Repurposing promethazine hydrochloride to inhibit biofilm formation against Burkholderia thailandensis.

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.

Burkholderia thailandensis uses a type VI secretion system to lyse protrusions without triggering host cell responses.

To spread within a host, intracellular Burkholderia form actin tails to generate membrane protrusions into neighboring host cells and use type VI secretion system-5 (T6SS-5) to induce cell-cell fusions. Here, we show that B. thailandensis also uses T6SS-5 to lyse protrusions to directly spread from cell to cell. Dynamin-2 recruitment to the membrane near a bacterium was followed by a short burst of T6SS-5 activity. This resulted in the polymerization of the actin of the newly invaded host cell and disruption of the protrusion membrane. Most protrusion lysis events were dependent on dynamin activity, caused no cell-cell fusion, and failed to be recognized by galectin-3. T6SS-5 inactivation decreased protrusion lysis but increased galectin-3, LC3, and LAMP1 accumulation in host cells. Our results indicate that B. thailandensis specifically activates T6SS-5 assembly in membrane protrusions to disrupt host cell membranes and spread without alerting cellular responses, such as autophagy.

Burkholderia ambifaria H8 as an effective biocontrol strain against maize stalk rot via producing volatile dimethyl disulfide.

BACKGROUND: Maize stalk rot (MSR) caused by Fusarium graminearum is the primary factor contributing to the reduction in maize yield and quality. However, this soil-borne disease presents a significant challenge for sustainable control through field management and chemical agents. The screening of novel biocontrol agents can aid in developing innovative and successful strategies for MSR control. RESULTS: A total of 407 strains of bacteria were isolated from the rhizosphere soil of a resistant maize inbred line. One strain exhibited significant antagonistic activity in plate and pot experiments, and was identified as Burkholderia ambifaria H8. The strain could significantly inhibit the mycelial growth and spore germination of F. graminearum, induce resistance to stalk rot, and promote plant growth. The volatile compounds produced by strain H8 and its secondary metabolites in the sterile fermentation broth exhibited antagonistic activity. The primary volatile compound produced by strain H8 was identified as dimethyl disulfide (DMDS) using gas chromatography tandem mass spectrometry. Through in vitro antagonistic activity assays and microscopic observation, it was confirmed that DMDS was capable of inhibiting mycelial growth and disrupting the mycelial structure of F. graminearum, suggesting it may be the major active compound for strain H8. The transcriptome data of F. graminearum further indicated that strain H8 and its volatile compounds could alter pathogenic fungi metabolism, influence the related metabolic pathways, and potentially induce cell apoptosis within F. graminearum. CONCLUSION: Our results showed that B. ambifaria H8 was capable of producing the volatile substance dimethyl disulfide, which influenced the synthesis and permeability of cell membranes in pathogens. Thus, B. ambifaria H8 was found to be a promising biological control agent against MSR. © 2024 Society of Chemical Industry.

[Two-stage Inhibition Effects of Burkholderia sp. Y4 Application on Cadmium Uptake and Transport in Wheat].

