Development of a recA gene-based identification approach for the entire Burkholderia genus

Burkholderia is an important bacterial genus containing species of ecological, biotechnological, and pathogenic interest. With their taxonomy undergoing constant revision and the phenotypic similarity of several species, correct identification of Burkholderia is difficult. A genetic scheme based on the recA gene has greatly enhanced the identification of Burkholderia cepacia complex species. However, the PCR developed for the latter approach was limited by its specificity for the complex. By alignment of existing and novel Burkholderia recA sequences, we designed new PCR primers and evaluated their specificity by testing a representative panel of Burkholderia strains. PCR followed by restriction fragment length polymorphism analysis of an 869-bp portion of the Burkholderia recA gene was not sufficiently discriminatory. Nucleotide sequencing followed by phylogenetic analysis of this recA fragment differentiated both putative and known Burkholderia species and all members of the B. cepacia complex. In addition, it enabled the design of a Burkholderia genus-specific recA PCR that produced a 385-bp amplicon, the sequence of which was also able to discriminate all species examined. Phylogenetic analysis of 188 novel recA genes enabled clarification of the taxonomic position of several important Burkholderia strains and revealed the presence of four novel B. cepacia complex recA lineages. Although the recA phylogeny could not be used as a means to differentiate B. cepacia complex strains recovered from clinical infection versus the natural environment, it did facilitate the identification of clonal strain types of B. cepacia, B. stabilis, and B. ambifaria capable of residing in both niches.     

The influence of bacteria of the Burkholderia cepacia and Pseudomonas aeruginosa complex on cell-mediated immune reactions in experimental animals

The evidence has been obtained that various species, as well as individual strains having pathogenicity factors, produced different effect on the functional activity of immunocompetent B and T lymphocytes of mice infected intraperitoneally. The injection of live P. aerruginosa PA 103 and B. cepacia 8240 cells resulted in imunosuppression of antibody-forming cells, synthesizing antibodies to heterologous antigens. On the contrary, in the animals infected with B. cepacia 8236 the functional activity of B lymphocytes increased. An increase in the proliferative activity of spleen cells was noted in the presence of T and B mitogens after the infection of mice with P. aeruginosa PA 103 in comparison with B. cepacia 8236 and B. cepacia 8240 which produced a faintly pronounced modulating effect. The pathogenesis mechanisms of infections induced by these microorganisms as well as the development of chronic, persisting forms of the infectious process are discussed.     

Interspecies communication between Burkholderia cepacia and Pseudomonas aeruginosa

Burkholderia cepacia and Pseudomonas aeruginosa are opportunistic pathogens that commonly cause pulmonary infections in cystic fibrosis patients and occasionally co-infect patients' lungs. Both organisms possess quorum-sensing systems dependent on N-acyl homoserine lactone (N-acyl-HSL). Cross-feeding assays demonstrated that P. aeruginosa and B. cepacia were able to utilize heterologous N-acyl-HSL signaling molecules. The ability of quorum-sensing genes from one species to complement the respective quorum-sensing mutations in the heterologous species was also examined. These studies suggest that B. cepacia CepR can use N-acyl-HSLs synthesized by RhlI and LasI and that P. aeruginosa LasR and RhlR can use N-acyl-HSLs synthesized by CepI. It is possible that a mixed bacterial population of B. cepacia and P. aeruginosa can coordinately regulate some of their virulence factors and influence the progression of lung disease due to infection with these organisms.     

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.     

Immunological and biological relationship among flagellin of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia

The flagellar protein (flagellin) was isolated and purified from strains of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. A significant difference was observed in the molecular weight of different flagellin preparations obtained from these bacterial isolates. Antiserum prepared against S. maltophilia flagellin did not react with flagellin of P. aeruginosa or/and B. cepacia on Immunoblot or in indirect ELISA. In addition the anti-flagellin did not agglutinate P. aeruginosa and B. cepacia. No inhibition of motility of P. aeruginosa and B. cepacia was observed in presence of antiserum; though the latter inhibited the motility of S. maltophilia. The results of the present study prove that no specific relationship existed among all the studied flagellar proteins obtained from closely related bacteria.     

A putative type III secretion gene cluster is widely distributed in the Burkholderia cepacia complex but absent from genomovar I

Using probes constructed from Ralstonia solanacearum and Burkholderia pseudomallei, putative type III secretion (TTS) genes were identified in Burkholderia cepacia J2315 (genomovar III). A cosmid clone containing DNA with homology to five TTS genes was sub-cloned and regions were sequenced in order to design oligonucleotides for polymerase chain reaction assays. These indicated that two putative TTS genes (bcscQ and bcscV) were present in all members of the B. cepacia complex with the exception of strains from genomovar I. Southern blot assays confirmed this observation, suggesting that the lack of a TTS gene cluster may define a major difference between B. cepacia genomovar I and other members of the B. cepacia complex, including genomovar III. In contrast to TTS gene clusters in other bacteria, a putative gene homologous to the virB1 gene of Brucella suis was located directly downstream of bcscQR.     

