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Production of common bean (Phaseolus vulgaris) is limited by the occurrence of damping off (rhizoctoniosis), which is caused by the fungus Rhizoctonia solani. However, the co-inoculation of plant growth-promoting rhizobacteria (PGPR) involved in biological control along with diatomic nitrogen (N2)-fixing rhizobia can enhance N nutrition and increase production. In this context, finding microorganisms with synergistic effects that perform these two roles is of fundamental importance to ensure adequate yield levels. The aim of this study was to evaluate the effects of co-inoculation of nodule endophytic strains of the genera Bacillus, Paenibacillus, Burkholderia, and Pseudomonas with Rhizobium tropici CIAT 899, an N2-fixing rhizobial strain, on the biocontrol of damping off and growth promotion in common bean plants. Greenhouse experiments were conducted under axenic conditions using the common bean cultivar Perola. The first experiment evaluated the potential of the 14 rhizobacterial strains, which were inoculated alone or in combination with CIAT 899, for the control of R. solani. The second experiment evaluated the ability of these 14 rhizobacterial strains to promote plant growth with three manners of N supply: co-inoculation with CIAT 899 at low mineral N supply (5.25 mg N mL−1), low mineral N supply (5.25 mg N mL−1), and high mineral N supply (52.5 mg N mL−1). The use of rhizobacteria combined with rhizobia contributed in a synergistic manner to the promotion of growth and the control of damping off in the common bean. Co-inoculation of the strains UFLA 02-281/03-18 (Pseudomonas sp.), UFLA 02-286 (Bacillus sp.), and UFLA 04-227 (Burkholderia fungorum) together with CIAT 899 effectively controlled damping off. For the common bean, mineral N supply can be replaced by the co-inoculation of CIAT 899 with plant growth-promoting strains UFLA 02-281/02-286/02-290/02-293. Nodule endophytes UFLA 02-281/02-286 are promising for co-inoculation with CIAT 899 in the common bean, promoting synergy with rhizobial inoculation and protection against disease.

On average less than 1% of the total phosphorous present in soils is available to plants, making phosphorous one of the most limiting macronutrients for crop productivity worldwide. The aim of this work was to isolate and select phosphate solubilizing bacteria (PSB) from the barley rhizosphere, which has other growth promoting traits and can increase crop productivity. A total of 104 different bacterial isolates were extracted from the barley plant rhizosphere. In this case, 64 strains were able to solubilize phosphate in agar plates. The 24 strains exhibiting the highest solubilizing index belonged to 16 different species, of which 7 isolates were discarded since they were identified as putative phytopathogens. The remaining nine strains were tested for their ability to solubilize phosphate in liquid medium and in pot trials performed in a greenhouse. Several of the isolated strains (Advenella mimigardefordensis, Bacillus cereus, Bacillus megaterium and Burkholderia fungorum) were able to significantly improve levels of assimilated phosphate, dry weight of ears and total starch accumulated on ears compared to non-inoculated plants. Since these strains were able to increase the growth and productivity of barley crops, they could be potentially used as microbial inoculants (biofertilizers).

In natural environments, bacteria often exist in close association with surfaces and interfaces by establishing biofilms. Here, we report on the ability of Burkholderia fungorum strains DBT1 and 95 to survive in high concentrations of hydrocarbons, and we compare their growth as a biofilm vs. planktonic cells. The 2 compounds tested were dibenzothiophene (DBT) and a mixture of naphthalene, phenanthrene, and pyrene (5:2:1) as representative compounds of thiophenes and polycyclic aromatic hydrocarbons (PAHs), respectively. The results showed that both strains were able to degrade DBT and to survive in the presence of up to a 2000 mg·L−1concentration of this compound both as a biofilm and as free-living cells. Moreover, B. fungorum DBT1 showed reduced tolerance towards the mixed PAHs (2000 mg·L−1naphthalene, 800 mg·L−1phenanthrene, and 400 mg·L−1pyrene) both as a biofilm and as free-living cells. Conversely, biofilms of B. fungorum 95 enhanced resistance against these toxic compounds compared with planktonic cells (P < 0.05). Visual observation through confocal laser scanning microscopy showed that exposure of biofilms to DBT and PAHs altered their structure: high concentrations of DBT triggered an aggregation of biofilm cells. These findings provide new perspectives on the effectiveness of using DBT-degrading bacterial strains in bioremediation of hydrocarbon-contaminated sites.

The novel bacterial strains Burkholderia fungorum T3A13001 and Caulobacter sp T2A12002, which were previously isolated from oil-contaminated sites and identified via 16S RNA sequences, were tested for their hydrocarbon degrading efficiency. Media spiked with 100 ppm pyrene were incubated at 25 °C and 37 °C. The bacterial isolates' pyrene-degrading capability was assessed in acidic (pH 5.0) and alkaline (pH 9.0) media. Caulobacter sp degraded 21% and 24% of the pyrene at pH 9.0 and 5.0, respectively; thus, the strain was more active at pH 5.0 than pH 9.0. B. fungorum was active in a wide range of pH values, because it biodegraded 59%–62% of the provided pyrene in acidic and alkaline media. Caulobacter sp degraded 35%–36% of the provided pyrene in cultures at 37 °C and 25 °C. B. fungorum degraded 56% and 59% of the provided pyrene at 37 °C and 25 °C, respectively. Given that B. fungorum biodegraded over the half-life of the initial 100 ppm pyrene in the cultures, the strain may be suitable for bioremediation of high molecular weight PAHs. The study provides important insight into possible PAH biodegradation and bioremediation of hydrocarbon-polluted sites using novel bacterial strains.

