Complete genome sequence of the fenitrothion-degrading Burkholderia sp. strain YI23

Burkholderia species are ubiquitous in soil environments. Many Burkholderia species isolated from various environments have the potential to biodegrade man-made chemicals. Burkholderia sp. strain YI23 was isolated from a golf course soil and identified as a fenitrothion-degrading bacterium. In this study, we report the complete genome sequence of Burkholderia sp. strain YI23.     

A comparative synteny map of Burkholderia species links large-scale genome rearrangements to fine-scale nucleotide variation in prokaryotes

Genome rearrangement events, including inversions and translocations, are frequently observed across related microbial species, but the impact of such events on functional diversity is unclear. To clarify this relationship, we compared 4 members of the Gram-negative Burkholderia family (Burkholderia pseudomallei, Burkholderia mallei, Burkholderia thailandensis, and Burkholderia cenocepacia) and identified a core set of 2,590 orthologs present in all 4 species (metagenes). The metagenes were organized into 255 synteny blocks whose relative order has been altered by a predicted minimum of 242 genome rearrangement events. Functionally, metagenes within individual synteny blocks were often related. The molecular divergence of metagenes adjacent to synteny breakpoints (boundary metagenes) was significantly greater compared with metagenes within blocks, suggesting an association between breakpoint locations and local fine-scale nucleotide alterations. This phenomenon, referred to as boundary element associated divergence, was also observed in Pseudomonas and Shigella, suggesting that this is a common phenomenon in prokaryotes. We also observed preferential localization of species-specific genes and insertion sequence element to synteny breakpoints in Burkholderia. Our results suggest that in prokaryotes, genome rearrangements may influence functional diversity through the enhanced divergence of boundary genes and the creation of foci for acquiring and deleting species-specific genes.     

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.     

The Burkholderia Genome Database: facilitating flexible queries and comparative analyses

As the genome sequences of multiple strains of a given bacterial species are obtained, more generalized bacterial genome databases may be complemented by databases that are focused on providing more information geared for a distinct bacterial phylogenetic group and its associated research community. The Burkholderia Genome Database represents a model for such a database, providing a powerful, user-friendly search and comparative analysis interface that contains features not found in other genome databases. It contains continually updated, curated and tracked information about Burkholderia cepacia complex genome annotations, plus other Burkholderia species genomes for comparison, providing a high-quality resource for its targeted cystic fibrosis research community. AVAILABILITY: http://www.burkholderia.com. Source code: GNU GPL.     

Revised Genome Sequence of Burkholderia thailandensis MSMB43 with Improved Annotation

There is growing interest in discovery of novel bioactive natural products from Burkholderia thailandensis. Here we report a significantly improved genome sequence and reannotation of Burkholderia thailandensis MSMB43, which will facilitate the discovery of new natural products through genome mining and studies of the metabolic versatility of this bacterium.     

16S rRNA gene-based phylogenetic microarray for simultaneous identification of members of the genus Burkholderia

For cultivation-independent and highly parallel analysis of members of the genus Burkholderia , an oligonucleotide microarray (phylochip) consisting of 131 hierarchically nested 16S rRNA gene-targeted oligonucleotide probes was developed. A novel primer pair was designed for selective amplification of a 1.3 kb 16S rRNA gene fragment of Burkholderia species prior to microarray analysis. The diagnostic performance of the microarray for identification and differentiation of Burkholderia species was tested with 44 reference strains of the genera Burkholderia , Pandoraea , Ralstonia and Limnobacter . Hybridization patterns based on presence/absence of probe signals were interpreted semi-automatically using the novel likelihood-based strategy of the web-tool PhyloDetect. Eighty-eight per cent of the reference strains were correctly identified at the species level. The evaluated microarray was applied to investigate shifts in the Burkholderia community structure in acidic forest soil upon addition of cadmium, a condition that selected for Burkholderia species. The microarray results were in agreement with those obtained from phylogenetic analysis of Burkholderia 16S rRNA gene sequences recovered from the same cadmium-contaminated soil, demonstrating the value of the Burkholderia phylochip for determinative and environmental studies.     

