense-linked SA, jasmonic acid, ethylene, and abscisic acid pathways, defense-related gene regulation, and chromatin modification. These data support that SSG is an induced systemic resistance (ISR) trigger conferring plant protection upon pathogen encounter.

Contribution of K solubilising bacteria (Burkholderia sp.) promotes tea plant growth (Camellia sinesis) and leaf polyphenols content by improving soil available K level.

K+ availability is important for growth and quality of tea (Camellia sine sis L.). K solubilising bacteria convert insoluble K to available K. This study was conducted to screen K solubilising bacteria isolated from tea rhizosphere soil in Qimen county, Anhui province, China. The maximum K solubilisation colony (the ratio of diameter halo/colony was 2.54) was identified as Burkholderia sp. (storage number: M2021105) by biochemistry and molecular analysis. Pot experiments (Laterite) showed that the inoculation of Burkholderia sp. significantly improved tea plant height (Zhongcha108, 1 year old) and total polyphenols content by 21.14% and 21.58% compared with the control, respectively. Higher polyphenol level promoted the formation of theaflavin in the fermentation experiments. Further experiments showed that tartaric acid and pryuvic acid produced by Burkholderia sp. are important components associated with K solubilisation in vitro . Burkholderia sp. significantly increased soil available K by 15.12%; however, there was no significant difference in available N and P, and Cu, Mg, Zn and Ca compared with the control. K content in inoculated tea roots and leaves was significantly higher (50% and 10%, respectively) than the control. Compared with the control, exogenous supply of 60mgkg-1 K significantly increased levels of polyphenol (53.97%), theaflavin (16.31%), theaflavin-3-gallate (20%), theaflavin 3'-gallic acid ester (32.24%) and theaflavin 3,3'-gallic acid ester (40.95%). Due to its ability to enable higher available soil K, ur study indicated that Burkholderia sp. have potential to increase total polyphenols content be a bio-inoculant for biofortification of tea.

Leaf nodule endosymbiotic Burkholderia confer targeted allelopathy to their Psychotria hosts.

After a century of investigations, the function of the obligate betaproteobacterial endosymbionts accommodated in leaf nodules of tropical Rubiaceae remained enigmatic. We report that the α-D-glucose analogue (+)-streptol, systemically supplied by mature Ca. Burkholderia kirkii nodules to their Psychotria hosts, exhibits potent and selective root growth inhibiting activity. We provide compelling evidence that (+)-streptol specifically affects meristematic root cells transitioning to anisotropic elongation by disrupting cell wall organization in a mechanism of action that is distinct from canonical cellulose biosynthesis inhibitors. We observed no inhibitory or cytotoxic effects on organisms other than seed plants, further suggesting (+)-streptol as a bona fide allelochemical. We propose that the suppression of growth of plant competitors is a major driver of the formation and maintenance of the Psychotria-Burkholderia association. In addition to potential agricultural applications as a herbicidal agent, (+)-streptol might also prove useful to dissect plant cell and organ growth processes.

Chemical or Genetic Alteration of Proton Motive Force Results in Loss of Virulence of Burkholderia glumae, the Cause of Rice Bacterial Panicle Blight.

Rice is an important source of food for more than half of the world's population. Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. B. glumae synthesizes toxoflavin, an essential virulence factor that is required for symptoms of the disease. The products of the tox operons, ToxABCDE and ToxFGHI, are responsible for the synthesis and the proton motive force (PMF)-dependent secretion of toxoflavin, respectively. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Our previous work has demonstrated that absence of certain DedA family members results in pleiotropic effects, impacting multiple pathways that are energized by PMF. We have demonstrated that a member of the DedA family from Burkholderia thailandensis, named DbcA, is required for the extreme polymyxin resistance observed in this organism. B. glumae encodes a homolog of DbcA with 73% amino acid identity to Burkholderia thailandensis DbcA. Here, we created and characterized a B. glumae ΔdbcA strain. In addition to polymyxin sensitivity, the B. glumae ΔdbcA strain is compromised for virulence in several BPB infection models and secretes only low amounts of toxoflavin (∼15% of wild-type levels). Changes in membrane potential in the B. glumae ΔdbcA strain were reproduced in the wild-type strain by the addition of subinhibitory concentrations of sodium bicarbonate, previously demonstrated to cause disruption of PMF. Sodium bicarbonate inhibited B. glumae virulence in rice, suggesting a possible non-toxic chemical intervention for bacterial panicle blight. IMPORTANCE Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Here, we constructed a B. glumae mutant with a deletion in a DedA family member named dbcA and report a loss of virulence in models of BPB. Physiological analysis of the mutant shows that the proton motive force is disrupted, leading to reduction of secretion of the essential virulence factor toxoflavin. The mutant phenotypes are reproduced in the virulent wild-type strain without an effect on growth using sodium bicarbonate, a nontoxic buffer that has been reported to disrupt the PMF. The results presented here suggest that bicarbonate may be an effective antivirulence agent capable of controlling BPB without imposing an undue burden on the environment.

