The complete genome sequence and emendation of the hyperthermophilic, obligate iron-reducing archaeon “Geoglobus ahangari” strain 234T

Michael Manzella; Dawn Holmes; Jessica Rocheleau; Amanda Chung; Gemma Reguera; Kazem Kashefi

doi:10.1186/s40793-015-0035-8

The complete genome sequence and emendation of the hyperthermophilic, obligate iron-reducing archaeon “Geoglobus ahangari” strain 234T

Manzella, Michael; Holmes, Dawn; Rocheleau, Jessica; Chung, Amanda; Reguera, Gemma; Kashefi, Kazem 2015-10-09 00:00:00 “Geoglobus ahangari” strain 234 is an obligate Fe(III)-reducing member of the Archaeoglobales, within the archaeal phylum Euryarchaeota, isolated from the Guaymas Basin hydrothermal system. It grows optimally at 88 °C by coupling the reduction of Fe(III) oxides to the oxidation of a wide range of compounds, including long-chain fatty acids, and also grows autotrophically with hydrogen and Fe(III). It is the first archaeon reported to use a direct contact mechanism for Fe(III) oxide reduction, relying on a single archaellum for locomotion, numerous curled extracellular appendages for attachment, and outer-surface heme-containing proteins for electron transfer to the insoluble Fe(III) oxides. Here we describe the annotation of the genome of “G. ahangari” strain 234 and identify components critical to its versatility in electron donor utilization and obligate Fe(III) respiratory metabolism at high temperatures. The genome comprises a single, circular chromosome of 1,770,093 base pairs containing 2034 protein-coding genes and 52 RNA genes. In addition, emended descriptions of the genus “Geoglobus” and species “G. ahangari” are described. Keywords: Euryarchaeota, Archaeoglobales, Hydrothermal vent, Guaymas basin, Fe(III) respiration, Extracellular electron transfer, Autotroph Introduction oxidation of a wide range of carbon compounds includ- “Geoglobus ahangari” strain 234 is the type strain and ing long-chain fatty acids such as stearate and palmitate, one of only two known members of the Geoglobus genus which were previously not known to be used as elec- within the order Archaeoglobales and the family Archae- tron donors by archaea [1]. It was also the first hyper- oglobaceae. It is an obligate Fe(III)-reducing archaeon thermophile reported to fully oxidize acetate to CO ,a isolated from the Guaymas Basin hydrothermal system metabolic function once thought to occur solely in and grows at temperatures ranging from 65–90 °C, with mesophilic environments [3]. Unlike the other two an optimum at about 88 °C [1]. It was the first isolate in genera in the order Archaeoglobales (Archaeoglobus a novel genus within the Archaeoglobales and the first and Ferroglobus), which can utilize acceptors such as example of a dissimilatory Fe(III)-reducer able to grow sulfate and nitrate [1, 4–10], the two cultured members autotrophically with H [1], a metabolic trait later shown of the genus Geoglobus can only use Fe(III) as an elec- to be conserved in many hyperthermophilic Fe(III) re- tron acceptor [1, 4]. The obligate nature of Fe(III) respir- ducers [2]. “G. ahangari” can also couple the reduction ation in Geoglobus spp. makes the genus an attractive of soluble and insoluble Fe(III) acceptors to the model to gain insights into the evolutionary mechanisms that may have led to the loss and/or gain of genes involved * Correspondence: [email protected] in the respiration of iron and other electron acceptors Department of Microbiology and Molecular Genetics, Michigan State such as sulfur- and nitrogen-containing compounds University, East Lansing, MI, USA within the Archaeoglobales. Full list of author information is available at the end of the article © 2015 Manzella et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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. Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 2 of 19 “G. ahangari” strain 234 also serves as a model or- that make this organism a good model system to study ganism for mechanistic studies of iron reduction at high Fe(III) reduction in hot environments and to gain in- (>85 °C) temperatures. Dissimilatory Fe(III) reduction sights into the evolution of Fe(III) respiration in the has been extensively studied in mesophilic bacteria family Archaeoglobales. (reviewed in references [11, 12]). By contrast, little is known about the mechanisms that allow (hyper)thermo- Organism information philic organisms to respire Fe(III) acceptors [13–18]. As Classification and features previously observed in the thermophilic Gram-positive “Geoglobus ahangari” strain 234 is a euryarchaeon ori- bacterium Carboxydothermus ferrireducens [13], “G. ginally isolated from samples obtained from a hydrother- ahangari” also needs to directly contact the insoluble mal chimney located within the Guaymas Basin (27° N, Fe(III) oxides to transfer respiratory electrons [14]. In 111° W) at a depth of 2000 m [1]. The sequence of the “G. ahangari”, cells are motile via a single archaellum, single 16S rRNA gene found in its genome was 99 % identi- which could help in locating the oxides, and also express cal to the previously published 16S rDNA sequence numerous curled extracellular appendages, which bind (AF220165). The full length 16S rRNA gene (1485 bp) was the mineral particles and position them close to heme- used to construct a phylogenetic tree in reference to 16S containing proteins on the outer surface of the cell to rRNA gene sequences from other hyperthermophilic ar- facilitate electron transfer [14]. A direct contact mechan- chaea using two thermophilic bacteria (“Aquifex aeolicus” ism such as this is predicted to confer on these organ- and Pseudothermotoga thermarum)asoutgroups (Fig.1). isms a competitive advantage over other organisms The closest known relative was Geoglobus acetivorans relying on soluble mediators such as metal chelators (97 % identical), the only other known member of the [19] and electron shuttles [20, 21], which are energetic- Geoglobus genus, which also is available in pure culture ally expensive to synthesize and are easily diluted or lost [4]. Closest relatives outside the genus were other hyper- in the environment once excreted [22]. This is particu- thermophilic archaea within the family Archaeoglobaceae larly important in hydrothermal vent systems such as such as the sulfate-reducing Archaeoglobus species A. ful- the Guaymas basin chimney where “G. ahangari” strain gidus and A. profundus (97 and 93 % identical, respect- 234 was isolated, as vent fluids in these systems can ively) and Ferroglobus placidus (94 % identical), which can flow through at rates as high as 2 m/s [23]. Here, we re- reduce Fe(III), thiosulfate, and nitrate [3, 5]. port the complete genome sequence of “G. ahangari” Cells of “G. ahangari” strain 234 are regular to irregu- strain 234 and summarize the physiological features lar cocci, 0.3 to 0.5 μm in diameter, and usually arranged Fig. 1 Phylogenetic tree. The phylogenetic tree was constructed with the maximum likelihood algorithm comparing the16S rRNA gene sequence from “G. ahangari” to other hyperthermophilic archaea. Bootstrap values were determined from 100 replicates and “Aquifex aeolicus” and Pseudothermotoga thermarum were used as outgroups Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 3 of 19 respiratory metabolisms in the Archaeoglobales,and, in particular, about hyperthermophilic iron reduction within the Archaea. The genome project information is listed in the Genomes OnLine Database (Gp0101274) [24] and the complete genome sequence has been deposited in GenBank (CP011267). A summary of the project infor- mation is shown in Table 2. Growth conditions and genomic DNA preparation “G. ahangari” strain 234 was from our private culture collection and is available at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM-27542), the Japan Collection of Microorganisms (JCM 12378), and the American Type Culture Collection (BAA-425). The strain was grown in marine medium [1] with 10 mM Fig. 2 Scanning electron micrograph of cells of “G. ahangari” strain 234 growing on insoluble Fe(III) oxides. Bar, 100 nm pyruvate as the electron donor and 56 mM ferric citrate as the electron acceptor. Cultures were incubated under a N :CO (80:20 %, v/v) atmosphere at 80 °C or 85 °C in the 2 2 dark. Strict anaerobic techniques were used throughout as single cells or in pairs (Fig. 2 and Table 1) [1]. Cells the culturing and sampling experiments [25]. are motile via a single archaellum [1], but also produce gDNA was extracted as previously reported for F. abundant extracellular curled filaments when grown placidus [26]. Alternatively, cells were lysed with an with both soluble and insoluble Fe(III) [14]. Though SDS-containing lysis buffer (5 % SDS, 0.125 M EDTA, optimum growth occurs at ca. 88 °C, growth is observed 0.5 M Tris, pH 9.4), as reported elsewhere for the between 65 and 90 °C [1]. Furthermore, growth was sup- preparation of whole cell extracts from “G. ahangari” ported at pH values between 5.0 and 7.6, with an [14], and gDNA was extracted using the MasterPure™ optimum at pH 7.0, and with NaCl concentrations ran- DNA Purification Kit (EPICENTRE® Biotechnologies), ging from 9 to 38 g/L, with an optimum at 19 g/L [1]. according to the manufacturer suggested guidelines. A distinctive feature of the metabolism of “G. ahan- gari” strain 234 is its obligate nature of Fe(III) respir- Genome sequencing and assembly ation, with both soluble and insoluble Fe(III) species The finished genome of “G. ahangari” strain 234 (CP supporting growth but the insoluble electron acceptor 011267) was generated from Illumina [27] draft sequences being preferred [1]. The obligate nature of Fe(III) reduc- generated independently at the Research Support and tion contrasts with the wide range of electron donors Training Facility at Michigan State University, the Deep that “G. ahangari” can oxidize [1]. Acetate, alongside a Sequencing Core Facility at the University of Massachu- number of other organic acids (such as propionate, bu- setts Medical School, and the Genomics Resource lab at tyrate, and valerate), several amino acids, and both the University of Massachusetts-Amherst. Table 2 pre- short-chain and long-chain fatty acids were completely sents the project information. oxidized to CO to support growth during Fe(III) respir- The sequencing project at the University of Massachusetts ation [1, 3]. Furthermore, “G. ahangari” strain 234 was facilities used gDNA suspended in 3 ml of sonication also able to grow autotrophically with H as the sole buffer (4.95 % glycerol, 10 mM Tris–HCl (pH 8.0), 1 mM electron donor and Fe(III) as the electron acceptor [1]. EDTA) and sonicated for 10 min (2 min on, 30 s off) using The main physiological features of the organism are a 550 Sonic Dismembrator (Fisher Scientific). The samples listed in Table 1. were then dispensed as equal volumes into 4 tubes and mixed with 150 μl TE buffer (10 mM Tris–HCl (pH 8.0), 1mM EDTA),100 μl ammonium acetate (5 M), 20 μl Genome sequencing and annotation glycogen (5 mg/ml), and 1 ml of cold (−20 °C) isopropa- Genome project history nol. Nucleic acids were precipitated at −30 °C for 1 h, as Based on its unique physiological characteristics [1] previously described [26] and suspended in EB buffer and use as a model system for mechanistic investiga- (Qiagen) before separating the DNA fragments in the tions of Fe(III) reduction by hyperthermophilic ar- sample by agarose gel electrophoresis. DNA fragments be- chaea [14], “G. ahangari” strain 234 was selected for tween 300–500 bp were then purified with the QiaQuick sequencing. Insights from its genome sequence and an- Gel Extraction Kit (Qiagen). All steps involved in end re- notation provide greater understanding of the evolution of pair, 3’ adenylation, adaptor ligation, and purification of Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 4 of 19 Table 1 Classification and general features of “G. ahangari” 234 according to the MIGS recommendations [106] MIGS ID Property Term Evidence code Current classification Domain Archaea TAS [107] Phylum Euryarchaeota TAS [107, 108] Class Archaeoglobi TAS [109] Order Archaeoglobales TAS [110] Family Archaeoglobaceae TAS [111] Genus Geoglobus TAS [1] Species “Geoglobus ahangari” TAS [1] Type strain 234 TAS [1] Gram stain Variable NAS Cell shape Irregular coccus TAS [1] Motility Motile TAS [1] Sporulation Non-sporulating NAS Temperature range 65−90 °C TAS [1] Optimal temperature 88 °C TAS [1] pH range; Optimum 5.0−7.6 (optimum 7.0) TAS [1] Carbon source CO TAS [1] Energy metabolism Chemolithoautotrophic, TAS [1] chemolithotrophic, chemoorganotrophic MIGS-6 Habitat Marine geothermally TAS [1] heated areas MIGS-6.3 Salinity 9.0−38 g/L NaCl TAS [1] MIGS-22 Oxygen requirement Anaerobe TAS [1] MIGS-15 Biotopic relationship Free-living TAS [1] MIGS-14 Pathogenicity Non-pathogen NAS Isolation Hydrothermal TAS [1] vent chimney MIGS-4 Geographic location Guaymas Basin TAS [1] hydrothermal system MIGS-5 Sample collection time Unknown NAS MIGS-4.1 Latitude 27° N TAS [1] MIGS-4.2 Longitude 111° W TAS [1] MIGS-4.3 Depth 2000 m TAS [1] MIGS-4.4 Altitude Not applicable Evidence 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). These evidence codes are from the Gene Ontology project [112] Illumina products were performed using reagents sup- 2.2.5). Reads were down-sampled to 500x coverage to plied by the TruSeq DNA Sample Prep Kit (Illumina). increase the efficacy of the Velvet assembler while the This resulted in the construction and sequencing of complete depth of reads was used to verify the final five independent 100 bp paired-end Illumina shotgun genome assembly. libraries which generated 7,970,036, 7,970,182, 7,973,896, A total of 1,780,565 bp were assembled into 25 scaf- 7,966,671 and 3,144,785 reads totaling 3.50 Gbp. The folds ranging in size from 207 bp to 510,180 bp. The Illumina draft sequences were assembled de novo with scaffolds were then connected by adaptor-PCR as previ- SeqMan NGen (DNASTAR) and Velvet [28] (version ously described [29]. “G. ahangari” gDNA was subse- 1.2.10) and optimized with VelvetOptimiser (version quently digested separately by four different restriction Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 5 of 19 Table 2 Genome sequencing project information Qiagen PCR purification kit and sent for sequencing at MIGS ID Property Term the University of Massachusetts (Amherst) sequencing fa- cility. This process was repeated until a single contig was MIGS-31 Finishing Finished quality obtained using SeqMan Pro assembly software. The assembly was then verified against a second, inde- MIGS-28 Libraries used 5 independent 100 bp paired-end Illumina shotgun libraries, 150 bp pendent genome assembly generated at Michigan State paired-end Illumina shotgun library University. Extracted gDNA was used to construct a sin- MIGS-29 Sequencing Illumina MiSeq gle Illumina shotgun library using the Illumina DNAseq platforms Library Kit, which was sequenced in two 150 bp paired- MIGS-31.2 Sequencing 1,977 × coverage (100 bp libraries) end runs with an Illumina MiSeq at the Research Sup- coverage 100 × (150 bp library) port and Training Facility at Michigan State University. MIGS-30 Assemblers SeqMan NGen, Velvet, SeqMan Pro The sequencing project generated 1,233,811 and 796,056 reads totaling 304.5 Mbp. Reads were quality trimmed MIGS-32 Gene calling JGI-ER, GLIMMER method using a combination of fastq-mcf [30] (ea-utils.1.1.2-537, using default parameters) and ConDeTri [31] (v2.2, using INSDC ID CP011267 default parameters with the exception of hq = 33 and Genbank May 11, 2015 Date of sc = 33) to remove low-quality reads and over-represented Release sequences. High-quality paired and unpaired reads GOLD ID Gp0101274 were assembled using Velvet [28] (v1.2.08 using de- fault parameters and a kmer value of 63) to generate NCBI project 258102 ID anew assembly (1.77Mbp,51contigs ≥ 1000 bp, and MIGS-13 Source ATCC BAA-425, DSMZ DSM-27542, JCM an N75 of 37,520 bp). The second assembly was then material JCM 12378 compared to the primary assembly to identify errors identifier and