Application of a recA gene-based identification approach to the maize rhizosphere reveals novel diversity in Burkholderia species

Burkholderia species are widely distributed in the natural environment. We evaluated the use of the recA gene in a cultivation-independent approach to examine the Burkholderia diversity associated with the maize rhizosphere. Two types of recA gene library were constructed, one with broad-specificity recA primers (BUR1 and BUR2) and a second from the products of nested PCRs using Burkholderia-specific primers (BUR3 and BUR4). The broad-specificity primer set provided near full-length recA sequences (869 bp) suitable for the creation of robust environmental sequence data sets; however, the nested PCR approach demonstrated the greatest specificity (84%) for detection of Burkholderia species recA genes. In addition, the screening approach was able to identify recA phylotypes matching Burkholderia cepacia complex species previously cultivated from the maize samples and discriminate these from other Burkholderia. The ecological benefit of Burkholderia species cultivated from maize rhizosphere is well documented, however, the fact that the majority of Burkholderia recA genes detected in this study (90%) were suggestive of novel taxa indicates that a wealth of potentially important interactions with uncultivated Burkholderia species remain unstudied in this habitat.     

Burkholderia thailandensis E125 harbors a temperate bacteriophage specific for Burkholderia mallei

Burkholderia thailandensis is a nonpathogenic gram-negative bacillus that is closely related to Burkholderia mallei and Burkholderia pseudomallei. We found that B. thailandensis E125 spontaneously produced a bacteriophage, termed phiE125, which formed turbid plaques in top agar containing B. mallei ATCC 23344. We examined the host range of phiE125 and found that it formed plaques on B. mallei but not on any other bacterial species tested, including B. thailandensis and B. pseudomallei. Examination of the bacteriophage by transmission electron microscopy revealed an isometric head and a long noncontractile tail. B. mallei NCTC 120 and B. mallei DB110795 were resistant to infection with phiE125 and did not produce lipopolysaccharide (LPS) O antigen due to IS407A insertions in wbiE and wbiG, respectively. wbiE was provided in trans on a broad-host-range plasmid to B. mallei NCTC 120, and it restored LPS O-antigen production and susceptibility to phiE125. The 53,373-bp phiE125 genome contained 70 genes, an IS3 family insertion sequence (ISBt3), and an attachment site (attP) encompassing the 3' end of a proline tRNA (UGG) gene. While the overall genetic organization of the phiE125 genome was similar to lambda-like bacteriophages and prophages, it also possessed a novel cluster of putative replication and lysogeny genes. The phiE125 genome encoded an adenine and a cytosine methyltransferase, and purified bacteriophage DNA contained both N6-methyladenine and N4-methylcytosine. The results presented here demonstrate that phiE125 is a new member of the lambda supergroup of Siphoviridae that may be useful as a diagnostic tool for B. mallei.     

Burkholderia semiarida sp. nov. and Burkholderia sola sp. nov., two novel B. cepacia complex species causing onion sour skin

Two putative novel Burkholderia cenocepacia lineages found in the semi-arid region of north-east Brazil causing onion sour skin were studied using genomic approaches to determine their taxonomic position. Four strains belonging to one novel lineage (CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171) and one strain (CCRMBC51) belonging to another novel lineage had their whole genome sequenced to carry out taxogenomic analyses. The phylogenomic tree built using the type (strain) genome server (TYGS) clustered the strains CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171 into the same clade, while grouped the strain CCRMBC51 separately. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) analysis showed values above 99.21 % and 93.2 %, respectively, among the strains CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171, while ANI and dDDH values between these strains and the strain CCRMBC51 were below 94.49 % and 56.6 %, respectively. All these strains showed ANI and dDDH values below 94.78 % and 58.8 % concerning type strains of the B. cepacia complex (Bcc) species. The phylogenetic maximum likelihood tree constructed based on the multilocus sequence analysis of core genes (cMLSA) clustered the strains CCRMBC16, CCRMBC33, CCRMBC74, and CCRMBC171 and the strain CCRMBC51 in two exclusive clades, which did not cluster with any known species of the Bcc. Therefore, combined data from TYGS, ANI, dDDH, and cMLSA demonstrated that the strains represent two novel species of the Bcc, which we classified as Burkholderia semiarida sp. nov. and Burkholderia sola sp. nov., and proposed the strains CCRMBC74^T (=IBSBF 3371 ^T = CBAS 905 ^T) and CCRMBC51^T (=IBSBF3370^T = CBAS 904 ^T) as type strains, respectively.     