Biodegradation of binary mixtures of octane with benzene, toluene, ethylbenzene or xylene (BTEX): insights on the potential of Burkholderia, Pseudomonas and Cupriavidus isolates.

The contamination of the environment by crude oil and its by-products, mainly composed of aliphatic and aromatic hydrocarbons, is a widespread problem. Biodegradation by bacteria is one of the processes responsible for the removal of these pollutants. This study was conducted to determine the abilities of Burkholderia sp. B5, Cupriavidus sp. B1, Pseudomonas sp. T1, and another Cupriavidus sp. X5 to degrade binary mixtures of octane (representing aliphatic hydrocarbons) with benzene, toluene, ethylbenzene, or xylene (BTEX as aromatic hydrocarbons) at a final concentration of 100 ppm under aerobic conditions. These strains were isolated from an enriched bacterial consortium (Yabase or Y consortium) that prefer to degrade aromatic hydrocarbon over aliphatic hydrocarbons. We found that B5 degraded all BTEX compounds more rapidly than octane. In contrast, B1, T1 and X5 utilized more of octane over BTX compounds. B5 also preferred to use benzene over octane with varying concentrations of up to 200 mg/l. B5 possesses alkane hydroxylase (alkB) and catechol 2,3-dioxygenase (C23D) genes, which are responsible for the degradation of alkanes and aromatic hydrocarbons, respectively. This study strongly supports our notion that Burkholderia played a key role in the preferential degradation of aromatic hydrocarbons over aliphatic hydrocarbons in the previously characterized Y consortium. The preferential degradation of more toxic aromatic hydrocarbons over aliphatics is crucial in risk-based bioremediation.

Effects of green synthesized and commercial nZVI on crystal violet degradation by Burkholderia vietnamiensis C09V: Dose-dependent toxicity and biocompatibility.

The increasingly common remedial application of nanoscale zero-valent iron (nZVI) to alleviate specific contaminant issues may inadvertently lead to nZVI accumulation in wastewater. This is a potential concern, because the effect of nZVI on the common microbes essential for wastewater biotreatment is not known. This is further complicated when there are many ways available to synthesize nZVI, which may interreact with bacteria differently. Thus, in this study, the different effects of nZVI synthesized by Eucalyptus leaves (EL-nZVI) and a commercially synthesized nZVI on the biodegradation of crystal violet by Burkholderia vietnamiensis C09V (B.V. C09V) was studied. At high dose (1000 mg/L), EL-nZVI and commercial nZVI both significantly inhibited the removal of crystal violet by B.V. C09V, decreasing removal rates by 10.5 and 13.1% respectively. Optical density (OD600) and soluble protein assays indicated that the growth of B.V. C09V improved under low doses (100 mg/L), but remained inhibited under high doses (500 and 1000 mg/L) of both commercial and EL-nZVI. Enzymes were also sensitive to nZVI, where the commercial variant exerted a greater effect on both the activity of lactate dehydrogenase (LDH) and superoxide dismutase (SOD) than EL-nZVI, indicating that EL-nZVI was less toxic than commercial nZVI. LIVE/DEAD staining also showed that the number of apoptotic cells was significantly higher when exposed to commercial nZVI rather than EL-nZVI. Furthermore, scanning electron microscopy (SEM) confirmed that direct contact between nZVI and cells at 1000 mg/L nZVI caused cell membrane disruption. Whereas, at 100 mg/L EL-nZVI, B.V. C09V grew better due to the formation of dense biofilms around the suspended EL-nZVI at a. Fourier transform infrared spectra (FTIR), confirmed an abundance of oxygen-containing functional groups on the surface of EL-nZVI which provided better biocompatibility than commercial nZVI. Overall, while dose was the most significant factor influencing nZVI toxicity, surface composition and morphology was also important. These new findings suggest chemical synthesis of metal nanoparticles should be replaced by biosynthetic routes to maintain viable microbial pollution during wastewater treatment.

