Draft genome sequence of Bosea sp. WAO an arsenite and sulfide oxidizer isolated from a pyrite rock outcrop in New Jersey
© The Author(s). 2018
Received: 19 July 2017
Accepted: 21 March 2018
Published: 10 April 2018
This genome report describes the draft genome and physiological characteristics of Bosea sp. WAO (=DSM 102914), a novel strain of the genus Bosea in the family Bradyrhizobiaceae. Bosea sp. WAO was isolated from pulverized pyritic shale containing elevated levels of arsenic. This aerobic, gram negative microorganism is capable of facultative chemolithoautotrophic growth under aerobic conditions by oxidizing the electron donors arsenite, elemental sulfur, thiosulfate, polysulfide, and amorphous sulfur. The draft genome is of a single circular chromosome 6,125,776 bp long consisting of 21 scaffolds with a G + C content of 66.84%. A total 5727 genes were predicted of which 5665 or 98.92% are protein-coding genes and 62 RNA genes. We identified the genes aioA and aioB, which encode the large and small subunits of the arsenic oxidase respectively. We also identified the genes for the complete sulfur oxidation pathway sox which is used to oxidize thiosulfate to sulfate.
Bosea sp. WAO (white arsenic oxidizer) was enriched from a pulverized sample of weathered black shale obtained from an outcropping near Trenton, NJ that contained high levels of arsenic . Bosea sp. WAO belongs to the class Alphaproteobacteria and family Bradyrhizobiaceae which currently consists of 12 genera: Bradyrhizobium , Afipia , Agromonas , Balneimonas , Blastobacter , Bosea , Nitrobacter , Oligotropha , Rhodoblastus , Rhodopseudomomonas, Salinarimonas , and Tardiphaga . This phenotypically diverse family is composed of microorganisms that are involved in nitrogen cycling, human diseases, phototropism in non-sulfur environments, plant commensalism, and chemolithoautotrophic growth . 16S rRNA gene analysis of the Bradyrhizobiaceae family indicates that the Bosea genus is most closely related to the genus Salinarimonas which currently consists of two species, Salinarimonas rosea and Salinarmonas ramus . The microorganisms belonging to the genus Bosea have been isolated from a variety of environments such as soils, sediments, hospital water systems, and digester sludge [3–5]. The type strain Bosea thiooxidans BI-42Tis capable of thiosulfate oxidation and the initial genus definition included this characteristic . In 2003 La Scola emended the genus description to remove thiosulfate oxidation as a key descriptor after isolation of several other Bosea spp. that were unable to oxidized thiosulfate . These organisms have a very diverse metabolism but their common characteristics include being Gram-negative, aerobic, rod shaped, motile, good growth between 25 to 35 °C, intolerant to salt concentrations above 6% NaCl and have been described to be heterotrophic [3–5]. Using selective enrichment and isolation techniques with arsenite [As(III)] as the sole electron donor Bosea sp. WAO was isolated under autotrophic conditions . Here we summarize the physiological features together with the draft genome sequence and data analysis of Bosea sp. WAO.
Classification and features
Classification and general features of Bosea sp. WAO 
Species Bosea sp.
Strain: WAO (DSM 102914)
pH range; Optimum
D-glucose, lactose, acetate, bicarbonate
Terrestrial, Black shale
No growth with > 3.5% NaCl (w/v)
Lockatong formation, New Jersey, USA
Extended feature descriptions
Strain WAO is a strict aerobe that can grow heterotrophically on acetate, glucose, and lactate in addition to autotrophically on carbon dioxide with the electron donors arsenite, thiosulfate, polysulfide, and elemental sulfur. The organism is also able to grow on the mineral arsenopyrite (FeAsS) by oxidizing both the arsenic and sulfur to produce sulfate and arsenate. No growth was observed under aerobic conditions with the aromatic compounds phenol, benzoate or ferulic acid or with the electron donors sulfite, ammonium, nitrite, selenite, or chromium(III). This organism was enriched from pulverized black shale that contained high levels of arsenic. The initial enrichment cultures using the shale material were amended with 5 mM arsenite and then serially diluted until purity was obtained .
Genome sequencing information
Genome project history
Illumina Genome Analyzer IIX
CLC Genomics Workbench 5.1
Gene calling method
GenBank Date of Release
January 8, 2016
Source Material Identifier
Environmental, biogeochemical cycling of arsenic and sulfur
Growth conditions and genomic DNA preparation
A culture of Bosea sp. WAO (GeneBank: DQ986321.1, DSM 102914) was grown in a dilute (50% normal strength) trypticase soy broth amended with 5 mM sodium arsenite and 5 mM sodium thiosulfate then incubated at 30 °C on an orbital shaker for maximum oxygen exchange. Once turbid genomic DNA was extracted using the MoBio Powersoil Kit following manufacturer’s directions with the modification that DNA was eluted into 100 uL water instead of buffer.