In the context of the pre-elimination of malaria, biological control may provide an alternative or additional tool to current malaria control strategies. During their various stages of development, mosquitoes undergo subsequent changes in their associated microbiota, depending on their environment and nutritional status. Although Anopheles gambiae s.l. and Anopheles funestus are the two major malaria vectors in Senegal, the composition of their microbiota is not yet well known. In this study, we explored the microbiota of mosquitoes naturally infected or not by Plasmodium falciparum (Pf) using the 16S ribosomal RNA gene-based bacterial metagenomic approach. In both vector species, the microbiota was more diverse in Pf-infected samples than in the noninfected ones, although the total number of reads appeared to be higher in noninfected mosquitoes. Overall, the microbiota was different between the two vector species. Noteworthy, the bacterial microbiota was significantly different between Pf-positive and Pf-negative groups whatever the species, but was similar between individuals of the same infection status within a species. Overall, the phylum of Proteobacteria was the most predominant in both species, with bacteria of the genus Burkholderia outweighing the others in noninfected vectors. The presence of some specific bacterial species such as Asaia bogorensis, Enterobacter cloacae, Burkholderia fungorum, and Burkholderia cepacia was also observed in Pf-free samples only. These preliminary observations pave the way for further characterization of the mosquito microbiota to select promising bacterial candidates for potential use in an innovative approach to controlling malaria and overcoming the challenges to achieving a malaria-free world.

OBJECTIVES To evaluate the impact of HIV infection on colonization resistance in the proximal gut. DESIGN It was a case-control study. METHODS We contrasted microbiota composition between eight HIV-1-infected patients and eight HIV-negative controls to characterize community alteration and detect exogenous bacteria in the esophagus, stomach, and duodenum, as well as the mouth using a universal 16s ribosomal RNA gene survey and correlated the findings with HIV serostatus and peripheral blood T-cell counts. RESULTS HIV infection was associated with an enrichment of Proteobacteria (P=0.020) and depletion of Firmicutes (P = 0.005) in the proximal gut. In particular, environmental species Burkholderia fungorum and Bradyrhizobium pachyrhizi colonized the duodenum of HIV patients who had abnormal blood CD4 T-cell counts but were absent in HIV-negative controls or HIV patients whose CD4 cell counts were normal. The two species coexisted and exhibited a decreasing trend proximally toward the stomach and esophagus and were virtually absent in the mouth. B. fungorum always outnumbered B. pachyrhizi in a ratio of approximately 15 to 1 regardless of the body sites (P

Multispecies biofilms are the dominant form of biofilms found in Nature. The application of fluorescence in situ hybridization (FISH)-based techniques to the discrimination of biofilm populations might contribute to the understanding of microorganism interactions in these structures, and might allow the development of efficient strategies to prevent or minimize biofilm-associated diseases. This work presents the first study that develops, optimizes and validates a multiplex FISH procedure using locked nucleic acid (LNA) and 2-O-methyl RNA (2OMe) oligonucleotides probes for the in vitro discrimination within mixed populations. As a case study, Escherichia coli, the major cause of urinary tract infections (UTIs), and three other atypical colonizers of urinary catheters (Delftia tsuruhatensis, Achromobacter xylosoxidans and Burkholderia fungorum) with unproven pathogenic potential, were selected. Specific probes for these species were designed and optimized for specific hybridization in multiplex experiments. Results showed that the LNA/2OMe-FISH method performed well in multiplex experiments and presented a good correlation with total and cultivability counts, regardless of the cells physiological state. In fact, the method was also able to report variations of viable but non-cultivable populations. Further analysis of mixed biofilm structures by confocal laser scanning microscopy provided a clear discrimination in three dimensions between the location of the different populations., This work was funded by FEDER funds through the Operational Programme for Competitiveness Factors - COMPETE, ON.2 - O Novo Norte -North Portugal Regional Operational Programme and National Funds through FCT - Foundation for Science and Technology under the projects: PEst-C/EQB/UI0511, NORTE-07-0124-FEDER-000025 - RL2_ Environment & Health and Project "DNA mimics" PIC/IC/82815/2007; PhD Fellowship [SFRH/BD/82663/2011]; and Postdoctoral Fellowship [SFRH/BPD/74480/2010]. The authors would like to thank to M. Fenice and A. Steinbuchel for kindly providing the D. tsuruhatensis BM90 and A. xylosoxidans B3 strains, respectively.

The interaction of a single bacterial species (Burkholderia fungorum) with basaltic rocks from the ICDP HSDP2 drill core and synthetic basaltic glasses was investigated in batch laboratory experiments to better understand the role of microbial activity on rock alteration and Fe mobilization. Incubation experiments were performed with drill core basaltic rock samples to investigate differences in the solution chemistry during biotic and abiotic alteration. Additionally, colonization experiments with synthetic basaltic glasses of different Fe redox states and residual stresses were performed to evaluate their influence on microbial activity and surface attachment of cells. In biotic incubation experiments bacterial growth was observed and the release of Fe and other major elements from drill core basaltic rocks to solution exceeded that of abiotic controls only when the rock sample assay was nutrient depleted. The concentration of dissolved major elements in solution in biotic colonization experiments with synthetic basaltic glasses increased with increasing residual stress and Fe(II) content. Furthermore, the concentration of dissolved Fe and Al increased similarly in biotic colonization experiments indicating that their dissolution might be triggered by microbial activity. Surface morphology imaging by SEM revealed that cells on basaltic rocks in incubation experiments were most abundant on the glass and surfaces with high roughness and almost absent on minerals. In colonization experiments, basaltic glasses with residual stress and high Fe(II) content were intensely covered with a cellular biofilm. In contrast, glasses with high Fe(III) content and no residual stress were sparsely colonized. We therefore conclude that structurally bound Fe is most probably used by B. fungorum as a nutrient. Furthermore, we assume that microbial activity overall increased rock dissolution as soon as the environment becomes nutrient depleted. Our results show that besides compositional effects, other factors such as redox state and residual stress can control microbial alteration of basaltic glasses.

