Cell envelope structural and functional contributions to antibiotic resistance in Burkholderia cenocepacia.

Antibiotic activity is limited by the physical construction of the Gram-negative cell envelope. Species of the Burkholderia cepacia complex (Bcc) are known as intrinsically multidrug-resistant opportunistic pathogens with low permeability cell envelopes. Here, we re-examined a previously performed chemical-genetic screen of barcoded transposon mutants in B. cenocepacia K56-2, focusing on cell envelope structural and functional processes. We identified structures mechanistically important for resistance to singular and multiple antibiotic classes. For example, susceptibility to novobiocin, avibactam, and the LpxC inhibitor, PF-04753299, was linked to the BpeAB-OprB efflux pump, suggesting these drugs are substrates for this pump in B. cenocepacia. Defects in peptidoglycan precursor synthesis specifically increased susceptibility to cycloserine and revealed a new putative amino acid racemase, while defects in divisome accessory proteins increased susceptibility to multiple ß-lactams. Additionally, disruption of the periplasmic disulfide bond formation system caused pleiotropic defects on outer membrane integrity and ß-lactamase activity. Our findings highlight the layering of resistance mechanisms in the structure and function of the cell envelope. Consequently, we point out processes that can be targeted for developing antibiotic potentiators.IMPORTANCEThe Gram-negative cell envelope is a double-layered physical barrier that protects cells from extracellular stressors, such as antibiotics. The Burkholderia cell envelope is known to contain additional modifications that reduce permeability. We investigated Burkholderia cell envelope factors contributing to antibiotic resistance from a genome-wide view by re-examining data from a transposon mutant library exposed to an antibiotic panel. We identified susceptible phenotypes for defects in structures and functions in the outer membrane, periplasm, and cytoplasm. Overall, we show that resistance linked to the cell envelope is multifaceted and provides new targets for the development of antibiotic potentiators.

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.

Challenges and mitigation strategies associated with Burkholderia cepacia complex contamination in pharmaceutical manufacturing.

Burkholderia cepacia complex (BCC) is a Gram-negative, non-spore-forming bacterium with more than 20 opportunistic pathogenic species, most commonly found in soil and water. Due to their rapid mutation rates, these organisms are adaptable and possess high genomic plasticity. BCC can cause life-threatening infections in immunocompromised individuals, such as those with cystic fibrosis, chronic granulomatous disease, and neonates. BCC contamination is a significant concern in pharmaceutical manufacturing, frequently causing non-sterile product recalls. BCC has been found in purified water, cosmetics, household items, and even ultrasound gel used in veterinary practices. Pharmaceuticals, personal care products, and cleaning solutions have been implicated in numerous outbreaks worldwide, highlighting the risks associated with intrinsic manufacturing site contamination. Regulatory compliance, product safety, and human health protection depend on testing for BCC in pharmaceutical manufacturing. Identification challenges exist, with BCC often misidentified as other bacteria like non-lactose fermenting Escherichia coli or Pseudomonas spp., particularly in developing countries where reporting BCC in pharmaceuticals remains limited. This review comprehensively aims to address the organisms causing BCC contamination, genetic diversity, identification challenges, regulatory requirements, and mitigation strategies. Recommendations are proposed to aid pharmaceutical chemists in managing BCC-associated risks and implementing prevention strategies within manufacturing processes.

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.

Non-coding regulatory sRNAs from bacteria of the Burkholderia cepacia complex.

Small non-coding RNAs (sRNAs) are key regulators of post-transcriptional gene expression in bacteria. Hundreds of sRNAs have been found using in silico genome analysis and experimentally based approaches in bacteria of the Burkholderia cepacia complex (Bcc). However, and despite the hundreds of sRNAs identified so far, the number of functionally characterized sRNAs from these bacteria remains very limited. In this mini-review, we describe the general characteristics of sRNAs and the main mechanisms involved in their action as regulators of post-transcriptional gene expression, as well as the work done so far in the identification and characterization of sRNAs from Bcc. The number of functionally characterized sRNAs from Bcc is expected to increase and to add new knowledge on the biology of these bacteria, leading to novel therapeutic approaches to tackle the infections caused by these opportunistic pathogens, particularly severe among cystic fibrosis patients. KEY POINTS: •Hundreds of sRNAs have been identified in Burkholderia cepacia complex bacteria (Bcc). •A few sRNAs have been functionally characterized in Bcc. •Functionally characterized Bcc sRNAs play major roles in metabolism, biofilm formation, and virulence.