Communication systems in the genus Burkholderia: global regulators and targets for novel antipathogenic drugs.

The genus Burkholderia not only contains the primary pathogens Burkholderia pseudomallei and Burkholderia mallei but also several species that have emerged as opportunistic pathogens in persons suffering from cystic fibrosis or chronic granulomatous disease and immunocompromised individuals. Burkholderia species utilize quorum-sensing (QS) systems that rely on N-acyl-homoserine lactone (AHL) signal molecules to express virulence factors and other functions in a population-density-dependent manner. Most Burkholderia species employ the CepIR QS system, which relies on N-octanoyl-homoserine lactone. However, some strains harbour multiple QS systems and produce numerous AHLs. QS systems have been demonstrated to be essential for full virulence in various infection models and, thus, these regulatory systems represent attractive targets for the development of novel therapeutics.     

Legume-nodulating betaproteobacteria: diversity, host range, and future prospects

Rhizobia form specialized nodules on the roots of legumes (family Fabaceae) and fix nitrogen in exchange for carbon from the host plant. Although the majority of legumes form symbioses with members of genus Rhizobium and its relatives in class Alphaproteobacteria, some legumes, such as those in the large genus Mimosa, are nodulated predominantly by betaproteobacteria in the genera Burkholderia and Cupriavidus. The principal centers of diversity of these bacteria are in central Brazil and South Africa. Molecular phylogenetic studies have shown that betaproteobacteria have existed as legume symbionts for approximately 50 million years, and that, although they have a common origin, the symbiosis genes in both subclasses have evolved separately since then. Additionally, some species of genus Burkholderia, such as B. phymatum, are highly promiscuous, effectively nodulating several important legumes, including common bean (Phaseolus vulgaris). In contrast to genus Burkholderia, only one species of genus Cupriavidus (C. taiwanensis) has so far been shown to nodulate legumes. The recent availability of the genome sequences of C. taiwanensis, B. phymatum, and B. tuberum has paved the way for a more detailed analysis of the evolutionary and mechanistic differences between nodulating strains of alpha- and betaproteobacteria. Initial analyses of genome sequences have suggested that plant-associated Burkholderia spp. have lower G+C contents than Burkholderia spp. that are opportunistic human pathogens, thus supporting previous suggestions that the plant- and human-associated groups of Burkholderia actually belong in separate genera.     

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.     

Diazotrophic burkholderia species associated with field-grown maize and sugarcane

Until recently, diazotrophy was known in only one of the 30 formally described species of Burkholderia. Novel N(2)-fixing plant-associated Burkholderia species such as B. unamae, B. tropica, and B. xenovorans have been described, but their environmental distribution is scarcely known. In the present study, the occurrence of N(2)-fixing Burkholderia species associated with different varieties of sugarcane and maize growing in regions of Mexico and Brazil was analyzed. Only 111 out of more than 900 isolates recovered had N(2)-fixing ability as demonstrated by the acetylene reduction assay. All 111 isolates also yielded a PCR product with primers targeting the nifH gene, which encodes a key enzyme in the process of nitrogen fixation. These 111 isolates were confirmed as belonging to the genus Burkholderia by using a new 16S rRNA-specific primer pair for diazotrophic species (except B. vietnamiensis) and closely related nondiazotrophic Burkholderia. In Mexico, many isolates of B. unamae (predominantly associated with sugarcane) and B. tropica (more often associated with maize) were recovered. However, in Brazil B. tropica was not identified among the isolates analyzed, and only a few B. unamae isolates were recovered from one sugarcane variety. Most Brazilian diazotrophic Burkholderia isolates (associated with both sugarcane and maize plants) belonged to a novel species, as revealed by amplified 16S rRNA gene restriction profiles, 16S rRNA gene sequencing, and protein electrophoresis. In addition, transmissibility factors such as the cblA and esmR genes, identified among clinical and environmental isolates of opportunistic pathogens of B. cenocepacia and other species of the B. cepacia complex, were not detected in any of the plant-associated diazotrophic Burkholderia isolates analyzed.     

