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.

Antimicrobial activity of essential oils against multidrug-resistant clinical isolates of the Burkholderia cepacia complex.

Members of the Burkholderia cepacia complex (Bcc) are an important cause of opportunistic or nosocomial infections that may be hard to treat due to a high incidence of multidrug resistance. We characterised a collection of 51 clinical isolates from this complex, assigning them to 18 sequence types using multi-locus sequence type analysis. Resistance to eight commonly used antibiotics was assessed using by using agar-dilution assays to calculate MICs and widespread and heterogeneous multidrug resistance was confirmed, with eight strains proving resistant to all antibiotics tested. Disc diffusion screening of antimicrobial activity of a range of plant essential oils against these Bcc isolates identified six oils with significant activity (lavender, lemongrass, marjoram, peppermint, tea tree and rosewood) and broth microdilution assays indicated that of these lemongrass and rosewood oils had the highest activity, with MIC50 values of 0.5% and MIC90 values of 1%. Comparison of MIC and MBC values showed that four of these six oils, including lemongrass and rosewood, were bacteriocidal rather than bacteriostatic in their effects. Qualitative analysis of the four bacteriocidal essential oils via GC/MS indicated the presence of 55 different component compounds, mostly monoterpenes. We assessed selected essential oil components as anti-Bcc agents and demonstrated that terpinen-4-ol and geraniol were effective with MICs of 0.125-0.5% (v/v) and 0.125-1% (v/v), respectively. Time-kill studies indicate that these two alcohols are effective against non-growing cells in an efflux-dependent manner. Analysis of bacterial leakage of potassium ions and 260 nm UV-absorbing material on treatment with terpinen-4-ol and geraniol suggested that the observed anti-Bcc activity was a consequence of membrane disruption. This finding was supported by a gas chromatography analysis of bacterial fatty acid methyl esters, which indicated changes in membrane fatty acid composition caused by terpinen-4-ol and geraniol. These essential oils or oil components may ultimately prove useful as therapeutic drugs, for example to treat Bcc infections in CF patients.

Antimicrobial activity of six essential oils against Burkholderia cepacia complex: insights into mechanism(s) of action.

AIM: To investigate the activity and mechanisms of action of six essential oils (EOs) against Burkholderia cepacia complex, opportunistic human pathogens highly resistant to antibiotics. MATERIALS & METHODS: Minimal inhibitory concentration of EOs alone, plus antibiotics or efflux pump inhibitors was determined. RESULTS: Origanum vulgare, Thymus vulgaris and Eugenia caryophyllata EOs resulted to be more active than the other EOs. EOs did not enhance antibiotic activity against the model strain B. cenocepacia J2315. EOs resulted more active in the presence of an efflux pump inhibitor acting on Resistance-Nodulation Cell Division efflux pumps and against B. cenocepacia J2315 Resistance-Nodulation Cell Division knocked-out mutants. CONCLUSION: EOs showed intracellular mechanisms of action and, thus, the efflux pumps inhibitor addition could boost their activity.

Characteristics of Organic Acid Secretion Associated with the Interaction between Burkholderia multivorans WS-FJ9 and Poplar Root System.

The objective of this study was to investigate whether plant-bacteria interaction affects the secretion of organic acids by both organisms and to assess whether the production of IAA by the bacterium increases the secretion of organic acids by root exudates, and if the stress produced by low available phosphorus (P) affects the production of organic acids by bacteria, by roots, or by root exudates in presence of bacterial cultures. With this purpose, we used as a biological model poplar plants and one strain of Burkholderia multivorans able to solubilize P. High performance liquid chromatography was utilized to measure organic acids. The tests, the inductive effects of exogenous indole-3-acetic acid (IAA) on secretion of organic acids, the 2 × 4 × 2 factorial design experiment, and the ability of organic acids to solubilize tricalcium phosphate were performed to investigate the interactive effects. The results showed that, after B. multivorans WS-FJ9 interacted with the poplar root system, the key phosphate-solubilizing driving force was gluconic acid (GA) which was produced in three ways: (1) secreted by the root system in the presence of IAA produced by B. multivorans WS-FJ9; (2) secreted by B. multivorans WS-FJ9; and (3) secreted by the poplar root system in the presence of phosphorus stress. When phosphorus stress was absent, the GA was produced as outlined in (1) and (2) above. These results demonstrated that inoculating B. multivorans WS-FJ9 into the poplar root system could increase the amount of GA secretion and implied that the interaction between B. multivorans WS-FJ9 and the poplar root system could contribute to the increase of P available fraction for poplar plants.

The type 2 secretion Pseudopilin, gspJ, is required for multihost pathogenicity of Burkholderia cenocepacia AU1054.

