Burkholderia thailandensis Isolated from Infected Wound, Southwest China, 2022.

We report a clinical isolate of Burkholderia thailandensis 2022DZh obtained from a patient with an infected wound in southwest China. Genomic analysis indicates that this isolate clusters with B. thailandensis BPM, a human isolate from Chongqing, China. We recommend enhancing monitoring and surveillance for B. thailandensis infection in both humans and livestock.

Molecular characterization and evaluation of novel management options for Burkholderia glumae BG1, the causative agent of panicle blight of rice (Oryza sativa L.).

BACKGROUND: Bacterial panicle blight, incited by Burkholderia glumae, has impacted rice production globally. Despite its significance, knowledge about the disease and the virulence pattern of the causal agent is very limited. Bacterial panicle blight is a major challenge in the rice-growing belts of North-western India, resulting in yield reduction. However, the management of B. glumae has become a challenge due to the lack of proper management strategies. METHODOLOGY AND RESULTS: Twenty-one BG strains have been characterized using the 16S rRNA and the gyrB gene-based sequence approach in the present study. The gyrB gene-based phylogenetic analysis resulted in geographic region-specific clustering of the BG isolates. The virulence screening of twenty-one BG strains by inoculating the pathogenic bacterial suspension of 1 × 10-8 cfu/ml at the booting stage (55 DAT) revealed the variation in the disease severity and the grain yield of rice plants. The most virulent BG1 strain resulted in the highest disease incidence (82.11%) and lowest grain yield (11.12 g/plant), and the least virulent BG10 strain resulted in lowest disease incidence of 18.94% and highest grain yield (24.62 g/plant). In vitro evaluation of various biocontrol agents and nano copper at different concentrations by agar well diffusion method revealed that nano copper at 1000 mg/L inhibited the colony growth of B. glumae. Under net house conditions, nano copper at 1000 mg/L reduced the disease severity to 21.23% and increased the grain yield by 20.91% (31.76 g per plant) compared to the positive control (COC 0.25% + streptomycin 200 ppm). Remarkably, pre-inoculation with nano copper at 1000 mg/L followed by challenge inoculation with B. glumae enhanced the activity of enzymatic antioxidants viz., Phenyl ammonia-lyase (PAL), Polyphenol oxidase (PPO) and Peroxidase (POX) and non-enzymatic antioxidant phenol. Additionally, we observed a substantial transcript level upregulation of six defense-related genes to several folds viz., OsPR2, OsPR5, OsWRKY71, OsPAL1, OsAPX1, and OsPPO1 in comparison to the pathogen control and healthy control. CONCLUSIONS: Overall, our study provides valuable insights into the potential and practical application of nano copper for the mitigation of bacterial panicle blight, offering promising prospects for commercial utilization in disease management.

The long-chain flavodoxin FldX1 improves the biodegradation of 4-hydroxyphenylacetate and 3-hydroxyphenylacetate and counteracts the oxidative stress associated to aromatic catabolism in Paraburkholderia xenovorans.

