Multiple chromosomes in Burkholderia cepacia and B. gladioli and their distribution in clinical and environmental strains of B. cepacia

Burkholderia cepacia is found in soils and waters, it can be used in biocontrol and bioremediation but is also a human pathogen. It is not yet clear what differentiates pathogenic from non-pathogenic strains of the organism. In this study the multiple replicon structure was investigated in 28 strains of B. cepacia by pulsed field gel electrophoresis. All strains examined, whether of clinical, environmental or plant pathogenic origin, were found to have two, three or four large (> 500 kbp) replicons. Many strains also contained small replicons. Clinical strains were more likely to have three or four large replicons than non-clinical strains. Multiple replicon structure was also demonstrated in B. gladioli and Alcaligenes eutrophus.     

Genomic complexity and plasticity of Burkholderia cepacia

Abstract Burkholderia cepacia has attracted attention because of its extraordinary degradative abilities and its potential as a pathogen for plants and for humans. This bacterium was formerly considered to belong to the genus Pseudomonas in the γ-subclass of the Proteobacteria , but recently has been assigned to the β-subclass based on rrn gene sequence analyses and other key phenotypic characteristics. The B. cepacia genome is comprised of multiple chromosomes and is rich in insertion sequences. These two features may have played a key role in the evolution of novel degradative functions and the unusual adaptability of this bacterium.     

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.     

Physical and genetic map of the genome of Vibrio parahaemolyticus: presence of two chromosomes in Vibrio species

We constructed a physical map of the genomic DNA (5.1 Mb) for Vibrio parahaemolyticus strain AQ4673 by combining 17 adjacent NotI fragments. This map shows two circular replicons of 3.2 and 1.9 Mb. Pulsed-field gel electrophoresis (PFGE) of undigested genomic DNA revealed two bands of corresponding sizes. Analysis both by NotI digestion and by Southern blot of the two isolated bands confirmed the existence of two replicons. The presence of genes for 16S rRNA on both the replicons indicates that the replicons are chromosomes rather than megaplasmids. The two bands were also seen after PFGE of undigested genomic DNA of V. parahaemolyticus strains other than AQ4673, and of strains belonging to other Vibrio species, such as V. vulnificus, V. fluvialis and various serovars and biovars of V. cholerae. It is noteworthy that V. cholerae O1 strain 569B, a classical biovar, was also shown to have two replicons of 2.9 and 1.2 Mb, which does not agree with a physical map proposed in a previous study. Our results suggest that a two-replicon structure is common throughout Vibrio species.     

Physical map and set of overlapping cosmid clones representing the genome of the archaeon Halobacterium sp. GRB.

We have constructed a complete, five-enzyme restriction map of the genome of the archaeon Halobacterium sp. GRB, based on a set of 84 overlapping cosmid clones. Fewer than 30 kbp, in three gaps, remain uncloned. The genome consists of five replicons: a chromosome (2038 kbp) and four plasmids (305, 90, 37, and 1.8 kbp). The genome of Halobacterium sp. GRB is similar in style to other halobacterial genomes by being partitioned among multiple replicons and by being mosaic in terms of nucleotide composition. It is unlike other halobacterial genomes, however, in lacking multicopy families of insertion sequences.     

Detailed physical map and set of overlapping clones covering the genome of the archaebacterium Haloferax volcanii DS2.

An integrated approach of "bottom up" and "top down" mapping has produced a minimal set of overlapping cosmid clones covering 96% of the 4140 kilobase-pairs (kbp) Haloferax volcanii DS2 genome and a completely closed physical map. This genome is partitioned into five replicons: a 2920 kbp chromosome and four plasmids, of 690 kbp (pHV4), 442 kbp (pHV3), 86 kbp(pHV1) and 6.4 kbp (pHV2). A restriction map for six infrequently-cutting restriction enzymes was constructed, representing a total of 903 sites in the cloned DNA. We have placed the two ribosomal RNA operons, the genes for 7 S RNA and for RNaseP RNA and 22 protein-coding genes on the map. Restriction site frequencies show significant variation in different portions of the genome. The regions of high site density correspond to halobacterial satellite or FII DNA which includes two small regions of the chromosome, the plasmids pHV1 and pHV2, and half of pHV4, but not pHV3.     

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.     

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.     

Bacteria of the Burkholderia cepacia complex: specific features of diagnostics,genome organization and metabolism

Modern data, related with the identification and typing of the complex B. cepacia bacteria, are analyzed in the article by using the poly-phase taxonomic approach. An optimal scheme for identifying and typing the complex B. cepacia bacteria, involving the microbiological and molecular-biological methods of laboratory diagnostics, is presented. The key and assumed factors of pathogenicity of the discussed bacteria are described. The possible phylogenetic relations of the complex B. cepacia bacteria with phytopathgens as well as with pathogenic bacteria of species Burkholderia, Pseudomonas, Escherichia, B. mallei, B. pdeudomallei, P. seruginosa and E. coli are described. A possible role of genome alterations and mutations in the genome of the complex B. cepacia bacteria (with the latter genome having unusual properties, i.e. a big size, and a considerable quantity of insertion sequences) in creating the conditions for the "pulsing" evolution "jerks", i.e. for a rapid change-over from saprophytism in the soil to a pathogenic causative agent of a viral-and-bacteriological infection. Such mechanism can be regarded as a rapid and radical adaptation of a microorganism under the conditions of changing ecological niches.     

