Development and application of an efficient recombineering system for Burkholderia glumae and Burkholderia plantarii

The lambda phage Red proteins Redα/Redβ/Redγ and Rac prophage RecE/RecT proteins are powerful tools for precise and efficient genetic manipulation but have been limited to only a few prokaryotes. Here, we report the development and application of a new recombineering system for Burkholderia glumae and Burkholderia plantarii based on three Rac bacteriophage RecET-like operons, RecEThe_BDU8, RecETh_TJI49 and RecETh1h2e_YI23, which were obtained from three different Burkholderia species. Recombineering experiments indicated that RecETh_TJI49 and RecETh1h2e_YI23 showed higher recombination efficiency compared to RecEThe_BDU8 in Burkholderia glumae PG1. Furthermore, all of the proteins currently categorized as hypothetical proteins in RecETh1h2e_YI23, RecETh_TJI49 and RecEThe_BDU8 may have a positive effect on recombination in B. glumae PG1 except for the h2 protein in RecETh1h2e_YI23. Additionally, RecET_YI23 combined with exonuclease inhibitors Pluγ or Redγ exhibited equivalent recombination efficiency compared to Redγβα in Escherichia coli, providing potential opportunity of recombineering in other Gram-negative bacteria for its loose host specificity. Using recombinase-assisted in situ insertion of promoters, we successfully activated three cryptic non-ribosomal peptide synthetase biosynthetic gene clusters in Burkholderia strains, resulting in the generation of a series of lipopeptides that were further purified and characterized. Compound 7 exhibited significant potential anti-inflammatory activity by inhibiting lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. This recombineering system may greatly enhance functional genome research and the mining of novel natural products in the other species of the genus Burkholderia after optimization of a protocol.     

Genes Involved in Degradation of para-Nitrophenol Are Differentially Arranged in Form of Non-Contiguous Gene Clusters in Burkholderia sp. strain SJ98

Biodegradation of para-Nitrophenol (PNP) proceeds via two distinct pathways, having 1,2,3-benzenetriol (BT) and hydroquinone (HQ) as their respective terminal aromatic intermediates. Genes involved in these pathways have already been studied in different PNP degrading bacteria. Burkholderia sp. strain SJ98 degrades PNP via both the pathways. Earlier, we have sequenced and analyzed a ~41 kb fragment from the genomic library of strain SJ98. This DNA fragment was found to harbor all the lower pathway genes; however, genes responsible for the initial transformation of PNP could not be identified within this fragment. Now, we have sequenced and annotated the whole genome of strain SJ98 and found two ORFs (viz., pnpA and pnpB) showing maximum identity at amino acid level with p-nitrophenol 4-monooxygenase (PnpM) and p-benzoquinone reductase (BqR). Unlike the other PNP gene clusters reported earlier in different bacteria, these two ORFs in SJ98 genome are physically separated from the other genes of PNP degradation pathway. In order to ascertain the identity of ORFs pnpA and pnpB, we have performed in-vitro assays using recombinant proteins heterologously expressed and purified to homogeneity. Purified PnpA was found to be a functional PnpM and transformed PNP into benzoquinone (BQ), while PnpB was found to be a functional BqR which catalyzed the transformation of BQ into hydroquinone (HQ). Noticeably, PnpM from strain SJ98 could also transform a number of PNP analogues. Based on the above observations, we propose that the genes for PNP degradation in strain SJ98 are arranged differentially in form of non-contiguous gene clusters. This is the first report for such arrangement for gene clusters involved in PNP degradation. Therefore, we propose that PNP degradation in strain SJ98 could be an important model system for further studies on differential evolution of PNP degradation functions.     

Genome Annotation of Burkholderia sp. SJ98 with Special Focus on Chemotaxis Genes

Burkholderia sp. strain SJ98 has the chemotactic activity towards nitroaromatic and chloronitroaromatic compounds. Recently our group published draft genome of strain SJ98. In this study, we further sequence and annotate the genome of stain SJ98 to exploit the potential of this bacterium. We specifically annotate its chemotaxis genes and methyl accepting chemotaxis proteins. Genome of Burkholderia sp. SJ98 was annotated using PGAAP pipeline that predicts 7,268 CDSs, 52 tRNAs and 3 rRNAs. Our analysis based on phylogenetic and comparative genomics suggest that Burkholderia sp. YI23 is closest neighbor of the strain SJ98. The genes involved in the chemotaxis of strain SJ98 were compared with genes of closely related Burkholderia strains (i.e. YI23, CCGE 1001, CCGE 1002, CCGE 1003) and with well characterized bacterium E. coli K12. It was found that strain SJ98 has 37 che genes including 19 methyl accepting chemotaxis proteins that involved in sensing of different attractants. Chemotaxis genes have been found in a cluster along with the flagellar motor proteins. We also developed a web resource that provides comprehensive information on strain SJ98 that includes all analysis data (http://crdd.osdd.net/raghava/genomesrs/burkholderia/).     

