Biological characteristics and salt-tolerant plant growth-promoting effects of an ACC deaminase-producing Burkholderia pyrrocinia strain isolated from the tea rhizosphere.

Plant growth-promoting rhizobacteria that produce 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase can promote plant growth and enhance abiotic stress tolerance. In this study, Burkholderia pyrrocinia strain P10, with an ACC deaminase activity of 33.01-µmol/h/mg protein, was isolated from the tea rhizosphere and identified based on morphological, biochemical, and molecular characteristics. In addition to its ACC deaminase activity at pH 5.0-9.0 and in response to 5% NaCl and 20% polyethylene glycol, strain P10 can also solubilize phosphorus compounds, produce indole-3-acetic acid, and secrete siderophores. Pot experiments revealed that strain P10 can significantly enhance peanut seedling growth under saline conditions (100- and 170-mmol/L NaCl). Specifically, it increased the fresh weight and root length of plants by 90.12% and 79.22%, respectively, compared with high-salt stress. These results provide new insights into the biological characteristics of Burkholderia pyrrocinia, which may be useful as a bio-fertilizer.

Bacterial endophytes from Artemisia nilagirica (Clarke) Pamp., with antibacterial efficacy against human pathogens.

A study was conducted to isolate and characterize endophytes from Artemisia nilagirica, a traditional medicinal plant. The plant was collected from Western Ghats, India. Endophytes isolated included Arthrobacter sp. WWAT1, Pseudomonas sp. WYAT2, Microbacterium sp. WYAT3, Psychrobacter sp. WBAT4, Enterobacter sp. WWAT5, Bacillus sp. WBAT6, Kosakonia cowanii WBAT7, Bacillus sp. WBAT8, Bacillus sp. WBAT9, Chromobacterium violaceum WVAT6, Serratia sp.WPAT8 and Burkholderia sp. WYAT7. Of these two bacteria, Chromobacterium violaceum strain WVAT6 and Burkholderia sp. strain WYAT exhibited antibacterial property against human pathogens. Similar to the environmental isolates, Burkholderia sp. WYAT7 showed pleomorphism and produced different enzymes, whereas like clinical strains they showed multidrug resistance, for their survival in different environmental conditions. Chromobacterium violaceum WVAT6 exhibited rod shape morphology and showed multiple drug resistance except to erythromycin, tetracycline and gentamicin antibiotics. Both produced biofilm and enzymes such as protease and lipase. The antimicrobial compounds from these endophytes may find application in the preparation of antimicrobial formulations.

Seasonal dynamics of the bacterioplankton community in a large, shallow, highly dynamic freshwater lake.

The spatiotemporal shifts of the bacterioplankton community can mirror their transition of functional traits in an aquatic ecosystem. However, the spatiotemporal variation of the bacterioplankton community composition structure (BCCS) within a large, shallow, highly dynamic freshwater lake is still poorly understood. Here, we examined the seasonal and spatial variability of the BCCs within Poyang Lake by sequencing the 16S rRNA gene amplicon to explore how hydrological changes affect the BCCs. Principal coordinate analysis showed that the BCCs varied significantly among four sampling seasons, but not spatially. The seasonal changes of the BCCs were mainly attributed to the differences between autumn and spring-winter. Higher α diversity indices were observed in autumn. Redundancy analysis indicated that the BCCs co-variated with water level, pH, temperature, total phosphorus, ammoniacal nitrogen, electrical conductivity, total nitrogen, and turbidity. Among them, water level was the key determinant separating autumn BCCs from the BCCs in other seasons. A significantly lower relative abundance of Burkholderiales (betI and betVII) and a higher relative abundance of Actinomycetales (acI, acTH1, and acTH2) were found in autumn than in other seasons. Overall, our results suggest that water level changes associated with pH, temperature, and nutrient status shaped the seasonal patterns of the BCCs within Poyang Lake.

Phylogeny and polycyclic aromatic hydrocarbons degradation potential of bacteria isolated from crude oil-contaminated site.