In order to evaluate the feasibility of using Burkholderia sp. Y4 as a cadmium （Cd）-reducing bacterial agent in contaminated wheat fields， the changes in the rhizosphere soil microbial community and Cd available state， as well as the content and transport characteristics of Cd in the wheat root， basal node， internode， and grain under the treatment of strain Y4 were tested using microbial high-throughput sequencing， step-by-step extraction， subcellular distribution， and occurrence analyses. The results showed that root application of strain Y4 significantly reduced the root and grain Cd content of wheat by 7.7% and 30.3%， respectively， compared with that in the control treatment. The Cd content and Cd transfer factor results in wheat vegetative organs showed that strain Y4 reduced the Cd transfer factor from basal node to internode by 79.3%， and Cd content in the wheat internode stem also decreased by 50.9%. The study of Cd occurrence morphology showed that strain Y4 treatment increased the proportion of residual Cd in roots and basal ganglia， decreased the contents of inorganic and water-soluble Cd in roots， and increased the content of residual Cd in basal ganglia. Further examination of the subcellular distribution of Cd showed that the Cd content in root cell walls and basal ganglia cell fluid increased by 21.3% and 98.2%， respectively， indicating that the Cd fixation ability of root cell walls and basal ganglia cell fluid was improved by the strain Y4 treatment. In the rhizosphere soil， it was found that the microbial community structure was changed by strain Y4 application. Under the Y4 treatment， the relative abundance of Burkholderia increased from 9.6% to 11.5%， whereas that of Acidobacteriota decreased. Additionally， the relative abundance of Gemmatimonadales， Pseudomonadales， and Chitinophagales were also increased by strain Y4 treatment. At the same time， the application of strain Y4 increased the pH value of rhizosphere soil by 8.3%. The contents of exchangeable Cd， carbonate-bound Cd， and iron-manganese oxide-bound Cd in the soil decreased by 44.4%， 21.7%， and 15.9%， respectively， whereas the proportion of residual Cd reached 53.6%. Root application of strain Y4 increased the contents of nitrate nitrogen and ammonium nitrogen in the soil by 22.0% and 21.4%， respectively， and the contents of alkaline nitrogen also increased to a certain extent. In conclusion， the root application of strain Y4 not only improved soil nitrogen availability but also inhibited Cd transport and accumulation from contaminated soil to wheat grains in a "two-stage" manner by reducing Cd availability in rhizosphere soil and improving Cd interception and fixation capacity of wheat roots and basal nodes. Therefore， Burkholderia Y4 has application potential as a Cd-reducing and growth-promoting agent in wheat.

Specialized digestive mechanism for an insect-bacterium gut symbiosis.

In Burkholderia-Riptortus symbiosis, the host bean bug Riptortus pedestris harbors Burkholderia symbionts in its symbiotic organ, M4 midgut, for use as a nutrient source. After occupying M4, excess Burkholderia symbionts are moved to the M4B region, wherein they are effectively digested and absorbed. Previous studies have shown that M4B has strong symbiont-specific antibacterial activity, which is not because of the expression of antimicrobial peptides but rather because of the expression of digestive enzymes, mainly cathepsin L protease. However, in this study, inhibition of cathepsin L activity did not reduce the bactericidal activity of M4B, indicating that there is an unknown digestive mechanism that renders specifically potent bactericidal activity against Burkholderia symbionts. Transmission electron microscopy revealed that the lumen of symbiotic M4B was filled with a fibrillar matter in contrast to the empty lumen of aposymbiotic M4B. Using chromatographic and electrophoretic analyses, we found that the bactericidal substances in M4B existed as high-molecular-weight (HMW) complexes that were resistant to protease degradation. The bactericidal HMW complexes were visualized on non-denaturing gels using protein- and polysaccharide-staining reagents, thereby indicating that the HMW complexes are composed of proteins and polysaccharides. Strongly stained M4B lumen with Periodic acid-Schiff (PAS) reagent in M4B paraffin sections confirmed HMW complexes with polysaccharide components. Furthermore, M4B smears stained with Periodic acid-Schiff revealed the presence of polysaccharide fibers. Therefore, we propose a key digestive mechanism of M4B: bacteriolytic fibers, polysaccharide fibers associated with digestive enzymes such as cathepsin L, specialized for Burkholderia symbionts in Riptortus gut symbiosis.

In Vitro Roles of Burkholderia Intracellular Motility A (BimA) in Infection of Human Neuroblastoma Cell Line.