BACKGROUND: Bacterial aromatic degradation may cause oxidative stress. The long-chain flavodoxin FldX1 of Paraburkholderia xenovorans LB400 counteracts reactive oxygen species (ROS). The aim of this study was to evaluate the protective role of FldX1 in P. xenovorans LB400 during the degradation of 4-hydroxyphenylacetate (4-HPA) and 3-hydroxyphenylacetate (3-HPA). METHODS: The functionality of FldX1 was evaluated in P. xenovorans p2-fldX1 that overexpresses FldX1. The effects of FldX1 on P. xenovorans were studied measuring growth on hydroxyphenylacetates, degradation of 4-HPA and 3-HPA, and ROS formation. The effects of hydroxyphenylacetates (HPAs) on the proteome (LC-MS/MS) and gene expression (qRT-PCR) were quantified. Bioaugmentation with strain p2-fldX1 of 4-HPA-polluted soil was assessed, measuring aromatic degradation (HPLC), 4-HPA-degrading bacteria, and plasmid stability. RESULTS: The exposure of P. xenovorans to 4-HPA increased the formation of ROS compared to 3-HPA or glucose. P. xenovorans p2-fldX1 showed an increased growth on 4-HPA and 3-HPA compared to the control strain WT-p2. Strain p2-fldX1 degraded faster 4-HPA and 3-HPA than strain WT-p2. Both WT-p2 and p2-fldX1 cells grown on 4-HPA displayed more changes in the proteome than cells grown on 3-HPA in comparison to glucose-grown cells. Several enzymes involved in ROS detoxification, including AhpC2, AhpF, AhpD3, KatA, Bcp, CpoF1, Prx1 and Prx2, were upregulated by hydroxyphenylacetates. Downregulation of organic hydroperoxide resistance (Ohr) and DpsA proteins was observed. A downregulation of the genes encoding scavenging enzymes (katE and sodB), and gstA and trxB was observed in p2-fldX1 cells, suggesting that FldX1 prevents the antioxidant response. More than 20 membrane proteins, including porins and transporters, showed changes in expression during the growth of both strains on hydroxyphenylacetates. An increased 4-HPA degradation by recombinant strain p2-fldX1 in soil microcosms was observed. In soil, the strain overexpressing the flavodoxin FldX1 showed a lower plasmid loss, compared to WT-p2 strain, suggesting that FldX1 contributes to bacterial fitness. Overall, these results suggest that recombinant strain p2-fldX1 is an attractive bacterium for its application in bioremediation processes of aromatic compounds. CONCLUSIONS: The long-chain flavodoxin FldX1 improved the capability of P. xenovorans to degrade 4-HPA in liquid culture and soil microcosms by protecting cells against the degradation-associated oxidative stress.

Impact of template denaturation prior to whole genome amplification on gene detection in high GC-content species, Burkholderia mallei and B. pseudomallei.

OBJECTIVE: In this study, we sought to determine the types and prevalence of antimicrobial resistance determinants (ARDs) in Burkholderia spp. strains using the Antimicrobial Resistance Determinant Microarray (ARDM). RESULTS: Whole genome amplicons from 22 B. mallei (BM) and 37 B. pseudomallei (BP) isolates were tested for > 500 ARDs using ARDM v.3.1. ARDM detected the following Burkholderia spp.-derived genes, aac(6), blaBP/MBL-3, blaABPS, penA-BP, and qacE, in both BM and BP while blaBP/MBL-1, macB, blaOXA-42/43 and penA-BC were observed in BP only. The method of denaturing template for whole genome amplification greatly affected the numbers and types of genes detected by the ARDM. BlaTEM was detected in nearly a third of BM and BP amplicons derived from thermally, but not chemically denatured templates. BlaTEM results were confirmed by PCR, with 81% concordance between methods. Sequences from 414-nt PCR amplicons (13 preparations) were 100% identical to the Klebsiella pneumoniae reference gene. Although blaTEM sequences have been observed in B. glumae, B. cepacia, and other undefined Burkholderia strains, this is the first report of such sequences in BM/BP/B. thailandensis (BT) clade. These results highlight the importance of sample preparation in achieving adequate genome coverage in methods requiring untargeted amplification before analysis.

Harnessing the Diversity of Burkholderia spp. Prophages for Therapeutic Potential.

Burkholderia spp. are often resistant to antibiotics, and infections with these organisms are difficult to treat. A potential alternative treatment for Burkholderia spp. infections is bacteriophage (phage) therapy; however, it can be difficult to locate phages that target these bacteria. Prophages incorporated into the bacterial genome have been identified within Burkholderia spp. and may represent a source of useful phages for therapy. Here, we investigate whether prophages within Burkholderia spp. clinical isolates can kill conspecific and heterospecific isolates. Thirty-two Burkholderia spp. isolates were induced for prophage release, and harvested phages were tested for lytic activity against the same 32 isolates. Temperate phages were passaged and their host ranges were determined, resulting in four unique phages of prophage origin that showed different ranges of lytic activity. We also analyzed the prophage content of 35 Burkholderia spp. clinical isolate genomes and identified several prophages present in the genomes of multiple isolates of the same species. Finally, we observed that Burkholdera cenocepacia isolates were more phage-susceptible than Burkholderia multivorans isolates. Overall, our findings suggest that prophages present within Burkholderia spp. genomes are a potentially useful starting point for the isolation and development of novel phages for use in phage therapy.