Immigration and emigration of Burkholderia cepacia and Pseudomonas aeruginosa between and within mixed biofilm communities

AIMS: To investigate the dynamics of binary culture biofilm formation through use of both the Sorbarod model of biofilm growth and the constant depth film fermenter (CDFF). METHODS AND RESULTS: Pseudo steady-state biofilm cultures of laboratory and clinical strains of Pseudomonas aeruginosa, selected on the basis of their ability to produce a Burkholderia cepacia growth-inhibitory substance, were established on Sorbarod filters and challenged with corresponding planktonic grown cultures of B. cepacia. Reverse challenges were also conducted. Both B. cepacia and P. aeruginosa were able to form steady-state monoculture biofilms after 48 h growth. When steady-state biofilms of B. cepacia NTCT 10661 were challenged with planktonically grown P. aeruginosa PAO1 known to produce a B. cepacia growth-inhibitory substance, the immigrant population was rapidly and almost completely bound to the biofilm, displacing B. cepacia. By contrast, established biofilms of P. aeruginosa PAO1 resisted immigration of B. cepacia 10661. Similar experiments conducted with a nongrowth inhibitory substance producing clinical pairing of P. aeruginosa 313113 and B. cepacia 313113 led to the formation of stable, mixed biofilm populations in both instances. Moreover, co-inoculation with these clinical isolates resulted in a stable, mixed steady-state biofilm. Similar observations were made for biofilms generated in CDFFs. In such instances following pan-swapping between two monoculture CDFFs, B. cepacia 313113 was able to integrate into an established P. aeruginosa 313113 biofilm to form a stable binary biofilm. CONCLUSIONS: Establishment of a mixed species community follows a specific sequence of inoculation that may either be due to some degree of match between co-colonizers or that P. aeruginosa predisposes uncolonized sections of the surface to permit B. cepacia colonization. SIGNIFICANCE AND IMPACT OF THE STUDY: Colonization of a surface with one bacterial species confers colonization resistance towards other species. Disinfection of a surface might well increase the probability of pathogen harbourage.     

Comparative analysis of total cell protein electrophoregram of pathogenic Burkholderia

Whole-cell proteins of 22 strain of Burkhoderia pseudomallei, including 13 B. mallei, 5 B. cepacia strains and 14 strains of opportunistically pathogenic Pseudomonas defined by 1D SDC-PAAG electrophoresis. Electrophoregrams contained 35 to 45 protein fractions sized 19 to 130 kDa, which were highly reproductive. On the basis of computer-aided comparative analysis of protein patterns the interspecies and intraspecies grouping of studied microorganisms was made. The cluster analysis of the similarity matrix of protein spectra made it possible to allocate two groups of strains at the level of similarity of 78%. Group I was formed by Burkholderia species that previously belonged to the II RNA-DNA homology group of Pseudomonas: B. pseudomallei, B. mallei, B. cepacia. All Pseudomonas species were added to the 2nd Group: P. aeruginosa, P. stutzeri, P. testosterone, P. fluorescens, P. putida, P. mendocina. Four phenons were isolated among the strains of B. pseudomallei and 2 phenons--among the strains of B. mallei at the threshold similarity level (89%). The authors conclude that the comparative analysis of electrophoregrams of whole-cell proteins can be useful in the identification and typing of pathogenic Burkholderia.     

Sensitivity of the Burkholderia cepacia complex and Pseudomonas aeruginosa to transducing bacteriophages

Burkholderia cepacia is now recognised as a life-threatening pathogen among several groups of immunocompromised patients. In this context, the proposed large-scale use of these bacteria in agriculture has increased the need for a better understanding of the genetics of the species forming the B. cepacia complex. Until now, little information has been available on the bacteriophages of the B. cepacia complex. Transducing phages, named NS1 and NS2, were derived from the lysogenic B. cepacia strains ATCC 29424 and ATCC 17616. The frequency of transduction per phage particle ranged from 1.0x10(-8) to 7.0x10(-6) depending on the phage and recipient strain used. The host range of NS1 and NS2 differed but in each case included environmental and clinical isolates, and strains belonging to several species and genomovars of the B. cepacia complex. The host range of both phages also included Pseudomonas aeruginosa. Some B. cepacia complex isolates were sensitive to the well-characterised P. aeruginosa transducing phages, B3, F116L and G101. The lytic activity of NS1 and NS2 was inhibited by B. cepacia lipopolysaccharide suggesting that this moiety is a binding site for both phages. The molecular size of the NS1 and NS2 genomes was approximately 48 kb.     