Burkolderia fungorum and Azospirillum brasilense are species commonly found in soils, and have been shown to be plant growth-promoting rhizobacteria (PGPR). This study examined the effect on the growth of the common bean plant of three strains belonging to B. fungorum and A. brasilense species inoculated alone or co-inoculated with Rhizobium tropici CIAT 899T (commercial inoculant strain). The ability of PGPR strains to improve bean growth in the presence of mineral nitrogen was also studied. CIAT 899T co-inoculated with B. fungorum UFLA 04-155 and UFLA 04-226 and A. brasilense BR 11001T did not show any benefits for bean growth. However, inoculation with B. fungorum UFLA 04-155 alone resulted in increased dry weight of the nodules and shoots, increased foliar contents of P, Mg, Cu, Fe, K, Ca, S, and Zn, and fostered nodulation by the indigenous rhizobia community. Plant growth promotion by B. fungorum UFLA 04-226 was stimulated in the presence of nitrogen fertilizer in experiments using potted plants. The results indicate the potential ability of B. fungorum to promote growth of the common bean plant under natural conditions.

Enrichment cultures inoculated with hydrothermally influenced nearshore sediment from Papua New Guinea led to the isolation of an arsenic-tolerant, acidophilic, facultatively aerobic bacterial strain designated PNG-AprilT. Cells of this strain were Gram-stain-negative, rod-shaped, motile and did not form spores. Strain PNG-AprilT grew at temperatures between 4 °C and 40 °C (optimum 30–37 °C), at pH 3.5 to 8.3 (optimum pH 5–6) and in the presence of up to 2.7 % NaCl (optimum 0–1.0 %). Both arsenate and arsenite were tolerated up to concentrations of at least 0.5 mM. Metabolism in strain PNG-AprilT was strictly respiratory. Heterotrophic growth occurred with O2 or nitrate as electron acceptors, and aerobic lithoautotrophic growth was observed with thiosulfate or nitrite as electron donors. The novel isolate was capable of N2-fixation. The respiratory quinones were Q-8 and Q-7. Phylogenetically, strain PNG-AprilT belongs to the genus Burkholderia and shares the highest 16S rRNA gene sequence similarity with the type strains of Burkholderia fungorum (99.8 %), Burkholderia phytofirmans (98.8 %), Burkholderia caledonica (98.4 %) and Burkholderia sediminicola (98.4 %). Differences from these related species in several physiological characteristics (lipid composition, carbohydrate utilization, enzyme profiles) and DNA–DNA hybridization suggested the isolate represents a novel species of the genus Burkholderia , for which we propose the name Burkholderia insulsa sp. nov. The type strain is PNG-AprilT ( = DSM 28142T = LMG 28183T).

Burkholderia fungorum DBT1, first isolated from settling particulate matter of an oil refinery wastewater, is a bacterial strain which has been shown capable of utilizing several polycyclic aromatic hydrocarbons (PAHs) including dibenzothiophene (DBT). In particular, this microbe is able to efficiently degrade DBT through the Kodama pathway. Previous investigations have lead to the identification of six genes, on a total of eight, required for DBT degradation. In the present study, a combined experimental/computational approach was adopted to identify and in silico characterize the two missing genes, namely a ferredoxin reductase and a hydratase-aldolase. Thus, the finding of all enzymatic components of the Kodama pathway in B. fungorum DBT1 makes this bacterial strain amenable for possible exploitation in soil bioremediation protocols.

Gram-negative bacteria exhibit lipopolysaccharides (LPSs) on their outer membrane surface. LPS is considered one of the most potent bacterial virulence factors. Here we report the elucidation of the LPS O-chain structure isolated from Burkholderia fungorum, a bacterium isolated from the white-rot fungus Phanerochaete chrysosporium that can act as a pathogen for plants and domesticated animals. The structure was determined by the employment of detailed chemical and NMR spectroscopy analyses as the following.

An extensive taxonomic analysis of the bacterial strain Burkholderia sp. DBT1, previously isolated from an oil refinery wastewater drainage, is discussed here. This strain is capable of transforming dibenzothiophene through the 'destructive' oxidative pathway referred to as the Kodama pathway. Burkholderia DBT1 has also been proved to use fluorene, naphthalene and phenanthrene as carbon and energy sources, although growth on the first two compounds requires a preinduction step. This evidence suggests that the strain DBT1 exerts a versatile metabolism towards polycyclic aromatic hydrocarbons other than condensed thiophenes. Phylogenetic characterization using a polyphasic approach was carried out to clarify the actual taxonomic position of this strain, potentially exploitable in bioremediation. In particular, investigations were focused on the possible exclusion of Burkholderia sp. DBT1 from the Burkholderia cepacia complex. Analysis of the sequences of 16S, recA and gyrB genes along with the DNA-DNA hybridization procedure indicated that the strain DBT1 belongs to the species Burkholderia fungorum, suggesting the proposal of the taxonomic denomination B. fungorum DBT1.

