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Complete Genome Sequence of Desulfurivibrio Alkaliphilus Strain

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Complete Genome Sequence of Desulfurivibrio Alkaliphilus Strain Melton et al. Standards in Genomic Sciences (2016) 11:67 DOI 10.1186/s40793-016-0184-4 EXTENDED GENOME REPORT Open Access Complete genome sequence of Desulfurivibrio alkaliphilus strain AHT2T,a haloalkaliphilic sulfidogen from Egyptian hypersaline alkaline lakes Emily Denise Melton1, Dimitry Y. Sorokin2,3, Lex Overmars1, Olga Chertkov4, Alicia Clum5, Manoj Pillay6, Natalia Ivanova5, Nicole Shapiro5, Nikos C. Kyrpides5,7, Tanja Woyke5, Alla L. Lapidus8 and Gerard Muyzer1* Abstract Desulfurivibrio alkaliphilus strain AHT2T is a strictly anaerobic sulfidogenic haloalkaliphile isolated from a composite sediment sample of eight hypersaline alkaline lakes in the Wadi al Natrun valley in the Egyptian Libyan Desert. D. alkaliphilus AHT2T is Gram-negative and belongs to the family Desulfobulbaceae within the Deltaproteobacteria. Here we report its genome sequence, which contains a 3.10 Mbp chromosome. D. alkaliphilus AHT2T is adapted to survive under highly alkaline and moderately saline conditions and therefore, is relevant to the biotechnology industry and life under extreme conditions. For these reasons, D. alkaliphilus AHT2T was sequenced by the DOE Joint Genome Institute as part of the Community Science Program. Keywords: Deltaproteobacteria, Soda lake, Sediment, Sulfur cycle, Sulfur disproportionation Abbreviations: acsA, Carbon monoxide dehydrogenase; acsB, Acetyl-CoA synthase; acsC, Corrinoid iron-sulfur protein large subunit; Formate DH, Formate dehydrogenase; fhs,Formyl-H4-folate synthase; folD,Formyl-H4folate cyclohydrolase/methylene-H4folate dehydrogenase; mthfr/acsD,Methylene-H4folate reductase/corrinoid iron-sulfur protein small subunit fusion; pulE, Type II secretory pathway ATPase PulE; THF, Tetrahydrofolate; WL, Wood Ljungdahl Introduction understanding how haloalkaliphilic organisms survive Soda lakes are extreme environments with high salinity and thrive under dual extreme conditions. Some meta- and highly alkaline pH values. They are formed in arid bolic processes within the reductive sulfur cycle are regions where high rates of evaporation lead to the ac- more favorable under alkaline pH conditions than cumulation of sodium carbonate salts, which are dom- under circumneutral conditions, such as the dispropor- inant in these distinctive lakes. Soda lakes support an tionation of elemental sulfur [2]. These sulfur redox active microbial sulfur cycle, enhanced by the stability processes are not only relevant in natural haloalkaline of intermediate sulfur species such as thiosulfate and environments, some wastewater and gas desulfurization polysulfides and much lower toxicity of sulfide at these treatment plants are often operated at high salt concen- elevated pH conditions. Correspondingly, a wide variety trations and pH values where haloalkaliphiles play a of anaerobic haloalkaliphiles active in the reductive role in the remediation of the affected areas. Thus, the sulfur cycle have been isolated from these lakes [1]. haloalkaliphile Desulfurivibrio alkaliphilus strain AHT2T Insights into sulfur redox processes will contribute to was sequenced for its relevance to sulfur cycling and the environmental biotechnology sector by the DOE-JGI Community Science Program. * Correspondence: [email protected] 1Microbial Systems Ecology, Department of Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands Full list of author information is available at the end of the article © 2016 The Author(s). 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. Melton et al. Standards in Genomic Sciences (2016) 11:67 Page 2 of 9 Organism information Genome sequencing information Classification and features Genome project history D. alkaliphilus AHT2T is the type strain of the Desul- D. alkaliphilus AHT2T was sequenced by the DOE Joint furivibrio alkaliphilus speciesandwasisolatedfroma Genome Institute [5] based on its relevance to the bio- mixed sediment sample from eight hypersaline alkaline technology industry. It is part of the Community Sci- lakes in the Wadi al Natrun valley in the Libyan Desert ence Program (CSP_788492) entitled ‘Haloalkaliphilic (Egypt) [3]. The cells are Gram-negative, non-motile, sulfate-, thiosulfate- and sulfur-reducing bacteria’.The curved rods that do not form spores (Fig. 1). D. alkali- project is registered in the Genomes Online Database philus AHT2T tolerates sodium carbonate concentra- (Ga0028523) [6] and the