Scholarly article on topic 'Working draft genome sequence of the mesophilic acetate oxidizing bacterium Syntrophaceticus schinkii strain Sp3'

Working draft genome sequence of the mesophilic acetate oxidizing bacterium Syntrophaceticus schinkii strain Sp3 Academic research paper on "Biological sciences"

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Academic research paper on topic "Working draft genome sequence of the mesophilic acetate oxidizing bacterium Syntrophaceticus schinkii strain Sp3"

Manzoor et al. Standards in Genomic Sciences (2015) 10:99 DOI 10.1186/s40793-015-0092-z

SHORT GENOME REPORT

Open Access

Working draft genome sequence of the mesophilic acetate oxidizing bacterium Syntrophaceticus schinkii strain Sp3

Shahid Manzoor1,3, Bettina Müller2*, Adnan Niazi1, Anna Schnürer2 and Erik Bongcam-Rudloff1

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Abstract

Syntrophaceticus schinkii strain Sp3 is a mesophilic syntrophic acetate oxidizing bacterium, belonging to the Clostridia class within the phylum Firmicutes, originally isolated from a mesophilic methanogenic digester. It has been shown to oxidize acetate in co-cultivation with hydrogenotrophic methanogens forming methane. The draft genome shows a total size of 3,196,921 bp, encoding 3,688 open reading frames, which includes 3,445 predicted protein-encoding genes and 55 RNA genes. Here, we are presenting assembly and annotation features as well as basic genomic properties of the type strain Sp3.

Keywords: Syntrophic acetate oxidizing bacteria, Acetogens, Syntrophy, Methanogens, Hydrogen producer, Methane production

Introduction

During anaerobic degradation of organic material, acetate is formed as a main fermentation product, which is further converted to methane. Two mechanisms for methane formation from acetate have been described: The first one is carried out by aceticlastic methanogens converting acetate to methane and CO2 under low ammonia conditions [1]. The second mechanism, dominating under high ammonia conditions, occurs in two steps, and is performed by acetate-oxidizing bacteria oxidizing acetate to H2 (formate) and CO2 and a methanogenic partner using the hydrogen (formate) to reduce CO2 to methane [2-4]. Most fascinating on this syntrophic relationship is, that the overall reaction operates with a AG"' of -36 kJ x mol-1 close to the thermodynamic equilibrium.

The number of isolated and characterized SAOB is restricted most likely due to their considerable differences in substrate utilization abilities and cultivation requirements. To date three mesophilic SAOB, namely Clostridium ultunense [5], Syntrophaceticus schinkii [6], " Tepidanaerobacter acetatoxydans" [7] and two thermophilic SAOB, namely Thermacetogenium phaeum

* Correspondence: Bettina.Muller@slu.se

2Department of Microbiology, Swedish University of AgriculturalSciences, BioCenter, Uppsala SE 750 07, Sweden

Fulllist of author information is available at the end of the article

[2] and Thermotoga lettingae [8] currently renamed to Pseudothermotoga lettingae have been isolated and characterized. Among those, two complete genome sequences of T. phaeum [9], "T. acetatoxydans" [10] and one draft genome sequence of C. ultunense [11] have been published, the later two by this working group. Here, we are presenting the draft genome sequence of the third mesophilic SAOB Syntrophaceticus schinkii strain Sp3. To date, strain Sp3 is the only isolated and characterized representative of the species S. schinkii and was recovered from an up flow anaerobic filter treating wastewater from a fishmeal factory [6]. This process was characterized by high ammonium concentration (6.4 g l- NH+). S. schinkii shows the least narrow substrate spectrum compared to all known SAOB, when growing heterotro-phically [6]. The main end product formed is acetate, what allocates the species to the physiological group of acetogens.

