- Short genome report
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Complete genome sequence and characterization of the haloacid–degrading Burkholderia caribensis MBA4
Standards in Genomic Sciencesvolume 10, Article number: 114 (2015)
Burkholderia caribensis MBA4 was isolated from soil for its capability to grow on haloacids. This bacterium has a genome size of 9,482,704 bp. Here we report the genome sequences and annotation, together with characteristics of the genome. The complete genome sequence consists of three replicons, comprising 9056 protein-coding genes and 80 RNA genes. Genes responsible for dehalogenation and uptake of haloacids were arranged as an operon. While dehalogenation of haloacetate would produce glycolate, three glycolate operons were identified. Two of these operons contain an upstream glcC regulator gene. It is likely that the expression of one of these operons is responsive to haloacetate. Genes responsible for the metabolism of dehalogenation product of halopropionate were also identified.
Human activities are thought to have great impact on the environment. While the development of industry has greatly improved our living condition, it has also escalates many environmental problems. Pollution has been an issue for a long time. Halogenated compounds have been used indiscriminately with the expansion of industrialization. Many of these compounds are found in the environment as disinfection by-product . Not only do they cause environmental problems they also have deleterious impact on our health .
Many bacteria are capable of transforming halogenated compounds and utilize them as carbon and energy sources. These bacteria are distinguished by their encoding enzymes known as dehalogenases which catalyze the breakdown of halogenated compounds through cleavage of the carbon-halogen bond . Burkholderia caribensis  MBA4 was isolated for its ability to mineralize 2-haloacids . The dehalogenase gene, deh4a, together with a downstream permease gene, deh4p, form an inducible operon that mediate the transformation and uptake of 2-haloacids, respectively, in MBA4 . The dehalogenase has been purified and characterized [5, 7, 8]. The permease has also been investigated . Moreover, MBA4 possesses a cryptic dehalogenase with a signal peptide [10, 11]. While proteomic analysis of the degradation of chloroacetate by MBA4 has been described, the identities of the differentially expressed proteins were hampered by the lack of a comprehensive protein database . The acquisition of a complete genomic sequence deems necessary. Here we describe the characterization of B. caribensis MBA4 and its complete genome sequence and annotation, with an emphasis on genomic features and genes related to degradation of haloacids.
Classification and features
Burkholderia caribensis MBA4 (=LMG 28094) is a Gram-negative, motile, rod-shaped bacterium (Fig. 1) in the order Burkholderiales  and class Betaproteobacteria . It grows poorly in traditional Luria-Bertani broth with NaCl but reasonably fast in LB‾ at 30 °C. The general features of this bacterium are shown in Table 1. MBA4 was isolated from forest soil collected from Chiang Mai, Thailand using monobromoacetic acid as an enrichment substrate . In addition to MBA, B. caribensis MBA4 is also capable of mineralizing monochloroacetate, 2-monobromopropionate and weakly on 2-monochloropropionate . MBA4 was initially classified as Pseudomonas cepacia  and subsequently as Burkholderia cepacia  based on its biochemical and phenotypic features. A polyphasic approach involving phenotypic, genotypic, and phylogenetic analysis was subsequently conducted to have a refined description. API 20NE and BIOLOG GN MicroPlate analyses were performed. These biochemical and substrate assimilation tests show that B. caribensis MBA4 failed to reduce nitrates to nitrites, nor from nitrates to nitrogen, incapable of producing indole from tryptophan, cannot acidify glucose and has no arginine dihydrolase nor urease. The bacterium possesses β-galactosidase but no α-glucosidase nor protease. It is able to assimilate glucose, arabinose, mannose, mannitol, N-acetyl-glucosamine, gluconate, caprate, malate and weakly on phenyl-acetate but not on maltose, citrate and adipate. Moreover, MBA4 is able to oxidize Tween-40, Tween-80, N-acetyl-D-galactosamine, adonitol, D-arabitol, D-fructose, L-fucose, m-inositol, L-rhamnose, D-sorbitol, D-trehalose, acetate, methylpyruvate, cis-aconitic acid, formic acid, D-galactonic acid lactone, D-galacturonic acid, D-glucosaminic acid, α-hydroxybutyric acid, β-hydroxybutyric acid, p-hydroxyphenylacetic acid, α-ketobutyric acid, α-ketoglutaric acid, α-ketovaleric acid, D,L-lactic acid, malonic acid, propionic acid, quinic acid, D-saccharic acid, bromosuccinic acid, alaninamide, D,L-alanine, L-alanyl-glycine, L-asparagine, L-aspartic acid, L-glutamic acid, L-histidine, hydroxy-L-proline, L-leucine, L-ornithine, L-phenylalanine, L-proline, L-pyroglutamic acid, L-serine, L-threonine, D,L-carnitine, γ-aminobutyric acid, 2-aminoethanol, glycerol, D,L-α-glycerolphosphate and glucose-6-phosphate and weakly on dextrin, glycogen, psicose, mono-methylsuccinate, succinic acid, succinamic acid, glucuronamide, D-serine and phenylethylamine. While whole cell fatty acid and whole cell protein SDS-PAGE profiles showed that MBA4 is closely related to certain Burkholderia species, BOX-PCR fingerprinting analysis  showed that the genomic structure of MBA4 is considerably different from other Burkholderia species . Phylogenetic analysis using 16S rRNA gene as a marker indicated that MBA4 is most closely related to B. caribensis , followed by B. hospita  and Burkholderia terrae  (Fig. 2). DNA-DNA hybridization values  were determined by the Belgian Coordinated Collections of Microorganisms using B. caribensis LMG 18531 T and B. hospita LMG 20598T as references. Hybridizations were conducted at 50 °C and the values are the mean of four or more tests. A DNA homology value of 74 and 62 % was obtained between MBA4 and LMG 18531 T, and LMG 20598T , respectively . It is thus concluded that MBA4 is a strain of B. caribensis .