low-coverage regions, which were subsequently Project Phylogenetic diversity, biotechnology, resolved by PCR-amplifying and sequencing the re- relevance evolution of metal respiration in gions of interest. hyperthermophiles, and anaerobic degradation of hydrocarbons Genome annotation Initial genome annotation was performed by the RAST enzymes (EcoRI, BamHI, BclI, and SalI). After a 1.5-h in- server [32], the IGS Annotation Engine [33] at the Uni- cubation at 37 °C (or 50 °C for SalI), the restriction di- versity Of Maryland School Of Medicine, and the IMG- gests were separated by agarose gel electrophoresis and ER platform [34]. The annotations were compared to fragments between 5–10 kb were isolated and purified manual annotations performed using GLIMMER [35] with the QiaQuick gel extraction kit (Qiagen). Adaptor for gene calls and DELTA-BLAST analysis to identify sequences with 3′ overhangs generated by EcoRI, BamHI, conserved domains and homology to known proteins. BclI, and SalI at the phosphorylated 5′ ends were then li- EC numbers and COG categories were determined with gated with T4 DNA ligase to the fragments purified from a combination of DELTA-BLAST analysis of each anno- the restriction digests of “G. ahangari” gDNA. The tated gene and the IMG-ER platform. Pseudogenes were adaptor sequences used were: EcoRI adaptor AATTCCC identified using the GenePRIMP pipeline [36]. The data TATAGTGAGTCGTATTAAC** (phosphorylated at 5′ were used to create a consensus annotation before the end); BclIand BamHI adaptor GATCCCCTATAGTGAG final assembled genome was uploaded onto the IMG-ER TCGTATTAAC**; and finally the SalI adaptor TCGACCC platform. IMG-ER annotations were manually curated TATAGTGAGTCGTATTAAC**. Further assembly was by comparison to the consensus annotation before sub- performed with SeqMan Pro (DNASTAR) and primers mitting the final genome annotation. were designed targeting the 3′ and 5′ ends of the 25 scaf- Potential c-type cytochromes were selected based on folds. The adaptor ligations were diluted 100-fold and 1 μl the presence of c-type heme binding motifs (CXXCH) of the diluted sample was used in PCR reactions with within the amino acid sequence as previously described AccuTaq™ LA DNA Polymerase (50 μl total volume) [37]. Predicted subcellular localization and the presence according to manufacturer specifications (Sigma-Aldrich). of signal peptides and/or an N-terminal membrane helix Fifty reactions were performed with “G. ahangari”-specific anchor [37] was investigated by PsortB [38], PRED-TAT primers designed from the various Illumina scaffolds and [39], TMPred [40], and the TMHMM Server (v. 2.0) a non-phosphorylated primer that complemented the [41]. Putative c-type cytochromes were then examined adaptor sequence on the gDNA (GTTAATACGACTCAC by BLAST analysis to determine homology to known c- TATAGGG). All PCR products were purified with the type cytochromes in the NCBI database. The molecular Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 6 of 19 weight of putative c-type cytochromes was estimated and their percentage representation are listed in Fig. 3 with the ExPASy ProtParam program [42]. The weight and Table 4. of the signal peptide was then subtracted from the pre- The preferred start codon is ATG (83.8 % of the dicted weight and 685 daltons were added for each genes), followed by GTG (10.4 %) and TTG (5.7 %). This heme-binding motif to estimate the molecular weight of distribution is similar to the start codon representation of the mature cytochrome. The predicted molecular weight the other member of the Geoglobus genus, G. acetivorans values and subcellular localization of the mature cyto- (79.4 % ATG, 11.6 % GTG, and 9.0 % TTG) [15] and the chromes were compared to the masses reported for mature closely related archaeon F. placidus (82.5 % ATG, 10.2 % heme-containing proteins present in whole cells and outer- GTG, 6.1 % TTG, and 1.3 % other) [26]. There is one copy surface protein preparations of “G. ahangari” [14]. of each of the rRNA genes but the genes are located in two different regions of the genome: the 16S rRNA (GAH_00462) and 23S rRNA (GAH_00460) genes are Genome properties in the same gene cluster and separated by a span of The genome of “G. ahangari” strain 234 comprises one 139 bp encoding a single tRNA whereas the 5S rRNA circular chromosome with a total size of 1,770,093 bp (GAH_02069) is located 205,273 bp away in a region and does not contain any plasmids. The genome size is with genes coding for proteins with functions unrelated within the range of those reported for other members of to ribosome function and biogenesis. the Archaeoglobales [15, 26, 43–45], and NC_015320.1. Almost all origins of DNA replication identified in Ar- The mol percent G + C is 53.1 %, which is lower than chaea to date are located in close proximity to genes the 58.7 % estimated experimentally via HPLC [1]. Out coding for a homologue of the eukaryotic Cdc6 and of the total 2072 genes annotated in the genome, 52 Orc1 proteins [46]. Interestingly, we identified two genes were identified as RNA genes and 2020 as protein- encoding Orc1/Cdc6 family replication initiation pro- coding genes (Table 3). There are 47 pseudogenes, com- teins (GAH_00094 and GAH_00965) in the genome of prising 2.3 % of the protein-coding genes. Furthermore, “G. ahangari”, thus raising the possibility that the gen- 76.5 % of the predicted genes (1557) are represented by ome contains more than one functional origin of replica- COG functional categories. Distribution of these genes tion. Many archaeal replication origins consist of long intergenic sequences upstream of the cdc6 gene contain- ing an A/T-rich duplex unwinding element flanked by Table 3 Nucleotide content and gene count levels of the several conserved repeat motifs known as ORBs [47]. A genome specific ORB could not be identified in the genome Attribute Value % of total when compared to other archaeal origins of replication Size (bp) 1,770,093 100.0 % available in the DoriC database [48]. However, the 320- Coding region (bp) 1,662,832 93.9 % bp long region upstream of GAH_00965, one of the G + C content (bp) 940,071 53.1 % Orc1/Cdc6 family replication initiation proteins, con- Number of replicons 1 tains a long (111 bp) non-coding intergenic region with Extrachromosomal 0 one AT-rich stretch and 8 direct repeats (3 TCGTGG, 3 elements CGTGGTC, and 2 GGGGATTA), which could function Total genes 2072 100.0 % as a replication origin. Furthermore, the 580-bp region RNA genes 52 2.5 % directly upstream of the other Orc1/Cdc6 family replica- tion initiation protein (GAH_00094) lacks a non-coding rRNA operons 2 intergenic region and/or AT-rich span but contains 8 Protein-coding genes 2034 100.0 % direct repeats (2 GGTTGAGAAG, 3 TGAGAAG, and 3 Pseudogenes 47 2.3 % AACATCCCG) and several “G-string” elements analo- Genes with function 1677 82.4 % gous to ori sites reported for haloarchaeal species [49]. prediction Genes in paralog clusters 1406 69.1 % Insights from the genome Genes assigned to COGs 1470 72.2 % Autotrophic growth with H as electron donor Genes assigned Pfam 1667 82.0 % “G. ahangari” strain 234 was the first dissimilatory Fe domains (III)-reducing hyperthermophile shown to grow autotro- Genes with signal peptides 55 2.7 % phically with H as an electron donor [1]. In its genome, Genes with transmembrane helices 409 20.1 % we identified genes required for the two branches of the reductive acetyl-CoA/Wood-Ljungdahl pathway [50–53], CRISPR repeats 7 a which other members of the Euryarchaeota [54], includ- The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome ing most members of the Archaeoglobales [8, 45, 55–58], Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 7 of 19 Fig. 3 Graphical circular map of the chromosome. From outside to the center: Genes on forward strand (colored by COG categories), genes on reverse strand (colored by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, and GC skew use for carbon fixation. A bifunctional carbon monoxide The genome of “G. ahangari” also contains 29 genes dehydrogenase/acetyl-CoA synthase complex (encoded encoding hydrogenase subunits, maturation proteins, by GAH_01139-01144, and two additional copies of the and a cluster of genes (hypA, hypB, hypC, hypD, and beta and maturation factors encoded by GAH_00919 hypE) involved in biosynthesis and assembly of Ni-Fe hy- and GAH_00306, respectively) are present within the drogenases (GAH_00190-00195). Genes coding for the genome, which could initiate carbon fixation. The bi- large, small, and b-type cytochrome subunits of a Ni-Fe functional nature of this enzyme also allows it to link me- hydrogenase I protein (GAH_00910-00912) were identi- thyl and carbonyl branches and enable acetyl-CoA fied in the genome. We also found a gene cluster biosynthesis, as reported for methanogenic archaea [59]. (GAH_00337-00347) encoding all subunits of a NADH- Complete enzymatic pathways for alternative means of car- quinone oxidoreductase, which transfers electrons to the bon fixation were not identified. quinone membrane pool and may function as the Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 8 of 19 Table 4 Number of genes associated with the 25 general COG functional categories Code Value % age Description J 155 7.6 % Translation, ribosomal structure and biogenesis A 2 0.1 % RNA processing and modification K 68 3.3 % Transcription L 58 2.9 % Replication, recombination and repair B 6 0.3 % Chromatin structure and dynamics D 20 1.0 % Cell cycle control, Cell division, chromosome partitioning Y 0 0.0 % Nuclear structure V 7 0.3 % Defense mechanisms T 24 1.2 % Signal transduction mechanisms M 35 1.7 % Cell wall/membrane biogenesis N 15 0.7 % Cell motility Z 0 0.0 % Cytoskeleton W 0 0.0 % Extracellular structures U 23 1.1 % Intracellular trafficking and secretion O 57 2.8 % Posttranslational modification, protein turnover, chaperones C 160 7.9 % Energy production and conversion G 37 1.8 % Carbohydrate transport and metabolism E 139 6.8 % Amino acid transport and metabolism F 49 2.4 % Nucleotide transport and metabolism H 103 5.1 % Coenzyme transport and metabolism I 55 2.7 % Lipid transport and metabolism P 86 4.2 % Inorganic ion transport and metabolism Q 16 0.8 % Secondary metabolites biosynthesis, transport and catabolism R 244 12.0 % General function prediction only S 198 9.7 % Function unknown - 477 23.5 % Not in COGs The total is based on the total number of protein coding genes in the genome primary generator of the proton-motive force [43]. An- in members of the Archaeoglobales [7–9, 55, 57] but other large cluster of hydrogenase genes (GAH_02036- not in the iron-respiring F. placidus [5] or in “G. ahan- 02044) codes for all coenzyme F hydrogenase subunits gari” [1]. Yet, the “G. ahangari” genome contains genes and proteins involved in recycling coenzyme F ,thus for all coenzyme subunits of the proteins coenzyme replenishing the cofactor for the reductive acetyl-CoA F -reducing hydrogenase (GAH_00337 and GAH_02 5 10 pathway [58, 60–62]. The presence of multiple hydroge- 036-02038), coenzyme F -dependent N ,N -methy- nases is not unusual in iron-reducing microorganisms lene tetrahydromethanopterin reductase (GAH_01605, and allows them to diversify the paths used to transfer GAH_01835), and F synthase (CofGH) (GAH_00662, electrons derived from the oxidation of H to their GAH_00663) [65]. Furthermore, although “G. ahangari” acceptors [63]. cannot produce methane when growing autotrophically Autotrophic growth in methanogens can also be sup- [1], its genome codes for nearly all enzymes responsible ported using reduced coenzyme F as an electron for the reduction of CO to methane [51]. Similar to A. 420 2 donor to produce methane [64]. The distinctive fluores- fulgidus, F. placidus, A. sulfaticallidus,and G. acetivorans, cence emission from this coenzyme has been detected “G. ahangari” has genes encoding all proteins involved in Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 9 of 19 the formation of 5-methyl-tetrahydromethanopterin and a Central metabolism gene coding for one of the 8 subunits (MtrH) of the en- Heterotrophic growth in “G. ahangari” is supported by zyme responsible for the transfer of a methyl group to co- a wide range of organic carbon compounds [1], which enzyme M (GAH_01245). Yet, the genome is missing all serve as electron donor for respiration while also pro- four genes required for a functional coenzyme M reduc- viding carbon for assimilation in the central pathways. tase, the enzyme responsible for the final step of methane Similar to other hyperthermophilic archaeal species [66], production by methanogenic archaea [51]. The fact that the “G. ahangari” genome contains a modified Embden- Archaeoglobale genomes have nearly all of the genes in- Meyherhof-Parnas glycolytic pathway (Fig. 4). The initial volved in methanogenesis and the high level of homology step of glycolysis (glucose phosphorylation to glucose 6- that exists between genes from the reductive acetyl-CoA phosphate) is carried out by an ATP-dependent archaeal pathway in both Archaeoglobales and the methanogenic ar- hexokinase (GAH_00546) belonging to the ROK family of chaea suggests that the Archaeoglobales mayhaveevolved proteins. A gene coding for the phosphoglucose isomerase from a methanogenic archaeon that lost its ability to reduce enzyme, which catalyzes the next reaction in the pathway CO and produce methane over time. (interconversion of the aldose in glucose 6-phosphate Fig. 4 Central metabolism in “Geoglobus ahangari” strain 234 Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 10 of 19 and the ketose in fructose 6-phosphate) was also iden- kinase protein is present in the close relative F. placidus tified (GAH_01135) and was most similar to cupin- (Ferp_0744), homologs were not identified in “G. ahan- type phosphoglucose isomerases from other anaerobic gari” or any other Archaeoglobale species. Instead, the ge- Euryarchaeota, including Archaeoglobus fulgidus [66]. nomes of “G. ahangari” (GAH_00154) and all other In A. fulgidus, fructose 6-phosphate is phosphorylated to sequenced Archaeoglobales species contain genes en- fructose 1,6-bisphosphate by an ADP-dependent phospho- coding PK_C superfamily proteins [15, 33, 50–52, and fructokinase protein (EC:2.7.1.11) [67]. However, homologs NC_015320.1], which have pyruvate kinase and alpha/ of this enzyme were not present in the genomes of “G. beta domains and are homologous to an A. fulgidus en- ahangari” or any other Archaeoglobale species sequenced zyme with pyruvate kinase activity in vitro [72]. Pyruvate to date. Instead, the genome of “G. ahangari” contained can then be converted into acetyl-CoA via pyruvate syn- two genes (GAH_00966 and GAH_01843) coding for thase (GAH_01438-01441 and GAH_02021-02024). proteins with pfkB-like domains and ATP-binding sites, As in the close relative F. placidus [26], “G. ahan- which are consistent with the ATP-dependent phos- gari” lacks genes from the oxidative pentose phosphate phofructokinases (PFK-B) of other hyperthermophilic pathway but is predicted to circumvent this limitation archaea such as Aeropyrum pernix and Desulfurococ- [73] viathe useofa complete RuMP pathway (GAH_0 cus amylolyticus [68, 69]. The genome also contains two 0051 and GAH_01859). The latter results in the accu- genes (GAH_00357 and GAH_01437) encoding archaeal mulation of formaldehyde [73], which in “G. ahangari” fructose 1,6-bisphosphatases, which catalyze the reverse re- could be removed by formaldehyde-activating enzymes action during gluconeogenesis but can also supply fructose (GAH_00575 and GAH_00673). Ribulose 5-phosphate 1,6-bisphosphate to the glycolytic pathway from dihydroxy- formed in the RuMP pathway could then be converted into acetone phosphate and D-glyceraldehyde 3-phosphate [70]. ribose-5-phosphate by a ribose 5-phosphate isomerase and Furthermore, a triosephosphate isomerase (GAH_00576) then into PRPP by ribose-phosphate pyrophosphokinase was identified in the genome to catalyze the isomerization enzymes (GAH_00743 and GAH_00557). This supplies PR of dihydroxyacetone