Since the recovery of S. schinkii we found it at high abundance in all mesophilic large scale and lab scale biogas producing process we have investigated so far. Genome analysis and comparative genomics might help us to understand general features of syntrophy in particular

O© 2015 Manzoor et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 BlORflCCl Central International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and

reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Fig. 1 Image. Phase-contrast micrograph of Syntrophaceticus schinkii strain Sp3

energy conservation and electron transfer mechanisms during syntrophic acetate oxidation.

The present study summarizes genome sequencing, assembly and annotation as well as general genomic properties of the Syntrophaceticus schinkii strain Sp3 genome.

Organism information

Classification and features

Syntrophaceticus schinkii Sp3 (Fig. 1) is an obligate anaerobic, endospores forming bacterium, whose cells were found to be Gram variable with changing shapes dependent on the growth condition (Table 1, [6]). No flagella have been observed under any condition tested. It can grow up to 0.6 M NH4Cl in pure culture between 25 °C and 40 °C. A more detailed physiological description can be found in Westerholm et al. [6]. Minimum Information about the Genome Sequence (MIGS) of S. schinkii strain Sp3 is provided in Table 1 and Table S1 (Additional file 1).

Phylogentic analysis of the single 16S rRNA gene copy affiliates S. schinkii strain Sp3 to the Clostridia class within the phylum Firmicutes. The RDP Classifier ([12] 2015-08-05) confirmed further the affiliation to Thermo-anaerobacteraceae as published by [6] in 2011 (Table 1). The comparison of the 16S rRNA gene sequence with the latest available databases from GenBank (2015-08-05) using NCBI BLAST [13] under default settings identified the thermophilic SAOB T. phaeum (NR_074723.1) as the closest characterized relative sharing 92.12 % identity (Fig. 2). S. schinkii is only distantly related to the characterized meso-philic SAOB C. ultunense ( 82.54 % identity), and "T.

- Clostridium ljungdahlii DSM 13528 (NR„074161)

- Clostridium magnum DSM 2767 (X77835) -Ruminococcus pmductus DSM 2950 (D14144)

87 I-Clostridium aceticum DSM 1496 (Y18183)

-Clostridium ultunense DSM 10521 (Z69293)

I-Eubacterium limosum ATCC 8486 (NR_044719)

100 I-Acetobacterium woodii DSM 1030 (NR_026323)

-Acetonema longum DSM 6540 (NR_041951)

I-Syntrophobacterpfennigii DSM 10092 (X82875)

100 I-Syntrophobacter wolinii DSM 2805 (X70905)

100 r Thermoanaerobacterium aotearoense DSM 10170 (X93359)

■ Thermoanaerobacterium saccharolyticum DSM 7060 (L09169)

- Thermoanaerobacterthermohydrosulfuricus DSM 567 (L09161)

— Thermoanaerobacter kivui DSM 2030 (L09160)

- Moorella thermoacetica DSM 2955T (AB572912)

! oo I-Thermacetogenium phaeum DSM 12270 (NR_024688)

'-Syntrophaceticus schinkii Sp3 (Sequenced)

-Thermotoga lettingae DSM 14385 (AF355615)

- Thermosediminibacter oceani DSM 16646 (AY703478)

■ Tepidanaerobacter syntrophicus DSM 15584 (AB106353)

100 1-Tepidanaerobacter acetatoxydans Re1 (NR_116298)

-Syntrophomonas sapovorans DSM 3441 (AF022249)

■ Syntrophothermus iipocalidus DSM 12680 (AB021305)

Fig. 2 Phylogentic tree. Phylogenetic tree highlighting the relationship of Syntrophaceticus schinkii Sp3 relative to known SAOB, acetogens, and other syntrophic operating bacteria. The 16S rRNA-based alignment was carried out using MUSCLE [32] and the phylogenetic tree was inferred from 1,521 aligned characteristics of the 16S rRNA gene sequence using the maximum-likelihood (ML) algorithm [33] with MEGA 6.06 [34, 35]. Bootstrap analysis [36] with 100 replicates was performed to assess the support of the clusters

Table 1 Classification and general features of Syntrophaceticus schinkii strain Sp3 according to the "minimum information about a