This study employed the use of 16S rRNA gene sequence analysis to identify three of four native bacterial strains isolated from crude oil-contaminated site in Poza Rica, Veracruz, Mexico. The identified bacteria were Ochrobactrum intermedium, Pandoraea pnomenusa and Ochrobactrum sp., but SA2-09 strain was not identified. The ability of the isolates to degrade polycyclic aromatic hydrocarbons (PAHs) was evaluated at 31.61 and 54.52 mg/kg PAHs in soil, when used as crude oil in soil microcosm during 80 days of incubation at 30°C. The results demonstrated that O. intermedium biodegraded many PAHs, including the high molecular weight (HMW) PAHs fluoranthene (100% equivalent 0.24 mg/kg), benzo [b] fluoranthene (81.8% equal 0.18 mg/kg), Benzo[a]pyrene (87.0%, 0.20 mg/kg) and Benzo[g,h,i]perylene (52.7%, 0.39 mg/kg). P. pnomenusa had a degradation profile of HMW PAHs, which was similar to O. intermedium, while Ochrobactrum sp. and the strain SA-09 exhibited lower degradation rates of HMW.

Insights into selenite reduction and biogenesis of elemental selenium nanoparticles by two environmental isolates of Burkholderia fungorum.

Microorganisms capable of transforming toxic selenium oxyanions into non-toxic elemental selenium (Se°) may be considered as biocatalysts for the production of selenium nanoparticles (SeNPs), eventually exploitable in different biotechnological applications. Two Burkholderia fungorum strains (B. fungorum DBT1 and B. fungorum 95) were monitored during their growth for both capacity and efficiency of selenite (SeO32-) reduction and elemental selenium formation. Both strains are environmental isolates in origin: B. fungorum DBT1 was previously isolated from an oil refinery drainage, while B. fungorum 95 has been enriched from inner tissues of hybrid poplars grown in a soil contaminated by polycyclic aromatic hydrocarbons. Our results showed that B. fungorum DBT1 is able to reduce 0.5mM SeO32- to Se° when cultured aerobically in liquid medium at 27°C, while B. fungorum 95 can reduce more than 1mM SeO32- to Se° within 96h under the same growth conditions, with the appearance of SeNPs in cultures of both bacterial strains. Biogenic SeNPs were spherical, with pH-dependent charge and an average hydrodynamic diameter of 170nm and 200nm depending on whether they were produced by B. fungorum 95 or B. fungorum DBT1, respectively. Electron microscopy analyses evidenced that Se nanoparticles occurred intracellularly and extracellularly. The mechanism of SeNPs formation can be tentatively attributed to cytoplasmic enzymatic activation mediated by electron donors. Biogenic nanoparticles were then probably released outside the bacterial cells as a consequence of a secretory process or cell lysis. Nevertheless, formation of elemental selenium nanoparticles under aerobic conditions by B. fungorum DBT1 and B. fungorum 95 is likely due to intracellular reduction mechanisms. Biomedical and other high tech sectors might exploit these biogenic nanoparticles in the near future, once fully characterized and tested for their multiple properties.

Purification, biochemical characterization, and genetic cloning of the phytase produced by Burkholderia sp. strain a13.

A phytase-producing bacterium, Burkholderia sp. a13 (JCM 30421), was isolated from Lake Kasumigaura by enrichment cultivation using minimum medium containing phytic acid as the sole phosphorus source. The phytase production by strain a13 was induced by the presence of phytic acid and repressed by the addition of glucose. The purified enzyme had a molecular weight of 44 kDa and a phytase activity of 174 µmol min(-1) mg(-1). The enzyme showed broad substrate specificity, but the highest activity was observed with phytic acid. The enzyme activity was strongly inhibited by Cu(2+), Zn(2+), Hg(2+), and iodoacetic acid, indicating the requirement of a thiol group for the activity. Genetic cloning reveals that the mature portion of this enzyme consists of 428 amino acids with a calculated molecular weight of 46 kDa. The amino acid sequence showed the highest similarity to the phytase produced by Hafnia alvei with 48% identity; it also contained histidine acid phosphatase (HAP) motifs (RHGXRXP and HD), indicating the classification of this enzyme in the HAP phytase family. We have successfully expressed the cloned gene in Escherichia coli from its putative initiation codon, showing that the gene actually encodes the phytase.

