A physical genome map of the Burkholderia cepacia type strain

Burkholderia cepacia (basonym Pseudomonas cepaci a), the type speciesof the new genus Burkholderia , is of interest, not only because of its broad catabolic capacity and its ability to antagonize soil-borne plant pathogens, but also because of its causative role in infections in man, which are particularly evident in patients with cystic fibrosis. A physical map of the 8.1 Mb genome of the B. cepacia type-strain ATCC 25416 was constructed by applying two-dimensional pulsed-field gel electrophoresis techniques. Placed onto the macrorestriction map were 38 Spel , 11 Swal , 11 Pacl , 11 Pmel and six l-Ceul sites, resulting in an average resolution of 1O5 kbp. Random single-hit linearization by irradiation and restriction mapping uncovered the presence of four circular replicons of 3.65 Mb, 3.17 Mb, 1.07 Mb and 200 kbp in size. The largest replicon harbours four rrn operons while the other two Megabase-size replicons each contain a single rrn operon, suggesting that the genome has three chromosomes and a large plasmid. Within the beta subdivision of proteobacteria, the existence of multiple replicons is not confined to B. cepacia . The phylogenetically related species Burkholderia glumae , Burkholderia pickettii , Burkholderia solanacearum , Alcaligenes eutrophus and the so far unassigned Pseudomonas glathei were also found to harbour more than one Megabase-size replicon.     

Multiple replicons constituting the genome of Pseudomonas cepacia 17616.

Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 17616, in conjunction with Southern hybridization experiments using junction fragments containing rare restriction enzyme sites as probes, indicated that this bacterium contains three large circular replicons of 3.4, 2.5, and 0.9 megabases (Mb). Inclusion of the 170-kb cryptic plasmid present in this strain gave an overall estimate of genome size of 7 Mb. Other Southern hybridization experiments indicated that the three large replicons contained rRNA genes as well as insertion sequence elements identified previously in this strain. The distribution of SwaI, PacI, and PmeI sites on the three replicons was determined. A derivative of Tn5-751 carrying a SwaI site was used to inactivate and map genes on the 2.5- and 3.4-Mb replicons. Mutants were isolated in which the 2.5- and 0.9-Mb replicons had been reduced in size to 1.8 and 0.65 Mb, respectively. The loss of DNA from the 2.5-Mb replicon was associated with lysine auxotrophy, beta-lactamase deficiency, and failure to utilize ribitol and trehalose as carbon and energy sources. DNA fragments corresponding in size to randomly linearized forms of the different replicons were detected in unrestricted DNA by pulsed-field gel electrophoresis. The results provide a framework for further genetic analysis of strain 17616 and for evaluation of the genomic complexities of other P. cepacia isolates.     

Burkholderia cepacia complex: distribution of genomovars among isolates from the maize rhizosphere in Italy

Burkholderia cepacia is a 'complex' in which seven genomic species or genomovars have so far been identified. It appears that all seven B. cepacia genomovars are capable of causing infections in vulnerable persons; in particular, the importance of Burkholderia multivorans (genomovar II) and B. cepacia genomovar III among cystic fibrosis isolates, especially epidemic ones, has been emphasized. In order to acquire a better comprehension of the genomovar composition of environmental populations of B. cepacia, 120 strains were isolated from the rhizosphere of maize plants cultivated in fields located in northern, central and southern Italy. The identification of the different genomovars was accomplished by a combination of molecular polymerase chain reaction (PCR)-based techniques, such as restriction fragment length polymorphism (RFLP) analysis of 16S rDNA (ARDRA), genomovar-specific PCR tests and RFLP analyses based on polymorphisms in the recA gene whole-cell protein electrophoresis. ARDRA analysis allowed us to distinguish between all B. cepacia genomovars except B. cepacia genomovar I, B. cepacia genomovar III and Burkholderia ambifaria (genomovar VII). The latter genomovars were differentiated by means of recA PCR tests and RFLP analyses. Among the rhizospheric isolates of B. cepacia, we found only B. cepacia genomovar I, B. cepacia genomovar III, Burkholderia vietnamiensis (genomovar V) and B. ambifaria. B. cepacia genomovars I and III and B. ambifaria were recovered from all three fields, whereas B. vietnamiensis was detected only in the population isolated from the field located in central Italy. Among strains isolated from northern and southern Italy, the most abundant genomovars were B. ambifaria and B. cepacia genomovar III respectively; in contrast, the population isolated in central Italy showed an even distribution of strains among genomovars. These results indicate that it is not possible to differentiate clinical and environmental strains, or pathogenic and non-pathogenic strains, of the B. cepacia complex simply on the basis of genomovar status, and that the environment may serve as a reservoir for B. cepacia genomovar III infections in vulnerable humans.     