Microorganisms play an important role in the transformation of material within the earth’s crust. The storage of CO2 could affect the composition of inorganic and organic components in the reservoir, consequently influencing microbial activities. To study the microbial induced processes together with geochemical, petrophysical and mineralogical changes, occurring during CO2 storage, long-term laboratory experiments under simulated reservoir P-T conditions were carried out. Clean inner core sections, obtained from the reservoir region at the CO2 storage site in Ketzin (Germany) from a depth of about 650 m, were incubated in high pressure vessels together with sterile synthetic formation brine under in situ P-T conditions of 5.5 MPa and 40 °C. A 16S rDNA based fingerprinting method was used to identify the dominant species in DNA extracts of pristine sandstone samples. Members of the α - and β -subdivisions of Proteobacteria and the Actinobacteria were identified. So far sequences belonging to facultative anaerobic, chemoheterotrophic bacteria (Burkholderia fungorum, Agrobacterium tumefaciens) gaining their energy from the oxidation of organic molecules and a genus also capable of chemolithoautotrophic growth (Hydrogenophaga) was identified. During CO2 incubation minor changes in the microbial community composition were observed. The majority of microbes were able to adapt to the changed conditions. During CO2 exposure increased concentrations of Ca2+, K+, Mg2+ and SO42− were observed. Partially, concentration rises are (i) due to equilibration between rock pore water and synthetic brine, and (ii) between rock and brine, and are thus independent on CO2 exposure. However, observed concentrations of Ca2+, K+, Mg2+ are even higher than in the original reservoir fluid and therefore indicate mineral dissolution due to CO2 exposure.

The Cd resistance mechanism of a Burkholderia strain isolated from paddy soil of an electronic waste recycling site was characterized for its potential application in metal bioremediation. 16S rRNA gene analysis revealed phylogenetic relatedness of this strain to Burkholderia fungorum. When grown in broth supplemented with Cd, this strain could decrease the bio-available Cd concentration through biosorption of Cd with its capsule or exopolysaccharide. Cd deposition in the cell envelope was confirmed by transmission electron microscopy and energy dispersive X-ray spectroscopy, but no Cd deposition was observed inside the cell. FT-IR revealed a major role of carboxyl, hydroxyl and amino groups along with a possible ion exchange mechanism in cation binding. X-ray powder diffraction analysis showed crystals formed on the exopolysaccharide from the Cd-supplemented culture. Thus our results indicated that sequestration of Cd by exopolysaccharide in the cell envelope might be one potential mechanism for Cd-detoxification in B. fungorum.

Aims: To search for culturable Burkholderia species associated with annual ryegrass in soils from natural pastures in Portugal, with plant growth-promoting effects. Methods and Results: Annual ryegrass seedlings were used to trap Burkholderia from two different soils in laboratory conditions. A combined approach using genomic fingerprinting and sequencing of 16S rRNA and recA genes resulted in the identification of Burkholderia strains belonging to the species Burkholderia graminis, Burkholderia fungorum and the Burkholderia cepacia complex. Most strains were able to solubilize mineral phosphate and to synthesize indole acetic acid; some of them could produce siderophores and antagonize the phytopathogenic oomycete, Phytophthora cinnamomi. A strain (G2Bd5) of B. graminis was selected for gnotobiotic plant inoculation experiments. The main effects were the stimulation of root growth and enhancement of leaf lipid synthesis and turnover. Fluorescence in situ hybridization and confocal laser microscopy evidenced that strain G2Bd5 is a rhizospheric and endophytic colonizer of annual ryegrass. Conclusions: This work revealed that annual ryegrass can naturally associate with members of the genus Burkholderia. A novel plant growth promoting strain of B. graminis was obtained. Significance and Impact of the Study: The novel strain belongs to the plant-associated Burkholderia cluster and is a promising candidate for exploitation as plant inoculant in field conditions.

A Gram-negative, non-sporulating, rod-shaped, motile bacterium, with a single polar flagellum, designated strain PsJNT, was isolated from surface-sterilized onion roots. This isolate proved to be a highly effective plant-beneficial bacterium, and was able to establish rhizosphere and endophytic populations associated with various plants. Seven related strains were recovered from Dutch soils. Based on 16S rRNA gene sequence data, strain PsJNT and the Dutch strains were identified as representing a member of the genus Burkholderia, as they were closely related to Burkholderia fungorum (98·7 %) and Burkholderia phenazinium (98·5 %). Analysis of whole-cell protein profiles and DNA–DNA hybridization experiments confirmed that all eight strains belonged to a single species. Strain PsJNT had a DNA G+C content of 61·0 mol%. Only low levels of DNA–DNA hybridization to closely related species were found. Qualitative and quantitative differences in fatty acid composition between strain PsJNT and closely related species were identified. The predominant fatty acids in strain PsJNT were 16 : 0, 18 : 1ω7c and summed feature 3 (comprising 16 : 1ω7c and/or iso-15 : 0 2-OH). Isolate PsJNT showed high 1-aminocyclopropane-1-carboxylate deaminase activity and is therefore able to lower the ethylene level in a developing or stressed plant. Production of the quorum-sensing signal compound 3-hydroxy-C8-homoserine lactone was detected. Based on the results of this polyphasic taxonomic study, strain PsJNT and the seven Dutch isolates are considered to represent a single, novel species, for which the name Burkholderia phytofirmans sp. nov. is proposed. The type strain is strain PsJNT (=LMG 22146T=CCUG 49060T).