Burkholderia ginsengiterrae sp. nov. and Burkholderia panaciterrae sp. nov., antagonistic bacteria against root rot pathogen Cylindrocarpon destructans, isolated from ginseng soil.

Strain DCY85(T) and DCY85-1(T), isolated from rhizosphere of ginseng, were rod-shaped, Gram-reaction-negative, strictly aerobic, catalase positive and oxidase negative. 16S rRNA gene sequence analysis revealed that strain DCY85(T) as well as DCY85-1(T) belonged to the genus Burkholderia and were closely related to Burkholderia fungorum KACC 12023(T) (98.1 and 98.0 % similarity, respectively). The major polar lipids of strain DCY85(T) and DCY85-1(T) were phosphatidylethanolamine, one unidentified aminolipid and two unidentified phospholipids. The major fatty acids of both strains are C16:0, C18:1 ω7c and summed feature 3 (C16:1 ω6c and/or C16:1 ω7c). The predominant isoprenoid quinone of each strain DCY85(T) and DCY85-1(T) was ubiquinone (Q-8) and the G+C content of their genomic DNA was 66.0 and 59.4 mol%, respectively, which fulfill the characteristic range of the genus Burkholderia. The polyamine content of both DCY85(T) and DCY85-1(T) was putrescine. Although both DCY85(T) and DCY85-1(T) have highly similar 16S rRNA and identical RecA and gyrB sequences, they show differences in phenotypic and chemotaxonomic characteristics. DNA-DNA hybridization results proved the consideration of both strains as two different species. Based on the results from our polyphasic characterization, strain DCY85(T) and DCY85-1(T) are considered novel Burkholderia species for which the name Burkholderia ginsengiterrae sp. nov and Burkholderia panaciterrae sp. nov are, respectively, proposed. An emended description of those strains is also proposed. DCY85(T) and DCY85-1(T) showed antagonistic activity against the common root rot pathogen of ginseng, Cylindrocarpon destructans. The proposed type strains are DCY85(T) (KCTC 42054(T) = JCM 19888(T)) and DCY85-1(T) (KCTC 42055(T) = JCM 19889(T)).

Rapid solubilization of insoluble phosphate by a novel environmental stress-tolerant Burkholderia vietnamiensis M6 isolated from ginseng rhizospheric soil.

We isolated and characterized novel insoluble phosphate (P)-solubilizing bacteria tolerant to environmental factors like high salt, low and high pHs, and low temperature. A bacterium M6 was isolated from a ginseng rhizospheric soil and confirmed to belong to Burkholderia vietnamiensis by BIOLOG system and 16S rRNA gene analysis. The optimal cultural conditions for the solubilization of P were 2.5% (w/v) glucose, 0.015% (w/v) urea, and 0.4% (w/v) MgCl(2).6H(2)O along with initial pH 7.0 at 35 degrees C. High-performance liquid chromatography analysis showed that B. vietnamiensis M6 produced gluconic and 2-ketogluconic acids. During the culture, the pH was reduced with increase in gluconic acid concentration and was inversely correlated with P solubilization. Insoluble P solubilization in the optimal medium was about 902 mg l(-1), which was approximately 1.6-fold higher than the yield in NBRIP medium (580 mg l(-1)). B. vietnamiensis M6 showed resistance against different environmental stresses like 10-45 degrees C, 1-5% (w/v) salt, and 2-11 pH range. The maximal concentration of soluble P produced by B. vietnamiensis M6 from Ca(3)(PO(4))(2), CaHPO(4), and hydroxyapatite was 1,039, 2,132, and 1,754 mg l(-1), respectively. However, the strain M6 produced soluble P with 20 mg l(-1) from FePO(4) after 2 days and 100 mg l(-1) from AlPO(4) after 6 days, respectively. Our results indicate that B. vietnamiensis M6 could be a potential candidate for the development of biofertilizer applicable to environmentally stressed soil.

Fosfomycin for the treatment of infections caused by multidrug-resistant non-fermenting Gram-negative bacilli: a systematic review of microbiological, animal and clinical studies.