Burkholderia cenocepacia AU1054 is an opportunistic pathogen isolated from the blood of a person with cystic fibrosis. AU1054 is a multihost pathogen causing rapid pathogenicity to Caenorhabditis elegans nematodes. Within 24 h, AU1054 causes greater than 50% mortality, reduced growth, emaciated body, distended intestinal lumen, rectal swelling, and prolific infection of the nematode intestine. To determine virulence mechanisms, 3,000 transposon mutants were screened for attenuated virulence in nematodes. Fourteen virulence-attenuated mutants were isolated, and the mutant genes were identified. These genes included paaA, previously identified as being required for full virulence of B. cenocepacia K56-2. Six mutants were restored in virulence by complementation with their respective wild-type gene. One of these contained an insertion in gspJ, predicted to encode a pseudopilin component of the type 2 secretion system (T2SS). Nematodes infected with AU1054 gspJ had fewer bacteria present in the intestine than those infected with the wild type but still showed rectal swelling. The gspJ mutant was also defective in pathogenicity to onion and in degradation of polygalacturonic acid and casein. This result differs from previous studies where no or little role was found for T2SS in Burkholderia virulence, although virulence factors such as zinc metalloproteases and polygalacturonase are known to be secreted by the T2SS. This study highlights strain specific differences in B. cenocepacia virulence mechanisms important for understanding what enables environmental microbes to function as opportunistic pathogens.

Plant host and sugar alcohol induced exopolysaccharide biosynthesis in the Burkholderia cepacia complex.

The species that presently constitute the Burkholderia cepacia complex (Bcc) have multiple roles; they include soil and water saprophytes, bioremediators, and plant, animal and human pathogens. Since the first description of pathogenicity in the Bcc was based on sour skin rot of onion bulbs, this study returned to this plant host to investigate the onion-associated phenotype of the Bcc. Many Bcc isolates, which were previously considered to be non-mucoid, produced copious amounts of exopolysaccharide (EPS) when onion tissue was provided as the sole nutrient. EPS production was not species-specific, was observed in isolates from both clinical and environmental sources, and did not correlate with the ability to cause maceration of onion tissue. Chemical analysis suggested that the onion components responsible for EPS induction were primarily the carbohydrates sucrose, fructose and fructans. Additional sugars were investigated, and all alcohol sugars tested were able to induce EPS production, in particular mannitol and glucitol. To investigate the molecular basis for EPS biosynthesis, we focused on the highly conserved bce gene cluster thought to be involved in cepacian biosynthesis. We demonstrated induction of the bce gene cluster by mannitol, and found a clear correlation between the inability of representatives of the Burkholderia cenocepacia ET12 lineage to produce EPS and the presence of an 11 bp deletion within the bceB gene, which encodes a glycosyltransferase. Insertional inactivation of bceB in Burkholderia ambifaria AMMD results in loss of EPS production on sugar alcohol media. These novel and surprising insights into EPS biosynthesis highlight the metabolic potential of the Bcc and show that a potential virulence factor may not be detected by routine laboratory culture. Our results also highlight a potential hazard in the use of inhaled mannitol as an osmolyte to improve mucociliary clearance in individuals with cystic fibrosis.

A biosurfactant from Burkholderia cenocepacia BSP3 and its enhancement of pesticide solubilization.

AIMS: To isolate a biosurfactant (BS)-producing bacterium, to characterize the BS properties and to evaluate its ability to enhance pesticide solubilization for further application in environmental remediation. METHODS AND RESULTS: Five BS-producing bacteria were isolated from fuel oil-contaminated soil. Among them, Burkholderia cenocepacia BSP3 exhibited the highest emulsification index and was chosen for further study. Glucose-containing medium supplemented with nitrate or sunflower seed oil provided suitable conditions for growth and BS production. The BS was identified as a glucolipid, having a critical micelle concentration (CMC) of 316 mg l(-1). It could lower the surface tension of deionized water to 25 +/- 0.2 mN m(-1) and exhibited good emulsion stability. Finally, the application of the BS to facilitate pesticide solubilization demonstrated that this BS at the concentration below and above its CMC could enhance the apparent water solubility of three pesticides, i.e. methyl parathion, ethyl parathion and trifluralin. CONCLUSIONS: Burkholderia cenocepacia BSP3 is a BS-producing bacterium isolated from oil-contaminated soil. The BS was identified as a glucolipid having a molecular mass of 550.4 g mol(-1). An apparent yield of the BS was 6.5 +/- 0.7 g l(-1). This glucolipid-type BS noticeably enhanced pesticide solubilization suggesting its role in environmental remediation. SIGNIFICANCE AND IMPACT OF THE STUDY: A glucolipid type BS normally found in marine micro-organisms was isolated from a soil-bacterium. Due to its surface active properties and good performance in enhancement of pesticide solubilization, it could be used as a solubilizing agent for environmental remediation and synergistic treatment with bioremediation of pesticide-contaminated soil.