Isolates belonging to six genera not previously known to oxidize CO were obtained from enrichments with aquatic and terrestrial plants. DNA from these and other isolates was used in PCR assays of the gene for the large subunit of carbon monoxide dehydrogenase ( coxL ). CoxL and putative coxL fragments were amplified from known CO oxidizers (e.g., Oligotropha carboxidovorans and Bradyrhizobium japonicum ), from novel CO-oxidizing isolates (e.g., Aminobacter sp. strain COX, Burkholderia sp. strain LUP, Mesorhizobium sp. strain NMB1, Stappia strains M4 and M8, Stenotrophomonas sp. strain LUP, and Xanthobacter sp. strain COX), and from several well-known isolates for which the capacity to oxidize CO is reported here for the first time (e.g., Burkholderia fungorum LB400, Mesorhizobium loti , Stappia stellulata , and Stappia aggregata ). PCR products from several taxa, e.g., O. carboxidovorans , B. japonicum , and B. fungorum , yielded sequences with a high degree (>99.6%) of identity to those in GenBank or genome databases. Aligned sequences formed two phylogenetically distinct groups. Group OMP contained sequences from previously known CO oxidizers, including O. carboxidovorans and Pseudomonas thermocarboxydovorans , plus a number of closely related sequences. Group BMS was dominated by putative coxL sequences from genera in the Rhizobiaceae and otherα - Proteobacteria . PCR analyses revealed that many CO oxidizers contained two coxL sequences, one from each group. CO oxidation by M. loti , for which whole-genome sequencing has revealed a single BMS-group putative coxL gene, strongly supports the notion that BMS sequences represent functional CO dehydrogenase proteins that are related to but distinct from previously characterized aerobic CO dehydrogenases.

The genus Burkholderia comprises over 28 species and species-specific, recA-based polymerase chain reaction (PCR) tests are available for several species, but not for some soil-inhabiting species including B. fungorum. Previous analysis of several novel rhizospheric, environmental isolates belonging to the B. cepacia complex suggested they may be closely related to B. fungorum. To discover any relationship between these isolates and B. fungorum we set out to clone and sequence a portion of the B. fungorum recA gene in order to design species-specific primer pairs for use in a recA-based PCR assay. Using a similar procedure we extended the recA-based PCR assay to identify B. sacchari and B. caledonica, two additional soil-inhabiting Burkholderia spp.

Polycyclic aromatic hydrocarbons (PAHs) degrading bacteria can be isolated from polluted coastal environments. They were close to Burkholderia fungorum and Mycobacterium gilvum, and had nidA, nidA3, pdoA2, and pcaH genes. In the pyrene and fluoranthene degradation experiment using a minimum medium, B. fungorum isolate no. 1 and M. gilvum isolate no. 13 were able to degrade 98.6% + 1.9 of pyrene after 20 days, and 99.9% ± 0.1 after a 16-day incubation period, respectively. Moreover, fluoranthene could be consumed by B. fungorum isolate no. 1 and M. gilvum isolate no. 13 at the level of 99.6% ± 0.7 after 24 days, and 100% after a 28-day incubation period, respectively.

Using a polyphasic approach (including cellular protein and fatty acid analysis, biochemical characterization, 16S ribosomal DNA sequencing, and DNA-DNA hybridizations), we characterized 51 bacterial isolates recovered from respiratory secretions of cystic fibrosis (CF) patients. Our analyses showed that 24 isolates belong to taxa that have so far not (or only rarely) been reported from CF patients. These taxa include Acinetobacter sp., Bordetella hinzii , Burkholderia fungorum , Comamonas testosteroni , Chryseobacterium sp., Herbaspirillum sp., Moraxella osloensis , Pandoraea genomospecies 4, Ralstonia gilardii , Ralstonia mannitolilytica , Rhizobium radiobacter , and Xanthomonas sp. In addition, one isolate most likely represents a novel Ralstonia species, whereas nine isolates belong to novel taxa within the α- Proteobacteria . Eight of these latter isolates are classified into the novel genus Inquilinus gen. nov. as Inquilinus limosus gen. nov., sp. nov., or as Inquilinus sp. The remaining 17 isolates are characterized as members of the family Enterobacteriaceae . The recovery of these species suggests that the CF lung is an ecological niche capable of supporting the growth of a wide variety of bacteria rarely seen in clinical samples. Elucidation of the factors that account for the association between these unusual species and the respiratory tract of CF patients may provide important insights into the pathophysiology of CF infection. Because accurate identification of these organisms in the clinical microbiology laboratory may be problematic, the present study highlights the utility of reference laboratories capable of identifying unusual species recovered from CF sputum.

The Department of Energy's Integrated Field-Scale Subsurface Research Challenge Site (IFRC) at Rifle, Colorado was created to address the gaps in knowledge on the mechanisms and rates of U(VI) bioreduction in alluvial sediments. Previous studies at the Rifle IFRC have linked microbial processes to uranium immobilization during acetate amendment. Several key bacteria believed to be involved in radionuclide containment have been described; however, most of the evidence implicating uranium reduction with specific microbiota has been indirect. Here, we report on the cultivation of a microorganism from the Rifle IFRC that reduces uranium and appears to utilize it as a terminal electron acceptor for respiration with acetate as electron donor. Furthermore, this bacterium constitutes a significant proportion of the subsurface sediment community prior to biostimulation based on TRFLP profiling of 16S rRNA genes. 16S rRNA gene sequence analysis indicates that the microorganism is a betaproteobacterium with a high similarity to Burkholderia fungorum. This is, to our knowledge, the first report of a betaproteobacterium capable of uranium respiration. Our results indicate that this microorganism occurs commonly in alluvial sediments located between 3-6 m below ground surface at Rifle and may play a role in the initial reduction of uranium at the site.

