Walczak et al. Standards in Genomic Sciences (2018) 13:6 https://doi.org/10.1186/s40793-018-0312-4 EXTENDED GENOME REPORT Open Access Draft genome sequence of Bosea sp. WAO an arsenite and sulfide oxidizer isolated from a pyrite rock outcrop in New Jersey Alexandra B. Walczak1*, Nathan Yee2 and Lily Y. Young2 Abstract 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. Keywords: Neutrophilic sulfur oxidizer, Sox, Arsenite oxidase gene, Aio,Geomicrobiology,Microbe-mineral interactions, carbon fixation RuBisCO Introduction the Bosea genus is most closely related to the genus Bosea sp. WAO (white arsenic oxidizer) was enriched Salinarimonas which currently consists of two from a pulverized sample of weathered black shale species, Salinarimonas rosea and Salinarmonas ramus obtained from an outcropping near Trenton, NJ that [2]. The microorganisms belonging to the genus contained high levels of arsenic [1]. Bosea sp. WAO Bosea have been isolated from a variety of environ- belongs to the class Alphaproteobacteria and family ments such as soils, sediments, hospital water Bradyrhizobiaceae which currently consists of 12 gen- systems, and digester sludge [3–5]. The type strain era: Bradyrhizobium, Afipia, Agromonas, Balneimonas, Bosea thiooxidans BI-42Tis capable of thiosulfate Blastobacter, Bosea, Nitrobacter, Oligotropha, Rhodo- oxidation and the initial genus definition included blastus, Rhodopseudomomonas, Salinarimonas,and this characteristic [3]. In 2003 La Scola emended the Tardiphaga [2]. This phenotypically diverse family is genus description to remove thiosulfate oxidation as a composed of microorganisms that are involved in key descriptor after isolation of several other Bosea nitrogen cycling, human diseases, phototropism in spp. that were unable to oxidized thiosulfate [4]. non-sulfur environments, plant commensalism, and These organisms have a very diverse metabolism but chemolithoautotrophic growth [2]. 16S rRNA gene their common characteristics include being Gram- analysis of the Bradyrhizobiaceae family indicates that negative, aerobic, rod shaped, motile, good growth be- tween 25 to 35 °C, intolerant to salt concentrations * Correspondence: [email protected] above 6% NaCl and have been described to be hetero- 1Division of Life Sciences, Rutgers University, The State University of New trophic [3–5]. Using selective enrichment and isola- Jersey, Piscataway, New Jersey, USA tion techniques with arsenite [As(III)] as the sole Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Walczak et al. Standards in Genomic Sciences (2018) 13:6 Page 2 of 12 electron donor Bosea sp. WAO was isolated under (98.88%), B. massiliensis 63287T (98.81%), B. autotrophic conditions [1]. Here we summarize the minatitlanensis AMX51T (98.48%) and B. lathyri R- physiological features together with the draft genome 46060T (98.18%). Phylogenetic analysis based on the sequence and data analysis of Bosea sp. WAO. 16S rRNA gene of Bosea spp. and phylogenetically related organisms placed Bosea sp. WAO closest to Organism information the type strain B. lupini DSM 26673T with B. Classification and features vestrisii 34635T and B. eneae 34614T in the same The genus Bosea has nine species with validly pub- cluster (Fig. 1,Table1). An average nucleotide lished names isolated from various environments: B. identity analysis (ANI) score between strain WAO thiooxidans BI-42T (AF508803) from agricultural soil and B. lupini DSM 26673T of 84.64% was computed [3], B. eneae 34614T (AF288300), B. vestrisii 34635T using IMG/ER [9]. This value is lower than the ANI (AF288306), and B. massiliensis 63287T (AF288309) species demarcation threshold range (95–96%) [10]. from a hospital water system [4], B. minatitlanensis To further identify Bosea sp. WAO to the species AMX51T (AF273081) from anaerobic digester sludge level phylogenic trees based on the housekeeping [5] B. lupini R-45681T (FR774992), B. lathyri R- genes atpD, dnaK, recA, gyrB and rpoB were 