The treatment of multidrug-resistant (MDR), extensively drug-resistant or pandrug-resistant non-fermenting Gram-negative bacterial infections constitutes a challenge in an era of few new antibiotic choices. This mandates the re-evaluation of already existing antibiotics such as fosfomycin. We systematically reviewed the literature to assess the clinical and microbiological effectiveness of fosfomycin in the treatment of these infections by searching PubMed, Scopus and the Cochrane Library databases. In 23 microbiological studies identified, 1859 MDR non-fermenting Gram-negative bacterial isolates were examined. The susceptibility rate to fosfomycin of MDR Pseudomonas aeruginosa isolates was >or=90% and 50-90% in 7/19 and 4/19 relevant studies, respectively. Cumulatively, 511/1693 (30.2%) MDR P. aeruginosa isolates were susceptible to fosfomycin. Serotype O12 isolates exhibited greater susceptibility. Only 3/85 (3.5%) MDR Acinetobacter baumannii and 0/31 MDR Burkholderia spp. isolates were susceptible to fosfomycin. Variable criteria of susceptibility were used in the included studies. Fosfomycin was synergistic in combination with a beta-lactam, aminoglycoside or ciprofloxacin in 46/86 (53.5%) MDR P. aeruginosa isolates. One animal study found a good therapeutic effect of the combination fosfomycin/gentamicin against MDR P. aeruginosa endocarditis. In six clinical studies, 33 patients with MDR P. aeruginosa infections (mainly pulmonary exacerbations of cystic fibrosis) received fosfomycin (25/33 in combination with other antibiotics); 91% of the patients clinically improved. In conclusion, fosfomycin could have a role as a therapeutic option against MDR P. aeruginosa infections. Further research is needed to clarify the potential utility of this agent.

Modified sublimation to isolate phenanthrene-degrading bacteria of the genera Sphingomonas and Burkholderia from Xiamen oil port.

Sublimation was developed by Alley and Brown (2000) in order to isolate bacterial strains that were capable of degrading water insoluble compounds. In this study, sublimation was modified by the use of nutritional agar plates, instead of mineral salt agar, to isolate phenanthrene-degrading bacteria from a mixed culture that had been enriched under the selective pressure of high phenanthrene content. Five strains were obtained with different morphology and degradation ability. Based on the 16S rDNA sequence, two of them were classified as species of the genus Sphingomonas; the others as species of the genus Burkholderia. Denaturing gradient gel electrophoresis (DGGE) was introduced to detect dynamic changes in the bacterial community during enrichment batch culture, and to determine any correlation between the five isolates and the phenanthrene-degrading consortium. The DGGE profile indicated that these five isolates corresponded to four dominant bands of the consortium. Compared to traditional means of isolation, we concluded that modified sublimation is effective and more convenient.

Isolation and characterization of a lipid-degrading bacterium and its application to lipid-containing wastewater treatment.

To construct an efficient lipid-containing wastewater treatment system, microorganisms that degrade lipids efficiently were isolated from various environmental sources. Strain DW2-1 showed the highest rate of degradation of 1% (w/v) salad oil among the isolated strains. Strain DW2-1 was identified as Burkholderia sp. and designated Burkholderia sp. DW2-1. The rate of degradation of salad oil, olive oil, sesame oil, and beef tallow by strain DW2-1 were 96.7%, 92.3%, 90.1% and 77.4%, respectively, during a 48-h cultivation. Strain DW2-1 grew well in a synthetic wastewater medium (>1 x 10(10) colony forming unit [CFU]/ml) between 20 degrees C and 38 degrees C, and its rate of degradation of salad oil was above 90% after a 48-h cultivation. The lipase and biosurfactant (BSF) activities of strain DW2-1 after a 48-h cultivation were 1720 U/l and 480 U/ml, respectively. In continuous cultures for lipid-containing wastewater treatment, DW2-1 was stably maintained and degraded more than 90% of salad oil during a 7-d cultivation.

Identification of bacteria isolated from diseased Neungee mushroom, Sarcodon aspratus.