Genome Sequence" (MIGS) specification [22]

MIGS ID Property Term Evidence codea

Classification Domain Bacteria TAS [23, 24]

Phylum Firmicutes TAS [25]

Class Clostridia TAS [26, 27]

Order Thermoanaerobacterales TAS [26] (p132), [28]

Family Thermoanaerobacteraceae TAS [26] (p132), [29]

Genus Syntrophaceticus TAS [6, 30]

Species Syntrophaceticus schinki TAS [6, 30]

Strain Sp3 TAS [6]

Gram stain Variable TAS [6]

Cell shape Variable b TAS [6]

Motility Non motile TAS [6]

Sporulation Terminalendospores TAS [6]

Temperature range Mesophilic TAS [6]

Optimum temperature 37-40 °C TAS [6]

Carbon source Heterotroph TAS [6]

Energy source Chemoheterotroph TAS [6]

MIGS-6 Habitat Anaerobic sludge TAS [6]

MIGS-6.3 Salinity Up to 0.6 M NH4Cl TAS [6]

MIGS-22 Oxygen Obligate anaerob TAS [6]

MIGS-15 Biotic relationship Syntrophy (beneficial) TAS [6]

MIGS-14 Pathogenicity Not reported NAS

MIGS-4 Geographic location Spain NAS

MIGS-5 Sample collection time 1992 NAS

MIGS-4.1 Latitude 42.851329 NAS

MIGS-4.2 Longitude -8.475933 NAS

MIGS-4.3 Depth Not reported NAS

MIGS-4.4 Altitude Not reported NAS

aEvidence codes—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). Evidence codes are from the Gene Ontology project [31]. bShape of cells varies between cocci and straight or slightly curved rods depend on NH4Cl concentration [6]

acetatoxydans" (84.1 % identity) and the thermophilic P. lettingae (79.64 %). Although S. schinkii has been physiologically affiliated to the group of acetogens, Fig. 2 illustrates a distant relationship to this group, as represented by e.g. the model acetogen Moorella thermoacetica (89.15 % identity).

Genome sequencing information

Genome project history

Syntrophaceticus schinkii strain Sp3 was sequenced and annotated by the SLU-Global Bioinformatics Centre at the Swedish University of Agricultural Sciences, Uppsala, Sweden. The genome project is deposited in the Genomes OnLine Database [14] with GOLD id Gi0035837 and the working draft genome is deposited in the European Nucleotide Archive database with accession number ERP005192. The SAOB was selected for sequencing

on the basis of environmental relevance to issues in global carbon cycling, alternative energy production, and biochemical importance. Table 2 contains the summary of project information.

Growth conditions and genomic DNA preparation

Since isolation by our research group, the strain has been kept in liquid cultures and a live culture and medium have been sent to DSMZ, (DSM21860). For DNA isolation batch cultures were grown in basal medium supplemented with 20 mM betaine as described by Westerholm et al. [6]. Cells were grown for 4 weeks at 37 °C without shaking and harvested at 5000 x g. DNA was isolated using the Blood & Tissue Kit from Qiagen (Hilden, Germany) according to the standard protocol recommended by the manufacturer.

Table 2 Genome sequencing project information for the Syntrophaceticus schinkii Sp3 genome

MIGS ID Property Term

MIGS-31 Finishing quality Draft

MIGD-28 Libraries used Ion Torrent single end reads

MIGS-29 Sequencing platform Ion Torrent PGM Systems

MIGS-31.2 Sequencing coverage 35x

MIGS-30 Assemblers Newbler 2.8 and MIRA 4.0

MIGS-32 Gene calling method PRODIGAL and AMIGene

Locus Tag SSCH

Genbank ID CDRZ00000000

GenBank Data of release March 21, 2014

GOLD ID Gi0035837

BIOPROJECT PRJNA224116

MIGS 13 Source Materialldentifier DSM 21860

Project relevance Biogas production

Genome sequencing and assembly

The genome of Syntrophaceticus schinkii was sequenced at the SciLifeLab Uppsala, Sweden using Ion torrent PM systems with the mean length of 206 bp, longest read length 392 bp and a total of final library reads of 2,985,963 for single end reads. All general aspects of sequencing performed can be found at Scilifelab website [15]. The FastQC software package [16] was used for reads quality assessment. After preassembly quality checking, the reads were assembled with MIRA 4.0 and Newbler 2.8 assemblers. Possible miss-assemblies were corrected manually by using Tablet, a graphical viewer for visualization of assemblies and read mappings [17].