Genomic editing in Burkholderia multivorans by CRISPR/Cas9.

Burkholderia cepacia complex bacteria have emerged as opportunistic pathogens in patients with cystic fibrosis and immunocompromised individuals, causing life-threatening infections. Because of the relevance of these microorganisms, genetic manipulation is crucial for explaining the genetic mechanisms leading to pathogenesis. Despite the availability of allelic exchange tools to obtain unmarked gene deletions in Burkholderia, these require a step of merodiploid formation and another of merodiploid resolution through two independent homologous recombination events, making the procedure long-lasting. The CRISPR/Cas9-based system could ease this constraint, as only one step is needed for allelic exchange. Here, we report the modification of a two-plasmid system (pCasPA and pACRISPR) for genome editing in Burkholderia multivorans. Several modifications were implemented, including selection marker replacement, the optimization of araB promoter induction for the expression of Cas9 and λ-Red system encoding genes, and the establishment of plasmid curing procedures based on the sacB gene or growth at a sub-optimal temperature of 18°C-20°C with serial passages. We have shown the efficiency of this CRISPR/Cas9 method in the precise and unmarked deletion of different genes (rpfR, bceF, cepR, and bcsB) from two strains of B. multivorans, as well as its usefulness in the targeted insertion of the gfp gene encoding the green fluorescence protein into a precise genome location. As pCasPA was successfully introduced in other Burkholderia cepacia complex species, this study opens up the possibility of using CRISPR/Cas9-based systems as efficient tools for genome editing in these species, allowing faster and more cost-effective genetic manipulation.IMPORTANCEBurkholderia encompasses different species of bacteria, some of them pathogenic to animals and plants, but others are beneficial by promoting plant growth through symbiosis or as biocontrol agents. Among these species, Burkholderia multivorans, a member of the Burkholderia cepacia complex, is one of the predominant species infecting the lungs of cystic fibrosis patients, often causing respiratory chronic infections that are very difficult to eradicate. Since the B. multivorans species is understudied, we have developed a genetic tool based on the CRISPR/Cas9 system to delete genes efficiently from the genomes of these strains. We could also insert foreign genes that can be precisely placed in a chosen genomic region. This method, faster than other conventional strategies based on allelic exchange, will have a major contribution to understanding the virulence mechanisms in B. multivorans, but it can likely be extended to other Burkholderia species.

Characterization of a novel high-efficiency cracking Burkholderia gladiolus phage vB_BglM_WTB and its application in black fungus.

Burkholderia gladiolus (B. gladiolus) is foodborne pathogenic bacteria producing bongkrekic acid (BA), which causes food poisoning and has a mortality rate of up to 40 % or more. However, no drugs have been reported in the literature for the prevention and treatment of this infection. In this study, a phage was identified to control B. gladiolus. The novel phage vB_BglM_WTB (WTB), which lyse B. gladiolus with high efficiency, was isolated from sewage of Huaihe Road Throttle Well Sewage Treatment Plant in Hefei. Transmission electron microscopy showed that WTB had an icosahedral head (69 ± 2 nm) and a long retractable tail (108 ± 2 nm). Its optimal temperature and pH ranges to control B. gladiolus were 25 °C -65 °C and 3-11 respectively. The phage WTB was identified as a linear double-stranded DNA phage of 68, 541 bp with 60.04 % G + C content, with a long latent period of 60 min. Phylogenetic analysis and comparative genetic analysis indicated that phage WTB has low identity (<50 %) with other phages, with the highest similarity to Burkholderia phage Maja (25.7 %), which showed that it does not belong to any previous genera recognized by the International Committee on Taxonomy of Viruses (ICTV) and was a candidate for a new genus within the Caudoviricetes. We have submitted a new proposal to ICTV to create a new genus, Bglawtbvirus. No transfer RNA (tRNA), virulence associated and antibiotic resistance genes were detected in phage WTB. Experimental results indicated that WTB at 4 °C and 25 °C had excellent inhibition activity against B. gladiolus in the black fungus, with an inhibition efficiency of over 99 %. The amount of B. gladiolus in the black fungus was reduced to a minimum of 89 CFU/mL when treated by WTB at 25 °C for 2 h. The inhibition rate remained at 99.97 % even after 12 h. The findings showed that the phage WTB could be applied as a food-cleaning agent for enhancing food safety and contributed to our understanding of phage biology and diversity.