Selection of nitrogen-fixing deficient Burkholderia vietnamiensis strains by cystic fibrosis patients: involvement of nif gene deletions and auxotrophic mutations.

Burkholderia vietnamiensis is the third most prevalent species of the Burkholderia cepacia complex (Bcc) found in cystic fibrosis (CF) patients. Its ability at fixing nitrogen makes it one of the main Bcc species showing strong filiations with environmental reservoirs. In this study, 83% (29 over 35) of the B. vietnamiensis CF isolates and 100% of the environmental ones (over 29) were found expressing the dinitrogenase complex (encoded by the nif cluster) which is essential in N(2) fixation. Among the deficient strains, two were found growing with ammonium chloride suggesting that they were defective in N(2) fixation, and four with amino acids supplements suggesting that they were harbouring auxotrophic mutations. To get insights about the genetic events that led to the emergence of the N(2)-fixing defective strains, a genetic analysis of B. vietnamiensis nitrogen-fixing property was undertaken. A 40-kb-long nif cluster and nif regulatory genes were identified within the B. vietnamiensis strain G4 genome sequence, and analysed. Transposon mutagenesis and nifH genetic marker exchanges showed the nif cluster and several other genes like gltB (encoding a subunit of the glutamate synthase) to play a key role in B. vietnamiensis ability at growing in nitrogen-free media. nif cluster DNA probings of restricted genomic DNA blots showed a full deletion of the nif cluster for one of the N(2)-fixing defective strain while the other one showed a genetic organization similar to the one of the G4 strain. For 17% of B. vietnamiensis clinical strains, CF lungs appeared to have favoured the selection of mutations or deletions leading to N(2)-fixing deficiencies.

Burkholderia cenocepacia utilizes ferritin as an iron source.

Burkholderia cenocepacia is a member of the Burkholderia cepacia complex, a group of genetically similar species that inhabit a number of environmental niches, including the lungs of patients with cystic fibrosis (CF). To colonize the lung, this bacterium requires a source of iron to satisfy its nutritional requirements for this important metal. Because of the high potential for damage in lung tissue resulting from oxygen-iron interactions, this metal is sequestered by a number of mechanisms that render it potentially unavailable to invading micro-organisms. Such mechanisms include the intracellular and extracellular presence of the iron-binding protein ferritin. Ferritin has a highly stable macromolecular structure and may contain up to 4500 iron atoms per molecule. To date, there has been no known report of a pathogenic bacterial species that directly utilizes iron sequestered by this macromolecule. To examine the ability of ferritin to support growth of B. cenocepacia J2315, iron-deficient media were supplemented with different concentrations of ferritin and the growth kinetics characterized over a 40 h period. The results indicated that B. cenocepacia J2315 utilizes iron bound by ferritin. Further studies examining the mechanisms of iron uptake from ferritin indicated that iron utilization results from a proteolytic degradation of this otherwise stable macromolecular structure. Since it is known that the ferritin concentration is significantly higher in the CF lung than in healthy lungs, this novel iron-acquisition mechanism may contribute to infection by B. cenocepacia in people with CF.

Involvement of a plasmid-encoded type IV secretion system in the plant tissue watersoaking phenotype of Burkholderia cenocepacia.

Burkholderia cenocepacia strain K56-2, a representative of the Burkholderia cepacia complex, is part of the epidemic and clinically problematic ET12 lineage. The strain produced plant tissue watersoaking (ptw) on onion tissue, which is a plant disease-associated trait. Using plasposon mutagenesis, mutants in the ptw phenotype were generated. The translated sequence of a disrupted gene (ptwD4) from a ptw-negative mutant showed homology to VirD4-like proteins. Analysis of the region proximal to the transfer gene homolog identified a gene cluster located on the 92-kb resident plasmid that showed homology to type IV secretion systems. The role of ptwD4, ptwC, ptwB4, and ptwB10 in the expression of ptw activity was determined by conducting site-directed mutagenesis. The ptw phenotype was not expressed by K56-2 derivatives with a disruption in ptwD4, ptwB4, or ptwB10 but was observed in a derivative with a disruption in ptwC. Complementation of ptw-negative K56-2 derivatives in trans resulted in complete restoration of the ptw phenotype. In addition, analysis of culture supernatants revealed that the putative ptw effector(s) was a secreted, heat-stable protein(s) that caused plasmolysis of plant protoplasts. A second chromosomally encoded type IV secretion system with complete homology to the VirB-VirD system was identified in K56-2. Site-directed mutagenesis of key secretory genes in the VirB-VirD system did not affect expression of the ptw phenotype. Our findings indicate that in strain K56-2, the plasmid-encoded Ptw type IV secretion system is responsible for the secretion of a plant cytotoxic protein(s).