The bacterial pathogen Burkholderia pseudomallei causes human melioidosis, which can infect the brain, leading to encephalitis and brain abscesses. Infection of the nervous system is a rare condition but is associated with an increased risk of mortality. Burkholderia intracellular motility A (BimA) was reported to play an important role in the invasion and infection of the central nervous system in a mouse model. Thus, to gain insight of the cellular mechanisms underlying the pathogenesis of neurological melioidosis, we explored the human neuronal proteomics to identify the host factors that are up- and downregulated during Burkholderia infection. When infected the SH-SY5Y cells with B. pseudomallei K96243 wild-type (WT), 194 host proteins showed a fold change of >2 compared with uninfected cells. Moreover, 123 proteins showed a fold change of >2 when infected with a knockout bimA mutant (ΔbimA) mutant compared with WT. The differentially expressed proteins were mainly associated with metabolic pathways and pathways linked to human diseases. Importantly, we observed the downregulation of proteins in the apoptosis and cytotoxicity pathway, and in vitro investigation with the ΔbimA mutant revealed the association of BimA with the induction of these pathways. Additionally, we disclosed that BimA was not required for invasion into the neuron cell line but was necessary for effective intracellular replication and multinucleated giant cell (MNGC) formation. These findings show the extraordinary capacity of B. pseudomallei in subverting and interfering with host cellular systems to establish infection and extend our understanding of B. pseudomallei BimA involvement in the pathogenesis of neurological melioidosis. IMPORTANCE Neurological melioidosis, caused by Burkholderia pseudomallei, can result in severe neurological damage and enhance the mortality rate of melioidosis patients. We investigate the involvement of the virulent factor BimA, which mediates actin-based motility, in the intracellular infection of neuroblastoma SH-SY5Y cells. Using proteomics-based analysis, we provide a list of host factors exploited by B. pseudomallei. The expression level of selected downregulated proteins in neuron cells infected with the ΔbimA mutant was determined by quantitative reverse transcription-PCR and was consistent with our proteomic data. The role of BimA in the apoptosis and cytotoxicity of SH-SY5Y cells infected by B. pseudomallei was uncovered in this study. Additionally, our research demonstrates that BimA is required for successful intracellular survival and cell fusion upon infection of neuron cells. Our findings have significant implications for understanding the pathogenesis of B. pseudomallei infections and developing novel therapeutic strategies to combat this deadly disease.

NosP Detection of Heme Modulates Burkholderia thailandensis Biofilm Formation.

Aggregated bacteria embedded within self-secreted extracellular polymeric substances, or biofilms, are resistant to antibiotics and cause chronic infections. As such, they are a significant public health threat. Heme is an abundant iron source for pathogenic bacteria during infection; many bacteria have systems to detect heme assimilated from host cells, which is correlated with the transition between acute and chronic infection states. Here, we investigate the heme-sensing function of a newly discovered multifactorial sensory hemoprotein called NosP and its role in biofilm regulation in the soil-dwelling bacterium Burkholderia thailandensis, the close surrogate of Bio-Safety-Level-3 pathogen Burkholderia pseudomallei. The NosP family protein has previously been shown to exhibit both nitric oxide (NO)- and heme-sensing functions and to regulate biofilms through NosP-associated histidine kinases and two-component systems. Our in vitro studies suggest that BtNosP exhibits heme-binding kinetics and thermodynamics consistent with a labile heme-responsive protein and that the holo-form of BtNosP acts as an inhibitor of its associated histidine kinase BtNahK. Furthermore, our in vivo studies suggest that increasing the concentration of extracellular heme decreases B. thailandensis biofilm formation, and deletion of nosP and nahK abolishes this phenotype, consistent with a model that BtNosP detects heme and exerts an inhibitory effect on BtNahK to decrease the biofilm.

An IS-mediated, RecA-dependent, bet-hedging strategy in Burkholderia thailandensis.

Adaptation to fluctuating environmental conditions is difficult to achieve. Phase variation mechanisms can overcome this difficulty by altering genomic architecture in a subset of individuals, creating a phenotypically heterogeneous population with subpopulations optimized to persist when conditions change, or are encountered, suddenly. We have identified a phase variation system in Burkholderia thailandensis that generates a genotypically and phenotypically heterogeneous population. Genetic analyses revealed that RecA-mediated homologous recombination between a pair of insertion sequence (IS) 2-like elements duplicates a 208.6 kb region of DNA that contains 157 coding sequences. RecA-mediated homologous recombination also resolves merodiploids, and hence copy number of the region is varied and dynamic within populations. We showed that the presence of two or more copies of the region is advantageous for growth in a biofilm, and a single copy is advantageous during planktonic growth. While IS elements are well known to contribute to evolution through gene inactivation, polar effects on downstream genes, and altering genomic architecture, we believe that this system represents a rare example of IS element-mediated evolution in which the IS elements provide homologous sequences for amplification of a chromosomal region that provides a selective advantage under specific growth conditions, thereby expanding the lifestyle repertoire of the species.