Mass Production of Arbuscular Mycorrhizal Fungi on the Sorghum Plants Inoculated with Burkholderia arboris Using Soybean Mill Waste and Vermicompost-Amended Soil-Sand Substrate.

Arbuscular mycorrhizal (AM) fungi are being used as a new generation of biofertilizers to increase plant growth by improving plant nutrition and bio-protection. However, because of the obligatory nature of the plant host, large-scale multiplication of AM propagules is challenging, which limits its applicability. This study evaluates the ability of Burkholderia arboris to increase AM production in soybean mill waste and vermicompost amended by soil-sand mixture planted with sorghum as a host plant. The experiment was conducted in a nursery using a completely randomized design with four inoculation treatments (B. arboris, AM fungi, B. arboris + AM fungi, and control) under sterilized and unsterilized conditions. AM production was investigated microscopically (spore density and root colonization), and biochemically (AM-specific lipid biomarker, 16:1ω5cis derived from neutral lipid fatty acid (NLFA), and phospholipid fatty acid (PLFA) fractions from both soil and roots). Integrating B. arboris with AM fungi in organically amended pots was found to increase AM fungal production by 62.16 spores g-1 soil and root colonization by 80.85%. Biochemical parameters also increased with B. arboris inoculation: 5.49 nmol PLFA g-1 soil and 692.68 nmol PLFA g-1 root and 36.72 nmol NLFA g-1 soil and 3147.57 nmol NLFA g-1 root. Co-inoculation also increased glomalin-related soil protein and root biomass. Principal component analysis (PCA) further supported the higher contribution of B. arboris to AM fungi production under unsterilized conditions. In conclusion, inoculation of AM plant host seeds with B. arboris prior to sowing into organic potting mix could be a promising and cost-effective approach for increasing AM inoculum density for commercial production. Furthermore, efforts need to be made for up-scaling the AM production with different plant hosts and soil-substrate types.

Computational design of prospective molecular targets for Burkholderia cepacia complex by molecular docking and dynamic simulation studies.

The study aimed to screen prospective molecular targets of BCC and potential natural lead candidates as effective binders by computational modeling, molecular docking, and dynamic (MD) simulation studies. Based on the virulent functions, tRNA 5-methylaminomethyl-2-thiouridine biosynthesis bifunctional protein (mnmC) and pyrimidine/purine nucleoside phosphorylase (ppnP) were selected as the prospective molecular targets. In the absence of experimental data, the three-dimensional (3D) structures of these targets were computationally predicted. After a thorough literature survey and database search, the drug-likeness, and pharmacokinetic properties of 70 natural molecules were computationally predicted and the effectual binding of the best lead molecules against both the targets was predicted by molecular docking. The stabilities of the best-docked complexes were validated by MD simulation and the binding energy calculations were carried out by MM-GBSA approaches. The present study revealed that the hypothetical models of mnmC and ppnP showed stereochemical accuracy. The study also showed that among 70 natural compounds subjected to computational screening, Honokiol (3',5-Di(prop-2-en-1-yl) [1,1'-biphenyl]-2,4'-diol) present in Magnolia showed ideal drug-likeness, pharmacokinetic features and showed effectual binding with mnmC and ppnP (binding energies -7.3 kcal/mol and -6.6 kcal/mol, respectively). The MD simulation and GBSA calculation studies showed that the ligand-protein complexes stabilized throughout tMD simulation. The present study suggests that Honokiol can be used as a potential lead molecule against mnmC and ppnP targets of BCC and this study provides insight into further experimental validation for alternative lead development against drug resistant BCC.