Wild mushrooms as potential reservoirs of plant pathogenic bacteria: a case study on Burkholderia gladioli.

Fruit bodies (sporocarps) of wild mushrooms growing in natural environments play a substantial role in the preservation of microbial communities, for example, clinical and food-poisoning bacteria. However, the role of wild mushrooms as natural reservoirs of plant pathogenic bacteria remains almost entirely unknown. Furthermore, bacterial transmission from a mushroom species to agricultural plants has rarely been recorded in the literature. In September 2021, a creamy-white Gram-negative bacterial strain was isolated from the sporocarp of Suillus luteus (slippery jack) growing in Bermuda grass (Cynodon dactylon) lawn in Southern Iran. A similar strain was isolated from the same fungus in the same area in September 2022. Both strains were identified as Burkholderia gladioli based on phenotypic features as well as phylogeny of 16S rRNA and three housekeeping genes. The strains were not only pathogenic on white button mushrooms (Agaricus bisporus) but also induced hypersensitive reaction (HR) on tobacco and common bean leaves and caused soft rot on a set of diverse plant species, that is, chili pepper, common bean pod, cucumber, eggplant, garlic, gladiolus, narcissus, onion, potato, spring onion, okra, kohlrabi, mango, and watermelon. Isolation of plant pathogenic B. gladioli strains from sporocarp of S. luteus in two consecutive years in the same area could be indicative of the role of this fungus in the preservation of the bacterium in the natural environment. B. gladioli associated with naturally growing S. luteus could potentially invade neighboring agricultural crops, for example, vegetables and ornamentals. The potential role of wild mushrooms as natural reservoirs of phytopathogenic bacteria is further discussed.IMPORTANCEThe bacterial genus Burkholderia contains biologically heterogeneous strains that can be isolated from diverse habitats, that is, soil, water, diseased plant material, and clinical specimens. In this study, two Gram-negative pectinolytic bacterial strains were isolated from the sporocarps of Suillus luteus in September 2021 and 2022. Molecular phylogenetic analyses revealed that both strains belonged to the complex species Burkholderia gladioli, while the pathovar status of the strains remained undetermined. Biological investigations accomplished with pathogenicity and host range assays showed that B. gladioli strains isolated from S. luteus in two consecutive years were pathogenic on a set of diverse plant species ranging from ornamentals to both monocotyledonous and dicotyledonous vegetables. Thus, B. gladioli could be considered an infectious pathogen capable of being transmitted from wild mushrooms to annual crops. Our results raise a hypothesis that wild mushrooms could be considered as potential reservoirs for phytopathogenic B. gladioli.

Genome-guided purification of high amounts of the siderophore ornibactin and detection of potentially novel burkholdine derivatives produced by Burkholderia catarinensis 89T.

AIM: The increased availability of genome sequences has enabled the development of valuable tools for the prediction and identification of bacterial natural products. Burkholderia catarinensis 89T produces siderophores and an unknown potent antifungal metabolite. The aim of this work was to identify and purify natural products of B. catarinensis 89T through a genome-guided approach. MATERIALS AND METHODS: The analysis of B. catarinensis 89T genome revealed 16 clusters putatively related to secondary metabolism and antibiotics production. Of particular note was the identification of a nonribosomal peptide synthetase (NRPS) cluster related to the production of the siderophore ornibactin, a hybrid NRPS-polyketide synthase Type 1 cluster for the production of the antifungal glycolipopeptide burkholdine, and a gene cluster encoding homoserine lactones (HSL), probably involved in the regulation of both metabolites. We were able to purify high amounts of the ornibactin derivatives D/C6 and F/C8, while also detecting the derivative B/C4 in mass spectrometry investigations. A group of metabolites with molecular masses ranging from 1188 to 1272 Da could be detected in MS experiments, which we postulate to be new burkholdine analogs produced by B. catarinensis. The comparison of B. catarinensis BGCs with other Bcc members corroborates the hypothesis that this bacterium could produce new derivatives of these metabolites. Moreover, the quorum sensing metabolites C6-HSL, C8-HSL, and 3OH-C8-HSL were observed in LC-MS/MS analysis. CONCLUSION: The new species B. catarinensis is a potential source of new bioactive secondary metabolites. Our results highlight the importance of genome-guided purification and identification of metabolites of biotechnological importance.

