Complete genome sequence of Desulfarculus baarsii type strain (2st14).

Desulfarculus baarsii (Widdel 1981) Kuever et al. 2006 is the type and only species of the genus Desulfarculus, which represents the family Desulfarculaceae and the order Desulfarculales. This species is a mesophilic sulfate-reducing bacterium with the capability to oxidize acetate and fatty acids of up to 18 carbon atoms completely to CO(2). The acetyl-CoA/CODH (Wood-Ljungdahl) pathway is used by this species for the complete oxidation of carbon sources and autotrophic growth on formate. The type strain 2st14(T) was isolated from a ditch sediment collected near the University of Konstanz, Germany. This is the first completed genome sequence of a member of the order Desulfarculales. The 3,655,731 bp long single replicon genome with its 3,303 protein-coding and 52 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.

Introduction

Most sulfate reducing bacteria, available in pure culture, oxidize organic electron donors incompletely to acetate, whereas species that oxidize acetate and other carbon compounds completely to CO2, using sulfate as an electron acceptor, are less frequently isolated. Sulfate reducers with the latter type of metabolism are of special interest, because it is assumed that they are dominant in anoxic marine sediments [1]. Sulfate reducing prokaryotes with the ability to mineralize organic compounds to CO2 are phylogenetically dispersed and can be found within the Proteobacteria, Firmicutes and Euryarchaeota. At the time of writing, representatives of this type of metabolism, for which a completely sequenced genome exists include Desulfobacterium autotrophicum [2], Desulfotomaculum acetoxidans [3] and Archaeoglobus fulgidus [4]. In the present work, the complete genome sequence of Desulfarculus baarsii a completely oxidizing sulfate reducing bacterium representing the order Desulfarculales within the Deltaproteobacteria, was determined. The original description of D. baarsii was based on strain 1st1 (= “Göttingen”) [5], which was probably subsequently lost and replaced by the designated type strain 2st14T (= “Konstanz”) [6]. Strain 2st14T (= DSM 2075 = ATCC 33931 = LMG 7858) was enriched from anoxic mud from a ditch near the University of Konstanz, Germany, in a medium supplemented with stearate and sulfate and subsequently isolated in an anaerobic agar dilution series with formate plus sulfate [7,8]. D. baarsii strain 2st14T is the first member of the family Desulfarculaceae within the order Desulfarculales with a sequenced genome. The presented sequence data will enable interesting genome comparisons with other sulfate reducing bacteria of the class Deltaproteobacteria.

Classification and features

The species D. baarsii represents a separate lineage within the Deltaproteobacteria which is only distantly related to most other members of this class. The closest relatives based on 16S rRNA gene sequence similarity values are the type strains of Desulfomonile tiedjei (87.6% sequence identity) and Desulfomonile liminaris (87.2%), both belonging to the family Syntrophaceae within the order Syntrophobacterales [9]. The most similar cloned 16S rRNA gene, EUB-42 [10] shared only 95.5% sequence similarity with D. baarsii and was retrieved from anaerobic sludge. Strain 2st14T represents the only strain of this species available from a culture collection, thus far. Currently available data from cultivation independent studies (environmental screening and genomic surveys) did not surpass 86% sequence similarity, indicating that members of this species are restricted to distinct habitats which are currently undersampled in most environments or are in low abundance, (status October 2010). The single genomic 16S rRNA sequence of strain 2st14T was compared using BLAST with the most resent release of the Greengenes database [11] and the relative frequencies of taxa and keywords, weighted by BLAST scores, were determined. The five most frequent genera were Desulfovibrio (43.3%), Syntrophobacter (14.4%), Desulfomonile (11.8%), Desulfarculus (9.6%) and Desulfatibacillum (7.5%). The species yielding the highest score was D. baarsii. The five most frequent keywords within the labels of environmental samples which yielded hits were 'sediment' (4.5%), 'microbial' (4.5%), 'lake' (1.7%), 'depth' (1.7%) and 'sea' (1.6%). Environmental samples which yielded hits of a higher score than the highest scoring species were not found.

Figure 1 shows the phylogenetic neighborhood of D. baarsii 2st14T in a 16S rRNA based tree. The sequence of the single 16S rRNA gene in the genome differs by one nucleotide from the previously published 16S rRNA gene sequence generated from DSM 2075 (AF418174) which contains five ambiguous base calls. Genbank entry M34403 from 1989 is also annotated as 16S rRNA sequence of strain 2st14T, but differs in 45 positions (3.2%) from the actual sequence. This difference probably reflects more the progress in sequencing technology than biological differences.

Figure 1

Phylogenetic tree highlighting the position of D. baarsii relative to the other type strains of related genera within the class Deltaproteobacteria. The tree was inferred from 1,465 aligned characters [12,13] of the 16S rRNA gene sequence under the maximum likelihood criterion [14] and rooted in accordance with the current taxonomy. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1,000 bootstrap replicates [15] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [16] are shown in blue, published genomes [17] and INSDC accession CP000478 for Syntrophobacter fumaroxidans in bold.