Currently known high grade easily-acquirable phosphate rock reserves are depleting. The objective of this study was to evaluate the potential application of bioleaching technology to the recovery of phosphorus from different phosphate ore materials, and isolate indigenous phosphorus-solubilising bacterial strains from them. In the bacterial enrichment and isolation experiments no growth was detected in the medium designed for acidophiles. Heterotrophic phosphorus solubilising isolates were enriched from the fluorapatite concentrate, and all four isolates were most closely related to Burkholderia fungorum. Bioleaching of phosphorus from low grade fluorapatite ore containing 8.2% P2O5 and from fluorapatite concentrate containing 29.8% P2O5 was carried out in shake flasks. Supplemental elemental sulphur was added as an energy source for acid generation. Mixed and pure acidophilic bacterial cultures consisting of iron- and/or sulphur-oxidizing bacteria Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans were used in the experiments. These acidophiles are commonly used in bioleaching of sulphide minerals, but their application to phosphorus solubilisation has until now been limited. Phosphorus leaching was shown to be a pH-dependent phenomenon. Phosphorus bioleaching yields of up to 97% and 28% were obtained in 21 d for low grade fluorapatite ore and concentrate, respectively, in solutions with a 1% solid content. With adaptation, the bacterial mixture was acclimated to suspensions with a 10-30% solid content. These results indicate a potential for the application of bioleaching to phosphorus extraction of low grade materials.

We report the first case of community-acquired bacteremia with Burkholderia fungorum, a newly described member of the Burkholderia cepacia complex. A 9-year-old girl sought treatment with septic arthritis in her right knee and ankle with soft tissue involvement. Commercial identification systems did not identify the causative microorganism.

The aim of this study was to enrich phosphorus solubilizing microorganisms from high-phosphorus iron ores, apatite ores and phosphogypsum waste. Phosphorus solubilizing microorganisms can be utilized in dephosphorization of high-phosphorus iron ores and in phosphorus leaching from fluorapatite ores. Low grade fluorapatite ore (3.6% P, pH 6.8), fluorapatite concentrate (13% P, pH 8.3), phosphogypsum waste (0.7% P, pH 2.3), iron ore 1 (0.19% P, pH 7.6) and iron ore 2 (0.18% P, pH 7.6) were used as potential sources of phosphorus solubilizing microorganisms. The samples were cultured in NBRIP media at pH 5 and 8 with either glucose or sucrose as a carbon source, and in modified 9K media at pH 1.5 and 2.5 for 3 weeks. Phosphorus solubilizing bacteria were enriched only from the fluorapatite concentrate at the pH of 8. The four obtained heterotrophic isolates were identified by 16S rRNA gene sequencing, and were shown to be closest related to Burkholderia fungorum. These results indicate that the diversity of culturable phosphorus solubilizing bacteria present in apatite and iron ores is relatively low. The isolated Burkholderia strain showed phosphorus solubilizing potential.

Burkholderia fungorum DBT1 is a bacterial strain isolated from an oil refinery discharge and capable of transforming dibenzothiophene, phenanthrene, naphthalene, and fluorene. In order to evaluate the influence of a policyclic aromatic hydrocarbon (PAH)-transforming bacterial strain on the phytoremediation of organic contaminants, B. fungorum DBT1 was inoculated into hybrid poplar (Populus deltoides×Populus nigra). The poplar plants were grown for 18-wk with or without naphthalene, phenanthrene, fluorene and dibenzothiophene (488mgkg(-1) soil each) in non-sterile sand-peat substrate. Evidences were gained that B. fungorum DBT1 was present in high concentration in poplar root tissues (2.9-9.5×10(3)CFUg(-1)), while the strain was not detected in stem, leaves and rhizosphere. When poplar was planted in uncontaminated substrate, the infection caused negative effects on biomass index, leaves and stem dry weight, without showing however any disease symptoms. On the other hand, plants inoculated with the strain DBT1 resulted in better tolerance against the toxic effects of PAHs, in terms of root dry weight. Although the presence of plants acted as the main effective treatment for PAH dissipation (82-87%), the inoculum with DBT1 strain lead to the highest PAH abatement (up to 99%). In the present study, an environmental isolate with proper metabolic features was demonstrated to be possibly suitable as a poplar endophyte for improving microbe-assisted phytoremediation in PAH contaminated matrices.

A halobenzene-degrading bacterium, Burkholderia fungorum FLU100 (DSM 23736), was isolated due to its outstanding trait to degrade fluorobenzene. Besides fluorobenzene, it utilizes, even in random mixtures, chlorobenzene, bromobenzene, iodobenzene, benzene, and toluene as sole sources of carbon and energy. FLU100 mineralizes mono-halogenated benzenes almost stoichiometrically (according to halide balance); after a lag phase, it also degrades 3-fluorophenol and 3-chlorophenol completely. The FLU100-derived transposon Tn5-mutant FLU100-P14R22 revealed 3-halocatechol to be a central metabolite of this new halobenzene degradation pathway. In FLU100, halocatechols are—as expected—strictly subject to ortho-cleavage of the catechol ring, with meta-cleavage never been observed. The strain is able to completely mineralize 3-fluorocatechol, the principal catecholic metabolite being nearly exclusively formed from fluorobenzene. The temporarily excreted 2-fluoromuconate formed thereof in turn is subsequently metabolized completely. This important finding falsifies the customary opinion of the persistence of 2-fluoromuconate valid up to now. The degradation of 4-fluorocatechol, however, being a very minor intermediate in FLU100, is substantially slower and incomplete and leads to the accumulation of uncharacterized derivatives of muconic acid and muconolactone in the medium. This branch therefore does not seem to be productive. To our knowledge, this represents the first example of the complete metabolism of 3-fluorocatechol via 2-fluoromuconate, a metabolite hitherto described as a dead-end metabolite in fluoroaromatic degradation.