Genome sequencing and assembly
A paired-end library was constructed using an Illumina Nextera Kit and sequenced using an Illumina Genome Analyzer IIX (Illumina Inc., San Diego, CA). The sequence assembly was performed using the CLC Genomics Workbench 5.1 (CLC Bio, Cambridge, MA). An average coverage of 240× and a mean read length of 106 bp was obtained. The genome was assembled into 42 contigs with no additional gap closures.
Genes were identified using the standard operating procedures of the DOE-JGI Microbial Genome Annotation pipeline  and The RAST Server: Rapid Annotation using subsystem technology [11, 12]. JGI-IMG/ER was used to obtain COG identities and overall statistics of the genome. RAST was used to identify functional genes of interest involved in sulfur and arsenic metabolism.
% of total
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Comparison of basic genome features of Bosea spp.
Genome Size (Mbp)
G + C Content (%)
No. of protein coding genes w/ function prediction
No. of protein coding genes in COGs
IMG Taxon ID
Bosea sp. WAO 
Bosea sp. LC85 
Bosea sp. UNC402CLCol
Bosea lupini DSM 26673 
Bosea sp. OK403
Bosea sp. AAP25
Bosea lathyri DSM 26656 
Bosea sp. 117
Bosea sp. AAP35
Bosea vaviloviae strain SD260
Bosea thiooxidans CGMCC 9174 V5_1
Reduced sulfur compound oxidation
Additional metabolic pathways
The Calvin Cycle consists of 13 enzymatic reactions with the enzyme ribulose-1,5 bisphosphate carboxylase/oxygenase (RuBisCO) responsible for the carbon fixation step . For the initial publication of Bosea sp. WAO the type II ribulose-1,5’bisphosphate carboxylase/oxygenase (RuBisCO) was amplified by traditional PCR [1, 16]. Analysis for the remaining genes of the Calvin-Benson-Bassham Cycle for carbon fixation indicated that all the other required genes were present for carbon fixation to occur. Nine of the available genomes have a match for strain WAO’s ribulose 1,5-bisphosphate carboxylase amino acid sequence: B. thiooxidans CGMCC 9174 V5_1, (85%), B. lathyri DSM 26656T, (86%), B. lupini DSM 26673T, (82%), B. vaviloviae strain SD260, (85%), Bosea sp. 117, (72%), Bosea sp. UNC402CLCol, (85%), Bosea sp. LC85, (84%), Bosea sp. OK403, (87%), and Bosea sp. AAP35, (84%). Since RuBisCO is considered a biomarker for the Calvin Cycle this suggests carbon fixation maybe be widespread in this genus despite the limited experimental evidences.
Additional KEGG analysis indicated incomplete pathways for nitrogen reduction. Bosea sp. WAO possesses some genes for each of the reductive pathways but each is incomplete supporting the observation that no growth occurred when nitrate was provided as an electron acceptor. No genes involved in ammonia oxidation were identified again supporting the absence of growth when cultivated under those conditions . Using IMG/ER Pipeline analysis Bosea sp. WAO was determined to be prototrophic for L-aspartate, L-glutamate, and glycine; auxotrophic for L-lysine, L-alanine, L-phenylalanine, L-tyrosine, L-tryptophan, L-histine, L-arginine, L-isoleucine, L-leucine, and L-valine; and not able to synthesize selenocycteine synthesizer or biotin based on the draft of the genome . Using the SEED viewer Bosea sp. WAO has complete pathways for the: tricarboxylic acid cycle, pentose phosphate pathway, acetyl-coA acetogenesis pathway, methylglyoxal metabolism, dihydroxyacetone kinases, catechol branch of beta-ketoadipate pathway, glycerol and clycerol-3-phosphate uptake and utilization, D-ribose utilization, deoxyribose and deoxynucleoside catabolism, and lactate utilization.
Bosea sp. WAO is able to grow chemolithoautotrophically on both arsenite and reduced sulfur compounds. It was originally enriched from pyritic shale obtained from a rock outcropping containing arsenic in the Lockatong geological formation in the Newark Basin near Trenton, New Jersey . The draft genome is 6.1 Mbps and a G + C content of 66.84%. COG analysis for Bosea sp. WAO assigned a large number of genes to amino acid transport and metabolism (13.76%), transcription (8.13%), inorganic ion transport and metabolism (8.06%), and energy production and conservation (6.97%). Bosea sp. WAO has 53 genes encoding for cytochromes alone. Strain WAO is able to engage in the oxidative part of biogeochemical cycling and grow autotrophically when nutrient conditions are low. When conditions favor heterotrophic growth, however, the organism is able to rapidly increase in biomass and maintain its population under the varying conditions that expected to prevail at an oxic mineral surface.
Technical support was provided by the Rutgers School of Environmental and Biological Sciences Genome Cooperative.
This work was supported in part by the New Jersey Water Resources Research Institute at Rutgers University.
ABW performed the laboratory experiments, analysed the assembled genome sequence data and wrote the draft manuscript. NY and LYY participated in the design of the study and revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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