Differences in chromosome number and genome rearrangements in the genus Brucella

We have studied the genomic structure and constructed the Spe I, Pac I and I- Ceu I restriction maps of the four biovars of the pathogenic bacterium Brucella suis . B . suis biovar 1 has two chromosomes of 2.1 Mb and 1.15 Mb, similar to those of the other Brucella species: B . melitensis , B . abortus , B . ovis and B . neotomae . Two chromosomes were also observed in the genome of B . suis biovars 2 and 4, but with sizes of 1.85 Mb and 1.35 Mb, whereas only one chromosome with a size of 3.1 Mb was found in B . suis biovar 3. We show that the differences in chromosome size and number can be explained by rearrangements at chromosomal regions containing the three rrn genes. The location and orientation of these genes confirmed that these rearrangements are due to homologous recombination at the rrn loci. This observation allows us to propose a scheme for the evolution of the genus Brucella in which the two chromosome-containing strains can emerge from an hypothetical ancestor with a single chromosome, which is probably similar to that of B . suis biovar 3. As the genus Brucella is certainly monospecific, this is the first time that differences in chromosome number have been observed in strains of the same bacterial species.     

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.     

Burkholderia spp. alter Pseudomonas aeruginosa physiology through iron sequestration

Pseudomonas aeruginosa and members of the Burkholderia cepacia complex often coexist in both the soil and the lungs of cystic fibrosis patients. To gain an understanding of how these different species affect each other's physiology when coexisting, we performed a screen to identify P. aeruginosa genes that are induced in the presence of Burkholderia: A random gene fusion library was constructed in P. aeruginosa PA14 by using a transposon containing a promoterless lacZ gene. Fusion strains were screened for their ability to be induced in the presence of Burkholderia strains in a cross-streak assay. Three fusion strains were induced specifically by Burkholderia species; all three had transposon insertions in genes known to be iron regulated. One of these fusion strains, containing a transposon insertion in gene PA4467, was used to characterize the inducing activity from Burkholderia: Biochemical and genetic evidence demonstrate that ornibactin, a siderophore produced by nearly all B. cepacia strains, can induce P. aeruginosa PA4467. Significantly, PA4467 is induced early in coculture with an ornibactin-producing but not an ornibactin-deficient B. cepacia strain, indicating that ornibactin can be produced by B. cepacia and detected by P. aeruginosa when the two species coexist.     

Burkholderia anthina sp. nov. and Burkholderia pyrrocinia,two additional Burkholderia cepacia complex bacteria,may confound results of new molecular diagnostic tools

Nineteen Burkholderia cepacia-like isolates of human and environmental origin could not be assigned to one of the seven currently established genomovars using recently developed molecular diagnostic tools for B. cepacia complex bacteria. Various genotypic and phenotypic characteristics were examined. The results of this polyphasic study allowed classification of the 19 isolates as an eighth B. cepacia complex genomovar (Burkholderia anthina sp. nov.) and to design tools for its identification in the diagnostic laboratory. In addition, new and published data for Burkholderia pyrrocinia indicated that this soil bacterium is also a member of the B. cepacia complex. This highlights another potential source for diagnostic problems with B. cepacia-like bacteria.     

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.     

Biological activity of Burkholderia (Pseudomonas) cepacia lipopolysaccharide

Abstract Burkholderia cepacia has emerged as an important multiresistant pathogen in cystic fibrosis (CF), associated in 20% of colonised patients with a rapid and fatal decline in lung function. Although knowledge of B. cepacia epidemiology has improved, the mechanisms involved in pathogenesis remain obscure. In this study, B. cepacia lipopolysaccharide (LPS) was assessed for endotoxic potential and the capacity to induce tumour necrosis factor (TNF). LPS preparations from clinical and environmental isolates of B. cepacia and from the closely related species Burkholderia gladioli exhibited a higher endotoxic activity and more pronounced cytokine response in vitro compared to preparations from the major CF pathogen Pseudomonas aeruginosa . This study may help to explain the vicious host immune response observed during pulmonary exacerbations in CF patients colonised by B. cepacia and lead to therapeutic advances in clinical management.     

Susceptibility of Burkholderia cepacia and other pathogens of importance in cystic fibrosis to u.v. light

To investigate the potential usefulness of u.v. germicidal irradiation (UVGI) in preventing the spread of Burkholderia cepacia, an important pathogen in cystic fibrosis (CF), the in-vitro susceptibility of B. cepacia to UVGI was determined. Five strains were exposed to UVGI from a 7.2-W source. Burkholderia cepacia was less susceptible to UVGI than other important CF-related pathogens, namely Staphylococcus aureus and Pseudomonas aeruginosa, but was more susceptible than Stenotrophomonas maltophilia. No strain of B. cepacia survived longer than an 8 s exposure to UVGI, with doses required to achieve 1 log reduction in bacterial numbers ranging from 28.3 to 57.5 J m(-2).     

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.     

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.     

The eroded genome of a Psychotria leaf symbiont: hypotheses about lifestyle and interactions with its plant host

Several plant species of the genus Psychotria (Rubiaceae) harbour Burkholderia sp. bacteria within specialized leaf nodules. The bacteria are transmitted vertically between plant generations and have not yet been cultured outside of their host. This symbiosis is also generally described as obligatory because plants devoid of symbionts fail to develop into mature individuals. We sequenced for the first time the genome of the symbiont of Psychotria kirkii in order to shed some light on the nature of their symbiotic relationship. We found that the 4?Mb genome of Candidatus Burkholderia kirkii (B.?kirkii) is small for a Burkholderia species and displays features consistent with ongoing genome erosion such as large proportions of pseudogenes and transposable elements. Reductive genome evolution affected a wide array of functional categories that may hinder the ability of the symbiont to be free-living. The genome does not encode functions commonly found in plant symbionts such as nitrogen fixation or plant hormone metabolism. Instead, a collection of genes for secondary metabolites' synthesis is located on the 140?kb plasmid of B.?kirkii and suggests that leaf nodule symbiosis benefits the host by providing protection against herbivores or pathogens.     

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.