Bacterial populations are often diverse, even when originating from a single cell. This diversity helps microbes survive in fluctuating environmental conditions by increasing the odds of population survival. For example, if environmental conditions change such that only a subpopulation with unique abilities survives, the entire population will be saved. Genomes are naturally dynamic. For example, mobile sections of DNA, called transposable elements, can change their position within a genome. If a transposable element jumps into a gene, it can harm the cell. But if it moves into a different site, it may provide an organism with new features that can help it survive. Most organisms contain multiple copies of transposable elements in their DNA. For example, a subtype of the soil bacterium Burkholderia thailandensis, strain E264, has two identical transposable elements that book-end a region of DNA that contains 157 genes. Lowrey et al. studied this bacterial strain in different environmental conditions to find out more. The experiments revealed that in growing populations of E264, some bacteria had one copy of the region, while others had two or three. In a rich environment, most bacteria had just one copy of the region. However, when grown in challenging conditions, most bacteria contained two or three copies of the region. Moreover, bacteria required at least two copies to form dense communities known as biofilms, which are advantageous for bacterial survival in challenging conditions. Bacteria with only one copy, however, were better adapted to a free-swimming lifestyle. Lowrey et al. further showed that the DNA repair system was required for duplicating the region. Usually, this system finds and recombines identical DNA sequences to repair broken DNA. However, if two identical DNA sequences (a pair of transposable elements) are present, the repair system can recombine them during DNA replication, resulting in the duplication of the DNA between the identical sequences. The same system also reduces the copy number of the region from three or two to just one. Since the repair system is constantly working and DNA recombination is always occurring at a low level, B. thailandensis E264 maintains a genetically diverse population with bacteria containing different copy numbers of the region. This diversity ensures that the strain survives in fluctuating environmental conditions. Transposable elements are hotspots of evolution. They are known to interrupt genes and shrink genomes. Lowrey et al. showed that transposable elements also influence evolution by providing DNA sequences that the DNA repair system can use to duplicate DNA. This process of duplicating genes is more frequent than random genetic mutations, expediting adaptation.

[Effects of Burkholderia sp. Y4 on Cadmium Damage and Uptake in Rice Seedlings].

In order to investigate the effects of Burkholderia sp. Y4 on rice seedlings under cadmium (Cd) stress, seed germination and vermiculite culture experiments were conducted using low Cd-accumulation xiangzaoxian 24 (X24) and high Cd-accumulation Tyou 705 (T705) varieties. The effects of Burkholderia sp. Y4 on rice growth, oxidative damage caused by Cd, and Cd accumulation were studied. Additionally, the Cd2+ flux rates in the elongation zone of rice roots under Burkholderia sp. Y4 application were detected using non-invasive micro-test technology. Burkholderia sp. Y4 alleviated the inhibition effect of Cd on rice seed germination by 13.8%. After inoculation with Burkholderia sp. Y4 for 7 d, the length of rice roots and buds increased by 83.3% and 12.2%, and their dry weight increased by 56.8% and 12.5%, respectively; those in the 10 d Y4 inoculation group increased by 28.6% and 20.0% in length and by 113.2% and 46.0% in dry weight, respectively. Burkholderia sp. Y4 inoculation also alleviated rice oxidative stress damage caused by Cd. The application of strain Y4 significantly reduced the content of the oxidative damage product malondialdehyde (MDA) in the shoots and roots of rice seedlings by 21.5% and 16.9%, respectively. Under Burkholderia sp. Y4 inoculation, the significant changes in antioxidant enzyme SOD and CAT activities caused by Cd stress disappeared in rice roots; those in shoots also decreased from 176.9% and 74.8% to 53.3% and 21.5%, respectively. Conversely, Burkholderia sp. Y4 inhibited Cd uptake by rice seedlings with different genotypes, including the low Cd-accumulation variety X24 and high Cd-accumulation variety T705. The root application of strain Y4 significantly reduced Cd accumulation in the shoots and roots of rice seedlings by 79.2% and 62.7% in T705 and by 57.3% and 24.1% in X24, respectively. The Cd2+ flux rate of high Cd-accumulation variety T705 was significantly higher than that of low Cd-accumulation variety X24. Under Burkholderia sp. Y4 inoculation, the yellow membrane was formed on the root surface of rice seedlings, and the Cd2+ flux rate in the elongation zone of T705 and X24 roots decreased by 36.0% and 35.0% in 3-day-old seedlings, as well as by 44.6% and 24.9% in 10-day-old seedlings, respectively. In conclusion, Burkholderia sp. Y4 inoculation inhibited the toxic effects of Cd on rice seedling growth through alleviating oxidative stress and damage caused by Cd. Furthermore, the root application of Burkholderia sp. Y4 effectively decreased the Cd2+ flux rate in the elongation zone of roots to inhibit the Cd uptake and accumulation in roots and shoots of rice seedlings. This study provides theoretical basis and data support for the application of Burkholderia sp. Y4 as a Cd-reducing and growth-promoting agent for rice in contaminated farmland.