Regulation of Glycine Cleavage and Detoxification by a Highly Conserved Glycine Riboswitch in Burkholderia spp.

The glycine riboswitch is a known regulatory element that is unique in having two aptamers that are joined by a linker region. In this study, we investigated a glycine riboswitch located in the 5' untranslated region of a glycine cleavage system homolog (gcvTHP) in Burkholderia spp. Structure prediction using the sequence generated a model with a glycine binding pocket composed of base-triple interactions (G62-A64-A86 and G65-U84-C85) that are supported by A/G minor interactions (A17-C60-G88 and G16-C61-G87, respectively) and two ribose-zipper motifs (C11-G12 interacting with A248-A247 and C153-U154 interacting with A79-A78) which had not been previously reported. The capacity of the riboswitch to bind to glycine was experimentally validated by native gel assays and the crucial role of interactions that make up the glycine binding pocket were proven by mutations of A17U and G16C which resulted in conformational differences that may lead to dysfunction. Using glycine supplemented minimal media, we were able to prove that the expression of the gcvTHP genes found downstream of the riboswitch responded to the glycine concentrations introduced thus confirming the role of this highly conserved Burkholderia riboswitch and its associated genes as a putative glycine detoxification system in Burkholderia spp.

Drug screening to identify compounds to act as co-therapies for the treatment of Burkholderia species.

Burkholderia pseudomallei is a soil-dwelling organism present throughout the tropics. It is the causative agent of melioidosis, a disease that is believed to kill 89,000 people per year. It is naturally resistant to many antibiotics, requiring at least two weeks of intravenous treatment with ceftazidime, imipenem or meropenem followed by 6 months of orally delivered co-trimoxazole. This places a large treatment burden on the predominantly middle-income nations where the majority of disease occurs. We have established a high-throughput assay for compounds that could be used as a co-therapy to potentiate the effect of ceftazidime, using the related non-pathogenic bacterium Burkholderia thailandensis as a surrogate. Optimization of the assay gave a Z' factor of 0.68. We screened a library of 61,250 compounds and identified 29 compounds with a pIC50 (-log10(IC50)) greater than five. Detailed investigation allowed us to down select to six "best in class" compounds, which included the licensed drug chloroxine. Co-treatment of B. thailandensis with ceftazidime and chloroxine reduced culturable cell numbers by two orders of magnitude over 48 hours, compared to treatment with ceftazidime alone. Hit expansion around chloroxine was performed using commercially available compounds. Minor modifications to the structure abolished activity, suggesting that chloroxine likely acts against a specific target. Finally, an initial study demonstrates the utility of chloroxine to act as a co-therapy to potentiate the effect of ceftazidime against B. pseudomallei. This approach successfully identified potential co-therapies for a recalcitrant Gram-negative bacterial species. Our assay could be used more widely to aid in chemotherapy to treat infections caused by these bacteria.

Biological characteristics and salt-tolerant plant growth-promoting effects of an ACC deaminase-producing Burkholderia pyrrocinia strain isolated from the tea rhizosphere.

Plant growth-promoting rhizobacteria that produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase can promote plant growth and enhance abiotic stress tolerance. In this study, Burkholderia pyrrocinia strain P10, with an ACC deaminase activity of 33.01-µmol/h/mg protein, was isolated from the tea rhizosphere and identified based on morphological, biochemical, and molecular characteristics. In addition to its ACC deaminase activity at pH 5.0-9.0 and in response to 5% NaCl and 20% polyethylene glycol, strain P10 can also solubilize phosphorus compounds, produce indole-3-acetic acid, and secrete siderophores. Pot experiments revealed that strain P10 can significantly enhance peanut seedling growth under saline conditions (100- and 170-mmol/L NaCl). Specifically, it increased the fresh weight and root length of plants by 90.12% and 79.22%, respectively, compared with high-salt stress. These results provide new insights into the biological characteristics of Burkholderia pyrrocinia, which may be useful as a bio-fertilizer.