Table 3 Genomic statistics for the Syntrophaceticus schinkii strain Sp3 genome

Attribute Value % of total

Genome size (bp) 3,196,921 100.00

DNA Coding (bp) 2,399,289 75.05

DNA G + C content (bp) 1,489,445 46.59

Number of scaffolds 215 -

Totalgenes 3,441 100.00

Protein coding genes 3,281 95.35

RNA genes 55 1.59

Pseudo gene 90 2.61

Genes in internal clusters 2,086 60.62

Genes with function prediction 2,099 61.00

Genes assigned to COGs 2,583 75.07

Genes with Pfam domains 2,749 79.88

Genes with signal peptides 57 1.65

CRISPR repeats 8 .23

A comparison of two assemblies obtained from both of the assemblers was used to fill the gaps between contigs. The multiple genome alignment tool Mauve was used for this purpose [18]. The working draft genome sequence of S. schinkii Sp3 contains 3,196,921 bp based on the analysis done with the tools summarized above.

Genome annotation

Automated gene modeling was completed by MaGe [19] a bacterial genome annotation system. Genes were identified using Prodigal [20] and AMIGene [21] as part of MaGe genome annotation pipeline. The predicted CDSs were translated and used to search the NCBI nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases using BLASTP. Predicted coding sequences were subjected to manual analysis using MaGe web-based platform, which also provides functional information of proteins, and which was used to assess and correct genes predicted through the automated pipeline. The predicted functions were also further analyzed by the MaGe annotation system (Fig. 4).

Genome properties

The working draft genome comprises 301 contigs in 215 scaffolds with a total size of 3,196,921 bp and a calculated GC content of 46.59 %. The genome shows a protein coding density of 75.21 % with an average intergenic length of 230.2 bp. The genome encodes further 50 tRNA genes and 5 rRNA genes, more precisely three 5S genes, one 16S and one 23S rRNA gene (Table 3, Fig. 3).

The genome of S. schinkii genome contains 3,441 predicted protein-encoding genes, of which 2,099 (61 %) have been assigned tentative functions. The remaining 1,346 ORFs are hypothetical / unknown proteins. 2,586 (app. 75 %) of all predicted protein-encoding genes could be allocated to the 22 functional COGs. This is in the same range as described for other acetogenic bacteria such as Acetobacterium woodii WB1 and M. thermoace-tica ATCC39073, acetate oxidizing sulfate reducers such as Desulfobacterium autotrophicum HRM2 and Desulfoto-maculum kuznetsovii, and the SAOB P. lettingae TMO. Analysis of COGs revealed that ~28 % of all protein-encoding genes fall into four main categories: amino acid transport and metabolism (9.8 %), replication, recombination and repair (6.6 %), energy metabolism (5.9 %), and coenzyme transport and metabolism (4.9 %) (Table 4).