Generation of Distinct Differentially Culturable Forms of Burkholderia following Starvation at Low Temperature.

Bacteria have developed unique mechanisms to adapt to environmental stresses and challenges of the immune system. Here, we report that Burkholderia pseudomallei, the causative agent of melioidosis, and its laboratory surrogate, Burkholderia thailandensis, utilize distinct mechanisms for surviving starvation at different incubation temperatures. At 21°C, Burkholderia are present as short rods which can rapidly reactivate and form colonies on solid media. At 4°C, Burkholderia convert into coccoid forms that cannot be cultured on solid agar but can be resuscitated in liquid media supplemented with supernatant obtained from logarithmic phase cultures of B. thailandensis, or catalase and Tween 80, thus displaying characteristics of differentially culturable bacteria (DCB). These DCB have low intensity fluorescence when stained with SYTO 9, have an intact cell membrane (propidium iodide negative), and contain 16S rRNA at levels comparable with growing cells. We also present evidence that lytic transglycosylases, a family of peptidoglycan-remodeling enzymes, are involved in the generation of coccoid forms and their resuscitation to actively growing cells. A B. pseudomallei ΔltgGCFD mutant with four ltg genes deleted did not produce coccoid forms at 4°C and could not be resuscitated in the liquid media evaluated. Our findings provide insights into the adaptation of Burkholderia to nutrient limitation and the generation of differentially culturable bacteria. IMPORTANCE Bacterial pathogens exhibit physiologically distinct forms that enable their survival in an infected host, the environment and following exposure to antimicrobial agents. B. pseudomallei causes the disease melioidosis, which has a high mortality rate and is difficult to treat with antibiotics. The bacterium is endemic to several countries and detected in high abundance in the environment. Here, we report that during starvation at low temperature, B. pseudomallei produces coccoid forms that cannot grow in standard media and which, therefore, can be challenging to detect using common tools. We provide evidence that the formation of these cocci is mediated by cell wall-specialized enzymes and lytic transglycosylases, and that resuscitation of these forms occurs following the addition of catalase and Tween 80. Our findings have important implications for the disease control and detection of B. pseudomallei, an agent of both public health and defense interest.

Skin models for cutaneous melioidosis reveal Burkholderia infection dynamics at wound's edge with inflammasome activation, keratinocyte extrusion and epidermal detachment.

ABSTRACTMelioidosis is a serious infectious disease endemic in Southeast Asia, Northern Australia and has been increasingly reported in other tropical and subtropical regions in the world. Percutaneous inoculation through cuts and wounds on the skin is one of the major modes of natural transmission. Despite cuts in skin being a major route of entry, very little is known about how the causative bacterium Burkholderia pseudomallei initiates an infection at the skin and the disease manifestation at the skin known as cutaneous melioidosis. One key issue is the lack of suitable and relevant infection models. Employing an in vitro 2D keratinocyte cell culture, a 3D skin equivalent fibroblast-keratinocyte co-culture and ex vivo organ culture from human skin, we developed infection models utilizing surrogate model organism Burkholderia thailandensis to investigate Burkholderia-skin interactions. Collectively, these models show that the bacterial infection was largely limited at the wound's edge. Infection impedes wound closure, triggers inflammasome activation and cellular extrusion in the keratinocytes as a potential way to control bacterial infectious load at the skin. However, extensive infection over time could result in the epidermal layer being sloughed off, potentially contributing to formation of skin lesions.