One of the most cultivated and consumed vegetables in Brazil is the common bean, Phaseolus vulgaris L. The symbiosis of this plant species with nitrogen-fixing bacteria that are adapted to the stresses commonly found in tropical soils can increase production. The aim of this study was to evaluate the symbiotic effectiveness of bacterial strains from soils under different land uses in the Amazon region. Further, rhizobia tolerance to acidity and aluminium and the involvement of some possible physiological mechanisms of such tolerance were also investigated. In assessing the efficiency of biological nitrogen fixation, inoculation with strains UFLA04-195, UFLA04-173 and UFLA04-202, belonging to the genus Rhizobium, resulted in greater plant growth, higher shoot nitrogen content and good nodulation compared to the inoculation with the strain CIAT 899 (R. tropici), and to the mineral nitrogen control or Burkholderia fungorum strains that nodulated or not bean plants. These efficient strains grew better at pH 5.0 than at pH 6.0 or pH 6.9; they also tolerated up to 1 mmol l(-1) of Al(3+) and showed an increased production of exopolysaccharides where the growing rates were less (pH 6.0 and pH 6.9). With respect to aluminium, the highest production of EPS produced greater tolerance to this element. Taken together, these results indicate that the strains evaluated in this study were tolerant to acidity and aluminium; they appeared to have developed resistance mechanisms such as EPS production and a resistant cell outer membrane (indicated by resistance to polymyxin and methyl violet). As these strains also gave increased yields of the host species, further studies on whether to recommend these strains as inoculants are already underway.

Thirty-three endophytic diazotrophs were isolated from surface-sterilized leaves, stem, and roots of wild rice Oryza alta. The SDS-PAGE profile of total protein and insertion sequence-based polymerase chain reaction (IS-PCR) fingerprinting grouped the isolates into four clusters (I-IV). The 16S rRNA gene sequence homology of the representative strains B21, B31, B1, and B23 of clusters I, II, III, and IV were assigned to Pseudomonas oleovorans (99.2% similarity), Burkholderia fungorum (99.4% similarity), Enterobacter cloacae (98.9% similarity), and Acinetobacter johnsonii (98.4% similarity), respectively. The results showed wide genetic diversity of the putative diazotrophic strains of the wild rice, O. alta, and the strains of cluster IV are the first report of nitrogen-fixing Acinetobacter species. The cell size, phenotypic characters, total protein profile, genomic DNA fingerprinting, DNA-DNA hybridization, and antibiotic resistance differentiated strain B23(T) from its closest relatives A. johnsonii LMG999(T) and Acinetobacter haemolyticus LMG996(T). The DNA-DNA hybridization also distinguished the strain B23(T) from the closely related Acinetobacter species. Based on these data, a novel species, Acinetobacter oryzae sp. nov., and strain B23(T) (=LMG25575(T) = CGMCC1.10689(T)) as the type strain were proposed.

We previously demonstrated that purified polyclonal and monoclonal anti-dsDNA antibodies bind a 15-mer peptide ASPVTARVLWKASHV in ELISA and Dot blot. This 15-mer peptide partial sequence ARVLWKASH shares similarity with burkholderia bacterial cytochrome B 561 partial sequence ARVLWRATH. In this study, we show that purified anti-dsDNA antibodies react with burkholderia fungorum bacterial cell lysates in Western blot. We used anti-dsDNA antibodies to make an anti-dsDNA antibodies affinity column and used this column to purify the burkholderia fungorum bacterial protein. Purified anti-dsDNA antibodies bind specifically to purified bacterial antigen and purified bacterial antigen blocked the anti-dsDNA antibodies binding to dsDNA antigen. Sera with anti-dsDNA antibodies bind specifically to purified bacterial antigen. We obtained protein partial sequence of RAGTDEGFG which is shared with burkholderia bacterial transcription regulator protein sequence. Sera with anti-dsDNA antibodies bind to RAGTDEGFG peptide better than control groups. These data support our hypothesis that the origin of anti-dsDNA antibodies in SLE may be associated with burkholderia bacterial infection.

Summary Strain AC1100 is well-known for its ability to degrade a variety of recalcitrant xenobiotics, including 2,4,5-trichlorophenoxyacetic acid. We performed a polyphasic-taxonomic study to determine its taxonomic position. The G+C content of strain AC1100 was 62.6 mol%. On the basis of 16S rRNA gene sequence similarity, strain AC1100 belonged to the b-Proteobacteria and was most closely related to Burkholderia fungorum (98.3% similarity). DNA-DNA hybridisations, comparison of protein profiles, cellular fatty acid analysis and biochemical tests allowed genotypic and phenotypic differentiation of strain AC1100 from other Burkholderia species. Our data show that strain AC1100 represents a novel species for which the name Burkholderia phenoliruptrix sp. nov. is proposed. The type strain is AC1100 T (= LMG 22037 T = CCUG 48558 T ).

Burkholderia species are free-living bacteria with a versatile metabolic lifestyle. The genome of B. fungorum LB400 is predicted to encode three different pathways for formaldehyde oxidation: an NAD-linked, glutathione (GSH)-independent formaldehyde dehydrogenase; an NAD-linked, GSH-dependent formaldehyde oxidation system; and a tetrahydromethanopterin-methanofuran-dependent formaldehyde oxidation system. The other Burkholderia species for which genome sequences are available, B. mallei , B. pseudomallei , and B. cepacia , are predicted to contain only the first two of these pathways. The roles of the three putative formaldehyde oxidation pathways in B. fungorum LB400 have been assessed via knockout mutations in each of these pathways, as well as in all combinations of knockouts. The resulting mutants have the expected loss of enzyme activities and exhibit defects of varying degrees of severity during growth on choline, a formaldehyde-producing substrate. Our data suggest that all three pathways are involved in formaldehyde detoxification and are functionally redundant under the tested conditions.