phosphate to D-glyceraldehyde 3- PP to various anabolic pathways such as the biosynthesis phosphate. Alternatively, GAH_01502 and GAH_01751, of histidine and purine/pyrimidine nucleotides. which encode proteins homologous to archaeal type class Similar to other Archaeoglobale species, a complete I fructose 1,6-bisphosphate aldolase proteins, could catalyze TCA cycle is present within the “G. ahangari” All enzymes the conversion of fructose 1,6-bisphosphate into D-glyceral- involved in the formation of oxaloacetate from acetyl-CoA dehyde 3-phosphate. (GAH_00258, GAH_01703, GAH_01110, GAH_02012-020 The next steps in the pathway involve the oxidation 13, GAH_00784-00784, GAH_00779-00782, GAH_00526- of D-glyceraldehyde 3-phosphate and formation of 3- 00527, and GAH_00039), including putative aconitase phosphoglycerate. The “G. ahangari” genome contains proteins (GAH_00857-00858) [74], were identified in a homolog (GAH_00413) of a GAPOR, which in A. the genome. Also present is a phosphoenolpyruvate fulgidus and many other archaeal species catalyzes the carboxylase (GAH_01652), which could catalyze the irreversible oxidation of D-glyceraldehyde-3-phosphate to reversible carboxylation of phosphoenolpyruvate to 3-phospho-D-glycerate bypassing the formation of the oxaloacetate, a precursor metabolite of many amino intermediate 1,3-bisphospho-D-glycerate [66]. In addition, acids. the genome of “G. ahangari” contains genes coding for an archaeal specific type II glyceraldehyde-3-phosphate de- Fatty acids as electron donors hydrogenase (GAH_01734) and a phosphoglycerate kinase “G. ahangari” strain 234 was the first hyperthermophile (GAH_01571), which could catalyze the formation of 3- reported to completely oxidize long-chain fatty acids phosphoglycerate via the 1,3-diphosphoglycerate inter- anaerobically, an unsuspected capability of hyperthermo- mediate. These two enzymes are unidirectional and philic microorganisms prior to this discovery [1]. Long- involved in formation of glyceraldehyde-3-phosphate chain fatty acids are abundant in sedimentary environ- from 3-phosphoglycerate during gluconeogenesis in most ments where they accumulate as byproducts of the hy- hyperthermophilic archaea [66]. drolysis of complex organic matter and the anaerobic As in A. fulgidus [71], “G. ahangari” has 2 genes coding degradation of alkanes [75, 76]. Long-chain fatty acids are for cofactor-independent phosphoglycerate mutase proteins also major components of crude oil [77], which is often (GAH_00739 and GAH_01116), which can catalyze the present in environments inhabited by Archaeoglobale spe- interconversion of 3-phospho-D-glycerate to 2-phospho-D- cies [78]. Consistent with the ability of Archaeoglobale glycerate. Phosphoenolpyruvate is then formed by an members to oxidize long-chain fatty acids, the genomes of enolase protein (GAH_00972), which is subsequently “G. ahangari” and other members of the Archaeoglobales dephosphorylated to pyruvate by pyruvate kinase. Al- (F. placidus, G. acetivorans, A. fulgidus, and others) con- though a gene coding for the well-characterized pyruvate tain a large number of genes coding for β-oxidation Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 11 of 19 pathway enzymes [15, 26, 43]. The A. fulgidus genome, for Degradation of aromatic compounds and n-alkanes example, contains 57 genes encoding the 5 core proteins F. placidus, a member of the Archaeoglobales closely re- (discussed below) involved in β-oxidation [43]. All of these lated to “G. ahangari”, can couple the complete oxidation genes were used as BLAST queries against the genomes of aromatic hydrocarbons to Fe(III) reduction [79–81]. of F. placidus [26],G. acetivorans [15], and “G. ahan- The “G. ahangari” genome does not contain any benzoate gari” and identified 39 homologous proteins in the ge- degradation genes, further supporting the observation that nomes of the F. placidus and G. acetivorans and 32 in it cannot utilize aromatic compounds as electron donors the genome of “G. ahangari”. for growth [1]. Interestingly, G. acetivorans, the other mem- Fatty acid degradation in the Archaeoglobales is thought ber of the Geoglobus genus, has homologues of all genes to occur in a manner similar to bacteria and mitochondria coding for proteins of the benzoyl-CoA ligation pathway [43], with the initial step involving activation of a long present in F. placidus, yet as in “G. ahangari” growth on chain fatty acid to a fatty acyl CoA by a fatty acyl CoA syn- aromatic hydrocarbons has not been observed in G. aceti- thetase/ligase. We identified seven genes in “G. ahangari” vorans [4, 15]. coding for fatty acid CoA synthetase proteins (GAH_00420, Another member of the Archaeoglobales, A. fulgidus, GAH_00623, GAH_01111, GAH_01124, GAH_01769, GA can also couple the oxidation of n-alkanes and n-alkenes H_01899, and GAH_02051). The next step in the pathway with sulfur respiration [82, 83]. This archaeon uses an involves the oxidation of the fatty acyl-CoA to a trans-2- alkylsuccinate synthase and an activating protein (AssD/ enoyl-CoA by acyl-CoA dehydrogenase proteins, which BssD; AF1449-1450) to oxidize saturated hydrocarbons in “G. ahangari” are putatively encoded by 11 genes (n-alkanes in the range of C -C ) [83]. We identified 10 21 (GAH_00179, GAH_00421, GAH_00484, GAH_00591, homologs of both of these proteins in the genome of GAH_01331, GAH_01442, GAH_01601, GAH_01810, “G. ahangari” (GAH_01645-01646) and G. acetivorans and GAH_02050). A water molecule is then added to (Gace_0420-0421). A. fulgidus can also oxidize long trans-2-enoyl-CoA to form (3S)-3-hydroxyacyl-CoA in a chain n-alk-1-enes (C to C ) when thiosulfate is 12:1 21:1 reaction catalyzed by an enoyl-CoA hydratase, which in provided as the terminal electron acceptor [82]. Al- “G. ahangari” could be encoded by 4 genes (GAH_00487, though enzymes involved in the activation of alkenes GAH_00802, GAH_01332, and GAH_01602). Two of these by A. fulgidus have not been characterized, the genome genes (GAH_00487 and GAH_01602) are in fact hybrid of A. fulgidus contains a homologue of a Mo-Fe-S con- proteins containing an enoyl-CoA hydratase domain fused taining enzyme (AF0173-AF0176) [82], which in Azoar- to a 3-hydroxyacyl-CoA dehydrogenase domain. Hybrid cus sp. EBN1 anaerobically hydroxylates a branched enoyl-CoA hydratase/dehydrogenase proteins such as these alkene [84]. The Mo-Fe-S enzyme consists of 4 sub- have been identified in other archaeal species including the units including a chaperonin-like protein, a membrane Archaeoglobales species G. acetivorans, F. placidus,and A. anchor heme-b binding subunit, an Fe-S binding subunit, fulgidus [15, 26, 43]. and a molybdopterin-binding subunit [85]. This gene clus- The next step in the β-oxidation pathway leads to the ter was identified in the genomes of “G. ahangari” formation of 3-oxoacyl-CoA in an oxidation reaction that (GAH_01285-01288) and F. placidus (Ferp_0121-0123), generates NADH and is catalyzed by a 3-hydroxyl-CoA but not in the other member of the Geoglobus genus, dehydrogenase protein, which in “G. ahangari” is likely G. acetivorans. encoded by several genes (GAH_00328, GAH_00487, GA H_01600, GAH_01602 – again noting the hybrid nature of Nitrogen compounds as electron acceptors GAH_00487 and GAH_01602). Finally, acetyl-CoA is re- Except for F. placidus [5, 57], all of the Archaeoglobales, moved from the 3-oxo-acyl-CoA molecule by an acetyl- including “G. ahangari” [1], are unable to use nitrate or ni- CoA acetyltransferase and is free to enter the TCA cycle. trite as electron acceptors for respiration [4, 6–10] There are 8 genes in the “G. ahangari” genome that (Table 5). Yet, surprisingly, the genome of “G. ahangari” could catalyze this reaction (GAH_00292, GAH_00485, contains several 4Fe-4S domain-containing nitrate and GAH_00625, GAH_00626, GAH_01327, GAH_01328, sulfite reductase proteins (GAH_01242 and GAH_02063) GAH_01886, and GAH_02049). Additional proteins in- volved in fatty-acid metabolism include the alpha (GA Table 5 Terminal electron acceptors in the Archaeoglobales H_01318) and beta (GAH_01319) subunits of a 3- Electron acceptors oxoacid CoA-transferase. The large number of genes dedicated to β-oxidation in “G. ahangari” and other spe- Organism Sulfate Sulfite Thiosulfate Nitrate Fe(III) cies within the Archaeoglobales provides genomic evi- Geoglobus spp. -- - - + dence supporting the notion that long- and short-chain Ferroglobus placidus -- + + + fatty acid oxidation is a conserved metabolic feature Archaeoglobus spp. +/− ++ - - within the family. Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 12 of 19 as well as all four subunits (NarGHIJ) of a nitrate reduc- pathway, and together with the GDH pathway, function as tase (GAH_01285-01288). A nitrate/nitrite transporter is the two major paths for ammonium assimilation in ar- also annotated in the genome (GAH_00501), though it chaea [86]. While the GDH pathway does not use ATP as does not cluster with genes involved in nitrate/nitrite res- an energy source, as the GS-GOGAT pathway does, it has piration and thus may function in the transport of alterna- a lower affinity for ammonium [86]. The presence of these tive compounds. In addition, we identified a gene in this enzymes and two ammonium transporter proteins region of the genome (GAH_01290) coding for an unchar- (GAH_00438 and GAH_01767) for the formation of 2- acterized channel protein, which could potentially func- oxoglutarate and glutamate from ammonium, is consistent tion as a nitrate transport protein. The presence of genes with the notion that “G. ahangari” is under pressure to as- encoding both nitrate reductase proteins (NarGHIJ and similate ammonium for anabolic processes. NirA) combined with the inability of “G. ahangari” to use nitrate for respiration [1] suggests a role for these Sulfur compounds as electron acceptors proteins in assimilatory, rather than dissimilatory, ni- Most members of the Archaeoglobales are dissimilatory trate reduction [86]. sulfate-reducing organisms and able to use several Similar to F. placidus,the “G. ahangari” genome does sulfur-containing compounds as electron acceptors to not contain any nir or nrf genes (for the NADH- and fuel their metabolism [5–10] (Table 5). By contrast, “G. formate-dependent nitrite reductase proteins, respect- ahangari” cannot couple the oxidation of electron do- ively), with the exception of several homologues of nors that supported Fe(III) reduction to the respiration NirA (GAH_00501, GAH_00506, GAH_01242, and GA of commonly considered sulfur-containing electron ac- H_02063), a nitrite reductase protein that catalyzes the ceptors such as sulfate, thiosulfate, sulfite, or S [1]. reduction of nitrite to ammonia and is involved in as- Interestingly, the genome of “G. ahangari” contains two similatory nitrate reduction in other organisms [86]. genes (GAH_02067 and GAH_01481) coding for sulfate Also missing are genes coding for nitric and nitrous adenylyltransferase, which can initiate the first step in oxide reductase proteins, which the genome of F. placi- both the dissimilatory and assimilatory sulfate reduction dus contains [26], again supporting the observation that pathways by catalyzing the formation of APS from ATP “G. ahangari” is not capable of dissimilatory nitrate re- and inorganic sulfate. The enzyme is also present in the duction [1]. The lack of these enzymes helps explain genome of F. placidus which, like “G. ahangari”, is un- the physiological separation of “G. ahangari” from its able to respire sulfate [5] (Table 5). APS can then be closephylogeneticrelative F. placidus, which is capable used as substrate in the assimilatory [90–93] or dissimi- of dissimilatory nitrate reduction to N O[57]. Further- latory [94, 95] pathway, depending on the needs and more, it is unlikely that the reduction of nitrogen- capabilities of the microorganism [92]. The assimilatory containing compounds exerts any significant selective pathway converts APS to the intermediate PAPS in a re- pressure on hydrothermal vent microorganisms, as con- action catalyzed by an adenylsulfate kinase, which in “G. centrations of these compounds are often low in vent ahangari” is encoded by GAH_01478. The genome of systems [87]. “G. ahangari” contains genes coding for both the alpha N gas, on the other hand, is the largest reservoir of and beta subunits of adenylsulfate reductase (GAH_ nitrogen in the ocean [87, 88] and nitrogen fixation sup- 02065-02066), an FAD dependent oxidoreductase protein plies hydrothermal vent systems with nitrogen sources that reduces APS to sulfite in the dissimilatory pathway. for assimilatory growth [87]. Ammonium is particularly However, the genome lacks genes coding for a dissimilatory abundant in the heavily sedimented Guaymas Basin sulfite reductase (dsrAB), which catalyzes the reduction of hydrothermal system [89], from which “G. ahangari” sulfite to hydrogen sulfide in the final step of the dissimila- was isolated [1], and this could select for organisms with tory sulfate reduction pathway [96]. Strong matches could assimilatory rather than dissimilatory nitrogen metabo- not be found even when the alpha (AAB17213.1) and beta lisms and inhibit nitrogen fixation. Not surprisingly, the (AEY99618.1) subunits of the sulfite reductase from annotated genome of “G. ahangari” and homology A. fulgidus were used as queries in manual searches. searches for the primary enzymes from the nitrogen fix- It is interesting to note that, despite the absence of ation pathway (nifH, nifD,and nifK) provided no signifi- dsrAB genes in the genome, “G. ahangari” does have a cant hits, as previously reported for other members of nitrite and sulfite reductase 4Fe-4S domain-containing the Archaeoglobales. protein (GAH_02063) located in a cluster of genes in- The genome does contain genes coding for a glutam- volved in sulfur metabolism (GAH_02063-02067). ine synthetase (GAH_01658), a glutamate synthase (GAH_ Whether these genes code for functional proteins of the 01667-01669), and a glutamate dehydrogenase (GAH_ dissimilatory pathway, perhaps with electron donor/ac- 00573 and GAH_01931). The enzymes glutamine syn- ceptor pairs not tested yet, remains to be elucidated. F. thetase-glutamate synthase comprise the GS-GOGAT placidus, for example, has homologs of all of these Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 13 of 19 genes, except for dsrAB, and it grows with thiosulfate and GAH_01279) and an oligosaccharyltransferase (GAH_ as the sole electron acceptor when hydrogen is pro- 01455), which could glycosylate the growing archaellum vided as an electron donor [5]. This capability may be [99] and post-translationally modify surface proteins, as is due to the presence of several molybdopterin oxidore- commonly observed in the Archaea [100]. However, ductase proteins within the genome of F. placidus that chemotaxis proteins, which are present in nearly all se- show high similarity to a predicted thiosulfate reductase quenced members of the Archaeoglobales [15, 26, 43, 44], (NP_719592.1) from Shewanella oneidensis.However, and NC_015320.1, with the exception of A. sulfaticallidus strong homologs of this protein were not present in the [45], and are typically found immediately upstream or genome of “G. ahangari”. downstream of the fla gene cluster, were absent in “G. ahangari”. The lack of chemotaxis genes in “G. ahangari” Fe(III) as the sole electron acceptor for respiration contrasts with their presence in most Archaeoglobales ge- The most distinctive physiological feature of “G. ahangari” nomes, including G. acetivorans [15], the other member strain 234 is its dependence on Fe(III) as an electron ac- of the genus. Both Geoglobus species were isolated from ceptor for respiration [1]. Both insoluble Fe(III) oxides hydrothermal vent chimneys: G. acetivorans from the and soluble species of Fe(III), such as Fe(III) citrate, sup- Ashadze field on the Mid-Atlantic Ridge at a depth of port growth, though the original isolate did not grow read- 4100 m [4] and “G. ahangari” from a Guaymas Basin ily with the soluble electron acceptor and required