The whole cell fatty acid profile of B. caribensis MBA4 (cells grown on tryptic soy agar) was determined by Department of Biology, The Chinese University of Hong Kong with a Sherlock® Microbial Identification System (Microbial IDentification Inc) using four replicates. The relative abundance for the fatty acids were 14:0 (4.5 %), 16:0 (19.9 %), 16:0 2-OH (1.5 %), 16:0 3-OH (4.2 %), 16:1 2-OH (1.6 %), 17:0 cyclo (12.5 %), 18:0 (1 %), 19:0 ω8c cyclo (6.1 %), summed feature 2 (14:0 3OH, 16:1 iso I, unidentified fatty acid with equivalent chain length value 10.928, 12:0 ALDE, or any of these combination, 6.3 %), summed feature 3 (16:1 ω7c, 15 iso 2OH, or any of their combination, 14.2 %), and summed feature 7 (18:1 ω7c, 18:1 ω9t, 18:1 ω12t, or any of these combination, 26.4 %).
Genome sequencing information
Genome project history
The genome of MBA4 was selected for sequencing in order to unravel the genetic background of the bacterium to utilize haloacids. MBA4 has a genome larger than most Burkholderia species with a size of more than 9.4 Mbp. Preliminary pulsed-field gel electrophoresis analysis showed that it contains three replicons with sizes of ca. 2.6, 3.5 and 3.7 Mbp (unpublished observations). The high-quality draft genome sequences with annotation were achieved and presented for public access in January 2014. Annotation was updated for the contigs in April 2014. The draft genome sequences was deposited in DDBJ/EMBL/GenBank under the accession number AXDD00000000. The three replicons of the complete genome sequence of MBA4 were finished in October 2015 and have been deposited in GenBank under accession numbers: CP012746, CP012747 and CP012748. Table 2 shows the project information and its association with MIGS version 2.0 compliance .
Growth conditions and DNA preparation
MBA4 was cultivated in 2 ml LB‾ with shaking at 30 °C. The culture was harvested at late exponential phase with an OD600 value of ca. 1.8. The cells were collected by centrifugation at 4000 rpm, 4 °C for 25 min. Genomic DNA was isolated with G-spin™ Genomic DNA extraction kit (iNtRON Biotechnology) according to the manufacturer’s protocol. The yield was about 40 μg and the 260/230 and the 260/280 ratios were 1.9. The concentration of the DNA used for library preparation was 258 ng/μl.
Genome sequencing and annotation
The genome of MBA4 was sequenced using Illumina HisSeq 2000, 454 GS FLX Titanium and PacBio System. Four sets of Illumina paired-end libraries (insert sizes: 100, 300, 500, and 2000 bp), a set of 454 library and a set of PacBio long read library were constructed. Collectively, the data furnished a coverage of about 850-fold. The raw reads for 500- and 2000-bp paired-end data were obtained from Beijing Genomics Institute while the 100- and 300-bp paired-end data and the 454 reads were obtained from Centre for Genome Sciences (previously Genome Research Centre), The University of Hong Kong. The PacBio long reads were obtained from Groken Bioscience. Bar codes were trimmed and low quality reads were filtered using the commercial software CLC Genomic Workbench 6.0.1 (CLC bio, Aarhus, Denmark). After trimming and filtering, Illumina paired-end and 454 reads were de novo assembled through CLC Genomic Workbench 6.0.1 with default setting. Scaffolds were then generated from the contigs with SSPACE basic 2.0  using information derived from the paired-end reads. De novo assembled transcripts from nine sets of RNA-seq paired-end raw data were mapped to the scaffolds to remove some of the internal gaps and ambiguous bases, and to join the scaffolds together. Standard PCR and Sanger-sequencing technology were employed to fill the gaps inside the scaffolds. Multiplex PCR was used to amplify unknown regions between scaffolds, and some scaffolds were linked after subsequent cloning and sequencing. Clean PacBio reads were assembled by SMRT Analysis v2.3.0 HGAP.2 with pre-assembled high-quality draft genome as reference sequences. Ambiguous base and inserted/deleted regions between PacBio-assembled and preassembled high quality draft sequences were manually corrected using consensus sequences derived from nine sets of transcriptome data.