Burkholderia multivorans requires species-specific GltJK for entry of a contact-dependent growth inhibition system protein.

Interbacterial antagonism and communication are driving forces behind microbial community development. In many Gram-negative bacteria, contact-dependent growth inhibition (CDI) systems contribute to these microbial interactions. CDI systems deliver the toxic C-terminus of a large surface exposed protein to the cytoplasm of neighboring bacteria upon cell-contact. Termed the BcpA-CT, import of this toxic effector domain is mediated by specific, yet largely unknown receptors on the recipient cell outer and inner membranes. In this study, we demonstrated that cytoplasmic membrane proteins GltJK, components of a predicted ABC-type transporter, are required for entry of CDI system protein BcpA-2 into Burkholderia multivorans recipient cells. Consistent with current CDI models, gltJK were also required for recipient cell susceptibility to a distinct BcpA-CT that shared sequences within the predicted "translocation domain" of BcpA-2. Strikingly, this translocation domain showed low sequence identity to the analogous region of an Escherichia coli GltJK-utilizing CDI system protein. Our results demonstrated that recipient bacteria expressing E. coli gltJK were resistant to BcpA-2-mediated interbacterial antagonism, suggesting that BcpA-2 specifically recognizes Burkholderia GltJK. Using a series of chimeric proteins, the specificity determinant was mapped to Burkholderia-specific sequences at the GltK C-terminus, providing insight into BcpA transport across the recipient cell cytoplasmic membrane.

Piperacillin triggers virulence factor biosynthesis via the oxidative stress response in Burkholderia thailandensis.

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 ß-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 ß-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.

Investigation of arsenic-resistant, arsenite-oxidizing bacteria for plant growth promoting traits isolated from arsenic contaminated soils.

The problem of arsenic (As) pollution being severe warrants opting for low-cost microbial remediation strategies. The present study of identifying suitable bacterial strains led to the isolation of eleven As-tolerant strains from the As-contaminated rhizosphere soils of West Bengal, India. They were found to oxidize/reduce 55-31.6% of 5 mM As(III) and 73-37.6% of 5 mM As(V) within 12 h. The four isolates (BcAl-1, JN 73, LAR-2, and AR-30) had a high level of As(III) oxidase activity along with a higher level of As(V) and As(III) resistance. The agar diffusion assay of the isolates further confirmed their ability to endure As stress. The presence of aoxB gene was observed in these four As(III) oxidizing isolates. Evaluation of plant growth-promoting characteristics revealed that BcAl-1 (Burkholderia cepacia), JN 73 (Burkholderia metallica), AR-30 (Burkholderia cenocepacia), and LAR-2 (Burkholderia sp.) had significant plant growth-promoting characteristics (PGP), including the ability to solubilize phosphate, siderophore production, indole acetic acid-like molecules production, ACC deaminase production, and nodule formation under As stressed condition. BcAl-1 and JN 73 emerged as the most promising traits in As removal as well as plant growth promotion.

A link between pH homeostasis and colistin resistance in bacteria.

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.

A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans.