chimney at a depth of 2000 m [1]. The hydrothermal prolonged adaptation under laboratory conditions to grow fluids spewed from chimneys within the Guaymas in its presence [1]. Key to the ability of “G. ahangari” to Basin system are likely enriched in nutrients after respire the insoluble Fe(III) oxides is the ability of the cells passage through the 300–400 m thick, organic-rich to locate the oxides, attach to them, and position electron sediments underneath [101]. Furthermore, hydrothermal carriers of the outer surface close enough to favor the circulation at this site is high [23], which would rapidly re- transfer of electrons [14]. Hence, we examined the gen- plenish nutrients, both electron donors and fresh Fe(III) ome of “G. ahangari” for genes that code for cellular com- oxides, and thus organisms living in this environment may ponents that could be involved in motility and attachment not need to utilize chemotactic mechanisms to seek out and extracellular electron transfer. these nutrients. By contrast, hydrothermal fluids from off- Motility in this organism is enabled by a single flagel- shore spreading systems, such as the Ashadze field, flow lum [1], which in archaea is designated as an archaellum through thin sediment layers before reaching the chimney to reflect its distinct evolutionary origin [97]. Archaeal [101]. This likely increases the selective pressure on resi- flagellar genes can be organized into one of two very dent microbes to evolve chemotactic mechanisms to lo- well conserved clusters (fla1 and fla2) based on the type cate nutrients. and order of genes in the cluster: flaBC(D/E)FGHIJ in The genome of “G. ahangari” also encodes proteins fla1 and flaBGFHIJ in fla2 [98]. The fla1 clusters are ex- potentially involved in the assembly of extracellular pro- clusively found in Euryarchaea while fla2 clusters are tein appendages such as pili. We identified, for example, a generally associated with the Crenarchaea, which in- prepilin peptidase (GAH_00760), numerous type II secre- cludes the Desulfurococcales and Sulfolobales orders, tion system proteins (GAH_01195-01196, GAH_00173, and are also present within the Euryarchaeal order GAH_00290, GAH_01412-01413), and a putative twitch- Archaeoglobales [98]. Interestingly, the Archaeoglobales ing motility pilus retraction ATPase (GAH_00960). Hom- have members with both types. We identified, for ex- ologous genes are also present in the genomes of G. ample, a fla1 gene cluster in the genome of “G. ahan- acetivorans [15], F. placidus [26], and A. fulgidus [43]. In gari” (GAH_01994-02001), as in F. placidus (Ferp_14 addition, “G. ahangari” has two genes encoding proteins 56-1463) [26], while the flagellar genes of Archaeoglo- with DUF1628 or DUF1628-like domains (GAH_01202, bus spp. [15, 26, 43–45], and NC_015320.1 and G. acet- GAH_01671), which are associated with previously de- ivorans [15] were of the fla2 type. It has been suggested scribed archaeal pilin proteins [102] and present in all se- that a horizontal gene transfer (HGT) event occurred quenced members of the Archaeoglobales [15, 26, 43–45], in the Ferroglobus lineage after divergence from the and NC_015320.1. Any of these proteins could be involved Archaeoglobus and Geoglobus lineages [15]. Yet, the in the assembly of the curled extracellular appendages that presence of a fla1 gene cluster in the flagellated and “G. ahangari” produces to attach to Fe(III) oxides and fa- motile “G. ahangari” [1], when compared to the fla2 cilitate the transfer of electrons from electron carriers gene cluster found in the non-motile and non-flagellated located on the outer surface to the insoluble electron G. acetivorans [4], would lend credence to a possible sec- acceptor [14]. ond HGT event within the family. “G. ahangari” uses heme-containing proteins to trans- Thegenomeof “G. ahangari” also encodes several port electrons across the cell envelope and to the insoluble glycosyltransferase genes (GAH_00218, GAH_00870, Fe(III) oxides [14]. The most common heme-containing Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 14 of 19 proteins used by mesophilic Fe(III) reducers for extracellu- and F. placidus (Ferp_1362 and Ferp_1364). All of these lar electron transport are c-type cytochromes [103]. Ar- proteins contain cysteine-rich motifs consisting of chaea are known to have a variant form of the cytochrome LX[S,N]C[E,D,H]C but lack the LRCXXC motif char- c maturation (Ccm) system, whereby the CcmE protein acteristic of most CcmH proteins. However, they all has a CXXXY-type motif, rather than the HXXXY motif flank a duplicate CcmF-encoding gene found only in found in eukaryotic and most bacterial c-cytochromes, and “G. ahangari” (GAH_01093), G. acetivorans [15], and CcmH is absent [37]. Similar to other sequenced Archaeo- F. placidus [26]. globales, “G. ahangari” has an archaeal-type CcmE protein In addition to having a distinct cytochrome c biogenesis (GAH_01977), a CcmC (GAH_00620) with a tryptophan- pathway, the iron-reducing Archaeoglobales, Geoglobus and rich motif (WG[S,T][F,Y]WNWDPRET), a CcmF protein Ferroglobus species, also have more c-type cytochromes (GAH_01976 and GAH_01093) with the motif than any other archaeon, and many of these c-type cyto- WGGXWFWDPVEN, and a gene coding for a CcmB chromes have multiple heme groups [15, 18, 26]. The gen- homolog (GAH_00449) lacking the conserved FXXDX ome of “G. ahangari” contains 21 genes (Table 6) encoding XDGSL motif. Although previously reported archaeal cyto- putative c-type cytochromes, 7 of which have more than 5 chrome maturation pathways do not contain CcmH [37], heme groups; F. placidus has 30 c-type cytochromes (12 we identified two putative CcmH proteins in the genomes with more than 5 heme groups); and G. acetivorans has 16 of not only “G. ahangari” (GAH_01092 and GAH_01094), c-type cytochromes (8 with more than 5 heme groups). By but also in G. acetivorans (GACE_2070 and GACE_2068) contrast, Archaeoglobus species, which do not use Fe(III) Table 6 Putative c-type cytochromes Gene ID: Annotation: # of heme Calculated TM binding motifs: molecular weight: domains: GAH_00015 Hypothetical protein 4 58.4 0 GAH_00283 Cytochrome c7 4 21.2 1 GAH_00286 Nitrate/TMAO reductases, 12 39.1 0 membrane-bound tetraheme cytochrome c subunit GAH_00301 Putative redox-active protein 2 31.5 3 (C_GCAxxG_C_C) GAH_00504 Hypothetical protein 10 54.5 1 GAH_00505 Hypothetical protein 4 26.8 2 GAH_00506 Cytochrome c3 9 48.6 0 GAH_00507 Cytochrome c7 4 27.4 1 GAH_00508 Hypothetical protein 5 28.5 1 GAH_00510 Hypothetical protein 4 27.3 1 GAH_00817 Seven times multi-haem 8 53.7 1 cytochrome CxxCH GAH_01091 Hypothetical protein 1 11.7 1 GAH_01235 Hypothetical protein 5 21.5 0 GAH_01236 Hypothetical protein 5 22.3 0 GAH_01253 Hypothetical protein 4 16.9 0 GAH_01256 NapC/NirT cytochrome c family, 10 43.6 1 N-terminal region GAH_01296 Cytochrome c family protein 4 17.2 1 GAH_01297 Seven times multi-haem 8 61.0 1 cytochrome CxxCH GAH_01306 Class III cytochrome C family 8 46.3 0 GAH_01534 Hypothetical protein 1 18.5 1 GAH_01700 Hypothetical protein 3 9.9 0 No signal peptide detected Signal peptide detected by PRED-SIGNAL Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 15 of 19 electron acceptors (Table 5), have significantly fewer c-type number of c-type cytochromes within and on the cell cytochromes. Within this genus, the greatest number of surface, as well as other redox-active proteins such as c-type cytochrome encoding genes was found in the thermostable ferredoxin and Fe-S proteins. The paucity genome of A. veneficus, which has 16 c-type cytochromes of c-type cytochromes within non-Fe(III) respiring mem- (3 with more than 5 hemes). Other species such as A. bers of the Archaeoglobales (Archaeoglobus species) is profundus and A. sulfaticallidus have only 1 monoheme consistent with the physiological separation between these c-type cytochrome and A. fulgidus has 3 c-type cyto- archaea and F. placidus, G. acetivorans, and “G. ahangari”, chromes (none of which have more than 5 heme groups). which can gain energy for growth from the reduction of The subcellular localization of the putative c-type cy- Fe(III) electron acceptors. Additionally, some genes re- tochromes of “G. ahangari” was also investigated. The quired for both dissimilatory sulfate and nitrate metabo- ExPASy TMPred program [42] revealed that a majority lisms are absent in “G. ahangari” and G. acetivorans. This (62 %) of the c-type cytochromes have at least 1 trans- supports the physiological separation of Geoglobus spp. membrane helix, consistent with their association to from F. placidus, which is capable of Fe(III)-, thiosulfate-, the cytoplasmic membrane. One of these c-type cyto- and nitrate respiration, and from Archaeoglobus species chrome proteins (GAH_00504) was predicted to be which are primarily sulfur-respiring organisms. Genomic extracellular. We also identified several c-type cyto- data also support the reported physiological similarities chromes (GAH_01306, GAH_00286, GAH_01534, and between “G. ahangari” and other Archaeoglobales such as GAH_01253) with predicted sizes once in mature form autotrophic growth with H via the reductive acetyl-CoA/ (46.3, 39.1, 18.5, and 16.9 kDa, respectively) matching Wood-Ljungdahl pathway and the use of similar electron those reported for outer-surface heme-containing pro- donors, including short- and long-chain fatty acids. teins required for the reduction of insoluble Fe(III) ox- Noteworthyis thefact that genomicevidencesupports ides, but not soluble Fe(III) citrate, by “G. ahangari” the synthesis of the methanogenic coenzyme-F in [14] (Table 6). Hence, these 4 c-type cytochromes likely “G. ahangari”, which is responsible for the characteris- function as the terminal electron carriers between the tic fluorescence detected in all Archaeoglobus spp. ex- cells and the oxides. cept for “G. ahangari” or F. placidus. Hence, the In addition to c-type cytochromes, we identified other genome sequence of “G. ahangari” provides valuable potential electron carriers such as quinones, flavoproteins, insights into its physiology and ecology as well as into and various Fe-S proteins (i.e. ferredoxins). We identified the evolution of respiration within the Archaeoglobales. a number of ub iquinone/menaquinone biosynthesis pro- teins in the genome of “G. ahangari” (Additional file 1), Taxonomic note which could create a quinone pool in the membrane to The initial publication [1] of the “Geoglobus” genus and promote electron transfer. The genome also contains a “Geoglobus ahangari” species was accepted for publica- great number of Fe-S binding domain proteins and ferre- tion with extenuating circumstances at several culture- doxins, which could participate in electron transfer path- collection agencies. Thus, upon the original publication ways (Additional file 2). Fe-S proteins and ferredoxins “G. ahangari” strain 234 was accepted only at a single were also abundant in the genome of G. acetivorans and agency. In addition, the G + C mol% determined from F. placidus, which, like “G. ahangari”, also utilize Fe(III) the complete genome sequence (53.1 mol%) differs from respiration as their primary metabolism. Fe-S proteins and that originally published (58.7 mol%), representing a dis- ferredoxins are regarded as some of the most ancient of crepancy of over 5 mol%. This publication thus warrants electron transfer carriers [104] and also have high thermo- an emended description of the genus Geoglobus and the stability [105], which is critical to ensure maximum rates type species, “Geoglobus ahangari”. of electron transfer in the hot hydrothermal vent systems. Thus, the abundance of electron carrier proteins, some Emended description of “Geoglobus” Kashefi et al. known to have increased thermostability, and c-type cyto- The description of the genus “Geoglobus” is the one pro- chromes, some of them localized to the outer surface, is vided by Kashefi et al. [1], with the following modifica- consistent with a mechanism evolved for efficient extracel- tions. In addition to the single monopolar flagellum, lular electron transfer in hot environments. numerous curled filaments can be seen per cell [14]. The G + C content of the genomic DNA of the type spe- Conclusions cies is 53.1 mol%. “G. ahangari” strain 234 is only one of three members of the Archaeoglobales capable of dissimilatory Fe(III) Emended description of “Geoglobus ahangari” Kashefi et respiration. Furthermore, it is an obligate Fe(III) reducer al. that grows better with insoluble than soluble Fe(III) spe- The description of the species “Geoglobus ahangari” is the cies. Consistent with this, the genome contains a large one provided by Kashefi et al. [1, 2], with the following Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 16 of 19 modifications. The type strain is strain 234 and has been 2002;52:719–28. Available at: http://ijs.sgmjournals.org/cgi/content/abstract/52/ 3/719. Accessed January 13, 2015. deposited at three culture collection agencies, which in- 2. Kashefi K. Hyperthermophiles: Metabolic diversity and biotechnological clude the Deutsche Sammlung von Mikroorganismen und applications. 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Int J Syst Evol Microbiol. The authors declare that they have no competing interests. 2010;60:2745–52. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 20061497. Accessed January 13, 2015. Authors’ contributions 10. Burggraf S, Jannasch HW, Nicolaus B, Stetter KO. Archaeoglobus MPM, DEH, JMR, and AC sequenced, assembled and annotated the genome. profundus sp. nov., represents a new species within the sulfate-reducing MPM, DEH, GR, JMR, and KK analyzed the data and drafted the manuscript. archaebacteria. Syst Appl Microbiol. 1990;13:24–8. Available at: http:// All authors read and approved the final manuscript. linkinghub.elsevier.com/retrieve/pii/S0723202011801761. Accessed January 13, 2015. Acknowledgements 11. Lovley DR. Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol Rev. The authors gratefully acknowledge Tracy K. Teal at Michigan State for 1991;55:259–87. Available at: http://www.pubmedcentral.nih.gov/ assistance with assembly and annotation and Abigail Vanderberg at the articlerender.fcgi?artid=372814&tool=pmcentrez&rendertype=abstract. Center for Advanced Microscopy at Michigan State for help with scanning Accessed January 13, 2015. electron microscopy. We also acknowledge technical support provided by 12. Weber KA, Achenbach LA, Coates JD. Microorganisms pumping iron: the Research Technology Support Facility and the Hypercomputing Center anaerobic microbial iron oxidation and reduction. Nat Rev Microbiol. at Michigan State University, the Deep Sequencing Core Facility at the 2006;4:752–64. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16980937. University of Massachusetts Medical School, and the Genomics Resource lab Accessed January 13, 2015. at the University of Massachusetts-Amherst. This work was supported with 13. Gavrilov SN, Lloyd JR, Kostrikina NA, Slobodkin AI. Fe(III) oxide reduction by funds from a Strategic Partnership Grant from the MSU Foundation to GR a Gram-positive thermophile: Physiological mechanisms for dissimilatory and KK, a GAANN fellowship by the Department of Education and a DuVall reduction of poorly crystalline Fe(III) oxide by a thermophilic Gram-positive Award to MPM, and a faculty grant from Western New England University to bacterium Carboxydothermus ferrireducens. Geomicrobiol J. 2012;29:804–19. DEH and JMR. Available at: http://www.tandfonline.com/doi/abs/10.1080/ 01490451.2011.635755. Accessed January 13, 2015. Author details 14. Manzella MP, Reguera G, Kashefi K. Extracellular electron transfer to Fe(III) Department of Microbiology and Molecular Genetics, Michigan State oxides by the hyperthermophilic archaeon Geoglobus ahangari via a direct University, East Lansing, MI, USA. Department of Physical and Biological contact mechanism. Appl Environ Microbiol. 2013;79:4694–700. 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Publisher: Springer Journals
Copyright: Copyright © 2015 by Manzella et al.