A draft genome was annotated automatically with the Rapid Annotations using Subsystems Technology server [23–25] and the Prokaryotic Genomes Automatic Annotation Pipeline from NCBI . Subsequent annotation of the complete genome was based on the annotated draft sequences. Minor corrections were conducted manually.
The complete genome is represented by three replicons. The total size of the genome is 9,482,704 bp with a GC content of 62.46 % . A total of 9151 genes were predicted for the genome, including 15 pseudo genes. As for RNA genes, 18 rRNA and 62 tRNA genes were identified. About 80.07 % of the total genes are protein coding with known function while 1729 genes were annotated as hypothetical protein . Among the total, 6596 genes were assigned to COGS. The properties and the statistics of the genome are described in Table 3. The distribution of the genes in COG functional categories  is shown in Table 4. Circular genome maps, showed in Fig. 3, were generated using CGview  based on ORFs with COG information, tRNA, rRNA and GC content.
Insights from the genome sequence
The haloacid utilizing operon, comprising dehalogenase deh4a and permease deh4p genes, was found in replicon CP012747. Besides deh4a, eight other genes are annotated as haloacid dehalogenase or haloacid dehalogenase-like protein for the whole genome. However, in previous studies, when MBA4 was grown in medium containing MCA as the sole carbon and energy source, only Deh4a was detected. A BLASTN analysis showed that these other genes have relatively different nucleotide sequences and which suggested that they are not homologs of deh4a. It would be interesting to investigate whether these putative dehalogenases have similar function as Deh4a. When MCA is taken into the cell and processed by Deh4a hydrolytically, glycolate will be produced. Further transformation of glycolate will be mediated by glycolate oxidase, an enzyme that consists of three subunits, viz GlcD, E and F. The genes encoding for glycolate oxidase are clustered as an operon. In MBA4, three glycolate oxidase operons were identified. One of these is located downstream of deh4a, in replicon CP012747. This operon has a downstream malate synthase gene, glcB, and an upstream regulator gene, glcC, in the opposite strand. Another glcDEF, also containing an upstream glcC, was discovered in replicon CP012748. A third glycolate oxidase operon, located in replicon CP012746, has neither glcC nor glcB in the neighborhood (Fig. 4). It is apparent that glycolate could be utilized in three ways after transformation to glyoxylate by glycolate oxidase. Whether these three glycolate oxidases are responsible for three different courses awaits further investigation.
For other features of the genome, 612 tandem repeats were found in the genome by Tandem Repeats Finder . There are at least 58 genomic islands being predicted by IslandViewer . On-line CRISPRFinder  has identified ten CRISPR regions with one confirmed and nine questionable CRISPRs. Four incomplete and one questionable prophage regions were identified using PHAST .
In this study, we report the complete genome sequence of Burkholderia caribensis MBA4 which was isolated for its ability to utilize haloacetates. Examination of genes such as dehalogenases and glycolate oxidases have provided insight on the metabolism of the bacterium in transforming haloacetates for carbon and energy source. Further analysis on genes related to conversion of halopropionate would be fruitful.
Luria-Bertani broth without NaCl
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We thank M. C. Fung, Y. P. Chan, S. Lok, A. Tong, N. Lin, J. Jiang, F. C. C. Leung, and the University Centre for Genomic Sciences (previously Genome Research Centre) for advice. We thank Department of Biology, The Chinese University of Hong Kong for fatty acid profile analysis. This work has been supported by grants from the University Small Project Funding 2010 and 2013, and the General Research Fund (project number HKU 780511) of the Research Grants Council of the Hong Kong Special Administrative Region, China.
The authors declare that they have no competing interests.
KFK carried out the molecular biology study. YP conducted the assembly, annotation, data analysis and draft the manuscript. JSHT conducted the data analysis, conceived of the study, and participated in its design and coordination and draft the manuscript. All authors read and approved the final manuscript.