The cells of D. baarsii 2st14T are vibrioid, Gram-negative and 0.5-0.7 by 1.5–4 µm in size (Figure 2, Table 1). Motility is conferred by a single polar flagellum (not visible in Figure 2) [5]. The temperature range for growth is 20-39°C with an optimum around 35°C. The pH range for growth is 6.5–8.2, with an optimum at 7.3. The strain grows optimally in the presence of 7–20 g/l NaCl and 1.2–3g/l MgCl2 × 6 H2O, but growth is nearly as rapid at lower concentrations [7]. D. baarsii strain 2st14T is a strictly anaerobic, non-fermentative, chemoorganotrophic sulfate-reducer that oxidizes organic substrates completely to CO2. Sulfate, sulfite and thiosulfate serve as terminal electron acceptors and are reduced to H2S, but sulfur, fumarate and nitrate cannot be utilized. The following compounds are utilized as electron donors and carbon sources: formate, acetate, propionate, butyrate, iso-butyrate, 2-methylbutyrate, valerate, iso-valerate, and higher fatty acids up to 18 carbon atoms. Growth on formate does not require an additional organic carbon source [5,7]. A high activity of carbon monoxide dehydrogenase is observed in D. baarsii, indicating the operation of the anaerobic C1-pathway (Wood-Ljungdahl pathway) for formate assimilation and CO2 fixation or complete oxidation of acetyl-CoA [27].

Figure 2

Scanning electron micrograph of D. baarsii 2st14T

Table 1

Classification and general features of D. baarsii strain 2st14T in according to the MIGS recommendations [18].

MIGS ID	Property	Term	Evidence code
	Current classification	Domain Bacteria	TAS [19]
		Phylum Proteobacteria	TAS [20]
		Class Deltaproteobacteria	TAS [21,22]
		Order Desulfarculales	TAS [21,23]
		Family Desulfarculaceae	TAS [7,21,23, 24]
		Genus Desulfarculus	TAS [7,21]
		Species Desulfarculus baarsii	TAS [6,7,21]
		Type strain 2st14	TAS [6]
	Gram stain	negative	TAS [5]
	Cell shape	vibrio-shaped	TAS [5]
	Motility	motile (single polar flagellum)	TAS [5]
	Sporulation	non-sporulating	TAS [5]
	Temperature range	20-39°C	TAS [5]
	Optimum temperature	35°C	TAS [5]
	Salinity	optimum growth at 7–20 g/l NaCl	TAS [5,7]
MIGS-22	Oxygen requirement	strictly anaerobic	TAS [5]
	Carbon source	CO2, formate, acetate, propionate, butyrate,    higher fatty acids	TAS [5]
	Energy source	formate, acetate, propionate, butyrate,    higher fatty acids	TAS [5]
MIGS-6	Habitat	anoxic freshwater or brackish sediments	TAS [5]
MIGS-15	Biotic relationship	free living	NAS
MIGS-14	Pathogenicity	none	TAS [25]
	Biosafety level	1	TAS [25]
	Isolation	mud from a ditch	TAS [7]
MIGS-4	Geographic location	Konstanz, Germany	TAS [7]
MIGS-5	Sample collection time	1981 or before	NAS
MIGS-4.1MIGS-4.2	Latitude   Longitude	47.7    9.2	NAS
MIGS-4.3	Depth	not reported
MIGS-4.4	Altitude	about 406 m	NAS

Evidence codes - IDA: Inferred from Direct Assay (first time in publication); 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 of the Gene Ontology project [26]. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.

The oxygen detoxification system of D. baarsii was analyzed in some detail. It could be shown that a genomic region encoding a putative rubredoxin oxidoreductase (rbo) and rubredoxin (rub) of D. baarsii is able to suppress deleterious effects of reactive oxygen species (ROS) in Escherichia coli mutants lacking superoxide dismutase [28]. The cloned genes were identified in the whole genome sequence as Deba_2049 (rub) and Deba_2050 (rbo) and found in close proximity to a gene encoding rubrerythrin (Deba_2051), which is supposed to play an important role in the oxygen tolerance of anaerobic bacteria [29]. The product of the recombinant rbo gene of D. baarsii was later further characterized and designated as desulfoferrodoxin (Dfx), because no evidence for a rubredoxin oxidoreductase could be demonstrated. Instead, a function as superoxide reductase was proposed [30].

Chemotaxonomy

The cellular fatty acid pattern of D. baarsii strain 2st14T is dominated by saturated straight chain fatty acids (43.0% C14:0, 9.9% C16:0, and 2.3% C18:0), followed by saturated iso- and anteiso-branched fatty acids (21.3% i-C14:0, 12.3% ai-C15:0, and 2.8% i-C15:0). Occurrence of the dimethylacetal (DMA) derivates C15:0 DMA (1.8%) and i-C15:0 DMA (0.6%) represents a distinctive trait of this strain, because these compounds are rarely found in Desulfovibrio species [31]. A comparison of the fatty acid profiles of D. baarsii and various Gram-negative sulfate-reducers by cluster analysis indicated a separate position of D. baarsii [31], corroborating the distinct phylogenetic position of the species as shown based on the 16S rRNA sequence analysis (Figure 1). Unfortunately, besides the cellular fatty acid composition no further chemotaxonomic data are available for this species.