Characterization of DC1, a broad-host-range Bcep22-like podovirus

Bcep22-like phages are a recently described group of podoviruses that infect strains of Burkholderia cenocepacia. We have isolated and characterized a novel member of this group named DC1. This podovirus shows many genomic similarities to BcepIL02 and Bcep22, but it infects strains belonging to multiple Burkholderia cepacia complex (BCC) species.     

Complete genome sequence of Burkholderia rhizoxinica, an Endosymbiont of Rhizopus microsporus

Burkholderia rhizoxinica is an intracellular symbiont of the phytopathogenic fungus Rhizopus microsporus. The vertically transmitted endosymbiont not only delivers the antimitotic macrolide rhizoxin to its host but is also essential for vegetative spore formation of the fungus. To shed light on the genetic equipment of this model organism, we sequenced the whole genome of B. rhizoxinica HKI 0454, thus providing the first genomic insight into an intracellular mutualist of a fungal species. The 3.75-Mb genome consists of a chromosome and two strain-specific plasmids. The primary metabolism appears to be specialized for the uptake of fungal metabolites. Besides the rhizoxin biosynthesis gene cluster, there are 14 loci coding for nonribosomal peptide synthetase (NRPS) assembly lines, which represent novel targets for genomic mining of cryptic natural products. Furthermore, the endosymbionts are equipped with a repertoire of virulence-related factors, which can now be studied to elucidate molecular mechanisms underlying bacterial-fungal interaction.     

Genome characterization of a novel Burkholderia cepacia complex genomovar isolated from dieback affected mango orchards

We characterized the genome of the antibiotic resistant, caseinolytic and non-hemolytic Burkholderia sp. strain TJI49, isolated from mango trees (Mangifera indica L.) with dieback disease. This isolate produced severe disease symptoms on the indicator plants. Next generation DNA sequencing and short-read assembly generated the 60X deep 7,631,934 nucleotide draft genome of Burkholderia sp. TJI49 which comprised three chromosomes and at least one mega plasmid. Genome annotation studies revealed a total 8,992 genes, out of which 8,940 were protein coding genes. Comparative genomics and phylogenetics identified Burkholderia sp. TJI49 as a distinct species of Burkholderia cepacia complex (BCC), closely related to B. multivorans ATCC17616. Genome-wide sequence alignment of this isolate with replicons of BCC members showed conservation of core function genes but considerable variations in accessory genes. Subsystem-based gene annotation identified the active presence of wide spread colonization island and type VI secretion system in Burkholderia sp. TJI49. Sequence comparisons revealed (a) 28 novel ORFs that have no database matches and (b) 23 ORFs with orthologues in species other than Burkholderia, indicating horizontal gene transfer events. Fold recognition of novel ORFs identified genes encoding pertactin autotransporter-like proteins (a constituent of type V secretion system) and Hap adhesion-like proteins (involved in cell–cell adhesion) in the genome of Burkholderia sp. TJI49. The genomic characterization of this isolate provided additional information related to the ‘pan-genome’ of Burkholderia species.     