Phenazines and toxoflavin act as interspecies modulators of resilience to diverse antibiotics.

Bacterial opportunistic pathogens make diverse secondary metabolites both in the natural environment and when causing infections, yet how these molecules mediate microbial interactions and their consequences for antibiotic treatment are still poorly understood. Here, we explore the role of three redox-active secondary metabolites, pyocyanin, phenazine-1-carboxylic acid, and toxoflavin, as interspecies modulators of antibiotic resilience. We find that these molecules dramatically change susceptibility levels of diverse bacteria to clinical antibiotics. Pyocyanin and phenazine-1-carboxylic acid are made by Pseudomonas aeruginosa, while toxoflavin is made by Burkholderia gladioli, organisms that infect cystic fibrosis and other immunocompromised patients. All molecules alter the susceptibility profile of pathogenic species within the "Burkholderia cepacia complex" to different antibiotics, either antagonizing or potentiating their effects, depending on the drug's class. Defense responses regulated by the redox-sensitive transcription factor SoxR potentiate the antagonistic effects these metabolites have against fluoroquinolones, and the presence of genes encoding SoxR and the efflux systems it regulates can be used to predict how these metabolites will affect antibiotic susceptibility of different bacteria. Finally, we demonstrate that inclusion of secondary metabolites in standard protocols used to assess antibiotic resistance can dramatically alter the results, motivating the development of new tests for more accurate clinical assessment.

Examining the activity of cefepime-taniborbactam against Burkholderia cepacia complex and Burkholderia gladioli isolated from cystic fibrosis patients in the United States.

The novel clinical-stage ß-lactam-ß-lactamase inhibitor combination, cefepime-taniborbactam, demonstrates promising activity toward many Gram-negative bacteria producing class A, B, C, and/or D ß-lactamases. We tested this combination against a panel of 150 Burkholderia cepacia complex (Bcc) and Burkholderia gladioli strains. The addition of taniborbactam to cefepime shifted cefepime minimum inhibitory concentrations toward the provisionally susceptible range in 59% of the isolates tested. Therefore, cefepime-taniborbactam possessed similar activity as first-line agents, ceftazidime and trimethoprim-sulfamethoxazole, supporting further development.

Activity of ETX0462 toward Some Burkholderia spp.

Burkholderia cepacia complex (Bcc) and Burkholderia gladioli are opportunistic human pathogens that are inherently multidrug resistant, limiting treatment options for infections. Here, a novel diazabicyclooctane, ETX0462, was evaluated for activity against Bcc and B. gladioli. Ninety-eight percent of the isolates examined in this study were susceptible. ETX0462 was found to demonstrate in vitro activity superior to that of currently available treatment options (e.g., trimethoprim-sulfamethoxazole and ceftazidime).

In vitro and in vivo activity of cefiderocol against Achromobacter spp. and Burkholderia cepacia complex, including carbapenem-non-susceptible isolates.

Achromobacter spp. and Burkholderia cepacia complex (Bcc) are rare but diverse opportunistic pathogens associated with serious infections, which are often multidrug resistant. This study compared the in vitro antibacterial activity of the siderophore antibiotic cefiderocol against Achromobacter spp. and Bcc isolates with that of other approved antibacterial drugs, including ceftazidime-avibactam, ciprofloxacin, colistin, imipenem-relebactam, and meropenem-vaborbactam. Isolates were collected in the SIDERO multinational surveillance program. Among 334 Achromobacter spp. isolates [76.6% from respiratory tract infections (RTIs)], cefiderocol had minimum inhibitory concentration (MIC)50/90 of 0.06/0.5 µg/mL overall and 0.5/4 µg/mL against 52 (15.6%) carbapenem-non-susceptible (Carb-NS) isolates. Eleven (3.3%) Achromobacter spp. isolates overall and 6 (11.5%) Carb-NS isolates were not susceptible to cefiderocol. Among 425 Bcc isolates (73.4% from RTIs), cefiderocol had MIC50/90 of ≤0.03/0.5 µg/mL overall and ≤0.03/1 µg/mL against 184 (43.3%) Carb-NS isolates. Twenty-two (5.2%) Bcc isolates overall and 13 (7.1%) Carb-NS isolates were not susceptible to cefiderocol. Cumulative MIC distributions showed cefiderocol to be the most active of the agents tested in vitro against both Achromobacter spp. and Bcc. In a neutropenic murine lung infection model and a humanized pharmacokinetic immunocompetent rat lung infection model, cefiderocol showed significant bactericidal activity against two meropenem-resistant Achromobacter xylosoxidans strains compared with untreated controls (P < 0.05) and vehicle-treated controls (P < 0.05), respectively. Meropenem, piperacillin-tazobactam, ceftazidime, and ciprofloxacin comparators showed no significant activity in these models. The results suggest that cefiderocol could be a possible treatment option for RTIs caused by Achromobacter spp. and Bcc.