46060T (FR774993), and B. robiniae R-46070T produced from available Bosea and related (FR774994) from the root nodules of legumes [6], Bradyrhizobiaceae type strains using MEGA7 (Figs. 2, and B. vaviloviae Vaf-18T(KJ848741) from the root 3, 4, 5, 6 and 7). Strain WAO did not consistently nodules of Vavilovia formosa [7]. Strain WAO’s group with any of the type strains for all five genes previously published identity was confirmed using further suggesting that it is a separate species. The ability the EzTaxon server [8]. The highest 16S rRNA of B. lupini to oxidize thiosulfate has not been determined pairwise similarities for strain WAO were found with [6]; however, B. vestrisii, B. eneae,andB. massiliensis have thetypestrainsB. vestrisii 34635T (99.72%), B. eneae been determined to not oxidize thiosulfate to sulfate [4]. 34614T (99.65%), B. lupini R-45681T (99.65%), B. These results suggest that strain WAO represents a thiooxidans BI-42T (99.24%), B. robiniae R-46070T distinct species in the genus Bosea. Fig. 1 Molecular Phylogenetic analysis by Maximum Likelihood method of the 16S rRNA gene. A Phylogenetic tree highlighting the position of Bosea sp. WAO relative to the other Bosea spp. based on the 16 s rRNA gene. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model [19]. The tree with the highest log likelihood (− 4792.5378) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 19 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1376 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [20]. Type strains are indicated with a superscript T Walczak et al. Standards in Genomic Sciences (2018) 13:6 Page 3 of 12 Table 1 Classification and general features of Bosea sp. WAO [22] MIGS ID Property Term Evidence codea Classification Domain Bacteria TAS [23, 24] Phylum Proteobacteria TAS [25] Class Alphaproteobacteria TAS [26, 27] Order Rhizobiales TAS [27, 28] Family Bradyrhizobiaceae TAS [27, 29] Genus Bosea TAS [3, 30] Species Bosea sp. TAS [24] Strain: WAO (DSM 102914) TAS [1] Gram stain Negative IDA Cell shape Rod TAS [1] Motility Motile TAS [1] Sporulation Not reported NAS Temperature range Mesophile IDA Optimum temperature 25–30 °C IDA pH range; Optimum 6–9; 8 IDA Carbon source D-glucose, lactose, acetate, bicarbonate TAS [1] MIGS-6 Habitat Terrestrial, Black shale TAS [1] MIGS-6.3 Salinity No growth with > 3.5% NaCl (w/v) IDA MIGS-22 Oxygen requirement Aerobic TAS [1] MIGS-15 Biotic relationship free-living TAS [1] MIGS-14 Pathogenicity Not reported NAS MIGS-4 Geographic location Lockatong formation, New Jersey, USA TAS [1] MIGS-5 Sample collection 2005 IDA MIGS-4.1 Latitude 40.289329 IDA MIGS-4.2 Longitude − 74.814366 IDA MIGS-4.4 Altitude 60 m IDA These evidence codes are from the Gene Ontology project [31] aEvidence codes IDA Inferred from Direct Assay, TAS Traceable Author Statement (i.e., a direct report exists in the literature), NAS Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence) Extended feature descriptions The organism is also able to grow on the mineral arseno- Bosea sp. WAO cells are Gram-negative, aerobic, motile, pyrite (FeAsS) by oxidizing both the arsenic and sulfur to and rod shaped. Colonies on trypticase soy agar are produce sulfate and arsenate. No growth was observed smooth, mucoid, round, convex, and beige with a diameter under aerobic conditions with the aromatic compounds as large as 10 mm after 2 weeks at 30 °C. Colonies on min- phenol, benzoate or ferulic acid or with the electron donors imal salts medium supplemented with 5 mM sodium thio- sulfite, ammonium, nitrite, selenite, or chromium(III). This sulfate are smooth, round, white and only grow to a organism was enriched from pulverized black shale that diameter of 2 mm after 2 weeks at 30 °C. Optimal growth contained high levels of arsenic. The initial enrichment cul- occurs at a temperature range from + 25 to 30 °C and pH 6 tures using the shale material were amended with 5 mM ar- to9withanoptimumatpH8(Table1).