complete genome sequence is tions ranging from 0.2 - 2.5 M total Na+ and grows deposited in GenBank (GCA_000092205). Sequencing within a pH range of 8.5 - 10.3 (optimum at pH 9.5) and assembly were performed at the DOE Joint Genome [3]. Phylogenetic analysis showed that the strain be- Institute using state of the art sequencing technology longs to the family Desulfobulbaceae within the Delta- [7]. A summary of the project information is shown proteobacteria and is most closely related to a, so far in Table 2. undescribed, haloalkaliphilic chemoautotrophic sulfur- disproportionator within the same genus: Desulfurivi- Growth conditions and genomic DNA preparation brio sp. strain AMeS2 [2]. Strains AMeS2 and AHT2T D. alkaliphilus AHT2T was grown anaerobically at 30 °C are, so far, the only known representatives of the Desul- in Na-carbonate buffered mineral medium containing + furivibrio genus (Fig. 2). The closest sequenced relative 0.6 M total Na with a pH of 10. 4 mM NH4Cl, 1 mM −1 to this novel genus, is another soda lake isolate delta MgCl2 x6H2O, 1 ml L trace element solution [8], 2 mM proteobacterium sp. MLMS-1, which has been enriched Na-acetate as C-source and ~5 g/L powdered sulfur (elec- as an arsenate-dependent sulfide oxidizer [4]. D. alkali- tron acceptor) were added after sterilization. 2 L culture philus AHT2T is able to reduce thiosulfate and elemen- was grown in a 10 L bottle mounted on a magnetic stirrer tal sulfur [3] and plays a roleinthereductivesulfur with an 0.5 bar H2 (electron donor) overpressure head- cycle in soda lake environments [1]. D. alkaliphilus space. The cells from 1 L culture were harvested by centri- AHT2T is also capable of chemolithoautotrophic fugation at 13,000 g for 30 min, washed with 1 M NaCl growth through the disproportionation of elemental and stored at −80 °C. The DNA was extracted and purified sulfur under alkaline pH conditions without iron(III) from frozen pellets by the phenol-chloroform method oxides [2], which are normally required by neutrophilic after pre-treatment with SDS-proteinase K according to sulfur disproportionators. More classifications and fea- Murmur [9]. The purity and molecular weight of the tures are listed in Table 1. DNA was checked by UV spectroscopy and gel electro- phoresis, respectively. Genome sequencing and assembly A The total size of the D. alkaliphilus AHT2T genome sequence assembly was 3.1 Mbp. The draft genome of D. alkaliphilus AHT2T was generated at the DOE Joint Genome Institute using a combination of Illumina [10] and 454 DNA sequencing technologies [11]. An Illumina 10 µm GAii shotgun library was constructed, which generated 3,998,684 reads and a 454 Titanium standard library, which generated 517,041 reads totalling 123.6 Mb of 454 data. The initial draft assembly contained 57 contigs in 1 B scaffold. The 454 Titanium data were assembled with Newbler, 2.0.00.20-PostRelease-11-05-2008-gcc-3.4.6. The Newbler consensus sequences were computationally shredded into 2 kb overlapping fake reads (shreds). Illumina sequencing data was assembled with VELVET, version 1.0.13 [12], and the consensus sequences were 1 µm computationally shredded into 1.5 kb overlapping fake reads (shreds). We integrated the 454 Newbler consensus Fig. 1 Morphology of D. alkaliphilus AHT2T. a A phase contrast shreds and the Illumina VELVET consensus shreds using T micrograph of the D. alkaliphilus AHT2 cells. b A scanning parallel Phrap, version SPS - 4.24 (High Performance electron microscope image of the D. alkaliphilus AHT2T cells Software, LLC). The software Consed [13] was used in Melton et al. Standards in Genomic Sciences (2016) 11:67 Page 3 of 9 Bootstrap > 80 % Desulforhopalus singaporensis (AF118453) Desulforhopalus vacuolatus (L42613) Desulfotalea arctica (AF099061) Desulfopila aestuarii (AB110542) Desulfofustis glycolicus (X99707) Desulfocapsa sulfexigens DSM 10523 (CP003985) Desulfocapsa thiozymogenes (X95181) Desulfobulbus elongatus (X95180) 0.01 Desulfobulbus propionicus (AY548789) Desulfobulbus japonicus (AB110549) Desulfobulbus mediterraneus (AJ866934) Desulfurivibrio sp. AMeS2 (KF148062) Desulfurivibrio alkaliphilus AHT2T (EF422413) Delta proteobacterium MLMS1 (AY459365) Desulfosarcina variabilis (M26632) Desulfonema magnum ( U45989) Desulfatibacillum alkenivorans (AY493562) Desulfosalsimonas propionicica (DQ067422) Syntrophobacter fumaroxidans MPOB (CP000478) Syntrophobacter pfennigii (X82875) Syntrophobacter sulfatireducens (AY651787) Fig. 2 Neighbour joining tree based on 16S rRNA gene sequences showing the phylogenetic position of
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