Burkholderia cepacia complex bacteria comprise opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. These microorganisms produce an exopolysaccharide named cepacian, which is considered a virulence determinant. To find genes implicated in the regulation of cepacian biosynthesis, we characterized an evolved nonmucoid variant (17616nmv) derived from the ancestor, Burkholderia multivorans ATCC 17616, after prolonged stationary phase. Lack of cepacian biosynthesis was correlated with downregulation of the expression of bce genes implicated in its biosynthesis. Furthermore, genome sequencing of the variant identified the transposition of the mobile element IS406 upstream of the coding sequence of an hns-like gene (Bmul_0158) encoding a histone-like nucleoid structuring (H-NS) protein, a known global transcriptional repressor. This insertion sequence (IS) element upregulated the expression of Bmul_0158 by 4-fold. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes in genes implicated in motility, pilus synthesis, type VI secretion, and chromosome-associated functions. Concomitant with these differences, the nonmucoid variant displays reduced adherence to a CF lung bronchial cell line and reduced surface hydrophobicity and forms smaller cellular aggregates but has an increase in swimming and swarming motilities. Finally, analysis of the GC content of the upstream region of differentially expressed genes led to the identification of various genomic regions, possibly acquired by horizontal gene transfer, which were transcriptionally repressed by the increased expression of the Bmul_0158 gene in the 17616nmv strain. Taken together, the results revealed a significant role for this H-NS protein in the regulation of B. multivorans persistence- and virulence-associated genes. IMPORTANCE Members of the histone-like nucleoid structuring (H-NS) family of proteins, present in many bacteria, are important global regulators of gene expression. Many of the regulated genes were acquired horizontally and include pathogenicity islands and prophages, among others. Additionally, H-NS can play a structural role by bridging and compacting DNA, fulfilling a crucial role in cell physiology. Several virulence phenotypes have been frequently identified in several bacteria as dependent on H-NS activity. Here, we describe an H-NS-like protein of the opportunistic pathogen Burkholderia multivorans, a species commonly infecting the respiratory tract of cystic fibrosis patients. Our results indicate that this protein is involved in regulating virulence traits such as exopolysaccharide biosynthesis, adhesion to biotic surfaces, cellular aggregation, and motility. Furthermore, this H-NS-like protein is one out of eight orthologs present in the B. multivorans ATCC 17616 genome, posing relevant questions to be investigated on how these proteins coordinate the expression of virulence traits.

Phosphate-solubilizing bacterium Burkholderia sp. strain N3 facilitates the regulation of gene expression and improves tomato seedling growth under cadmium stress.

Cadmium (Cd) is among the most toxic heavy metals in soils. The ways by which tomato plants inoculated with a phosphate-solubilizing bacterium (PSB) respond to Cd and regulate gene expression remain unclear. We investigated hormone metabolism and genes involved in Cd resistance in tomato seedlings inoculated with the PSB strain N3. Cd inhibited tomato plant growth and nutrient uptake and increase in dry weight. Compared with Cd treatment, N3 inoculation inhibited the accumulation of Cd in the shoots and roots, and the root dry weight significantly increased by 30.50% (P < 0.05). The nitrogen and potassium contents in the roots of seedlings treated with N3 increased, and the phosphorus levels were the same as those in the control. N3 decreased the rate of Zn2+ absorption but increased Fe3+ absorption in the roots, and the amount of accumulated Cd increased with Zn2+ uptake. The concentrations of hormones (indole-3-acetic acid, IAA; zeatin, ZEA; and jasmonic acid, JA) increased under Cd stress, whereas inoculation with N3 reduced IAA and ZEA levels. In the comparison between N3 + Cd and Cd treatments, the highest number of up- and downregulated genes was obtained. Pathways involved in signaling response, photosynthesis, phenylpropanoid biosynthesis, and DNA replication and the photosynthesis-antenna proteins pathway play important roles in the responses and adaptation of seedlings to Cd. Inoculation with N3 alleviates Cd stress in tomato seedlings. The present study provides new insights into the differentially expressed genes related to interaction between PSB and tomato exposed to Cd in soils.

A biological agent modulates the physiology of barley infected with Drechslera teres.

Recognized as the causal agent of net blotch, Drechslera teres is responsible for major losses of barley crop yield. The consequences of this leaf disease are due to the impact of the infection on the photosynthetic performance of barley leaves. To limit the symptoms of this ascomycete, the use of beneficial bacteria known as "Plant Growth Promoting Rhizobacteria" constitutes an innovative and environmentally friendly strategy. A bacterium named as strain B25 belonging to the genus Burkholderia showed a strong antifungal activity against D. teres. The bacterium was able to limit the development of the fungus by 95% in detached leaves of bacterized plants compared to the non-bacterized control. In this study, in-depth analyses of the photosynthetic performance of young barley leaves infected with D. teres and/or in the presence of the strain B25 were carried out both in and close to the necrotic area. In addition, gas exchange measurements were performed only near the necrotic area. Our results showed that the presence of the beneficial bacterium reduced the negative impact of the fungus on the photosynthetic performance and modified only the net carbon assimilation rate close to the necrotic area. Indeed, the presence of the strain B25 decreased the quantum yield of regulated non-photochemical energy loss in PSII noted as Y(NPQ) and allowed to maintain the values stable of maximum quantum yield of PSII photochemistry known as Fv/Fm and close to those of the control in the presence of D. teres. To the best of our knowledge, these data constitute the first study focusing on the impact of net blotch fungus and a beneficial bacterium on photosynthesis and respiratory parameters in barley leaves.