Insights from the genome sequence

Synteny-based analyses with all bacterial genomes present in the NCBI Reference Sequence database confirmed again that T. phaeum is the closest relative of S. schinkii having approximately 50 % of the total genome size in synteny (Fig. 4). A comparison of all inferred

Fig. 3 Circular map. Circular map of the Syntrophaceticus schinkii Sp3 genome (from the outside to the center): (1) GC percent deviation (GC window—mean GC) in a 1000-bp window. (2) Predicted CDSs transcribed in the clockwise direction. (3) Predicted CDSs transcribed in the counterclockwise direction. (4) GC skew (G + C/G-C) in a 1000-bp window. (5) rRNA (blue), tRNA (green), misc_RNA (orange), Transposable elements (pink) and pseudogenes (grey)

proteins of S. schinkii with all proteins collected in the NCBI RefSeq database revealed the highest number of orthologous (1788: 51.90 %) with T. phaeum. Both S. schinkii and T. phaeum, are known as syntrophic acetate oxidizing bacteria able to oxidize acetate in co-culture with a hydrogenotrophic methanogenic partner, but differ clearly in their substrate utilization patterns [2, 6] Moreover, in contrast to the thermophilic T. phaeum, S. schinkii possess mesophilic characteristics and cannot switch to a chemolithoautotrophic lifestyle.

The genome has been analyzed regarding general phenotypic features such as sporulation, oxygen tolerance, secreted and selenocystein-containing proteins and motility. The genome contains the master regulator Spo0A (SSCH_630004) needed for sporulation but lacks genes encoding the phosphorelays Spo0F and Spo0B as it has been observed in other clostridia. All the sporulation-specific sigma factors SigE (SSCH_460001), SigG (SSCH_1070017), and SigK (SSCH_700028) were

predicted except for SigF. Two putative manganese containing catalases (SSCH_1760003, SSCH_2560004) and two putative rubrerythrin encoding genes (SSCH_590006, SSCH_180042) identified within the genome give reasons to believe, that this organism posses the ability to tolerate small amounts of oxygen. According to the observed immobility S. schinkii does not harbor any flagellum related genes including hook-associated proteins (FlgE, FlgK, FlgL), basal and hook proteins (FlgE), capping proteins (FliD), biosynthesis secretory proteins (FlhA, FlhB, FliF, FliH and FliI), flagella formation proteins, motor proteins (FliG and FliM) and the basal proteins (FlgC and FlgB).

Genes encoding key components of the selenocysteine-decoding (SelA, SelB, SelC, SelD) machinery are widely distributed in bacterial genomes. Also S. schinkii appears to have the ability to express selenocysteine proteins: The genome contains a single copy of the L-selenocysteinyl-tRNASec transferase (selA: SSCH_110005/ 6), monoselenophosphate synthase (selD: SSCH_970007),

Table 4 Number of genes associated with the general COG functional categories

Code Value % age Description

J 156 4.53 Translation, ribosomalstructure and biogenesis

A 0 0.00 RNA processing and modification

K 211 6.12 Transcription

L 230 6.68 Replication, recombination and repair

B 1 0.03 Chromatin structure and dynamics

D 59 1.71 Cellcycle control, celldivision, chromosome partitioning

Y 0 0.00 Nuclear structure

V 117 3.39 Defense mechanisms

T 136 3.95 Signaltransduction mechanisms

M 169 4.90 Cellwall/membrane/envelope biogenesis

N 37 1.07 Cellmotility

Z 1 0.02 Cytoskeleton

W 1 0.03 Extracellular structures

U 61 1.77 Intracellular trafficking, secretion, and vesicular transport

O 101 2.93 Posttranslationalmodification, protein turnover, chaperones

C 204 5.92 Energy production and conversion

G 138 4.00 Carbohydrate transport and metabolism

E 339 9.84 Amino acid transport and metabolism

F 70 2.03 Nucleotide transport and metabolism

H 172 4.99 Coenzyme transport and metabolism

I 52 1.51 Lipid transport and metabolism

P 206 5.98 Inorganic ion transport and metabolism

Q 54 1.57 Secondary metabolites biosynthesis, transport and catabolism

R 369 10.71 Generalfunction prediction only

S 219 6.36 Function unknown

342 9.93 Not in COGs

Syntrophaceticus schinkii Sp3

Thermacetogenium phaeum

Fig. 4 Synteny comparison. Synteny comparison of S. schinkii genome with the closely related genome of T. phaeum. Linear comparison of all predicted gene loci from S. schinkii with T. phaeum was perfomed using built-in tool in MaGe Platform with the synton size of> = 3 genes. The lines indicate syntons between two genomes. Red lines show inversions around the origin of replication. Verticalbars on the boarder line indicate different elements in genomes such as pink: transposases or insertion sequences: blue: rRNA and green: tRNA