Niches of two polysaccharide-degrading Polaribacter isolates from the North Sea during a spring diatom bloom

Members of the flavobacterial genus Polaribacter thrive in response to North Sea spring phytoplankton blooms. We analyzed two respective Polaribacter species by whole genome sequencing, comparative genomics, substrate tests and proteomics. Both can degrade algal polysaccharides but occupy distinct niches. The liquid culture isolate Polaribacter sp. strain Hel1_33_49 has a 3.0-Mbp genome with an overall peptidase:CAZyme ratio of 1.37, four putative polysaccharide utilization loci (PULs) and features proteorhodopsin, whereas the agar plate isolate Polaribacter sp. strain Hel1_85 has a 3.9-Mbp genome with an even peptidase:CAZyme ratio, eight PULs, a mannitol dehydrogenase for decomposing algal mannitol-capped polysaccharides but no proteorhodopsin. Unlike other sequenced Polaribacter species, both isolates have larger sulfatase-rich PULs, supporting earlier assumptions that Polaribacter take part in the decomposition of sulfated polysaccharides. Both strains grow on algal laminarin and the sulfated polysaccharide chondroitin sulfate. For strain Hel1_33_49, we identified by proteomics (i) a laminarin-induced PUL, (ii) chondroitin sulfate-induced CAZymes and (iii) a chondroitin-induced operon that likely enables chondroitin sulfate recognition. These and other data suggest that strain Hel1_33_49 is a planktonic flavobacterium feeding on proteins and a small subset of algal polysaccharides, while the more versatile strain Hel1_85 can decompose a broader spectrum of polysaccharides and likely associates with algae.     

Draft Genome Sequence of the Xylan-Degrading Marine Bacterium Strain S124, Representing a Novel Species of the Genus Oceanicola

We isolated a xylan-degrading bacterium from seawater of Micronesia and identified it as Oceanicola sp. strain S124. We sequenced the Oceanicola sp. S124 genome using GSFLX 454 pyrosequencing and predicted 4,433 open reading frames (ORFs) including putative saccharification and phage-related genes.     

Genome sequence of the acid-tolerant Burkholderia sp. strain WSM2232 from Karijini National Park,Australia

Burkholderia sp. strain WSM2232 is an aerobic, motile, Gram-negative, non-spore-forming acid-tolerant rod that was trapped in 2001 from acidic soil collected from Karijini National Park (Australia) using Gastrolobium capitatum as a host. WSM2232 was effective in nitrogen fixation with G. capitatum but subsequently lost symbiotic competence during long-term storage. Here we describe the features of Burkholderia sp. strain WSM2232, together with genome sequence information and its annotation. The 7,208,311 bp standard-draft genome is arranged into 72 scaffolds of 72 contigs containing 6,322 protein-coding genes and 61 RNA-only encoding genes. The loss of symbiotic capability can now be attributed to the loss of nodulation and nitrogen fixation genes from the genome. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.     

Genome sequence of the acid-tolerant Burkholderia sp. strain WSM2230 from Karijini National Park,Australia

Burkholderia sp. strain WSM2230 is an aerobic, motile, Gram-negative, non-spore-forming acid-tolerant rod isolated from acidic soil collected in 2001 from Karijini National Park, Western Australia, using Kennedia coccinea (Coral Vine) as a host. WSM2230 was initially effective in nitrogen-fixation with K. coccinea, but subsequently lost symbiotic competence. Here we describe the features of Burkholderia sp. strain WSM2230, together with genome sequence information and its annotation. The 6,309,801 bp high-quality-draft genome is arranged into 33 scaffolds of 33 contigs containing 5,590 protein-coding genes and 63 RNA-only encoding genes. The genome sequence of WSM2230 failed to identify nodulation genes and provides an explanation for the observed failure of the laboratory grown strain to nodulate. The genome of this strain is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.     