As the first step in an investigation of the problem with quality deterioration seen in the Neungee mushroom (Sarcodon aspratus) due to bacterial overgrowth during its storage, an attempt to isolate bacterial strains was made using infected gills of Sarcodon aspratus. Five bacterial strains were isolated; one phototrophic cyanobacterial species and four heterotrophic Gram negative rods. The four heterotrophic bacterial isolates (strains P, S, R, and MK1) were subjected to identification based on biochemical characteristics using the Biolog system, cellular fatty acid analysis using the MIDI system, cytology by scanning microscopy, and 16s rDNA sequence analysis. A slow grower, the P strain (ca. 0.7 microm x 1.5 microm), which forms pink colonies on Tryptic Soy agar (TSA) and glucose minimal salt medium containing thiamine (MT medium), belongs to genus Methylobacterium, and is likely M. radiotolerans. The methanol-utilizing capacity of the P strain was confirmed by growth on methanol-supplemented medium as a sole carbon source. Both the S and R strains (ca. 0.5 microm x 0.8 microm) produced smooth and slightly rough white colonies, respectively, on TSA, MT, and potato dextrose (PD) agar are members of the Burkholderia cepacia complex. Although both strains showed some differences from each other in colony morphology, nitrogen fixation capacity, and denitrification, they were considered to be Burkholderia stabilis because their 16s rDNA sequences showed 99.93% similarity with those of B. stabilis LMG 14294T (NCBI AF 148554). The MK1 strain, a rod-shaped bacterium with a tapered end (ca. 0.6 microm x 1.8 microm), produces a copious mucoid substance on MT and PD agar, but not on TSA. Despite extensive identification studies, the M strain is not currently identifiable, which suggests that it is a novel bacterium.

Burkholderia soli sp. nov., isolated from soil cultivated with Korean ginseng.

A polyphasic study was carried out to clarify the taxonomic position of a Gram-negative bacterium isolated from soil cultivated with Korean ginseng in the Eumseong region of Korea. The novel strain, GP25-8(T), grew optimally at pH 6-7, 28 degrees C and 0-1 % NaCl (w/v). The major fatty acids were C(18 : 1)omega7c, summed feature 3 (C(16 : 1)omega7c/C(15 : 0) iso 2-OH) and C(16 : 0) (together representing 71.2 % of the total). The 16S rRNA gene sequence similarities between strain GP25-8(T) and members of the genus Burkholderia ranged from 94.7 to 97.4 %, indicating that this novel strain was phylogenetically related to members of that genus. The novel strain showed the highest sequence similarities to Burkholderia caryophylli ATCC 25418(T) (97.4 %) and Burkholderia phenazinium LMG 2247(T) (97.2 %); the levels of DNA-DNA hybridization with these strains were 28 and 12 %, respectively. These results support the conclusion that strain GP25-8(T) represents a novel species within the genus Burkholderia, for which the name Burkholderia soli sp. nov. is proposed. The type strain is GP25-8(T) (=KACC 11589(T)=DSM 18235(T)).

Burkholderia ginsengisoli sp. nov., a beta-glucosidase-producing bacterium isolated from soil of a ginseng field.

A bacterial strain (designated KMY03T) that possesses beta-glucosidase activity was isolated from soil from a ginseng field in South Korea and was characterized in order to determine its taxonomic position. The bacterium was found to comprise Gram-negative, rod-shaped, motile cells with unipolar polytrichous flagella. On the basis of 16S rRNA gene sequence similarity, strain KMY03T was shown to belong to the family Burkholderiaceae of the Betaproteobacteria, being most closely related to Burkholderia caledonica LMG 19076T (97.8%), Burkholderia terricola LMG 20594T (97.5%), Burkholderia xenovorans LMG 21463T (97.4%) and Burkholderia phytofirmans LMG 22146T (97.3%). Chemotaxonomic data (major ubiquinone, Q-8; major fatty acids, C17:0 cyclo, C16:0, C19:0 cyclo omega8c and summed feature 2) supported the affiliation of the novel strain with the genus Burkholderia. The results of DNA-DNA hybridizations and physiological and biochemical tests allowed the strain to be differentiated genotypically and phenotypically from Burkholderia species with validly published names. On the basis of these data, strain KMY03T represents a novel species of the genus Burkholderia, for which the name Burkholderia ginsengisoli sp. nov. is proposed. The type strain is KMY03T (=KCTC 12389T=NBRC 100965T).