the selenocysteinyl-tRNA specific elongation factor (selB: SSCH_110004) and potential selenocysteine-specific tRNASec (selC: SSCH_tRNA31). We found two potential selenocysteine containing glycine/sarcosine/betaine reduc-tase complexes encoded by the genome (SSCH_440002-8, SSCH_960012-15) consisting of selenoprotein subunit A, the substrate specific selenoprotein subunit B and acetyl phosphate forming subunit C. Since S. schinkii can only grow on betaine but not on glycine or sarcosine [6], this reductase complex might be specifically involved in betaine utilization. 57 CDSs were predicted to encode surface associated or secreted proteins identified by putative N-terminal signal peptides (signal peptide I and II).

Conclusions

Acetate oxidation under anoxic conditions is thermo-dynamically unfavorable and requires the metabolic cooperation of a partner organism in order to make endergonic reactions more exergonic through the efficient removal of the products. S. schinkii oxidizes acetate to hydrogen and/or formate, which is directly used by a hydrogenotrophic methanogen. Since the methanogenic partner has been isolated and sequenced S. schinkii appears to have great potential to serve as a model organism for studying methane producing syntrophic relationships. The working draft genome sequence presented here will open the door for understanding the preferred habitats, the metabolism behind different life styles, and the mechanisms initiating syntrophy. This knowledge will help us to trigger SAOB towards an efficient and stable hydrogen/ biogas production in engineered anaerobic digestion processes suffering high ammonia release.

Additional file

Additional file 1: Table S1. Associated MIGS record for Syntrophaceticus schinkii strain Sp3. (DOCX 111 kb)

Abbreviations

SAOB: Syntrophic acetate-oxidizing bacteria; DSMZ: Deutsche Sammlung für Mikroorganismen und Zellkulturen; MIRA: Mimicking Intelligent Read Assembly; MaGe: Magnifying Genomes; BLASTP: Basic local alignment search tool for proteins; NCBI: National Center for Biotechnology Information.

Competing interests

The authors declare that they have no competing interests. Authors' contributions

SM, BM and AS contributed to the conception and design of this project. SM was involved in the acquisition and initial analysis of the data. SM and BM were involved in the interpretation of the data. SM prepared the first draft of the manuscript. EBR and AS provided financial support. All authors were involved in the critical revision of the manuscript and have given final approval of the version to be published and agree to be accountable for all aspects of the work.

Acknowledgements

This work was supported by the Higher Education Commission, Pakistan, University of the Punjab, Lahore, Pakistan. Uppsala Genome Center performed sequencing supported by Science for Life Laboratory (Uppsala),

the Swedish Bioinformatics Infrastructure for the Life Sciences supporting the SGBC bioinformatics platform at SLU, University of the Punjab, Lahore, Pakistan and Uppsala Multidisciplinary Center for Advanced Computational Science, Uppsala, Sweden. The contribution of SM and EB-R was supported by EU-COST action BM1006-SeqAhead. EB-R was also partially supported by EU FP7 ALLBIO project, grant number 289452, The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We wish to thank Maria Westerholm, who isolated and characterized Syntrophaceticus schinkii strain Sp3 for providing the micrograph.

Author details

department of Animal Breeding and Genetics Science, Swedish University of Agricultural Science, SLU-Global Bioinformatics Centre, Uppsala SE 750 07, Sweden. 2Department of Microbiology, Swedish University of Agricultural Sciences, BioCenter, Uppsala SE 750 07, Sweden. 3University of the Punjab, Lahore, Pakistan.

Received: 24 May 2014 Accepted: 29 October 2015 Published online: 11 November 2015

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