The sequence of the extradiol dioxygenase ThnC, involved in tetralin biodegradation, was aligned with other extradiol dioxygenases involved in biodegradation of polycyclic compounds, and a three-dimensional model of ThnC, based on the structure of the previously crystallized 2,3-dihydroxybiphenyl dioxygenase fromBurkholderia fungorumLB400, was built. In order to assess the functional importance of some non-active-site residues whose relevance could not be established by structural information, a number of positions surrounding the substrate-binding site were mutated in ThnC. Ten mutant proteins were purified and their activity towards 1,2-dihydroxytetralin, 1,2-dihydroxynaphthalene and 2,3-dihydroxybiphenyl was characterized. N213H, Q198H, G206M, A282R and A282G mutants increasedkcat/Kmat least twofold using 1,2-dihydroxytetralin as the substrate, thus showing that activity of ThnC is not maximized for this substrate. N213H and Q198H mutants increasedkcat/Kmusing any of the substrates tested, thus showing the relevance for activity of these two histidines, which are highly conserved in dihydroxybiphenyl dioxygenases, but not present in dihydroxynaphthalene dioxygenases. Different substitutions in position 282 had different effects on general activity or substrate specificity, thus showing the functional importance of the most C-terminalβ-sheet of the protein. A251M and G206M mutants showed increased activity specifically for a particular substrate. N213H, G206M, A282R, A282G and Y177I substitutions resulted in enzymes more tolerant to acidic pH, the most striking effect being observed in mutant Y177I, which showed maximal activity at pH 5·5. In addition, Q198D and V175D mutants, which had alteredKm, also showed altered sensitivity to substrate inhibition, thus indicating that inhibition is exerted through the same binding site. This mutational analysis, therefore, identified conserved residues important for activity or substrate specificity, and also shed some light on the mechanism of substrate inhibition exhibited by extradiol dioxygenases.

The facultatively anaerobic bacterium Alcaligenes defragrans NKNTAU was found to oxidize taurine (2-aminoethanesulphonate) with nitrate as the terminal electron acceptor. Taurine was transaminated to 2-sulphoacetaldehyde. This was not converted into sulphite and acetate by a ‘sulphoacetaldehyde sulpho-lyase’ (EC 4.4.1.12), but into sulphite and acetyl phosphate, which was identified by three methods. The enzyme, which required the addition of phosphate, thiamin diphosphate and Mg2+ ions for activity, was renamed sulphoacetaldehyde acetyltransferase (Xsc; EC 2.3.1.-). Inducible Xsc was expressed at high levels, and a three-step 11-fold purification yielded an essentially homogeneous soluble protein, which was a homotetramer in its native form; the molecular mass of the subunit was found to be between about 63kDa (SDS/PAGE) and 65.3kDa (matrix-assisted laser-desorption ionization—time-of-flight MS). The N-terminal and two internal amino acid sequences were determined, and PCR primers were generated. The xsc gene was amplified and sequenced; the derived molecular mass of the processed protein was 65.0kDa. The downstream gene presumably encoded the inducible phosphate acetyltransferase (Pta) found in crude extracts. The desulphonative enzymes ('EC 4.4.1.12') from Achromobacter xylosoxidans NCIMB 10751 and Desulfonispora thiosulfatigenes GKNTAU were shown to be Xscs. We detected at least three subclasses of xsc in Proteobacteria and in Gram-positive bacteria, and they comprised a distinct group within the acetohydroxyacid synthase supergene family. Genome sequencing data revealed xsc genes in Burkholderia fungorum (80% sequence identity) and Sinorhizobium meliloti (61%) with closely linked pta genes. Different patterns of regulation for the transport and dissimilation of taurine were hypothesized for S. meliloti and B. fungorum.

Analysis of the genome sequence of the polyhydroxyalkanoate- (PHA) accumulating bacteriumRalstonia eutrophastrain H16 revealed three homologues (PhaP2, PhaP3 and PhaP4) of the phasin protein PhaP1. PhaP1 is known to constitute the major component of the layer at the surface of poly(3-hydroxybutyrate), poly(3HB), granules. PhaP2, PhaP3 and PhaP4 exhibited 42, 49 and 45 % identity or 61, 62 and 63 % similarity to PhaP1, respectively. The calculated molecular masses of PhaP1, PhaP2, PhaP3 and PhaP4 were 20·0, 20·2, 19·6 and 20·2 kDa, respectively. RT-PCR analysis showed thatphaP2,phaP3andphaP4were transcribed under conditions permissive for accumulation of poly(3HB). 2D PAGE of the poly(3HB) granule proteome and analysis of the detected proteins by MALDI-TOF clearly demonstrated that PhaP1, PhaP3 and PhaP4 are bound to the poly(3HB) granules in the cells. PhaP3 was expressed at a significantly higher level in PhaP1-negative mutants. Occurrence of an unknown protein with an N-terminal amino-acid sequence identical to that of PhaP2 in crude cellular extracts ofR. eutrophahad previously been shown by others. Although PhaP2 could not be localizedin vivoon poly(3HB) granules,in vitroexperiments clearly demonstrated binding of PhaP2 to these granules. Further analysis of complete or partial genomes of other poly(3HB)-accumulating bacteria revealed the existence of multiple phasin homologues inRalstonia solanacearum,Burkholderia fungorumandAzotobacter vinelandii. These new and unexpected findings should affect our current models of PHA-granule structure and may also have a considerable impact on the establishment of heterologous production systems for PHAs.