Burkholderia pseudomallei, B. thailandensis, and B. ambifaria produce 4-hydroxy-2-alkylquinoline analogues with a methyl group at the 3 position that is required for quorum-sensing regulation

4-Hydroxy-2-alkylquinolines (HAQs), especially 3,4-dihydroxy-2-heptylquinoline (Pseudomonas quinolone signal) and its precursor, 4-hydroxy-2-heptylquinoline, are attracting much attention, mainly because of their role as signaling molecules in Pseudomonas aeruginosa. The pqsABCDE operon is centrally involved in their biosynthesis. The presence of a homologous operon in Burkholderia pseudomallei and B. thailandensis was recently reported. Thus, we have investigated the abilities of 11 Burkholderia species to produce HAQ-like molecules by liquid chromatography/mass spectrometry. We have identified 29 different HAQ derivatives produced by the only three Burkholderia species where a pqsABCDE homologue was found among available sequenced Burkholderia species genomes, including B. ambifaria, a member of the Burkholderia cepacia complex. In contrast with those of P. aeruginosa, Burkholderia HAQs typically bear a methyl group, hence their designation as 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs). We identified three families of HMAQs with a saturated or unsaturated alkyl chain at the 2' position, in contrast with the 1' position of P. aeruginosa, including one with an N-oxide group. Furthermore, the operon in these species contains two more genes downstream of the pqsE homologue, resulting in the hmqABCDEFG operon. While the inactivation of hmqA inhibits the production of HMAQs, the methylation of the quinoline ring requires a putative methyltransferase encoded by hmqG. Interestingly, hmqA or hmqG mutations increase the production of acyl homoserine lactones and, consequently, phenotypes under the control of quorum sensing in B. ambifaria: antifungal activity, siderophore production, and proteolytic activity. These results indicate that only HAQs bearing a methyl group (HMAQs) are involved in quorum-sensing regulation.     

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.     

Identification of two metabolites induced by a butyrolactone autoregulator IM-2 in Streptomyces sp. FRI-5

Abstract In order to determine whether non-elastase-producing strains of Pseudomonas aeruginosa such as N-10, PA103 and IFO3080 can express foreign elastase genes, we introduced elastase genes from P. aeruginosa IFO3455 (elastase-producing) as well as from PA103 and N-10 into non-elastase-producing P. aeruginosa strains. Results suggested that gene expression, secretion, and precursor processing systems of elastase were essentially normal in P. aeruginosa N-10 and IFO3080. Our studies using various elastase genes showed that both the elastase structural gene and 5'-upstream regions of P. aeruginosa PA103 were also normal. This was confirmed by the finding that P. aeruginosa N-10 and IFO3080 which carry the PA103 elastase gene produced elastase. Several deleted or chimeric genes were constructed using the 5'-upstream regions of elastase genes from P. aeruginosa N-10 or PA103 and studies of expression revealed that two individual DNA bases seem to be important in suppressing P. aeruginosa N-10 elastase gene expression. Possible reasons for the lack of elastase in these non-elastase-producing strains are discussed.     

The tomato rhizosphere, an environment rich in nitrogen-fixing Burkholderia species with capabilities of interest for agriculture and bioremediation

Burkholderia strains are promising candidates for biotechnological applications. Unfortunately, most of these strains belong to species of the Burkholderia cepacia complex (Bcc) involved in human infections, hampering potential applications. Novel diazotrophic Burkholderia species, phylogenetically distant from the Bcc species, have been discovered recently, but their environmental distribution and relevant features for agro-biotechnological applications are little known. In this work, the occurrence of N2-fixing Burkholderia species in the rhizospheres and rhizoplanes of tomato plants field grown in Mexico was assessed. The results revealed a high level of diversity of diazotrophic Burkholderia species, including B. unamae, B. xenovorans, B. tropica, and two other unknown species, one of them phylogenetically closely related to B. kururiensis. These N2-fixing Burkholderia species exhibited activities involved in bioremediation, plant growth promotion, or biological control in vitro. Remarkably, B. unamae and B. kururiensis grew with aromatic compounds (phenol and benzene) as carbon sources, and the presence of aromatic oxygenase genes was confirmed in both species. The rhizospheric and endophyte nature of B. unamae and its ability to degrade aromatic compounds suggest that it could be used in rhizoremediation and for improvement of phytoremediation. B. kururiensis and other Burkholderia sp. strains grew with toluene. B. unamae and B. xenovorans exhibited ACC (1-aminocyclopropane-1-carboxylic acid) deaminase activity, and the occurrence of acdS genes encoding ACC deaminase was confirmed. Mineral phosphate solubilization through organic acid production appears to be the mechanism used by most diazotrophic Burkholderia species, but in B. tropica, there presumably exists an additional unknown mechanism. Most of the diazotrophic Burkholderia species produced hydroxamate-type siderophores. Certainly, the N2-fixing Burkholderia species associated with plants have great potential for agro-biotechnological applications.     