Distribution of Bacillus megaterium QM B1551 plasmids among other B. megaterium strains and Bacillus species

Bacillus megaterium QM B1551 contains seven plasmids. Two are small rolling circle plasmids and five are theta-replicating plasmids with cross-hybridizing replicons that define a new family of very homologous yet compatible theta replicons. Previous sequencing of several of the plasmids has shown genes with high similarity to those on the genomes and plasmids of other Gram-positive bacteria. To test the possible distribution of these plasmids, nine other B. megaterium strains and 20 other Bacillus or related species were tested for the presence of similar replicons, and specific flanking DNA by both hybridization and PCR. The theta replicons were widespread among the B. megaterium strains, and two had one or more of the rolling circle plasmids, but none of the plasmid replicon regions were observed in the other Bacillus or related species. It appears from the data that even though some plasmids carry genes suggesting horizontal transfer, their replicons seem to be unique to B. megaterium, or rarely present in related species.     

Species abundance and diversity of Burkholderia cepacia complex in the environment

Despite considerable interest in studying Burkholderia cepacia complex in the environment, we still do not have efficient methods to detect, isolate, and screen large numbers of B. cepacia isolates. To better describe the ecology and diversity of B. cepacia complex, a colony hybridization assay was developed to detect specifically all species of the complex based on polymorphism of the variable V3 region of the 16S rRNA sequence. The sensitivity of the assay was dramatically enhanced by using a probe consisting of three repeats of a B. cepacia complex-specific probe, each separated by a phosphoramidite spacer. In addition, a duplex PCR targeting B. cepacia complex-specific recA and 16S rRNA sequences was developed to enable a fast and reliable diagnostic assay for members of the complex. When applied to maize rhizosphere samples, colony hybridization results were in good agreement with those of most-probable-number duplex PCR, both indicating a >100-fold fluctuation of abundance between individual plants. Using restriction analysis of recA for a total of 285 confirmed isolates of the B. cepacia complex, up to seven B. cepacia complex species were identified; however, their diversity and abundance were not evenly distributed among individual plants, and several allelic variants were commonly found from the same rhizosphere sample. These results indicate that not only complex communities of B. cepacia complex species and closely related strains of the same species may coexist at high population levels but also species composition and abundance may dramatically vary between individual plants.     

Clinical and environmental isolates of Burkholderia cepacia exhibit differential cytotoxicity towards macrophages and mast cells

Burkholderia cepacia is an emerging opportunistic pathogen that causes fatal infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Various environmental isolates of B. cepacia are, however, capable of degrading environmental pollutants, such as trichloroethylene, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), etc., and are also highly effective in controlling plant diseases caused by nematodes and fungi. Such strains have therefore been proposed for environmental release to clean up toxic dump sites or as biopesticides. Various efforts to distinguish between clinical and environmental isolates of B. cepacia with regard to their virulence characteristics have produced ambiguous results, suggesting that newer methods are needed to test for the presence or absence of pathogenic potential in B. cepacia strains proposed for environmental release. We now report that several clinical strains of B. cepacia secrete cytotoxic factors that allow macrophage and mast cell death in the presence of external ATP. Several environmental strains had reduced activity in this regard. We also demonstrate that, while all the strains secrete enzymes that have nucleoside diphosphate kinase (Ndk), adenylate kinase (Ak) and 5'-nucleotidase activity, the level of secretion of the 5'-nucleotidase (and/or ATPase/phosphatase) appears to be lower in the environmental strains than in the clinical strains. The secretion of these enzymes is specifically activated in the presence of eukaryotic proteins such as alpha2-macroglobulin. As macrophage-or mast cell surface-associated P2Z receptors promote their cell death in the presence of mM concentrations of ATP, and as the secreted ATP-using enzymes generate various phosphorylated or non-phosphorylated adenine nucleotides that may even be better agonists than ATP in activating the P2Z receptors or may act through the activation of additional purinergic receptors, such enzymes may play an important role in allowing B. cepacia to evade host defence.     