Subject: Life Sciences; Microbial Genetics and Genomics; Plant Genetics & Genomics; Animal Genetics and Genomics
eISSN: 1944-3277
DOI: 10.1186/s40793-015-0035-8
pmid: 26457129
Publisher site: See Article on Publisher Site

Abstract

“Geoglobus ahangari” strain 234 is an obligate Fe(III)-reducing member of the Archaeoglobales, within the archaeal phylum Euryarchaeota, isolated from the Guaymas Basin hydrothermal system. It grows optimally at 88 °C by coupling the reduction of Fe(III) oxides to the oxidation of a wide range of compounds, including long-chain fatty acids, and also grows autotrophically with hydrogen and Fe(III). It is the first archaeon reported to use a direct contact mechanism for Fe(III) oxide reduction, relying on a single archaellum for locomotion, numerous curled extracellular appendages for attachment, and outer-surface heme-containing proteins for electron transfer to the insoluble Fe(III) oxides. Here we describe the annotation of the genome of “G. ahangari” strain 234 and identify components critical to its versatility in electron donor utilization and obligate Fe(III) respiratory metabolism at high temperatures. The genome comprises a single, circular chromosome of 1,770,093 base pairs containing 2034 protein-coding genes and 52 RNA genes. In addition, emended descriptions of the genus “Geoglobus” and species “G. ahangari” are described. Keywords: Euryarchaeota, Archaeoglobales, Hydrothermal vent, Guaymas basin, Fe(III) respiration, Extracellular electron transfer, Autotroph Introduction oxidation of a wide range of carbon compounds includ- “Geoglobus ahangari” strain 234 is the type strain and ing long-chain fatty acids such as stearate and palmitate, one of only two known members of the Geoglobus genus which were previously not known to be used as elec- within the order Archaeoglobales and the family Archae- tron donors by archaea [1]. It was also the first hyper- oglobaceae. It is an obligate Fe(III)-reducing archaeon thermophile reported to fully oxidize acetate to CO ,a isolated from the Guaymas Basin hydrothermal system metabolic function once thought to occur solely in and grows at temperatures ranging from 65–90 °C, with mesophilic environments [3]. Unlike the other two an optimum at about 88 °C [1]. It was the first isolate in genera in the order Archaeoglobales (Archaeoglobus a novel genus within the Archaeoglobales and the first and Ferroglobus), which can utilize acceptors such as example of a dissimilatory Fe(III)-reducer able to grow sulfate and nitrate [1, 4–10], the two cultured members autotrophically with H [1], a metabolic trait later shown of the genus Geoglobus can only use Fe(III) as an elec- to be conserved in many hyperthermophilic Fe(III) re- tron acceptor [1, 4]. The obligate nature of Fe(III) respir- ducers [2]. “G. ahangari” can also couple the reduction ation in Geoglobus spp. makes the genus an attractive of soluble and insoluble Fe(III) acceptors to the model to gain insights into the evolutionary mechanisms that may have led to the loss and/or gain of genes involved * Correspondence: [email protected] in the respiration of iron and other electron acceptors Department of Microbiology and Molecular Genetics, Michigan State such as sulfur- and nitrogen-containing compounds University, East Lansing, MI, USA within the Archaeoglobales. Full list of author information is available at the end of the article © 2015 Manzella et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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. Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 2 of 19 “G. ahangari” strain 234 also serves as a model or- that make this organism a good model system to study ganism for mechanistic studies of iron reduction at high Fe(III) reduction in hot environments and to gain in- (>85 °C) temperatures. Dissimilatory Fe(III) reduction sights into the evolution of Fe(III) respiration in the has been extensively studied in mesophilic bacteria family Archaeoglobales. (reviewed in references [11, 12]). By contrast, little is known about the mechanisms that allow (hyper)thermo- Organism information philic organisms to respire Fe(III) acceptors [13–18]. As Classification and features previously observed in the thermophilic Gram-positive “Geoglobus ahangari” strain 234 is a euryarchaeon ori- bacterium Carboxydothermus ferrireducens [13], “G. ginally isolated from samples obtained from a hydrother- ahangari” also needs to directly contact the insoluble mal chimney located within the Guaymas Basin (27° N, Fe(III) oxides to transfer respiratory electrons [14]. In 111° W) at a depth of 2000 m [1]. The sequence of the “G. ahangari”, cells are motile via a single archaellum, single 16S rRNA gene found in its genome was 99 % identi- which could help in locating the oxides, and also express cal to the previously published 16S rDNA sequence numerous curled extracellular appendages, which bind (AF220165). The full length 16S rRNA gene (1485 bp) was the mineral particles and position them close to heme- used to construct a phylogenetic tree in reference to 16S containing proteins on the outer surface of the cell to rRNA gene sequences from other hyperthermophilic ar- facilitate electron transfer [14]. A direct contact mechan- chaea using two thermophilic bacteria (“Aquifex aeolicus” ism such as this is predicted to confer on these organ- and Pseudothermotoga thermarum)asoutgroups (Fig.1). isms a competitive advantage over other organisms The closest known relative was Geoglobus acetivorans relying on soluble mediators such as metal chelators (97 % identical), the only other known member of the [19] and electron shuttles [20, 21], which are energetic- Geoglobus genus, which also is available in pure culture ally expensive to synthesize and are easily diluted or lost [4]. Closest relatives outside the genus were other hyper- in the environment once excreted [22]. This is particu- thermophilic archaea within the family Archaeoglobaceae larly important in hydrothermal vent systems such as such as the sulfate-reducing Archaeoglobus species A. ful- the Guaymas basin chimney where “G. ahangari” strain gidus and A. profundus (97 and 93 % identical, respect- 234 was isolated, as vent fluids in these systems can ively) and Ferroglobus placidus (94 % identical), which can flow through at rates as high as 2 m/s [23]. Here, we re- reduce Fe(III), thiosulfate, and nitrate [3, 5]. port the complete genome sequence of “G. ahangari” Cells of “G. ahangari” strain 234 are regular to irregu- strain 234 and summarize the physiological features lar cocci, 0.3 to 0.5 μm in diameter, and usually arranged Fig. 1 Phylogenetic tree. The phylogenetic tree was constructed with the maximum likelihood algorithm comparing the16S rRNA gene sequence from “G. ahangari” to other hyperthermophilic archaea. Bootstrap values were determined from 100 replicates and “Aquifex aeolicus” and Pseudothermotoga thermarum were used as outgroups Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 3 of 19 respiratory metabolisms in the Archaeoglobales,and, in particular, about hyperthermophilic iron reduction within the Archaea. The genome project information is listed in the Genomes OnLine Database (Gp0101274) [24] and the complete genome sequence has been deposited in GenBank (CP011267). A summary of the project infor- mation is shown in Table 2. Growth conditions and genomic DNA preparation “G. ahangari” strain 234 was from our private culture collection and is available at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM-27542), the Japan Collection of Microorganisms (JCM 12378), and the American Type Culture Collection (BAA-425). The strain was grown in marine medium [1] with 10 mM Fig. 2 Scanning electron micrograph of cells of “G. ahangari” strain 234 growing on insoluble Fe(III) oxides. Bar, 100 nm pyruvate as the electron donor and 56 mM ferric citrate as the electron acceptor. Cultures were incubated under a N :CO (80:20 %, v/v) atmosphere at 80 °C or 85 °C in the 2 2 dark. Strict anaerobic techniques were used throughout as single cells or in pairs (Fig. 2 and Table 1) [1]. Cells the culturing and sampling experiments [25]. are motile via a single archaellum [1], but also produce gDNA was extracted as previously reported for F. abundant extracellular curled filaments when grown placidus [26]. Alternatively, cells were lysed with an with both soluble and insoluble Fe(III) [14]. Though SDS-containing lysis buffer (5 % SDS, 0.125 M EDTA, optimum growth occurs at ca. 88 °C, growth is observed 0.5 M Tris, pH 9.4), as reported elsewhere for the between 65 and 90 °C [1]. Furthermore, growth was sup- preparation of whole cell extracts from “G. ahangari” ported at pH values between 5.0 and 7.6, with an [14], and gDNA was extracted using the MasterPure™ optimum at pH 7.0, and with NaCl concentrations ran- DNA Purification Kit (EPICENTRE® Biotechnologies), ging from 9 to 38 g/L, with an optimum at 19 g/L [1]. according to the manufacturer suggested guidelines. A distinctive feature of the metabolism of “G. ahan- gari” strain 234 is its obligate nature of Fe(III) respir- Genome sequencing and assembly ation, with both soluble and insoluble Fe(III) species The finished genome of “G. ahangari” strain 234 (CP supporting growth but the insoluble electron acceptor 011267) was generated from Illumina [27] draft sequences being preferred [1]. The obligate nature of Fe(III) reduc- generated independently at the Research Support and tion contrasts with the wide range of electron donors Training Facility at Michigan State University, the Deep that “G. ahangari” can oxidize [1]. Acetate, alongside a Sequencing Core Facility at the University of Massachu- number of other organic acids (such as propionate, bu- setts Medical School, and the Genomics Resource lab at tyrate, and valerate), several amino acids, and both the University of Massachusetts-Amherst. Table 2 pre- short-chain and long-chain fatty acids were completely sents the project information. oxidized to CO to support growth during Fe(III) respir- The sequencing project at the University of Massachusetts ation [1, 3]. Furthermore, “G. ahangari” strain 234 was facilities used gDNA suspended in 3 ml of sonication also able to grow autotrophically with H as the sole buffer (4.95 % glycerol, 10 mM Tris–HCl (pH 8.0), 1 mM electron donor and Fe(III) as the electron acceptor [1]. EDTA) and sonicated for 10 min (2 min on, 30 s off) using The main physiological features of the organism are a 550 Sonic Dismembrator (Fisher Scientific). The samples listed in Table 1. were then dispensed as equal volumes into 4 tubes and mixed with 150 μl TE buffer (10 mM Tris–HCl (pH 8.0), 1mM EDTA),100 μl ammonium acetate (5 M), 20 μl Genome sequencing and annotation glycogen (5 mg/ml), and 1 ml of cold (−20 °C) isopropa- Genome project history nol. Nucleic acids were precipitated at −30 °C for 1 h, as Based on its unique physiological characteristics [1] previously described [26] and suspended in EB buffer and use as a model system for mechanistic investiga- (Qiagen) before separating the DNA fragments in the tions of Fe(III) reduction by hyperthermophilic ar- sample by agarose gel electrophoresis. DNA fragments be- chaea [14], “G. ahangari” strain 234 was selected for tween 300–500 bp were then purified with the QiaQuick sequencing. Insights from its genome sequence and an- Gel Extraction Kit (Qiagen). All steps involved in end re- notation provide greater understanding of the evolution of pair, 3’ adenylation, adaptor ligation, and purification of Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 4 of 19 Table 1 Classification and general features of “G. ahangari” 234 according to the MIGS recommendations [106] MIGS ID Property Term Evidence code Current classification Domain Archaea TAS [107] Phylum Euryarchaeota TAS [107, 108] Class Archaeoglobi TAS [109] Order Archaeoglobales TAS [110] Family Archaeoglobaceae TAS [111] Genus Geoglobus TAS [1] Species “Geoglobus ahangari” TAS [1] Type strain 234 TAS [1] Gram stain Variable NAS Cell shape Irregular coccus TAS [1] Motility Motile TAS [1] Sporulation Non-sporulating NAS Temperature range 65−90 °C TAS [1] Optimal temperature 88 °C TAS [1] pH range; Optimum 5.0−7.6 (optimum 7.0) TAS [1] Carbon source CO TAS [1] Energy metabolism Chemolithoautotrophic, TAS [1] chemolithotrophic, chemoorganotrophic MIGS-6 Habitat Marine geothermally TAS [1] heated areas MIGS-6.3 Salinity 9.0−38 g/L NaCl TAS [1] MIGS-22 Oxygen requirement Anaerobe TAS [1] MIGS-15 Biotopic relationship Free-living TAS [1] MIGS-14 Pathogenicity Non-pathogen NAS Isolation Hydrothermal TAS [1] vent chimney MIGS-4 Geographic location Guaymas Basin TAS [1] hydrothermal system MIGS-5 Sample collection time Unknown NAS MIGS-4.1 Latitude 27° N TAS [1] MIGS-4.2 Longitude 111° W TAS [1] MIGS-4.3 Depth 2000 m TAS [1] MIGS-4.4 Altitude Not applicable Evidence 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). These evidence codes are from the Gene Ontology project [112] Illumina products were performed using reagents sup- 2.2.5). Reads were down-sampled to 500x coverage to plied by the TruSeq DNA Sample Prep Kit (Illumina). increase the efficacy of the Velvet assembler while the This resulted in the construction and sequencing of complete depth of reads was used to verify the final five independent 100 bp paired-end Illumina shotgun genome assembly. libraries which generated 7,970,036, 7,970,182, 7,973,896, A total of 1,780,565 bp were assembled into 25 scaf- 7,966,671 and 3,144,785 reads totaling 3.50 Gbp. The folds ranging in size from 207 bp to 510,180 bp. The Illumina draft sequences were assembled de novo with scaffolds were then connected by adaptor-PCR as previ- SeqMan NGen (DNASTAR) and Velvet [28] (version ously described [29]. “G. ahangari” gDNA was subse- 1.2.10) and optimized with VelvetOptimiser (version quently digested separately by four different restriction Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 5 of 19 Table 2 Genome sequencing project information Qiagen PCR purification kit and sent for sequencing at MIGS ID Property Term the University of Massachusetts (Amherst) sequencing fa- cility. This process was repeated until a single contig was MIGS-31 Finishing Finished quality obtained using SeqMan Pro assembly software. The assembly was then verified against a second, inde- MIGS-28 Libraries used 5 independent 100 bp paired-end Illumina shotgun libraries, 150 bp pendent genome assembly generated at Michigan State paired-end Illumina shotgun library University. Extracted gDNA was used to construct a sin- MIGS-29 Sequencing Illumina MiSeq gle Illumina shotgun library using the Illumina DNAseq platforms Library Kit, which was sequenced in two 150 bp paired- MIGS-31.2 Sequencing 1,977 × coverage (100 bp libraries) end runs with an Illumina MiSeq at the Research Sup- coverage 100 × (150 bp library) port and Training Facility at Michigan State University. MIGS-30 Assemblers SeqMan NGen, Velvet, SeqMan Pro The sequencing project generated 1,233,811 and 796,056 reads totaling 304.5 Mbp. Reads were quality trimmed MIGS-32 Gene calling JGI-ER, GLIMMER method using a combination of fastq-mcf [30] (ea-utils.1.1.2-537, using default parameters) and ConDeTri [31] (v2.2, using INSDC ID CP011267 default parameters with the exception of hq = 33 and Genbank May 11, 2015 Date of sc = 33) to remove low-quality reads and over-represented Release sequences. High-quality paired and unpaired reads GOLD ID Gp0101274 were assembled using Velvet [28] (v1.2.08 using de- fault parameters and a kmer value of 63) to generate NCBI project 258102 ID anew assembly (1.77Mbp,51contigs ≥ 1000 bp, and MIGS-13 Source ATCC BAA-425, DSMZ DSM-27542, JCM an N75 of 37,520 bp). The second assembly was then material JCM 12378 compared to the primary assembly to identify errors identifier and low-coverage regions, which were subsequently Project Phylogenetic diversity, biotechnology, resolved by PCR-amplifying and sequencing the re- relevance evolution of metal respiration in gions of interest. hyperthermophiles, and anaerobic degradation of hydrocarbons Genome annotation Initial genome annotation was performed by the RAST enzymes (EcoRI, BamHI, BclI, and SalI). After a 1.5-h in- server [32], the IGS Annotation Engine [33] at the Uni- cubation at 37 °C (or 50 °C for SalI), the restriction di- versity Of Maryland School Of Medicine, and the IMG- gests were separated by agarose gel electrophoresis and ER platform [34]. The annotations were compared to fragments between 5–10 kb were isolated and purified manual annotations performed using GLIMMER [35] with the QiaQuick gel extraction kit (Qiagen). Adaptor for gene calls and DELTA-BLAST analysis to identify sequences with 3′ overhangs generated by EcoRI, BamHI, conserved domains and homology to known proteins. BclI, and SalI at the phosphorylated 5′ ends were then li- EC numbers and COG categories were determined with gated with T4 DNA ligase to the fragments purified from a combination of DELTA-BLAST analysis of each anno- the restriction digests of “G. ahangari” gDNA. The