A computational approach to identify CRISPR-Cas loci in the complete genomes of the lichen-associated Burkholderia sp. PAMC28687 and PAMC26561

The genus Burkholderia and its strains PAMC28687 and PAMC26561 are lichen-associated bacteria isolated from the Antarctic region. Our study is the first to provide the genome sequence of the Burkholderia sp. PAMC26561 strain. The genus Burkholderia includes bacteria that are pathogenic to plants, animals, and humans. Computational analysis of complete genomes of strains from the uncategorized Burkholderia group was performed using the NCBI databank and PATRIC (for genome sequence information) and CRISPRCasFinder (online and offline versions) software in order to predict the CRISPR loci and Cas genes. The RNAfold Webserver online software was used to predict RNA secondary structures. Our study showed that strain MSMB0852 (plasmid) possesses CRISPR-Cas system Class 2, and two lichen-associated strains, PAMC28687 (chromosome I) and PAMC26561 (chromosome I), possess CRISPR-Cas system Class 1. Additionally, only the two lichen-associated strains possess a variety of Cas genes.     

Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125

The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15–20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a λ phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis. Received: April 17, 1998 / Accepted: June 23, 1998     

Non-contiguous finished genome sequence and description of Anaerococcus senegalensis sp. nov

Anaerococcus senegalensis strain JC48^T sp. nov. is the type strain of A. senegalensis sp. nov. a new species within the genus Anaerococcus. This strain whose genome is described here was isolated from the fecal flora of a healthy patient. A. senegalensis is an obligate anaerobic coccus. Here we describe the features of this organism together with the complete genome sequence and annotation. The 1,790,835 bp long genome (1 chromosome but no plasmid) contains 1,721 protein-coding and 53 RNA genes including 5 rRNA genes     

Characterization of Burkholderia bacteria clade compositions in soil and Riptortus pedestris (Hemiptera: Alydidae) in South Korea

Riptortus pedestris (Hemiptera: Alydidae) is known to acquire the genus Burkholderia, symbiotic bacteria, from soil. Therefore, symbiont acquisition of R. pedestris would be directly affected by bacterial diversity in soil. Soil typically harbors diverse microbes including different Burkholderia clades such as SBE (stinkbug-associated beneficial and environmental), PBE (plant-associated beneficial and environmental), and BCC (Burkholderia cepacia and complex). Nevertheless, little is known about Burkholderia acquisition patterns of R. pedestris in nature, especially in the context of bacteria clade compositions in soil. Therefore, based on diagnostic PCR analysis, we investigated Burkholderia clade compositions in field-collected soil itself and R. pedestris when the insects were provided with the soil. Also, wild R. pedestris were surveyed to characterize their Burkholderia compositions. First, 88.44% of soil samples were detected with the genus Burkholderia, and triple clade (SBE + PBE + BCC) was most frequently detected. Second, R. pedestris nymphs readily acquired Burkholderia bacteria from field-collected soil where 91.25% of the reared insects harbored the bacteria in their midguts. In contrast to soil, the detection of single BCC clade was the most dominant among the three identified Burkholderia clades. Third, from wild R. pedestris, 80.62% of the insects were found to harbor the genus Burkholderia, and single BCC clade was most frequently detected. Finally, 29.13% and 47.06% of the reared and wild R. pedestris were detected with unidentified Burkholderia clade, which does not belong to any of the three identified clades. Our findings provide baseline information to better understand ecological associations between R. pedestris and Burkholderia bacteria in different clades.     

Re-Annotation of Protein-Coding Genes in the Genome of Saccharomyces cerevisiae Based on Support Vector Machines

The annotation of the well-studied organism, Saccharomyces cerevisiae, has been improving over the past decade while there are unresolved debates over the amount of biologically significant open reading frames (ORFs) in yeast genome. We revisited the total count of protein-coding genes in S. cerevisiae S288c genome using a theoretical approach by combining the Support Vector Machine (SVM) method with six widely used measurements of sequence statistical features. The accuracy of our method is over 99.5% in 10-fold cross-validation. Based on the annotation data in Saccharomyces Genome Database (SGD), we studied the coding capacity of all 1744 ORFs which lack experimental results and suggested that the overall number of chromosomal ORFs encoding proteins in yeast should be 6091 by removing 488 spurious ORFs. The importance of the present work lies in at least two aspects. First, cross-validation and retrospective examination showed the fidelity of our method in recognizing ORFs that likely encode proteins. Second, we have provided a web service that can be accessed at http://cobi.uestc.edu.cn/services/yeast/, which enables the prediction of protein-coding ORFs of the genus Saccharomyces with a high accuracy.     

Nodulation in black locust by the Gammaproteobacteria Pseudomonas sp. and the Betaproteobacteria Burkholderia sp.