High-Yield Lasso Peptide Production in a Burkholderia Bacterial Host by Plasmid Copy Number Engineering.

The knotted configuration of lasso peptides confers thermal stability and proteolytic resistance, addressing two shortcomings of peptide-based drugs. However, low isolation yields hinder the discovery and development of lasso peptides. While testing Burkholderia sp. FERM BP-3421 as a bacterial host to produce the lasso peptide capistruin, an overproducer clone was previously identified. In this study, we show that an increase in the plasmid copy number partially contributed to the overproducer phenotype. Further, we modulated the plasmid copy number to recapitulate titers to an average of 160% relative to the overproducer, which is 1000-fold higher than previously reported with E. coli, reaching up to 240 mg/L. To probe the applicability of the developed tools for lasso peptide discovery, we targeted a new lasso peptide biosynthetic gene cluster from endosymbiont Mycetohabitans sp. B13, leading to the isolation of mycetolassin-15 and mycetolassin-18 in combined titers of 11 mg/L. These results validate Burkholderia sp. FERM BP-3421 as a production platform for lasso peptide discovery.

MALDI-TOF MS analysis of Burkholderia pseudomallei and closely related species isolated from soils and water in Khon Kaen, Thailand.

Melioidosis is caused by Burkholderia pseudomallei (Bp) acquired from the environment. Conventional identification methods for environmental Bp are challenging due to the presence of closely related species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is accurate for bacterial identification, but has been little used to identify Bp from environmental samples. This study aims to evaluate MALDI-TOF MS for the identification of Bp and closely related species isolated from environmental samples in Thailand using whole-genome sequencing (WGS) as the gold standard, including determining the best sample preparation method for this purpose. We identified Bp (n = 22), Burkholderia spp. (n = 28), and other bacterial species (n = 32) using WGS. MALDI-TOF analysis of all Bp isolates yielded results consistent with WGS. A decision-tree algorithm identified 16 important variable peaks, using the protein extraction method (PEM), demonstrating distinct MALDI-TOF profiles for the three categories (Bp, Burkholderia spp. and "other bacterial species"). Three biomarker peaks (4060, 5196, and 6553 Da) could discriminate Bp from other Burkholderia and closely related species with 100% sensitivity and specificity. Hence, the MALDI-TOF technique has shown its potential as a species discriminatory tool, providing results comparable to WGS for classification and surveillance of environmental Bp.

Identification of the lipodepsipeptide selethramide encoded in a giant nonribosomal peptide synthetase from a Burkholderia bacterium.

Bacterial natural products have found many important industrial applications. Yet traditional discovery pipelines often prioritize individual natural product families despite the presence of multiple natural product biosynthetic gene clusters in each bacterial genome. Systematic characterization of talented strains is a means to expand the known natural product space. Here, we report genomics, epigenomics, and metabolomics studies of Burkholderia sp. FERM BP-3421, a soil isolate and known producer of antitumor spliceostatins. Its genome is composed of two chromosomes and two plasmids encoding at least 29 natural product families. Metabolomics studies showed that FERM BP-3421 also produces antifungal aminopyrrolnitrin and approved anticancer romidepsin. From the orphan metabolome features, we connected a lipopeptide of 1,928 Da to an 18-module nonribosomal peptide synthetase encoded as a single gene in chromosome 1. Isolation and structure elucidation led to the identification of selethramide which contains a repeating pattern of serine and leucine and is cyclized at the side chain oxygen of the one threonine residue at position 13. A (R)-3-hydroxybutyric acid moiety decorates the N-terminal serine. Initial attempts to obtain deletion mutants to probe the role of selethramide failed. After acquiring epigenome (methylome) data for FERM BP-3421, we employed a mimicry by methylation strategy that improved DNA transfer efficiency. Mutants defective in selethramide biosynthesis showed reduced surfactant activity and impaired swarming motility that could be chemically complemented with selethramide. This work unveils a lipopeptide that promotes surface motility, establishes improved DNA transfer efficiency, and sets the stage for continued natural product identification from a prolific strain.