tated gene and the IMG-ER platform. Pseudogenes were adaptor sequences used were: EcoRI adaptor AATTCCC identified using the GenePRIMP pipeline [36]. The data TATAGTGAGTCGTATTAAC** (phosphorylated at 5′ were used to create a consensus annotation before the end); BclIand BamHI adaptor GATCCCCTATAGTGAG final assembled genome was uploaded onto the IMG-ER TCGTATTAAC**; and finally the SalI adaptor TCGACCC platform. IMG-ER annotations were manually curated TATAGTGAGTCGTATTAAC**. Further assembly was by comparison to the consensus annotation before sub- performed with SeqMan Pro (DNASTAR) and primers mitting the final genome annotation. were designed targeting the 3′ and 5′ ends of the 25 scaf- Potential c-type cytochromes were selected based on folds. The adaptor ligations were diluted 100-fold and 1 μl the presence of c-type heme binding motifs (CXXCH) of the diluted sample was used in PCR reactions with within the amino acid sequence as previously described AccuTaq™ LA DNA Polymerase (50 μl total volume) [37]. Predicted subcellular localization and the presence according to manufacturer specifications (Sigma-Aldrich). of signal peptides and/or an N-terminal membrane helix Fifty reactions were performed with “G. ahangari”-specific anchor [37] was investigated by PsortB [38], PRED-TAT primers designed from the various Illumina scaffolds and [39], TMPred [40], and the TMHMM Server (v. 2.0) a non-phosphorylated primer that complemented the [41]. Putative c-type cytochromes were then examined adaptor sequence on the gDNA (GTTAATACGACTCAC by BLAST analysis to determine homology to known c- TATAGGG). All PCR products were purified with the type cytochromes in the NCBI database. The molecular Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 6 of 19 weight of putative c-type cytochromes was estimated and their percentage representation are listed in Fig. 3 with the ExPASy ProtParam program [42]. The weight and Table 4. of the signal peptide was then subtracted from the pre- The preferred start codon is ATG (83.8 % of the dicted weight and 685 daltons were added for each genes), followed by GTG (10.4 %) and TTG (5.7 %). This heme-binding motif to estimate the molecular weight of distribution is similar to the start codon representation of the mature cytochrome. The predicted molecular weight the other member of the Geoglobus genus, G. acetivorans values and subcellular localization of the mature cyto- (79.4 % ATG, 11.6 % GTG, and 9.0 % TTG) [15] and the chromes were compared to the masses reported for mature closely related archaeon F. placidus (82.5 % ATG, 10.2 % heme-containing proteins present in whole cells and outer- GTG, 6.1 % TTG, and 1.3 % other) [26]. There is one copy surface protein preparations of “G. ahangari” [14]. of each of the rRNA genes but the genes are located in two different regions of the genome: the 16S rRNA (GAH_00462) and 23S rRNA (GAH_00460) genes are Genome properties in the same gene cluster and separated by a span of The genome of “G. ahangari” strain 234 comprises one 139 bp encoding a single tRNA whereas the 5S rRNA circular chromosome with a total size of 1,770,093 bp (GAH_02069) is located 205,273 bp away in a region and does not contain any plasmids. The genome size is with genes coding for proteins with functions unrelated within the range of those reported for other members of to ribosome function and biogenesis. the Archaeoglobales [15, 26, 43–45], and NC_015320.1. Almost all origins of DNA replication identified in Ar- The mol percent G + C is 53.1 %, which is lower than chaea to date are located in close proximity to genes the 58.7 % estimated experimentally via HPLC [1]. Out coding for a homologue of the eukaryotic Cdc6 and of the total 2072 genes annotated in the genome, 52 Orc1 proteins [46]. Interestingly, we identified two genes were identified as RNA genes and 2020 as protein- encoding Orc1/Cdc6 family replication initiation pro- coding genes (Table 3). There are 47 pseudogenes, com- teins (GAH_00094 and GAH_00965) in the genome of prising 2.3 % of the protein-coding genes. Furthermore, “G. ahangari”, thus raising the possibility that the gen- 76.5 % of the predicted genes (1557) are represented by ome contains more than one functional origin of replica- COG functional categories. Distribution of these genes tion. Many archaeal replication origins consist of long intergenic sequences upstream of the cdc6 gene contain- ing an A/T-rich duplex unwinding element flanked by Table 3 Nucleotide content and gene count levels of the several conserved repeat motifs known as ORBs [47]. A genome specific ORB could not be identified in the genome Attribute Value % of total when compared to other archaeal origins of replication Size (bp) 1,770,093 100.0 % available in the DoriC database [48]. However, the 320- Coding region (bp) 1,662,832 93.9 % bp long region upstream of GAH_00965, one of the G + C content (bp) 940,071 53.1 % Orc1/Cdc6 family replication initiation proteins, con- Number of replicons 1 tains a long (111 bp) non-coding intergenic region with Extrachromosomal 0 one AT-rich stretch and 8 direct repeats (3 TCGTGG, 3 elements CGTGGTC, and 2 GGGGATTA), which could function Total genes 2072 100.0 % as a replication origin. Furthermore, the 580-bp region RNA genes 52 2.5 % directly upstream of the other Orc1/Cdc6 family replica- tion initiation protein (GAH_00094) lacks a non-coding rRNA operons 2 intergenic region and/or AT-rich span but contains 8 Protein-coding genes 2034 100.0 % direct repeats (2 GGTTGAGAAG, 3 TGAGAAG, and 3 Pseudogenes 47 2.3 % AACATCCCG) and several “G-string” elements analo- Genes with function 1677 82.4 % gous to ori sites reported for haloarchaeal species [49]. prediction Genes in paralog clusters 1406 69.1 % Insights from the genome Genes assigned to COGs 1470 72.2 % Autotrophic growth with H as electron donor Genes assigned Pfam 1667 82.0 % “G. ahangari” strain 234 was the first dissimilatory Fe domains (III)-reducing hyperthermophile shown to grow autotro- Genes with signal peptides 55 2.7 % phically with H as an electron donor [1]. In its genome, Genes with transmembrane helices 409 20.1 % we identified genes required for the two branches of the reductive acetyl-CoA/Wood-Ljungdahl pathway [50–53], CRISPR repeats 7 a which other members of the Euryarchaeota [54], includ- The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome ing most members of the Archaeoglobales [8, 45, 55–58], Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 7 of 19 Fig. 3 Graphical circular map of the chromosome. From outside to the center: Genes on forward strand (colored by COG categories), genes on reverse strand (colored by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, and GC skew use for carbon fixation. A bifunctional carbon monoxide The genome of “G. ahangari” also contains 29 genes dehydrogenase/acetyl-CoA synthase complex (encoded encoding hydrogenase subunits, maturation proteins, by GAH_01139-01144, and two additional copies of the and a cluster of genes (hypA, hypB, hypC, hypD, and beta and maturation factors encoded by GAH_00919 hypE) involved in biosynthesis and assembly of Ni-Fe hy- and GAH_00306, respectively) are present within the drogenases (GAH_00190-00195). Genes coding for the genome, which could initiate carbon fixation. The bi- large, small, and b-type cytochrome subunits of a Ni-Fe functional nature of this enzyme also allows it to link me- hydrogenase I protein (GAH_00910-00912) were identi- thyl and carbonyl branches and enable acetyl-CoA fied in the genome. We also found a gene cluster biosynthesis, as reported for methanogenic archaea [59]. (GAH_00337-00347) encoding all subunits of a NADH- Complete enzymatic pathways for alternative means of car- quinone oxidoreductase, which transfers electrons to the bon fixation were not identified. quinone membrane pool and may function as the Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 8 of 19 Table 4 Number of genes associated with the 25 general COG functional categories Code Value % age Description J 155 7.6 % Translation, ribosomal structure and biogenesis A 2 0.1 % RNA processing and modification K 68 3.3 % Transcription L 58 2.9 % Replication, recombination and repair B 6 0.3 % Chromatin structure and dynamics D 20 1.0 % Cell cycle control, Cell division, chromosome partitioning Y 0 0.0 % Nuclear structure V 7 0.3 % Defense mechanisms T 24 1.2 % Signal transduction mechanisms M 35 1.7 % Cell wall/membrane biogenesis N 15 0.7 % Cell motility Z 0 0.0 % Cytoskeleton W 0 0.0 % Extracellular structures U 23 1.1 % Intracellular trafficking and secretion O 57 2.8 % Posttranslational modification, protein turnover, chaperones C 160 7.9 % Energy production and conversion G 37 1.8 % Carbohydrate transport and metabolism E 139 6.8 % Amino acid transport and metabolism F 49 2.4 % Nucleotide transport and metabolism H 103 5.1 % Coenzyme transport and metabolism I 55 2.7 % Lipid transport and metabolism P 86 4.2 % Inorganic ion transport and metabolism Q 16 0.8 % Secondary metabolites biosynthesis, transport and catabolism R 244 12.0 % General function prediction only S 198 9.7 % Function unknown - 477 23.5 % Not in COGs The total is based on the total number of protein coding genes in the genome primary generator of the proton-motive force [43]. An- in members of the Archaeoglobales [7–9, 55, 57] but other large cluster of hydrogenase genes (GAH_02036- not in the iron-respiring F. placidus [5] or in “G. ahan- 02044) codes for all coenzyme F hydrogenase subunits gari” [1]. Yet, the “G. ahangari” genome contains genes and proteins involved in recycling coenzyme F ,thus for all coenzyme subunits of the proteins coenzyme replenishing the cofactor for the reductive acetyl-CoA F -reducing hydrogenase (GAH_00337 and GAH_02 5 10 pathway [58, 60–62]. The presence of multiple hydroge- 036-02038), coenzyme F -dependent N ,N -methy- nases is not unusual in iron-reducing microorganisms lene tetrahydromethanopterin reductase (GAH_01605, and allows them to diversify the paths used to transfer GAH_01835), and F synthase (CofGH) (GAH_00662, electrons derived from the oxidation of H to their GAH_00663) [65]. Furthermore, although “G. ahangari” acceptors [63]. cannot produce methane when growing autotrophically Autotrophic growth in methanogens can also be sup- [1], its genome codes for nearly all enzymes responsible ported using reduced coenzyme F as an electron for the reduction of CO to methane [51]. Similar to A. 420 2 donor to produce methane [64]. The distinctive fluores- fulgidus, F. placidus, A. sulfaticallidus,and G. acetivorans, cence emission from this coenzyme has been detected “G. ahangari” has genes encoding all proteins involved in Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 9 of 19 the formation of 5-methyl-tetrahydromethanopterin and a Central metabolism gene coding for one of the 8 subunits (MtrH) of the en- Heterotrophic growth in “G. ahangari” is supported by zyme responsible for the transfer of a methyl group to co- a wide range of organic carbon compounds [1], which enzyme M (GAH_01245). Yet, the genome is missing all serve as electron donor for respiration while also pro- four genes required for a functional coenzyme M reduc- viding carbon for assimilation in the central pathways. tase, the enzyme responsible for the final step of methane Similar to other hyperthermophilic archaeal species [66], production by methanogenic archaea [51]. The fact that the “G. ahangari” genome contains a modified Embden- Archaeoglobale genomes have nearly all of the genes in- Meyherhof-Parnas glycolytic pathway (Fig. 4). The initial volved in methanogenesis and the high level of homology step of glycolysis (glucose phosphorylation to glucose 6- that exists between genes from the reductive acetyl-CoA phosphate) is carried out by an ATP-dependent archaeal pathway in both Archaeoglobales and the methanogenic ar- hexokinase (GAH_00546) belonging to the ROK family of chaea suggests that the Archaeoglobales mayhaveevolved proteins. A gene coding for the phosphoglucose isomerase from a methanogenic archaeon that lost its ability to reduce enzyme, which catalyzes the next reaction in the pathway CO and produce methane over time. (interconversion of the aldose in glucose 6-phosphate Fig. 4 Central metabolism in “Geoglobus ahangari” strain 234 Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 10 of 19 and the ketose in fructose 6-phosphate) was also iden- kinase protein is present in the close relative F. placidus tified (GAH_01135) and was most similar to cupin- (Ferp_0744), homologs were not identified in “G. ahan- type phosphoglucose isomerases from other anaerobic gari” or any other Archaeoglobale species. Instead, the ge- Euryarchaeota, including Archaeoglobus fulgidus [66]. nomes of “G. ahangari” (GAH_00154) and all other In A. fulgidus, fructose 6-phosphate is phosphorylated to sequenced Archaeoglobales species contain genes en- fructose 1,6-bisphosphate by an ADP-dependent phospho- coding PK_C superfamily proteins [15, 33, 50–52, and fructokinase protein (EC:2.7.1.11) [67]. However, homologs NC_015320.1], which have pyruvate kinase and alpha/ of this enzyme were not present in the genomes of “G. beta domains and are homologous to an A. fulgidus en- ahangari” or any other Archaeoglobale species sequenced zyme with pyruvate kinase activity in vitro [72]. Pyruvate to date. Instead, the genome of “G. ahangari” contained can then be converted into acetyl-CoA via pyruvate syn- two genes (GAH_00966 and GAH_01843) coding for thase (GAH_01438-01441 and GAH_02021-02024). proteins with pfkB-like domains and ATP-binding sites, As in the close relative F. placidus [26], “G. ahan- which are consistent with the ATP-dependent phos- gari” lacks genes from the oxidative pentose phosphate phofructokinases (PFK-B) of other hyperthermophilic pathway but is predicted to circumvent this limitation archaea such as Aeropyrum pernix and Desulfurococ- [73] viathe useofa complete RuMP pathway (GAH_0 cus amylolyticus [68, 69]. The genome also contains two 0051 and GAH_01859). The latter results in the accu- genes (GAH_00357 and GAH_01437) encoding archaeal mulation of formaldehyde [73], which in “G. ahangari” fructose 1,6-bisphosphatases, which catalyze the reverse re- could be removed by formaldehyde-activating enzymes action during gluconeogenesis but can also supply fructose (GAH_00575 and GAH_00673). Ribulose 5-phosphate 1,6-bisphosphate to the glycolytic pathway from dihydroxy- formed in the RuMP pathway could then be converted into acetone phosphate and D-glyceraldehyde 3-phosphate [70]. ribose-5-phosphate by a ribose 5-phosphate isomerase and Furthermore, a triosephosphate isomerase (GAH_00576) then into PRPP by ribose-phosphate pyrophosphokinase was identified in the genome to catalyze the isomerization enzymes (GAH_00743 and GAH_00557). This supplies PR of dihydroxyacetone phosphate to D-glyceraldehyde 3- PP to various anabolic pathways such as the biosynthesis phosphate. Alternatively, GAH_01502 and GAH_01751, of histidine and purine/pyrimidine nucleotides. which encode proteins homologous to archaeal type class Similar to other Archaeoglobale species, a complete I fructose 1,6-bisphosphate aldolase proteins, could catalyze TCA cycle is present within the “G. ahangari” All enzymes the conversion of fructose 1,6-bisphosphate into D-glyceral- involved in the formation of oxaloacetate from acetyl-CoA dehyde 3-phosphate. (GAH_00258, GAH_01703, GAH_01110, GAH_02012-020 The next steps in the pathway involve the oxidation 13, GAH_00784-00784, GAH_00779-00782, GAH_00526- of D-glyceraldehyde 3-phosphate and formation of 3- 00527, and GAH_00039), including putative aconitase phosphoglycerate. The “G. ahangari” genome contains proteins (GAH_00857-00858) [74], were identified in a homolog (GAH_00413) of a GAPOR, which in A. the genome. Also present is a phosphoenolpyruvate fulgidus and many other archaeal species catalyzes the carboxylase (GAH_01652), which could catalyze the irreversible oxidation of D-glyceraldehyde-3-phosphate to reversible carboxylation of phosphoenolpyruvate to 3-phospho-D-glycerate bypassing the formation of the oxaloacetate, a precursor metabolite of many amino intermediate 1,3-bisphospho-D-glycerate [66]. In addition, acids. the genome of “G. ahangari” contains genes coding for an archaeal specific type II glyceraldehyde-3-phosphate de- Fatty acids as electron donors hydrogenase (GAH_01734) and a phosphoglycerate kinase “G. ahangari” strain 234 was the first hyperthermophile (GAH_01571), which could catalyze the formation of 3- reported to completely oxidize long-chain fatty acids phosphoglycerate via the 1,3-diphosphoglycerate inter- anaerobically, an unsuspected capability of hyperthermo- mediate. These two enzymes are unidirectional and philic microorganisms prior to this discovery [1]. Long- involved in formation of glyceraldehyde-3-phosphate chain fatty acids are abundant in sedimentary environ- from 3-phosphoglycerate during gluconeogenesis in most ments where they accumulate as byproducts of the hy- hyperthermophilic