Evidence of transmission of Burkholderia cepacia, Burkholderia multivorans and Burkholderia dolosa among persons with cystic fibrosis

Previous studies have identified specific Burkholderia cepacia complex strains that are common to multiple persons with cystic fibrosis (CF). Such so-called epidemic strains have an apparent enhanced capacity for inter-patient spread and reside primarily in Burkholderia cenocepacia (formerly B. cepacia complex genomovar III). We sought to identify strains from B. cepacia complex species other than B. cenocepacia that are similarly shared by multiple CF patients. We performed genotype analysis of 360 recent sputum culture isolates from 360 persons residing in 29 cities by using repetitive extragenic palendromic polymerase chain reaction (rep-PCR) and pulsed field gel electrophoresis. The results indicate that sharing of a common Burkholderia multivorans strain occurs relatively infrequently; however, several small clusters of patients infected with the same strain were identified. A cluster of seven patients infected with the same B. cepacia (genomovar I) strain was found. We also identified a large group of 28 patients receiving care in the same treatment center and infected with the same Burkholderia dolosa strain. These observations suggest that B. cepacia complex strains in species other than B. cenocepacia may be spread among CF patients.     

Bacillus subtilis soil isolates: plasmid replicon analysis and construction of a new theta-replicating vector

We have searched for plasmids in a collection of 55 Bacillus subtilis strains isolated from various natural sources of the territory of Belarus. Twenty percent of the strains contained one or two plasmids of either 6-8 or approximately 90 kb. Small plasmids were shown to carry a rolling circle replicon of the pC194 type. Four out of the eight large plasmids contained a related theta replicon that has no homolog in databases as shown by sequence determination. A B. subtilis/Escherichia coli shuttle vector based on this replicon was constructed. It has a low copy number (6 units per chromosome) and is stably inherited in B. subtilis. It might thus be a useful tool for DNA cloning. These data extend previous observations, indicating that most of the small plasmids of B. subtilis replicate as rolling circles and belong to the pC194 family. On the contrary, large plasmids appear to form a large pool of theta-replicating determinants, since three different replicons have already been isolated from them.     

Identification and onion pathogenicity of Burkholderia cepacia complex isolates from the onion rhizosphere and onion field soil

Burkholderia cepacia complex strains are genetically related but phenotypically diverse organisms that are important opportunistic pathogens in patients with cystic fibrosis (CF,) as well as pathogens of onion and banana, colonizers of the rhizospheres of many plant species, and common inhabitants of bulk soil. Genotypic identification and pathogenicity characterization were performed on B. cepacia complex isolates from the rhizosphere of onion and organic soils in Michigan. A total of 3,798 putative B. cepacia complex isolates were recovered on Pseudomonas cepacia azelaic acid tryptamine and trypan blue tetracycline semiselective media during the 2004 growing season from six commercial onion fields located in two counties in Michigan. Putative B. cepacia complex isolates were identified by hybridization to a 16S rRNA gene probe, followed by duplex PCR using primers targeted to the 16S rRNA gene and recA sequences and restriction fragment length polymorphism analysis of the recA sequence. A total of 1,290 isolates, 980 rhizosphere and 310 soil isolates, were assigned to the species B. cepacia (160), B. cenocepacia (480), B. ambifaria (623), and B. pyrrocinia (27). The majority of isolates identified as B. cepacia (85%), B. cenocepacia (90%), and B. ambifaria (76%) were pathogenic in a detached onion bulb scale assay and caused symptoms of water soaking, maceration, and/or necrosis. A phylogenetic analysis of recA sequences from representative B. cepacia complex type and panel strains, along with isolates collected in this study, revealed that the B. cenocepacia isolates associated with onion grouped within the III-B lineage and that some strains were closely related to strain AU1054, which was isolated from a CF patient. This study revealed that multiple B. cepacia complex species colonize the onion rhizosphere and have the potential to cause sour skin rot disease of onion. In addition, the onion rhizosphere is a natural habitat and a potential environmental source of B. cenocepacia.     

Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis

The large pXO1 plasmid (181.6kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two separate regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication of the full-length pXO1 plasmid. We generated mutants of the full-length pXO1 plasmid in which either the RepX or the ORFs 14/16 replicon was inactivated by TargeTron insertional mutagenesis. Plasmid pXO1 derivatives containing only the RepX or the ORFs 14/16 replicon were able to replicate when introduced into a plasmid-less B. anthracis strain. Plasmid copy number analysis showed that the ORFs 14/16 replicon is more efficient than the RepX replicon. Our studies demonstrate that both the RepX and ORFs 14/16 replicons can independently support the replication of the full-length pXO1 plasmid.     