Nucleic Acid Amplification Free-QCM-DNA Biosensor for Burkholderia pseudomallei Detection.

Burkholderia pseudomallei is a gram-negative bacterium that causes the infectious disease melioidosis, a disease that can still be fatal despite appropriate treatment. The bacterium contains the gene clusters for the type III secretion system (TTSS), which are essential for its pathogenicity. This gene was often employed for accurate diagnosis through the laborious process of gene amplification. This work intends to develop a quartz crystal microbalance (QCM)-based TTSS gene detection method without gene amplification approaches to simplify the diagnosis process. In this study, it was demonstrated that a 540 bp sequence flanked by BglI restriction sites within the TTSS1 on the B. pseudomallei genome is an effective target for specific detection of the bacteria. After cultivation and genome extraction, the bacteria can be detected by digesting its genome with BglI in which the TTSS1 fragment is detected by a QCM-DNA biosensor, eliminating the need for nucleic acid amplification. A specific probe designed to bind to the TTSSI fragment was utilized as the receptor on the QCM-DNA biosensor which provided the ability to detect the fragment. The limit of detection of the QCM-DNA biosensor was 0.4 µM of the synthetic DNA target oligonucleotide. The system was also capable of specifically detecting the BglI digested-DNA fragment of B. pseudomallei species with significantly higher signal than B. thailandensis. This study provides evidence for an effective QCM-DNA biosensor that can identify B. pseudomallei without the need for nucleic acid amplification.

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.

Identification of two distinct phylogenomic lineages and model strains for the understudied cystic fibrosis lung pathogen Burkholderia multivorans.

Burkholderia multivorans is the dominant Burkholderia pathogen recovered from lung infection in people with cystic fibrosis. However, as an understudied pathogen there are knowledge gaps in relation to its population biology, phenotypic traits and useful model strains. A phylogenomic study of B. multivorans was undertaken using a total of 283 genomes, of which 73 were sequenced and 49 phenotypically characterized as part of this study. Average nucleotide identity analysis (ANI) and phylogenetic alignment of core genes demonstrated that the B. multivorans population separated into two distinct evolutionary clades, defined as lineage 1 (n=58 genomes) and lineage 2 (n=221 genomes). To examine the population biology of B. multivorans, a representative subgroup of 77 B. multivorans genomes (28 from the reference databases and the 49 novel short-read genome sequences) were selected based on multilocus sequence typing (MLST), isolation source and phylogenetic placement criteria. Comparative genomics was used to identify B. multivorans lineage-specific genes - ghrB_1 in lineage 1 and glnM_2 in lineage 2 - and diagnostic PCRs targeting them were successfully developed. Phenotypic analysis of 49 representative B. multivorans strains showed considerable inter-strain variance, but the majority of the isolates tested were motile and capable of biofilm formation. A striking absence of B. multivorans protease activity in vitro was observed, but no lineage-specific phenotypic differences were demonstrated. Using phylogenomic and phenotypic criteria, three model B. multivorans CF strains were identified, BCC0084 (lineage 1), BCC1272 (lineage 2a) and BCC0033 lineage 2b, and their complete genome sequences determined. B. multivorans CF strains BCC0033 and BCC0084, and the environmental reference strain, ATCC 17616, were all capable of short-term survival within a murine lung infection model. By mapping the population biology, identifying lineage-specific PCRs and model strains, we provide much needed baseline resources for future studies of B. multivorans.