Genetic dissection of the degradation pathways for the mycotoxin fusaric acid in Burkholderia ambifaria T16.

IMPORTANCE: Fusaric acid (FA) is an important virulence factor produced by several Fusarium species. These fungi are responsible for wilt and rot diseases in a diverse range of crops. FA is toxic for animals, humans and soil-borne microorganisms. This mycotoxin reduces the survival and competition abilities of bacterial species able to antagonize Fusarium spp., due to its negative effects on viability and the production of antibiotics effective against these fungi. FA biodegradation is not a common characteristic among bacteria, and the determinants of FA catabolism have not been identified so far in any microorganism. In this study, we identified genes, enzymes, and metabolic pathways involved in the degradation of FA in the soil bacterium Burkholderia ambifaria T16. Our results provide insights into the catabolism of a pyridine-derivative involved in plant pathogenesis by a rhizosphere bacterium.

Phage therapy in a lung transplant recipient with cystic fibrosis infected with multidrug-resistant Burkholderia multivorans.

BACKGROUND: There is increased interest in bacteriophage (phage) therapy to treat infections caused by antibiotic-resistant bacteria. A lung transplant recipient with cystic fibrosis and Burkholderia multivorans infection was treated with inhaled phage therapy for 7 days before she died. METHODS: Phages were given via nebulization through the mechanical ventilation circuit. Remnant respiratory specimens and serum were collected. We quantified phage and bacterial deoxyribonucleic acid (DNA) using quantitative polymerase chain reaction, and tested phage neutralization in the presence of patient serum. We performed whole genome sequencing and antibiotic and phage susceptibility testing on 15 B. multivorans isolates. Finally, we extracted lipopolysaccharide (LPS) from two isolates and visualized their LPS using gel electrophoresis. RESULTS: Phage therapy was temporally followed by a temporary improvement in leukocytosis and hemodynamics, followed by worsening leukocytosis on day 5, deterioration on day 7, and death on day 8. We detected phage DNA in respiratory samples after 6 days of nebulized phage therapy. Bacterial DNA in respiratory samples decreased over time, and no serum neutralization was detected. Isolates collected between 2001 and 2020 were closely related but differed in their antibiotic and phage susceptibility profiles. Early isolates were not susceptible to the phage used for therapy, while later isolates, including two isolates collected during phage therapy, were susceptible. Susceptibility to the phage used for therapy was correlated with differences in O-antigen profiles of an early versus a late isolate. CONCLUSIONS: This case of clinical failure of nebulized phage therapy highlights the limitations, unknowns, and challenges of phage therapy for resistant infections.

Microbial Epidemiology of the Cystic Fibrosis Airways: Past, Present, and Future.