Genotypic and phenotypic relationships in Burkholderia cepacia isolated from cystic fibrosis patients and the environment

Twenty-one strains of Burkholderia cepacia isolated from the environment, and 21 clinical strains isolated principally from sputum of cystic fibrosis (CF) patients, were characterized genotypically by macrorestriction analysis (genome fingerprinting) and PCR ribotyping, and phenotypically by susceptibility to antibiotics and the ability to macerate onion tissue. The plasmid content of the strains was also investigated. Environmental isolates showed a high degree of genetic variability, all strains differing from both one another and the CF isolates. The CF isolates were less variable, with common strains found in patients attending three geographically distinct CF centres. Phenotypic variation was found both within and between CF and environmental strains. Generally, CF isolates displayed higher levels of antibiotic resistance, while the ability to macerate onion tissue was more prevalent amongst environmental isolates. Plasmids were more frequently found in CF isolates, but were of similar size in both groups of strains. Such variability is not surprising in view of the existence of multiple genomovars within the B. cepacia complex.     

Complex of Burkholderia cepacia lipase with transition state analogue of 1-phenoxy-2-acetoxybutane: biocatalytic, structural and modelling study.

In a series of four racemic phenoxyalkyl-alkyl carbinols, 1-phenoxy-2-hydroxybutane (1) is enantioselectively acetylated by Burkholderia cepacia (formerly Pseudomonas cepacia) lipase with an E value > or = 200, whereas for the other three racemates E was found to be < or = 4. To explain the high preference of B. cepacia lipase for (R)-(+)-1, a precursor of its transition state analogue with a tetrahedral P-atom, (R(P),S(P))-O-(2R)-(1-phenoxybut-2-yl)methylphosphonic acid chloride was prepared and crystallized in complex with B. cepacia lipase. The X-ray structure of the complex was determined, allowing to compare the conformation of the inhibitor with results of molecular modelling.     

A quorum-quenching approach to investigate the conservation of quorum-sensing-regulated functions within the Burkholderia cepacia complex

Taxonomic studies of the past few years have shown that the Burkholderia cepacia complex, a heterogeneous group of B. cepacia-like organisms, consists of at least nine species. B. cepacia complex strains are ubiquitously distributed in nature and have been used for biocontrol, bioremediation, and plant growth promotion purposes. At the same time, B. cepacia complex strains have emerged as important opportunistic pathogens of humans, particularly those with cystic fibrosis. All B. cepacia complex species investigated thus far use quorum-sensing (QS) systems that rely on N-acylhomoserine lactone (AHL) signal molecules to express certain functions, including the production of extracellular proteases, swarming motility, biofilm formation, and pathogenicity, in a population-density-dependent manner. In this study we constructed a broad-host-range plasmid that allowed the heterologous expression of the Bacillus sp. strain 240B1 AiiA lactonase, which hydrolyzes the lactone ring of various AHL signal molecules, in all described B. cepacia complex species. We show that expression of AiiA abolished or greatly reduced the accumulation of AHL molecules in the culture supernatants of all tested B. cepacia complex strains. Phenotypic characterization of wild-type and transgenic strains revealed that protease production, swarming motility, biofilm formation, and Caenorhabditis elegans killing efficiency was regulated by AHL in the large majority of strains investigated.     

土壤中产脂肪酶洋葱伯克霍尔德菌的分离与鉴定

洋葱伯克霍尔德菌(Burkholderia cepacia)在生物防治、生物降解等农业领域有着广泛的应用,它产生的脂肪酶则在有机合成、精细化工等领域潜力巨大。采用改良的TB-T平板筛选法从土壤中初步筛选出300株洋葱伯克霍尔德菌,然后用脂肪酶活性检测平板对300株菌进行筛选,最终获得6株脂肪酶产量高的菌,通过发酵发现6株菌均有较好的产脂肪酶能力。随后通过16S rDNA比对的方法将6株全部鉴定为B.cepacia。在此基础上,采用HaeⅢ-recA RFLP和基因种特异性PCR对6株菌进行了基因种鉴定,结果表明JWT16、G63YL、WJ158和JWT137属于Burkholderia cenocepacia菌,JWP9属于Burkhold-eria vietnamiensis,JWT267则属于Burkholderia multivorans。     

Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia.

The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms. Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P. aeruginosa alone. The interactions between P. aeruginosa biofilms and B. cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship. Using the Calgary Biofilm Device (CBD), members of all genomovars of the B. cepacia complex were shown to form biofilms, including those isolated from CF lungs. Mixed species biofilm formation between CF isolates of P. aeruginosa and B. cepacia was readily achieved using the CBD. Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition. Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure. Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B. cepacia complex. Characterization of the soluble inhibitor suggested pyocyanin as the active compound.     