A membrane protein of the rice pathogen Burkholderia glumae required for oxalic acid secretion and quorum sensing.

Bacterial panicle blight is caused by Burkholderia glumae and results in damage to rice crops worldwide. Virulence of B. glumae requires quorum sensing (QS)-dependent synthesis and export of toxoflavin, responsible for much of the damage to rice. The DedA family is a conserved membrane protein family found in all bacterial species. B. glumae possesses a member of the DedA family, named DbcA, which we previously showed is required for toxoflavin secretion and virulence in a rice model of infection. B. glumae secretes oxalic acid as a "common good" in a QS-dependent manner to combat toxic alkalinization of the growth medium during the stationary phase. Here, we show that B. glumae ΔdbcA fails to secrete oxalic acid, leading to alkaline toxicity and sensitivity to divalent cations, suggesting a role for DbcA in oxalic acid secretion. B. glumae ΔdbcA accumulated less acyl-homoserine lactone (AHL) QS signalling molecules as the bacteria entered the stationary phase, probably due to nonenzymatic inactivation of AHL at alkaline pH. Transcription of toxoflavin and oxalic acid operons was down-regulated in ΔdbcA. Alteration of the proton motive force with sodium bicarbonate also reduced oxalic acid secretion and expression of QS-dependent genes. Overall, the data show that DbcA is required for oxalic acid secretion in a proton motive force-dependent manner, which is critical for QS of B. glumae. Moreover, this study supports the idea that sodium bicarbonate may serve as a chemical for treatment of bacterial panicle blight.

Paraburkholderia phytofirmans PsJN colonization of rice endosphere triggers an atypical transcriptomic response compared to rice native Burkholderia s.l. endophytes.

The plant microbiome has recently emerged as a reservoir for the development of sustainable alternatives to chemical fertilizers and pesticides. However, the response of plants to beneficial microbes emerges as a critical issue to understand the molecular basis of plant-microbiota interactions. In this study, we combined root colonization, phenotypic and transcriptomic analyses to unravel the commonalities and specificities of the response of rice to closely related Burkholderia s.l. endophytes. In general, these results indicate that a rice-non-native Burkholderia s.l. strain, Paraburkholderia phytofirmans PsJN, is able to colonize the root endosphere while eliciting a markedly different response compared to rice-native Burkholderia s.l. strains. This demonstrates the variability of plant response to microbes from different hosts of origin. The most striking finding of the investigation was that a much more conserved response to the three endophytes used in this study is elicited in leaves compared to roots. In addition, transcriptional regulation of genes related to secondary metabolism, immunity, and phytohormones appear to be markers of strain-specific responses. Future studies need to investigate whether these findings can be extrapolated to other plant models and beneficial microbes to further advance the potential of microbiome-based solutions for crop production.

Lactonase-mediated inhibition of quorum sensing largely alters phenotypes, proteome, and antimicrobial activities in Burkholderia thailandensis E264.

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 (acyl-homoserine 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.

Understanding Autologous Spliceostatin Transcriptional Regulation to Derive Parts for Heterologous Expression in a Burkholderia Bacterial Host.

Burkholderia ß-Proteobacteria are emerging sources of natural products. We are interested in developing Burkholderia sp. FERM BP-3421 into a synthetic biology chassis to facilitate natural product discovery. FERM BP-3421 produces autologous spliceostatins on gram per liter scale. We reasoned that transcription factors and promoters that regulate spliceostatin biosynthesis would provide valuable parts for heterologous expression. Herein we demonstrate that fr9A encodes a pathway-specific transcriptional activator of spliceostatin biosynthesis. In-frame deletion of fr9A abolished spliceostatin production, which was restored by genetic complementation. Using transcriptomics and green fluorescent protein (GFP) reporter assays, we identified four fr9 promoters, three of which are activated by LuxR-type regulator Fr9A. We then constructed an Fr9A-regulated promoter system that was compared to benchmarks and effectively applied for GFP and capistruin lasso peptide expression in an optimized host background. Our findings enrich the genetic toolbox for optimizing heterologous expression and promoting the discovery and development of natural products from Burkholderia bacteria.

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.