A ß-lactamase gene of Fusarium oxysporum alters the rhizosphere microbiota of soybean.

The rhizosphere is a multitrophic environment, and for soilborne pathogens such as Fusarium oxysporum, microbial competition in the rhizosphere is inevitable before reaching and infecting roots. This study established a tritrophic interaction among the plant growth-promoting rhizobacterium Burkholderia ambifaria, F. oxysporum and Glycine max (soybean) to study the effects of F. oxysporum genes on shaping the soybean microbiota. Although B. ambifaria inhibited mycelial growth and increased bacterial propagation in the presence of F. oxysporum, F. oxysporum still managed to infect soybean in the presence of B. ambifaria. RNA-Seq identified a putative F. oxysporum secretory ß-lactamase-coding gene, FOXG_18438 (abbreviated as Fo18438), that is upregulated during soybean infection in the presence of B. ambifaria. The ∆Fo18438 mutants displayed reduced mycelial growth towards B. ambifaria, and the complementation of full Fo18438 and the Fo18438 ß-lactamase domain restored mycelial growth. Using the F. oxysporum wild type, ∆Fo18438 mutants and complemented strains with full Fo18438, Fo18438 ß-lactamase domain or Fo18438 RTA1-like domain for soil inoculation, 16S rRNA amplicon sequencing revealed that the abundance of a Burkholderia operational taxonomic unit (OTU) was increased in the rhizosphere microbiota infested by the strains with Fo18438 ß-lactamase domain. Non-metric multidimensional scaling and PICRUSt2 functional analysis revealed differential abundance for the bacterial ß-lactam-related functions when contrasting the genotypes of F. oxysporum. These results indicated that the Fo18438 ß-lactamase domain provides F. oxysporum with the advantage of growing into the soybean rhizosphere, where ß-lactam antibiosis is involved in microbial competition. Accordingly, this study highlights the capability of an F. oxysporum gene for altering the soybean rhizosphere and taproot microbiota.

Functional Analysis of Phenazine Biosynthesis Genes in Burkholderia spp.

Burkholderia encompasses a group of ubiquitous Gram-negative bacteria that includes numerous saprophytes as well as species that cause infections in animals, immunocompromised patients, and plants. Some species of Burkholderia produce colored, redox-active secondary metabolites called phenazines. Phenazines contribute to competitiveness, biofilm formation, and virulence in the opportunistic pathogen Pseudomonas aeruginosa, but knowledge of their diversity, biosynthesis, and biological functions in Burkholderia is lacking. In this study, we screened publicly accessible genome sequence databases and identified phenazine biosynthesis genes in multiple strains of the Burkholderia cepacia complex, some isolates of the B. pseudomallei clade, and the plant pathogen B. glumae We then focused on B. lata ATCC 17760 to reveal the organization and function of genes involved in the production of dimethyl 4,9-dihydroxy-1,6-phenazinedicarboxylate. Using a combination of isogenic mutants and plasmids carrying different segments of the phz locus, we characterized three novel genes involved in the modification of the phenazine tricycle. Our functional studies revealed a connection between the presence and amount of phenazines and the dynamics of biofilm growth in flow cell and static experimental systems but at the same time failed to link the production of phenazines with the capacity of Burkholderia to kill fruit flies and rot onions.IMPORTANCE Although the production of phenazines in Burkholderia was first reported almost 70 years ago, the role these metabolites play in the biology of these economically important microorganisms remains poorly understood. Our results revealed that the phenazine biosynthetic pathway in Burkholderia has a complex evolutionary history, which likely involved horizontal gene transfers among several distantly related groups of organisms. The contribution of phenazines to the formation of biofilms suggests that Burkholderia, like fluorescent pseudomonads, may benefit from the unique redox-cycling properties of these versatile secondary metabolites.