Identification of aquatic Burkholderia (Pseudomonas) cepacia by hybridization with species-specific rRNA gene probes.

Burkholderia (Pseudomonas) cepacia is a common environmental bacterium which can be pathogenic for plants and humans. In this study, four strategies were used to identify aquatic isolates: API test strips, hybridization with species-specific DNA probes for the 16S and 23S rRNA genes, fatty acid methyl ester (FAME) profiles, and growth on selective medium (TB-T agar [C. Hagedorn, W. D. Gould, T. R. Bardinelli, and D. R. Gustarson, Appl. Environ. Microbiol. 53:2265-2268, 1987]). Only 59% of the isolates identified as B. cepacia with the API test strips were confirmed as B. cepacia by using fatty acid profiles. The 23S rRNA probe generated a few false-positive results but dramatically underestimated the number of B. cepacia isolates (i.e., 40% of the colonies that did not hybridize to the probe were B. cepacia, as determined by FAME). The 16S rRNA probe generated more false-positive results than the 23S rRNA probe but was effective in identifying the majority of the B. cepacia isolates. The selective medium was only partially successful in recovering B. cepacia. Use of the B. cepacia-specific 16S rRNA probe was the most efficient and accurate way of identifying B. cepacia.     

Surface characteristics of Pseudomonas cepacia

Two major surface characteristics of Pseudomonas cepacia were examined in this study: reactivity with lectins and hydrophobicity. The results indicated that the surfaces of P. cepacia strains are heterogeneous with regard to the distribution of lectin receptors. Only lima bean agglutinin was found to strongly agglutinate all strains. The strains were also heterogeneous with regard to hydrophobicity as determined by adhesion to hexadecane. The degree of hydrophobicity, however, was not significantly altered when selected strains were mixed with either fibronectin or bovine serum albumin. In addition, the strains exhibited no apparent affinities for buccal epithelial cells and gave no evidence for an ability to haemagglutinate human red cells.     

Plasmids of Pseudomonas cepacia strains of diverse origins.

Thirty-seven strains of Pseudomonas cepacia from clinical, pharmaceutical-industrial, and environmental origins were analyzed for the presence of plasmid DNA by a modification of the rapid alkaline extraction method of Birnboim (H. C. Birnboim, Methods Enzymol. 100:243-255, 1983). Plasmids were present in 31 strains (84%) from all sources, with no one source showing less than 75% plasmid carriage among its strains. The plasmid profiles indicated that the presence of large plasmids (146 to 222 kb) was the norm. Those strains with greater antibiotic resistance were mainly in the clinical and pharmaceutical groups and carried large plasmids (222 kb) that appeared essentially identical by restriction digest analysis. The ability for conjugative transfer was shown with the broad-host-range plasmid R751 carrying the gene for resistance to trimethoprim, one of the few antimicrobial agents effective against P. cepacia. The plasmid was transferred from Pseudomonas aeruginosa to P. cepacia strains as well as from P. cepacia transconjugants to other P. cepacia strains.     

Serum IgG response to Burkholderia cepacia outer membrane antigens in cystic fibrosis: Assessment of cross-reactivity with Pseudomonas aeruginosa

Abstract Burkholderia cepacia (Pseudomonas cepacia) is now recognised as an important pathogen in cystic fibrosis patients, and several reports have suggested that sputum-culture-proven colonisation occurs despite the presence of specific antibody. In an attempt to establish the use of antibody studies as diagnostic and prognostic indicators of B. cepacia infection, we have examined the IgG response to B. cepacia outer membrane proteins and lipopolysaccharide in patients also colonised with P. aeruginosa . The B. cepacia strains were grown in a modified iron-depleted chemically defined medium and outer membrane components examined by SDS-PAGE and immunoblotting. IgG antibodies were detected against B. cepacia outer membrane antigens, which were not diminished by extensive preadsorption with P. aeruginosa . The response to B. cepacia O-antigen could be readily removed by adsorption of serum either with B. cepacia whole cells or purified LPS, whereas we were unable to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells. The inability to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells maybe due to non-exposed surface epitopes. Several B. cepacia sputum-culture negative patients colonised with P. aeruginosa had antibodies directed against B. cepacia outer membrane protein. This study suggests that there is a specific anti- B. cepacia LPS IgG response, which is not due to antibodies cross-reactive with P. aeruginosa . Our studies indicate that much of the B. cepacia anti-outer membrane protein response is specific and not attributable to reactivity against co-migrating LPS.