archaea [66]. drolysis of complex organic matter and the anaerobic As in A. fulgidus [71], “G. ahangari” has 2 genes coding degradation of alkanes [75, 76]. Long-chain fatty acids are for cofactor-independent phosphoglycerate mutase proteins also major components of crude oil [77], which is often (GAH_00739 and GAH_01116), which can catalyze the present in environments inhabited by Archaeoglobale spe- interconversion of 3-phospho-D-glycerate to 2-phospho-D- cies [78]. Consistent with the ability of Archaeoglobale glycerate. Phosphoenolpyruvate is then formed by an members to oxidize long-chain fatty acids, the genomes of enolase protein (GAH_00972), which is subsequently “G. ahangari” and other members of the Archaeoglobales dephosphorylated to pyruvate by pyruvate kinase. Al- (F. placidus, G. acetivorans, A. fulgidus, and others) con- though a gene coding for the well-characterized pyruvate tain a large number of genes coding for β-oxidation Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 11 of 19 pathway enzymes [15, 26, 43]. The A. fulgidus genome, for Degradation of aromatic compounds and n-alkanes example, contains 57 genes encoding the 5 core proteins F. placidus, a member of the Archaeoglobales closely re- (discussed below) involved in β-oxidation [43]. All of these lated to “G. ahangari”, can couple the complete oxidation genes were used as BLAST queries against the genomes of aromatic hydrocarbons to Fe(III) reduction [79–81]. of F. placidus [26],G. acetivorans [15], and “G. ahan- The “G. ahangari” genome does not contain any benzoate gari” and identified 39 homologous proteins in the ge- degradation genes, further supporting the observation that nomes of the F. placidus and G. acetivorans and 32 in it cannot utilize aromatic compounds as electron donors the genome of “G. ahangari”. for growth [1]. Interestingly, G. acetivorans, the other mem- Fatty acid degradation in the Archaeoglobales is thought ber of the Geoglobus genus, has homologues of all genes to occur in a manner similar to bacteria and mitochondria coding for proteins of the benzoyl-CoA ligation pathway [43], with the initial step involving activation of a long present in F. placidus, yet as in “G. ahangari” growth on chain fatty acid to a fatty acyl CoA by a fatty acyl CoA syn- aromatic hydrocarbons has not been observed in G. aceti- thetase/ligase. We identified seven genes in “G. ahangari” vorans [4, 15]. coding for fatty acid CoA synthetase proteins (GAH_00420, Another member of the Archaeoglobales, A. fulgidus, GAH_00623, GAH_01111, GAH_01124, GAH_01769, GA can also couple the oxidation of n-alkanes and n-alkenes H_01899, and GAH_02051). The next step in the pathway with sulfur respiration [82, 83]. This archaeon uses an involves the oxidation of the fatty acyl-CoA to a trans-2- alkylsuccinate synthase and an activating protein (AssD/ enoyl-CoA by acyl-CoA dehydrogenase proteins, which BssD; AF1449-1450) to oxidize saturated hydrocarbons in “G. ahangari” are putatively encoded by 11 genes (n-alkanes in the range of C -C ) [83]. We identified 10 21 (GAH_00179, GAH_00421, GAH_00484, GAH_00591, homologs of both of these proteins in the genome of GAH_01331, GAH_01442, GAH_01601, GAH_01810, “G. ahangari” (GAH_01645-01646) and G. acetivorans and GAH_02050). A water molecule is then added to (Gace_0420-0421). A. fulgidus can also oxidize long trans-2-enoyl-CoA to form (3S)-3-hydroxyacyl-CoA in a chain n-alk-1-enes (C to C ) when thiosulfate is 12:1 21:1 reaction catalyzed by an enoyl-CoA hydratase, which in provided as the terminal electron acceptor [82]. Al- “G. ahangari” could be encoded by 4 genes (GAH_00487, though enzymes involved in the activation of alkenes GAH_00802, GAH_01332, and GAH_01602). Two of these by A. fulgidus have not been characterized, the genome genes (GAH_00487 and GAH_01602) are in fact hybrid of A. fulgidus contains a homologue of a Mo-Fe-S con- proteins containing an enoyl-CoA hydratase domain fused taining enzyme (AF0173-AF0176) [82], which in Azoar- to a 3-hydroxyacyl-CoA dehydrogenase domain. Hybrid cus sp. EBN1 anaerobically hydroxylates a branched enoyl-CoA hydratase/dehydrogenase proteins such as these alkene [84]. The Mo-Fe-S enzyme consists of 4 sub- have been identified in other archaeal species including the units including a chaperonin-like protein, a membrane Archaeoglobales species G. acetivorans, F. placidus,and A. anchor heme-b binding subunit, an Fe-S binding subunit, fulgidus [15, 26, 43]. and a molybdopterin-binding subunit [85]. This gene clus- The next step in the β-oxidation pathway leads to the ter was identified in the genomes of “G. ahangari” formation of 3-oxoacyl-CoA in an oxidation reaction that (GAH_01285-01288) and F. placidus (Ferp_0121-0123), generates NADH and is catalyzed by a 3-hydroxyl-CoA but not in the other member of the Geoglobus genus, dehydrogenase protein, which in “G. ahangari” is likely G. acetivorans. encoded by several genes (GAH_00328, GAH_00487, GA H_01600, GAH_01602 – again noting the hybrid nature of Nitrogen compounds as electron acceptors GAH_00487 and GAH_01602). Finally, acetyl-CoA is re- Except for F. placidus [5, 57], all of the Archaeoglobales, moved from the 3-oxo-acyl-CoA molecule by an acetyl- including “G. ahangari” [1], are unable to use nitrate or ni- CoA acetyltransferase and is free to enter the TCA cycle. trite as electron acceptors for respiration [4, 6–10] There are 8 genes in the “G. ahangari” genome that (Table 5). Yet, surprisingly, the genome of “G. ahangari” could catalyze this reaction (GAH_00292, GAH_00485, contains several 4Fe-4S domain-containing nitrate and GAH_00625, GAH_00626, GAH_01327, GAH_01328, sulfite reductase proteins (GAH_01242 and GAH_02063) GAH_01886, and GAH_02049). Additional proteins in- volved in fatty-acid metabolism include the alpha (GA Table 5 Terminal electron acceptors in the Archaeoglobales H_01318) and beta (GAH_01319) subunits of a 3- Electron acceptors oxoacid CoA-transferase. The large number of genes dedicated to β-oxidation in “G. ahangari” and other spe- Organism Sulfate Sulfite Thiosulfate Nitrate Fe(III) cies within the Archaeoglobales provides genomic evi- Geoglobus spp. -- - - + dence supporting the notion that long- and short-chain Ferroglobus placidus -- + + + fatty acid oxidation is a conserved metabolic feature Archaeoglobus spp. +/− ++ - - within the family. Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 12 of 19 as well as all four subunits (NarGHIJ) of a nitrate reduc- pathway, and together with the GDH pathway, function as tase (GAH_01285-01288). A nitrate/nitrite transporter is the two major paths for ammonium assimilation in ar- also annotated in the genome (GAH_00501), though it chaea [86]. While the GDH pathway does not use ATP as does not cluster with genes involved in nitrate/nitrite res- an energy source, as the GS-GOGAT pathway does, it has piration and thus may function in the transport of alterna- a lower affinity for ammonium [86]. The presence of these tive compounds. In addition, we identified a gene in this enzymes and two ammonium transporter proteins region of the genome (GAH_01290) coding for an unchar- (GAH_00438 and GAH_01767) for the formation of 2- acterized channel protein, which could potentially func- oxoglutarate and glutamate from ammonium, is consistent tion as a nitrate transport protein. The presence of genes with the notion that “G. ahangari” is under pressure to as- encoding both nitrate reductase proteins (NarGHIJ and similate ammonium for anabolic processes. NirA) combined with the inability of “G. ahangari” to use nitrate for respiration [1] suggests a role for these Sulfur compounds as electron acceptors proteins in assimilatory, rather than dissimilatory, ni- Most members of the Archaeoglobales are dissimilatory trate reduction [86]. sulfate-reducing organisms and able to use several Similar to F. placidus,the “G. ahangari” genome does sulfur-containing compounds as electron acceptors to not contain any nir or nrf genes (for the NADH- and fuel their metabolism [5–10] (Table 5). By contrast, “G. formate-dependent nitrite reductase proteins, respect- ahangari” cannot couple the oxidation of electron do- ively), with the exception of several homologues of nors that supported Fe(III) reduction to the respiration NirA (GAH_00501, GAH_00506, GAH_01242, and GA of commonly considered sulfur-containing electron ac- H_02063), a nitrite reductase protein that catalyzes the ceptors such as sulfate, thiosulfate, sulfite, or S [1]. reduction of nitrite to ammonia and is involved in as- Interestingly, the genome of “G. ahangari” contains two similatory nitrate reduction in other organisms [86]. genes (GAH_02067 and GAH_01481) coding for sulfate Also missing are genes coding for nitric and nitrous adenylyltransferase, which can initiate the first step in oxide reductase proteins, which the genome of F. placi- both the dissimilatory and assimilatory sulfate reduction dus contains [26], again supporting the observation that pathways by catalyzing the formation of APS from ATP “G. ahangari” is not capable of dissimilatory nitrate re- and inorganic sulfate. The enzyme is also present in the duction [1]. The lack of these enzymes helps explain genome of F. placidus which, like “G. ahangari”, is un- the physiological separation of “G. ahangari” from its able to respire sulfate [5] (Table 5). APS can then be closephylogeneticrelative F. placidus, which is capable used as substrate in the assimilatory [90–93] or dissimi- of dissimilatory nitrate reduction to N O[57]. Further- latory [94, 95] pathway, depending on the needs and more, it is unlikely that the reduction of nitrogen- capabilities of the microorganism [92]. The assimilatory containing compounds exerts any significant selective pathway converts APS to the intermediate PAPS in a re- pressure on hydrothermal vent microorganisms, as con- action catalyzed by an adenylsulfate kinase, which in “G. centrations of these compounds are often low in vent ahangari” is encoded by GAH_01478. The genome of systems [87]. “G. ahangari” contains genes coding for both the alpha N gas, on the other hand, is the largest reservoir of and beta subunits of adenylsulfate reductase (GAH_ nitrogen in the ocean [87, 88] and nitrogen fixation sup- 02065-02066), an FAD dependent oxidoreductase protein plies hydrothermal vent systems with nitrogen sources that reduces APS to sulfite in the dissimilatory pathway. for assimilatory growth [87]. Ammonium is particularly However, the genome lacks genes coding for a dissimilatory abundant in the heavily sedimented Guaymas Basin sulfite reductase (dsrAB), which catalyzes the reduction of hydrothermal system [89], from which “G. ahangari” sulfite to hydrogen sulfide in the final step of the dissimila- was isolated [1], and this could select for organisms with tory sulfate reduction pathway [96]. Strong matches could assimilatory rather than dissimilatory nitrogen metabo- not be found even when the alpha (AAB17213.1) and beta lisms and inhibit nitrogen fixation. Not surprisingly, the (AEY99618.1) subunits of the sulfite reductase from annotated genome of “G. ahangari” and homology A. fulgidus were used as queries in manual searches. searches for the primary enzymes from the nitrogen fix- It is interesting to note that, despite the absence of ation pathway (nifH, nifD,and nifK) provided no signifi- dsrAB genes in the genome, “G. ahangari” does have a cant hits, as previously reported for other members of nitrite and sulfite reductase 4Fe-4S domain-containing the Archaeoglobales. protein (GAH_02063) located in a cluster of genes in- The genome does contain genes coding for a glutam- volved in sulfur metabolism (GAH_02063-02067). ine synthetase (GAH_01658), a glutamate synthase (GAH_ Whether these genes code for functional proteins of the 01667-01669), and a glutamate dehydrogenase (GAH_ dissimilatory pathway, perhaps with electron donor/ac- 00573 and GAH_01931). The enzymes glutamine syn- ceptor pairs not tested yet, remains to be elucidated. F. thetase-glutamate synthase comprise the GS-GOGAT placidus, for example, has homologs of all of these Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 13 of 19 genes, except for dsrAB, and it grows with thiosulfate and GAH_01279) and an oligosaccharyltransferase (GAH_ as the sole electron acceptor when hydrogen is pro- 01455), which could glycosylate the growing archaellum vided as an electron donor [5]. This capability may be [99] and post-translationally modify surface proteins, as is due to the presence of several molybdopterin oxidore- commonly observed in the Archaea [100]. However, ductase proteins within the genome of F. placidus that chemotaxis proteins, which are present in nearly all se- show high similarity to a predicted thiosulfate reductase quenced members of the Archaeoglobales [15, 26, 43, 44], (NP_719592.1) from Shewanella oneidensis.However, and NC_015320.1, with the exception of A. sulfaticallidus strong homologs of this protein were not present in the [45], and are typically found immediately upstream or genome of “G. ahangari”. downstream of the fla gene cluster, were absent in “G. ahangari”. The lack of chemotaxis genes in “G. ahangari” Fe(III) as the sole electron acceptor for respiration contrasts with their presence in most Archaeoglobales ge- The most distinctive physiological feature of “G. ahangari” nomes, including G. acetivorans [15], the other member strain 234 is its dependence on Fe(III) as an electron ac- of the genus. Both Geoglobus species were isolated from ceptor for respiration [1]. Both insoluble Fe(III) oxides hydrothermal vent chimneys: G. acetivorans from the and soluble species of Fe(III), such as Fe(III) citrate, sup- Ashadze field on the Mid-Atlantic Ridge at a depth of port growth, though the original isolate did not grow read- 4100 m [4] and “G. ahangari” from a Guaymas Basin ily with the soluble electron acceptor and required chimney at a depth of 2000 m [1]. The hydrothermal prolonged adaptation under laboratory conditions to grow fluids spewed from chimneys within the Guaymas in its presence [1]. Key to the ability of “G. ahangari” to Basin system are likely enriched in nutrients after respire the insoluble Fe(III) oxides is the ability of the cells passage through the 300–400 m thick, organic-rich to locate the oxides, attach to them, and position electron sediments underneath [101]. Furthermore, hydrothermal carriers of the outer surface close enough to favor the circulation at this site is high [23], which would rapidly re- transfer of electrons [14]. Hence, we examined the gen- plenish nutrients, both electron donors and fresh Fe(III) ome of “G. ahangari” for genes that code for cellular com- oxides, and thus organisms living in this environment may ponents that could be involved in motility and attachment not need to utilize chemotactic mechanisms to seek out and extracellular electron transfer. these nutrients. By contrast, hydrothermal fluids from off- Motility in this organism is enabled by a single flagel- shore spreading systems, such as the Ashadze field, flow lum [1], which in archaea is designated as an archaellum through thin sediment layers before reaching the chimney to reflect its distinct evolutionary origin [97]. Archaeal [101]. This likely increases the selective pressure on resi- flagellar genes can be organized into one of two very dent microbes to evolve chemotactic mechanisms to lo- well conserved clusters (fla1 and fla2) based on the type cate nutrients. and order of genes in the cluster: flaBC(D/E)FGHIJ in The genome of “G. ahangari” also encodes proteins fla1 and flaBGFHIJ in fla2 [98]. The fla1 clusters are ex- potentially involved in the assembly of extracellular pro- clusively found in Euryarchaea while fla2 clusters are tein appendages such as pili. We identified, for example, a generally associated with the Crenarchaea, which in- prepilin peptidase (GAH_00760), numerous type II secre- cludes the Desulfurococcales and Sulfolobales orders, tion system proteins (GAH_01195-01196, GAH_00173, and are also present within the Euryarchaeal order GAH_00290, GAH_01412-01413), and a putative twitch- Archaeoglobales [98]. Interestingly, the Archaeoglobales ing motility pilus retraction ATPase (GAH_00960). Hom- have members with both types. We identified, for ex- ologous genes are also present in the genomes of G. ample, a fla1 gene cluster in the genome of “G. ahan- acetivorans [15], F. placidus [26], and A. fulgidus [43]. In gari” (GAH_01994-02001), as in F. placidus (Ferp_14 addition, “G. ahangari” has two genes encoding proteins 56-1463) [26], while the flagellar genes of Archaeoglo- with DUF1628 or DUF1628-like domains (GAH_01202, bus spp. [15, 26, 43–45], and NC_015320.1 and G. acet- GAH_01671), which are associated with previously de- ivorans [15] were of the fla2 type. It has been suggested scribed archaeal pilin proteins [102] and present in all se- that a horizontal gene transfer (HGT) event occurred quenced members of the Archaeoglobales [15, 26, 43–45], in the Ferroglobus lineage after divergence