Nodulation abilities of bacteria in the subclasses Gammaproteobacteria and Betaproteobacteria on black locust (Robinia pseudoacacia) were tested. Pseudomonas sp., Burkholderia sp., Klebsiella sp., and Paenibacillus sp. were isolated from surface-sterilized black locust nodules, but their nodulation ability is unknown. The aims of this study were to determine if these bacteria are symbiotic. The species and genera of the strains were determined by RFLP analysis and DNA sequencing of 16S rRNA gene. Inoculation tests and histological studies revealed that Pseudomonas sp. and Burkholderia sp. formed nodules on black locust and also developed differentiated nodule tissue. Furthermore, a phylogenetic analysis of nodA and a BLASTN analysis of the nodC, nifH, and nifHD genes revealed that these symbiotic genes of Pseudomonas sp. and Burkholderia sp. have high similarities with those of rhizobial species, indicating that the strains acquired the symbiotic genes from rhizobial species in the soil. Therefore, in an actual rhizosphere, bacterial diversity of nodulating legumes may be broader than expected in the Alpha-, Beta-, and Gammaproteobacteria subclasses. The results indicate the importance of horizontal gene transfer for establishing symbiotic interactions in the rhizosphere.     

Burkholderia sp. Induces Functional Nodules on the South African Invasive Legume Dipogon lignosus (Phaseoleae) in New Zealand Soils

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678^T which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.     

A Virulent Phage Infecting Lactococcus garvieae,with Homology to Lactococcus lactis Phages

A new virulent phage belonging to the Siphoviridae family and able to infect Lactococcus garvieae strains was isolated from compost soil. Phage GE1 has a prolate capsid (56 by 38 nm) and a long noncontractile tail (123 nm). It had a burst size of 139 and a latent period of 31 min. Its host range was limited to only two L. garvieae strains out of 73 tested. Phage GE1 has a double-stranded DNA genome of 24,847 bp containing 48 predicted open reading frames (ORFs). Putative functions could be assigned to only 14 ORFs, and significant matches in public databases were found for only 17 ORFs, indicating that GE1 is a novel phage and its genome contains several new viral genes and encodes several new viral proteins. Of these 17 ORFs, 16 were homologous to deduced proteins of virulent phages infecting the dairy bacterium Lactococcus lactis, including previously characterized prolate-headed phages. Comparative genome analysis confirmed the relatedness of L. garvieae phage GE1 to L. lactis phages c2 (22,172 bp) and Q54 (26,537 bp), although its genome organization was closer to that of phage c2. Phage GE1 did not infect any of the 58 L. lactis strains tested. This study suggests that phages infecting different lactococcal species may have a common ancestor.     

Analysis of the Saccharomyces cerevisiae proteome with PeptideAtlas

We present the Saccharomyces cerevisiae PeptideAtlas composed from 47 diverse experiments and 4.9 million tandem mass spectra. The observed peptides align to 61% of Saccharomyces Genome Database (SGD) open reading frames (ORFs), 49% of the uncharacterized SGD ORFs, 54% of S. cerevisiae ORFs with a Gene Ontology annotation of 'molecular function unknown', and 76% of ORFs with Gene names. We highlight the use of this resource for data mining, construction of high quality lists for targeted proteomics, validation of proteins, and software development.     

Polyphasic characterization of four soil-derived phenanthrene-degrading Acidovorax strains and proposal of Acidovorax carolinensis sp. nov.

Four bacterial strains identified as members of the Acidovorax genus were isolated from two geographically distinct but similarly contaminated soils in North Carolina, USA, characterized, and their genomes sequenced. Their 16S rRNA genes were highly similar to those previously recovered during stable-isotope probing (SIP) of one of the soils with the polycyclic aromatic hydrocarbon (PAH) phenanthrene. Heterotrophic growth of all strains occurred with a number of organic acids, as well as phenanthrene, but no other tested PAHs. Optimal growth occurred aerobically under mesophilic temperature, neutral pH, and low salinity conditions. Predominant fatty acids were C_16:1ω7c/C_16:1ω6c, C_16:0, and C_18:1ω7c, and were consistent with the genus. Genomic G + C contents ranged from 63.6 to 64.2%. A combination of whole genome comparisons and physiological analyses indicated that these four strains likely represent a single species within the Acidovorax genus. Chromosomal genes for phenanthrene degradation to phthalate were nearly identical to highly conserved regions in phenanthrene-degrading Delftia, Burkholderia, Alcaligenes, and Massilia species in regions flanked by transposable or extrachromosomal elements. The lower degradation pathway for phenanthrene metabolism was inferred by comparisons to described genes and proteins. The novel species Acidovorax carolinensis sp. nov. is proposed, comprising the four strains described in this study with strain NA3^T as the type strain (=LMG 30136, =DSM 105008).