Burkholderia caballeronis sp. nov., a nitrogen fixing species isolated from tomato (Lycopersicon esculentum) with the ability to effectively nodulate Phaseolus vulgaris

During a survey of Burkholderia species with potential use in agrobiotechnology, a group of 12 strains was isolated from the rhizosphere and rhizoplane of tomato plants growing in Mexico (Nepantla, Mexico State). A phylogenetic analysis of 16S rRNA gene sequences showed that the strains are related to Burkholderia kururiensis and Burkholderia mimosarum (97.4 and 97.1 %, respectively). However, they induced effective nitrogen-fixing nodules on roots of Phaseolus vulgaris. Based on polyphasic taxonomy, the group of strains represents a novel species for which the name Burkholderia caballeronis sp. nov. is proposed. The type species is TNe-841^T (= LMG 26416^T = CIP 110324^T).     

Characterization of the poly-β-1,6-N-acetylglucosamine polysaccharide component of Burkholderia biofilms

We demonstrated the production of poly-β-1,6-N-acetylglucosamine (PNAG) polysaccharide in the biofilms of Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia ambifaria, Burkholderia cepacia, and Burkholderia cenocepacia using an immunoblot assay for PNAG. These results were confirmed by further studies, which showed that the PNAG hydrolase, dispersin B, eliminated immunoreactivity of extracts from the species that were tested (B. cenocepacia and B. multivorans). Dispersin B also inhibited biofilm formation and dispersed preformed biofilms of Burkholderia species. These results imply a role for PNAG in the maintenance of Burkholderia biofilm integrity. While PNAG was present in biofilms of all of the wild-type test organisms, a ΔpgaBC mutant of B. multivorans (Mu5) produced no detectable PNAG, indicating that these genes are needed for Burkholderia PNAG formation. Furthermore, restoration of PNAG production in PNAG negative E. coli TRXWMGΔC (ΔpgaC) by complementation with B. multivorans pgaBCD confirmed the involvement of these genes in Burkholderia PNAG production. While the confocal scanning laser microscopy of untreated wild-type B. multivorans showed thick, multilayered biofilm, Mu5 and dispersin B-treated wild-type biofilms were thin, poorly developed, and disrupted, confirming the involvement of PNAG in B. multivorans biofilm formation. Thus, PNAG appears to be an important component of Burkholderia biofilms, potentially contributing to its resistance to multiple antibiotics and persistence during chronic infections, including cystic fibrosis-associated infection.     

Burkholderia, a genus rich in plant-associated nitrogen fixers with wide environmental and geographic distribution

The genus Burkholderia comprises 19 species, including Burkholderia vietnamiensis which is the only known N(2)-fixing species of this bacterial genus. The first isolates of B. vietnamiensis were recovered from the rhizosphere of rice plants grown in a phytotron, but its existence in natural environments and its geographic distribution were not reported. In the present study, most N(2)-fixing isolates recovered from the environment of field-grown maize and coffee plants cultivated in widely separated regions of Mexico were phenotypically identified as B. cepacia using the API 20NE system. Nevertheless, a number of these isolates recovered from inside of maize roots, as well as from the rhizosphere and rhizoplane of maize and coffee plants, showed similar or identical features to those of B. vietnamiensis TVV75(T). These features include nitrogenase activity with 10 different carbon sources, identical or very similar nifHDK hybridization patterns, very similar protein electrophoregrams, identical amplified 16S rDNA restriction (ARDRA) profiles, and levels of DNA-DNA reassociation higher than 70% with total DNA from strain TVV75(T). Although the ability to fix N(2) is not reported to be a common feature among the known species of the genus Burkholderia, the results obtained show that many diazotrophic Burkholderia isolates analyzed showed phenotypic and genotypic features different from those of the known N(2)-fixing species B. vietnamiensis as well as from those of B. kururiensis, a bacterium identified in the present study as a diazotrophic species. DNA-DNA reassociation assays confirmed the existence of N(2)-fixing Burkholderia species different from B. vietnamiensis. In addition, this study shows the wide geographic distribution and substantial capability of N(2)-fixing Burkholderia spp. for colonizing diverse host plants in distantly separated environments.     

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.