Progressive obstructive lung disease secondary to chronic airway infection, coupled with impaired host immunity, is the leading cause of morbidity and mortality in cystic fibrosis (CF). Classical pathogens found in the airways of persons with CF (pwCF) include Pseudomonas aeruginosa, Staphylococcus aureus, the Burkholderia cepacia complex, Achromobacter species, and Haemophilus influenzae. While traditional respiratory-tract surveillance culturing has focused on this limited range of pathogens, the use of both comprehensive culture and culture-independent molecular approaches have demonstrated complex highly personalized microbial communities. Loss of bacterial community diversity and richness, counteracted with relative increases in dominant taxa by traditional CF pathogens such as Burkholderia or Pseudomonas, have long been considered the hallmark of disease progression. Acquisition of these classic pathogens is viewed as a harbinger of advanced disease and postulated to be driven in part by recurrent and frequent antibiotic exposure driven by frequent acute pulmonary exacerbations. Recently, CF transmembrane conductance regulator (CFTR) modulators, small molecules designed to potentiate or restore diminished protein levels/function, have been successfully developed and have profoundly influenced disease course. Despite the multitude of clinical benefits, structural lung damage and consequent chronic airway infection persist in pwCF. In this article, we review the microbial epidemiology of pwCF, focus on our evolving understanding of these infections in the era of modulators, and identify future challenges in infection surveillance and clinical management.

Identification of Burkholderia cenocepacia non-coding RNAs expressed during Caenorhabditis elegans infection.

Small non-coding RNAs (sRNAs) are key regulators of post-transcriptional gene expression in bacteria. Despite the identification of hundreds of bacterial sRNAs, their roles on bacterial physiology and virulence remain largely unknown, as is the case of bacteria of the Burkholderia cepacia complex (Bcc). Bcc is a group of opportunistic pathogens with relatively large genomes that can cause lethal lung infections amongst cystic fibrosis (CF) patients. To characterise sRNAs expressed by Bcc bacteria when infecting a host, the nematode Caenorhabditis elegans was used as an infection model by the epidemic CF strain B. cenocepacia J2315. A total of 108 new and 31 previously described sRNAs with a predicted Rho independent terminator were identified, most of them located on chromosome 1. RIT11b, a sRNA downregulated under C. elegans infection conditions, was shown to directly affect B. cenocepacia virulence, biofilm formation, and swimming motility. RIT11b overexpression reduced the expression of the direct targets dusA and pyrC, involved in biofilm formation, epithelial cell adherence, and chronic infections in other organisms. The in vitro direct interaction of RIT11b with the dusA and pyrC messengers was demonstrated by electrophoretic mobility shift assays. To the best of our knowledge this is the first report on the functional characterization of a sRNA directly involved in B. cenocepacia virulence. KEY POINTS: • 139 sRNAs expressed by B. cenocepacia during C. elegans infection were identified • The sRNA RIT11b affects B. cenocepacia virulence, biofilm formation, and motility • RIT11b directly binds to and regulates dusA and pyrC mRNAs.

Subtractive sequence analysis aided druggable targets mining in Burkholderia cepacia complex and finding inhibitors through bioinformatics approach.

Burkholderia cepacia complex (BCC) is a group of gram-negative bacteria composed of at least 20 different species that cause diseases in plants, animals as well as humans (cystic fibrosis and airway infection). Here, we analyzed the proteomic data of 47 BCC strains by classifying them in three groups. Phylogenetic analyses were performed followed by individual core region identification for each group. Comparative analysis of the three individual core protein fractions resulted in 1766 ortholog/proteins. Non-human homologous proteins from the core region gave 1680 proteins. Essential protein analyses reduced the target list to 37 proteins, which were further compared to a closely related out-group, Burkholderia gladioli ATCC 10,248 strain, resulting in 21 proteins. 3D structure modeling, validation, and druggability step gave six targets that were subjected to further target prioritization parameters which ultimately resulted in two BCC targets. A library of 12,000 ZINC drug-like compounds was screened, where only the top hits were selected for docking orientations. These included ZINC01405842 (against Chorismate synthase aroC) and ZINC06055530 (against Bifunctional N-acetylglucosamine-1-phosphate uridyltransferase/Glucosamine-1-phosphate acetyltransferase glmU). Finally, dynamics simulation (200 ns) was performed for each ligand-receptor complex, followed by ADMET profiling. Of these targets, details of their applicability as drug targets have not yet been elucidated experimentally, hence making our predictions novel and it is suggested that further wet-lab experimentations should be conducted to test the identified BCC targets and ZINC scaffolds to inhibit them.