Detection of cultured and uncultured Burkholderia cepacia complex bacteria naturally occurring in the maize rhizosphere

The species composition of a Burkholderia cepacia complex population naturally occurring in the maize rhizosphere was investigated by using both culture-dependent and culture-independent methods. B. cepacia complex isolates were recovered from maize root slurry on the two selective media Pseudomonas cepacia azelaic acid tryptamine (PCAT) and trypan blue tetracycline (TB-T) and subjected to identification by a combination of restriction fragment length polymorphism (RFLP) analysis and species-specific polymerase chain reaction (PCR) tests of the recA gene. DNA extracted directly from root slurry was examined by means of nested PCR to amplify recA gene with species-specific B. cepacia complex primers and to obtain a library of PCR amplified recA genes. Using the culture-dependent method the species Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia ambifaria and Burkholderia pyrrocinia were identified, whereas using the culture-independent method also the species Burkholderia vietnamiensis was detected. The latter method also allowed us to highlight a higher diversity within the B. cenocepacia species. In fact, by using the culture-independent method the species B. cenocepacia recA lineages IIIA and IIID besides B. cenocepacia recA lineage IIIB were detected. Moreover, higher heterogeneity of recA RFLP patterns was observed among clones assigned to the species B. cenocepacia than among B. cenocepacia isolates from selective media.     

Sequence heterogeneity of the ferripyoverdine uptake (fpvA), but not the ferric uptake regulator (fur), genes among strains of the fluorescent pseudomonads Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida

Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida are of importance to medicine, agriculture and biocycling. These microbes acquire ferric ion via the use of the siderophores pyochelin and the family known as the pyoverdines or pseudobactins. The ferric uptake regulator (fur) gene is responsible, at least in part, for the regulation of siderophore synthesis and uptake in P. aeruginosa.To determine whether the organisms contain single or multiple homologues of the siderophore-related genes fpvA (ferripyoverdine uptake) and fur, and whether these homologues displayed sequence heterogeneity, their chromosomal DNAs were probed with fur and fpvA sequences. As a representative of a non-fluorescent pseudomonad, the bacterium Burkholderia (Pseudomonas) cepacia was also examined.The pseudomonads all contained fpvA- and fur-like homologues, and heterogeneity was observed among the different species. The presence of two or more fpvA-like genes is indicated in all of the fluorescent pseudomonads surveyed. In contrast, B. cepacia DNA either did not hybridize to these probes, or did so only very weakly, suggesting that fur- and fpvA-like homologues are either absent or significantly different in B. cepacia compared to the fluorescent pseudomonads examined.     

Quorum-Sensing Signals and Quorum-Sensing Genes in Burkholderia vietnamiensis

Acyl-homoserine lactone (acyl-HSL) quorum sensing is common to many Proteobacteria including a clinical isolate of Burkholderia cepacia. The B. cepacia isolate produces low levels of octanoyl-HSL. We have examined an environmental isolate of Burkholderia vietnamiensis. This isolate produced several acyl-HSLs. The most abundant species was decanoyl-HSL. Decanoyl-HSL in B. vietnamiensis cultures reached concentrations in excess of 20 microM. We isolated a B. vietnamiensis DNA fragment containing a gene for the synthesis of decanoyl-HSL (bviI) and an open reading frame that codes for a putative signal receptor (bviR). A B. vietnamiensis bviI mutant did not produce detectable levels of decanoyl-HSL.     

Role of Quinolinate Phosphoribosyl Transferase in Degradation of Phthalate by Burkholderia cepacia DBO1

Two distinct regions of DNA encode the enzymes needed for phthalate degradation by Burkholderia cepacia DBO1. A gene coding for an enzyme (quinolinate phosphoribosyl transferase) involved in the biosynthesis of NAD+ was identified between these two regions by sequence analysis and functional assays. Southern hybridization experiments indicate that DBO1 and other phthalate-degrading B. cepacia strains have two dissimilar genes for this enzyme, while non-phthalate-degrading B. cepacia strains have only a single gene. The sequenced gene was labeled ophE, due to the fact that it is specifically induced by phthalate as shown by lacZ gene fusions. Insertional knockout mutants lacking ophE grow noticeably slower on phthalate while exhibiting normal rates of growth on other substrates. The fact that elevated levels of quinolinate phosphoribosyl transferase enhance growth on phthalate stems from the structural similarities between phthalate and quinolinate: phthalate is a competitive inhibitor of this enzyme and the phthalate catabolic pathway cometabolizes quinolinate. The recruitment of this gene for growth on phthalate thus gives B. cepacia an advantage over other phthalate-degrading bacteria in the environment.