from the and NC_015320.1. Any of these proteins could be involved Archaeoglobus and Geoglobus lineages [15]. Yet, the in the assembly of the curled extracellular appendages that presence of a fla1 gene cluster in the flagellated and “G. ahangari” produces to attach to Fe(III) oxides and fa- motile “G. ahangari” [1], when compared to the fla2 cilitate the transfer of electrons from electron carriers gene cluster found in the non-motile and non-flagellated located on the outer surface to the insoluble electron G. acetivorans [4], would lend credence to a possible sec- acceptor [14]. ond HGT event within the family. “G. ahangari” uses heme-containing proteins to trans- Thegenomeof “G. ahangari” also encodes several port electrons across the cell envelope and to the insoluble glycosyltransferase genes (GAH_00218, GAH_00870, Fe(III) oxides [14]. The most common heme-containing Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 14 of 19 proteins used by mesophilic Fe(III) reducers for extracellu- and F. placidus (Ferp_1362 and Ferp_1364). All of these lar electron transport are c-type cytochromes [103]. Ar- proteins contain cysteine-rich motifs consisting of chaea are known to have a variant form of the cytochrome LX[S,N]C[E,D,H]C but lack the LRCXXC motif char- c maturation (Ccm) system, whereby the CcmE protein acteristic of most CcmH proteins. However, they all has a CXXXY-type motif, rather than the HXXXY motif flank a duplicate CcmF-encoding gene found only in found in eukaryotic and most bacterial c-cytochromes, and “G. ahangari” (GAH_01093), G. acetivorans [15], and CcmH is absent [37]. Similar to other sequenced Archaeo- F. placidus [26]. globales, “G. ahangari” has an archaeal-type CcmE protein In addition to having a distinct cytochrome c biogenesis (GAH_01977), a CcmC (GAH_00620) with a tryptophan- pathway, the iron-reducing Archaeoglobales, Geoglobus and rich motif (WG[S,T][F,Y]WNWDPRET), a CcmF protein Ferroglobus species, also have more c-type cytochromes (GAH_01976 and GAH_01093) with the motif than any other archaeon, and many of these c-type cyto- WGGXWFWDPVEN, and a gene coding for a CcmB chromes have multiple heme groups [15, 18, 26]. The gen- homolog (GAH_00449) lacking the conserved FXXDX ome of “G. ahangari” contains 21 genes (Table 6) encoding XDGSL motif. Although previously reported archaeal cyto- putative c-type cytochromes, 7 of which have more than 5 chrome maturation pathways do not contain CcmH [37], heme groups; F. placidus has 30 c-type cytochromes (12 we identified two putative CcmH proteins in the genomes with more than 5 heme groups); and G. acetivorans has 16 of not only “G. ahangari” (GAH_01092 and GAH_01094), c-type cytochromes (8 with more than 5 heme groups). By but also in G. acetivorans (GACE_2070 and GACE_2068) contrast, Archaeoglobus species, which do not use Fe(III) Table 6 Putative c-type cytochromes Gene ID: Annotation: # of heme Calculated TM binding motifs: molecular weight: domains: GAH_00015 Hypothetical protein 4 58.4 0 GAH_00283 Cytochrome c7 4 21.2 1 GAH_00286 Nitrate/TMAO reductases, 12 39.1 0 membrane-bound tetraheme cytochrome c subunit GAH_00301 Putative redox-active protein 2 31.5 3 (C_GCAxxG_C_C) GAH_00504 Hypothetical protein 10 54.5 1 GAH_00505 Hypothetical protein 4 26.8 2 GAH_00506 Cytochrome c3 9 48.6 0 GAH_00507 Cytochrome c7 4 27.4 1 GAH_00508 Hypothetical protein 5 28.5 1 GAH_00510 Hypothetical protein 4 27.3 1 GAH_00817 Seven times multi-haem 8 53.7 1 cytochrome CxxCH GAH_01091 Hypothetical protein 1 11.7 1 GAH_01235 Hypothetical protein 5 21.5 0 GAH_01236 Hypothetical protein 5 22.3 0 GAH_01253 Hypothetical protein 4 16.9 0 GAH_01256 NapC/NirT cytochrome c family, 10 43.6 1 N-terminal region GAH_01296 Cytochrome c family protein 4 17.2 1 GAH_01297 Seven times multi-haem 8 61.0 1 cytochrome CxxCH GAH_01306 Class III cytochrome C family 8 46.3 0 GAH_01534 Hypothetical protein 1 18.5 1 GAH_01700 Hypothetical protein 3 9.9 0 No signal peptide detected Signal peptide detected by PRED-SIGNAL Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 15 of 19 electron acceptors (Table 5), have significantly fewer c-type number of c-type cytochromes within and on the cell cytochromes. Within this genus, the greatest number of surface, as well as other redox-active proteins such as c-type cytochrome encoding genes was found in the thermostable ferredoxin and Fe-S proteins. The paucity genome of A. veneficus, which has 16 c-type cytochromes of c-type cytochromes within non-Fe(III) respiring mem- (3 with more than 5 hemes). Other species such as A. bers of the Archaeoglobales (Archaeoglobus species) is profundus and A. sulfaticallidus have only 1 monoheme consistent with the physiological separation between these c-type cytochrome and A. fulgidus has 3 c-type cyto- archaea and F. placidus, G. acetivorans, and “G. ahangari”, chromes (none of which have more than 5 heme groups). which can gain energy for growth from the reduction of The subcellular localization of the putative c-type cy- Fe(III) electron acceptors. Additionally, some genes re- tochromes of “G. ahangari” was also investigated. The quired for both dissimilatory sulfate and nitrate metabo- ExPASy TMPred program [42] revealed that a majority lisms are absent in “G. ahangari” and G. acetivorans. This (62 %) of the c-type cytochromes have at least 1 trans- supports the physiological separation of Geoglobus spp. membrane helix, consistent with their association to from F. placidus, which is capable of Fe(III)-, thiosulfate-, the cytoplasmic membrane. One of these c-type cyto- and nitrate respiration, and from Archaeoglobus species chrome proteins (GAH_00504) was predicted to be which are primarily sulfur-respiring organisms. Genomic extracellular. We also identified several c-type cyto- data also support the reported physiological similarities chromes (GAH_01306, GAH_00286, GAH_01534, and between “G. ahangari” and other Archaeoglobales such as GAH_01253) with predicted sizes once in mature form autotrophic growth with H via the reductive acetyl-CoA/ (46.3, 39.1, 18.5, and 16.9 kDa, respectively) matching Wood-Ljungdahl pathway and the use of similar electron those reported for outer-surface heme-containing pro- donors, including short- and long-chain fatty acids. teins required for the reduction of insoluble Fe(III) ox- Noteworthyis thefact that genomicevidencesupports ides, but not soluble Fe(III) citrate, by “G. ahangari” the synthesis of the methanogenic coenzyme-F in [14] (Table 6). Hence, these 4 c-type cytochromes likely “G. ahangari”, which is responsible for the characteris- function as the terminal electron carriers between the tic fluorescence detected in all Archaeoglobus spp. ex- cells and the oxides. cept for “G. ahangari” or F. placidus. Hence, the In addition to c-type cytochromes, we identified other genome sequence of “G. ahangari” provides valuable potential electron carriers such as quinones, flavoproteins, insights into its physiology and ecology as well as into and various Fe-S proteins (i.e. ferredoxins). We identified the evolution of respiration within the Archaeoglobales. a number of ub iquinone/menaquinone biosynthesis pro- teins in the genome of “G. ahangari” (Additional file 1), Taxonomic note which could create a quinone pool in the membrane to The initial publication [1] of the “Geoglobus” genus and promote electron transfer. The genome also contains a “Geoglobus ahangari” species was accepted for publica- great number of Fe-S binding domain proteins and ferre- tion with extenuating circumstances at several culture- doxins, which could participate in electron transfer path- collection agencies. Thus, upon the original publication ways (Additional file 2). Fe-S proteins and ferredoxins “G. ahangari” strain 234 was accepted only at a single were also abundant in the genome of G. acetivorans and agency. In addition, the G + C mol% determined from F. placidus, which, like “G. ahangari”, also utilize Fe(III) the complete genome sequence (53.1 mol%) differs from respiration as their primary metabolism. Fe-S proteins and that originally published (58.7 mol%), representing a dis- ferredoxins are regarded as some of the most ancient of crepancy of over 5 mol%. This publication thus warrants electron transfer carriers [104] and also have high thermo- an emended description of the genus Geoglobus and the stability [105], which is critical to ensure maximum rates type species, “Geoglobus ahangari”. of electron transfer in the hot hydrothermal vent systems. Thus, the abundance of electron carrier proteins, some Emended description of “Geoglobus” Kashefi et al. known to have increased thermostability, and c-type cyto- The description of the genus “Geoglobus” is the one pro- chromes, some of them localized to the outer surface, is vided by Kashefi et al. [1], with the following modifica- consistent with a mechanism evolved for efficient extracel- tions. In addition to the single monopolar flagellum, lular electron transfer in hot environments. numerous curled filaments can be seen per cell [14]. The G + C content of the genomic DNA of the type spe- Conclusions cies is 53.1 mol%. “G. ahangari” strain 234 is only one of three members of the Archaeoglobales capable of dissimilatory Fe(III) Emended description of “Geoglobus ahangari” Kashefi et respiration. Furthermore, it is an obligate Fe(III) reducer al. that grows better with insoluble than soluble Fe(III) spe- The description of the species “Geoglobus ahangari” is the cies. Consistent with this, the genome contains a large one provided by Kashefi et al. [1, 2], with the following Manzella et al. Standards in Genomic Sciences (2015) 10:77 Page 16 of 19 modifications. The type strain is strain 234 and has been 2002;52:719–28. Available at: http://ijs.sgmjournals.org/cgi/content/abstract/52/ 3/719. Accessed January 13, 2015. deposited at three culture collection agencies, which in- 2. Kashefi K. Hyperthermophiles: Metabolic diversity and biotechnological clude the Deutsche Sammlung von Mikroorganismen und applications. In: Anitori RP, editor. Extremophiles: Microbiology and Zellkulturen (DSM-27542), the Japan Collection of Micro- Biotechnology. Norfolk, UK.: Caister Academic Press; 2012. 3. Tor JM, Kashefi K, Lovley DR. Acetate oxidation coupled to Fe(III) reduction in organisms (JCM 12378), and the American Type Culture hyperthermophilic microorganisms. Appl Environ Microbiol. 2001;67:1363–5. Collection (BAA-425). Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC92735/. Accessed January 13, 2015. 4. Slobodkina G, Kolganova T, Querellou J, Bonch-Osmolovskaya E, Slobodkin Additional files A. Geoglobus acetivorans sp. nov., an iron(III)-reducing archaeon from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol. 2009;59:2880–3. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19628601. Accessed Additional file 1: Ub iquinone and menaquinone biosynthesis January 13, 2015. proteins present in the genome of G. ahangari. Ub iquinone and menaquinone biosynthesis proteins identified within the genome of G. 5. Hafenbradl D, Keller M, Dirmeier R, Rachel R, Roßnagel P, Burggraf S, et al. ahangari strain 234 . (DOCX 16 kb) Ferroglobus placidus gen. nov., sp. nov., a novel hyperthermophilic archaeum that oxidizes Fe2+ at neutral pH under anoxic conditions. Arch Microbiol. Additional file 2: Fe-S binding domain proteins and ferredoxins 1996;166:308–14. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8929276. within the genome of G. ahangari. Fe-S binding domain proteins and Accessed January 13, 2015. ferredoxins identified within the genome of G. ahangari strain 234 . 6. Beeder J, Nilsen RK, Rosnes JT, Torsvik T, Lien T. Archaeoglobus fulgidus (DOCX 16 kb) isolated from hot North Sea oil field waters. Appl Environ Microbiol. 1994;60:1227–31. Available at: http://aem.asm.org/content/60/4/1227.short. Accessed January 13, 2015. Abbreviations 7. Huber H, Jannasch H, Rachel R, Fuchs T, Stetter KO. Archaeoglobus veneficus SDS: Sodium dodecyl sulfate; EDTA: Ethylenediaminetetraacetic acid; TE: sp. nov., a novel facultative chemolithoautotrophic hyperthermophilic Tris-EDTA; EB: Elution buffer; RAST: Rapid Annotation using Subsystem sulfite reducer, isolated from abyssal black smokers. Syst Appl Microbiol. Technology; IGS: Institute for Genome Sciences; IMG-ER: Integrated Microbial 1997;20:374–80. Available at: http://www.sciencedirect.com/science/article/pii/ Genomes – Expert Review; EC: Enzyme Commission; COG: Clusters of S0723202097800057. Accessed January 13, 2015. Orthologous Group; HPLC: High performance liquid chromatography; 8. Mori K, Maruyama A, Urabe T, Suzuki K-I, Hanada S. Archaeoglobus infectus ORB: Origin recognition box; ROK: Repressor protein, open reading frame, sp. nov., a novel thermophilic, chemolithoheterotrophic archaeon isolated sugar kinase; GAPOR: Glyceraldehyde-3-phosphate:ferredoxin oxidoreductase; from a deep-sea rock collected at Suiyo Seamount, Izu-Bonin Arc, western RuMP: Ribulose monophosphate; PRPP: 5-phosphoribosyl diphosphate; Pacific Ocean. Int J Syst Evol Microbiol. 2008;58:810–6. Available at: http:// TCA: Tricarboxylic acid; Fe-S: Iron-sulfur; GS-GOGAT: Glutamine www.ncbi.nlm.nih.gov/pubmed/18398174. Accessed January 13, 2015. synthetase-glutamate synthase; GDH: Glutamate dehydrogenase; 9. Steinsbu BO, Thorseth IH, Nakagawa S, Inagaki F, Lever MA, Engelen B, APS: Adenosine 5′-phosphosulfate; PAPS: 3′-phosphoadenylyl sulfate. et al. Archaeoglobus sulfaticallidus sp. nov., a thermophilic and facultatively lithoautotrophic sulfate-reducer isolated from black rust Competing interests exposed to hot ridge flank crustal fluids. Int J Syst Evol Microbiol. The authors declare that they have no competing interests. 2010;60:2745–52. Available at: http://www.ncbi.nlm.nih.gov/pubmed/ 20061497. Accessed January 13, 2015. Authors’ contributions 10. Burggraf S, Jannasch HW, Nicolaus B, Stetter KO. Archaeoglobus MPM, DEH, JMR, and AC sequenced, assembled and annotated the genome. profundus sp. nov., represents a new species within the sulfate-reducing MPM, DEH, GR, JMR, and KK analyzed the data and drafted the manuscript. archaebacteria. Syst Appl Microbiol. 1990;13:24–8. Available at: http:// All authors read and approved the final manuscript. linkinghub.elsevier.com/retrieve/pii/S0723202011801761. Accessed January 13, 2015. Acknowledgements 11. Lovley DR. Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol Rev. The authors gratefully acknowledge Tracy K. Teal at Michigan State for 1991;55:259–87. Available at: http://www.pubmedcentral.nih.gov/ assistance with assembly and annotation and Abigail Vanderberg at the articlerender.fcgi?artid=372814&tool=pmcentrez&rendertype=abstract. Center for Advanced Microscopy at Michigan State for help with scanning Accessed January 13, 2015. electron microscopy. We also acknowledge technical support provided by 12. Weber KA, Achenbach LA, Coates JD. Microorganisms pumping iron: the Research Technology Support Facility and the Hypercomputing Center anaerobic microbial iron oxidation and reduction. Nat Rev Microbiol. at Michigan State University, the Deep Sequencing Core Facility at the 2006;4:752–64. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16980937. University of Massachusetts Medical School, and the Genomics Resource lab Accessed January 13, 2015. at the University of Massachusetts-Amherst. This work was supported with 13. Gavrilov SN, Lloyd JR, Kostrikina NA, Slobodkin AI. Fe(III) oxide reduction by funds from a Strategic Partnership Grant from the MSU Foundation to GR a Gram-positive thermophile: Physiological mechanisms for dissimilatory and KK, a GAANN fellowship by the Department of Education and a DuVall reduction of poorly crystalline Fe(III) oxide by a thermophilic Gram-positive Award to MPM, and a faculty grant from Western New England University to bacterium Carboxydothermus ferrireducens. Geomicrobiol J. 2012;29:804–19. DEH and JMR. Available at: http://www.tandfonline.com/doi/abs/10.1080/ 01490451.2011.635755. Accessed January 13, 2015. Author details 14. Manzella MP, Reguera G, Kashefi K. Extracellular electron transfer to Fe(III) Department of Microbiology and Molecular Genetics, Michigan State oxides by the hyperthermophilic archaeon Geoglobus ahangari via a direct University, East Lansing, MI, USA. Department of Physical and Biological contact mechanism. Appl Environ Microbiol. 2013;79:4694–700. 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The complete genome sequence and emendation of the hyperthermophilic, obligate iron-reducing archaeon “Geoglobus ahangari” strain 234T

The complete genome sequence and emendation of the hyperthermophilic, obligate iron-reducing archaeon “Geoglobus ahangari” strain 234T

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The complete genome sequence and emendation of the hyperthermophilic, obligate iron-reducing archaeon “Geoglobus ahangari” strain 234T

The complete genome sequence and emendation of the hyperthermophilic, obligate iron-reducing archaeon “Geoglobus ahangari” strain 234T

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