Development of a qPCR detection approach for pathogenic Burkholderia cenocepacia associated with fresh vegetables.

Natural environment serves as a reservoir for Burkholderia cepacia complex organisms, including the highly transmissible opportunistic human pathogen B. cenocepacia. Currently, there is a lack of an effective and quantitative method for B. cenocepacia detection in fresh food and other environmental niches. A quantitative real-time PCR (qPCR) detection method for B. cenocepacia bacteria was established in this study and validated using artificially inoculated fresh vegetable samples. Genome-wide comparative methods were applied to identify target regions for the design of species-specific primers. Assay specificity was measured with 12 strains of closely related Burkholderia bacteria and demonstrated the primer pair BCF6/R6 were 100% specific for detection of B. cenocepacia. The described qPCR assay evaluated B. cenocepacia with a 2 pg µl-1 limit of detection and appropriate linearity (R2 = 0.999). In 50 samples of experimentally infected produce (lettuce, onion, and celery), the assay could detect B. cenocepacia as low as 2.6 × 102 cells in each sample equal to 1 g. The established qPCR method quantitatively detects B. cenocepacia with high sensitivity and specificity, making it a promising technique for B. cenocepacia detection and epidemiological research on B. cepacia complex organisms from fresh vegetables.

Pangenome inventory of Burkholderia sensu lato, Burkholderia sensu stricto, and the Burkholderia cepacia complex reveals the uniqueness of Burkholderia catarinensis.

Here the pangenome analysis of Burkholderia sensu lato (s.l.) was performed for the first time, together with an updated analysis of the pangenome of Burkholderia sensu stricto, and Burkholderia cepacia complex (Bcc) focusing on the Bcc B. catarinensis specific features of its re-sequenced genome. The pangenome of Burkholderia s.l., Burkholderia s.s., and of the Bcc was open, composed of more than 96% of accessory genes, and more than 62% of unknown genes. Functional annotations showed that secondary metabolism genes belonged to the variable portion of genomes, which might explain their production of several compounds with varied bioactivities. Taken together, this work showed the great variability and uniqueness of these genomes and revealed an underexplored unknown potential in poorly characterized genes. Regarding B. catarinensis 89T, its genome harbors genes related to hydrolases production and plant growth promotion. This draft genome will be valuable for further investigation of its biotechnological potentials.

Optimization of Constitutive Promoters Using a Promoter-Trapping Vector in Burkholderia pyrrocinia JK-SH007.

Selecting suitable promoters to drive gene overexpression can provide significant insight into the development of engineered bacteria. In this study, we analyzed the transcriptome data of Burkholderia pyrrocinia JK-SH007 and identified 54 highly expressed genes. The promoter sequences were located using genome-wide data and scored using the prokaryotic promoter prediction software BPROM to further screen out 18 promoter sequences. We also developed a promoter trap system based on two reporter proteins adapted for promoter optimization in B. pyrrocinia JK-SH007: firefly luciferase encoded by the luciferase gene set (Luc) and trimethoprim (TP)-resistant dihydrofolate reductase (TPr). Ultimately, eight constitutive promoters were successfully inserted into the probe vector and transformed into B. pyrrocinia JK-SH007. The transformants were successfully grown on Tp antibiotic plates, and firefly luciferase expression was determined by measuring the relative light unit (RLU). Five of the promoters (P4, P9, P10, P14, and P19) showed 1.01-2.51-fold higher activity than the control promoter λ phage transcriptional promoter (PRPL). The promoter activity was further validated via qPCR analysis, indicating that promoters P14 and P19 showed stable high transcription levels at all time points. Then, GFP and RFP proteins were overexpressed in JK-SH007. In addition, promoters P14 and P19 were successfully used to drive gene expression in Burkholderia multivorans WS-FJ9 and Escherichia coli S17-1. The two constitutive promoters can be used not only in B. pyrrocinia JK-SH007 itself to gene overexpression but also to expand the scope of application.

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.