Microbial conversion of major ginsenoside rb(1) to pharmaceutically active minor ginsenoside rd.

More than seventy strains of aerobic bacteria showing beta-glucosidase activity were isolated from a ginseng field, using a newly designed Esculin-R2A agar, and identified by their 16S rRNA gene sequences. Of these microorganisms, twelve strains could convert the major ginsenoside, Rb(1), to the pharmaceutically active minor ginsenoside Rd. Three strains, Burkholderia pyrrocinia GP16, Bacillus megaterium GP27 and Sphingomonas echinoides GP50, were phylogenetically studied, and observed to be most potent at converting ginsenoside Rb(1) almost completely within 48 h, as shown by TLC and HPLC analyses.

Burkholderia phytofirmans sp. nov., a novel plant-associated bacterium with plant-beneficial properties.

A Gram-negative, non-sporulating, rod-shaped, motile bacterium, with a single polar flagellum, designated strain PsJN(T), 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 PsJN(T) 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 PsJN(T) 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 PsJN(T) and closely related species were identified. The predominant fatty acids in strain PsJN(T) were 16 : 0, 18 : 1omega7c and summed feature 3 (comprising 16 : 1omega7c and/or iso-15 : 0 2-OH). Isolate PsJN(T) 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 PsJN(T) 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 PsJN(T) (=LMG 22146(T) = CCUG 49060(T)).

Burkholderia unamae sp. nov., an N2-fixing rhizospheric and endophytic species.

It was shown recently that the genus Burkholderia is rich in N2-fixing bacteria that are associated with plants. A group of these diazotrophic isolates with identical or very similar 16S rDNA restriction patterns [designated amplified rDNA restriction analysis (ARDRA) genotypes 13, 14 and 15] was selected and a polyphasic taxonomic study was performed, which included new isolates that were recovered from rhizospheres, rhizoplanes or internal tissues of maize, sugarcane and coffee plants. Morphological, physiological and biochemical features, as well as multi-locus enzyme electrophoresis profiles and whole-cell protein patterns, of 20 strains were analysed. In addition, analysis of cellular fatty acid profiles, 16S rDNA sequence analysis and DNA-DNA reassociation experiments were performed with representative strains. The taxonomic data indicated that the strains analysed belong to a novel diazotrophic Burkholderia species, for which the name Burkholderia unamae sp. nov. is proposed. Strain MTl-641T (=ATCC BAA-744T=CIP 107921T), isolated from the rhizosphere of maize, was designated as the type strain. B. unamae was found as an endophyte of plants grown in regions with climates ranging from semi-hot subhumid to hot humid, but not from plants grown in regions with semi-hot or hot dry climates. Moreover, B. unamae was isolated from rhizospheres and plants growing in soils with pH values in the range 4.5-7.1, but not from soils with pH values higher than 7.5.

Diversity of Burkholderia isolates from woodland rhizosphere environments.

AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.

Incidence of the cblA major subunit pilin gene amongst Burkholderia species.

PCR-amplification has been used to screen 75 isolates of the Burkholderia cepacia complex for the cblA pilin gene. PCR-amplified products of the correct size (664 bp) were cloned and sequenced and the sequences compared. Apart from in the control, epidemic cystic fibrosis (CF)-associated B. cepacia lineage we also identified, for the first time, cblA genes in a unique, non-CF clinical isolate from France and a plant (onion) pathogenic isolate from Italy. The sequence of the cblA gene amplified from the clinical isolate was more diverged from the epidemic lineage than that amplified from the onion pathogenic isolate.

Detection and characterization of plasmid pJP4 transfer to indigenous soil bacteria.

Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism, Escherichia coli ATCC 15224, by plate mating with Ralstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2, 4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 10(7) and 10(8) transconjugants g of dry soil(-1) for samples supplemented with 500 and 1,000 microg of 2,4-D g of dry soil(-1), respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genus Burkholderia or the genus Pseudomonas. No mercury-resistant, 2, 4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.