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position [32], and is part of the enomic ncyclopedia of acteria and rchaea project [33]. The genome project is deposited in the Genome OnLine Database [16] and the complete genome sequence is deposited in GenBank Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Table 2

Genome sequencing project information.

MIGS ID	Property	Term
MIGS-31	Finishing quality	Finished
MIGS-28	Libraries used	Two 454 pyrosequence libraries, standard and pairs end (13 kb insert size)     and one Illumina standard library
MIGS-29	Sequencing platforms	454 Titanium, Illumina GAii
MIGS-31.2	Sequencing coverage	43.1 × 454 Titanium; 73.2 × Illumina
MIGS-30	Assemblers	Newbler, Velvet, phrap
MIGS-32	Gene calling method	Prodigal
	INSDC ID	CP002085
	GenBank Date of Release	August 6, 2010
	GOLD ID	Gc01335
	NCBI project ID	37955
	Database: IMG-GEBA	2502957037
MIGS-13	Source material identifier	DSM 2075
	Project relevance	GEBA

Growth conditions and DNA isolation

D. baarsii, strain 2st14T, DSM 2075, was grown anaerobically in DSMZ medium 208 (Desulfovibrio baarsii medium) [34] at 37°C. DNA was isolated from 0.5-1 g of cell paste using Jetflex Genomic DNA Purification Kit (GENOMED 600100) following the manufacturer’s instructions, but with 30 min incubation at 58°C with an additional 10 µl proteinase K for cell lysis.

Genome sequencing and assembly

The genome of was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [35]. Pyrosequencing reads were assembled using the Newbler assembler version 2.1-PreRelease-4-28-2009-gcc-3.4.6-threads (Roche). The initial Newbler assembly consisted of 42 contigs in two scaffolds and was converted into a phrap assembly by making fake reads from the consensus, collecting the read pairs in the 454 paired end library. Illumina GAii sequencing data (267.7Mb) were assembled with Velvet [36] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 157.7 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package [37] was used for sequence assembly and quality assessment in the following finishing process: After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution [35], Dupfinisher, or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) [38]. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F.Chang, unpublished). A total of 344 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [39]. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 116.3 × coverage of the genome. Final assembly contained 431,804 pyrosequence and 7,436,430 Illumina reads.

Genome annotation

Genes were identified using Prodigal [40] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [41]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review platform [42].

Genome properties

The genome is 3,655,731 bp long and comprises one main circular chromosome with an overall GC content of 65.7% (Table 3 and Figure 3). Of the 3,355 genes predicted, 3,303 were protein-coding genes, and 52 RNAs; 26 pseudogenes were also identified. The majority of the protein-coding genes (73.4%) were assigned a putative function while those remaining were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Table 3

Genome Statistics

Attribute	Value	% of Total
Genome size (bp)	3,655,731	100.00%
DNA coding region (bp)	3,313,356	90.63%
DNA G+C content (bp)	2,401,943	65.70%
Number of replicons	1
Extrachromosomal elements	0
Total genes	3,355	100.00%
RNA genes	52	1.55%
rRNA operons	1
Protein-coding genes	3,303	98.45%
Pseudo genes	26	0.77%
Genes with function prediction	2,463	73.41%
Genes in paralog clusters	481	14.34%
Genes assigned to COGs	2,466	73.50%
Genes assigned Pfam domains	2,613	77.88%
Genes with signal peptides	686	20.45%
Genes with transmembrane helices	768	22.89%
CRISPR repeats	3

Figure 3

Graphical circular map of the genome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

Table 4

Number of genes associated with the general COG functional categories

Code	value	%age	Description
J	155	5.7	Translation, ribosomal structure and biogenesis
A	1	0.0	RNA processing and modification
K	137	5.0	Transcription
L	109	4.0	Replication, recombination and repair
B	3	0.1	Chromatin structure and dynamics
D	32	1.2	Cell cycle control, cell division, chromosome partitioning
Y	0	0.0	Nuclear structure
V	39	1.4	Defense mechanisms
T	260	9.6	Signal transduction mechanisms
M	210	7.7	Cell wall/membrane biogenesis
N	103	3.8	Cell motility
Z	0	0.0	Cytoskeleton
W	0	0.0	Extracellular structures
U	74	2.7	Intracellular trafficking and secretion, and vesicular transport
O	84	3.1	Posttranslational modification, protein turnover, chaperones
C	228	8.4	Energy production and conversion
G	89	3.3	Carbohydrate transport and metabolism
E	180	6.6	Amino acid transport and metabolism
F	62	2.3	Nucleotide transport and metabolism
H	149	5.5	Coenzyme transport and metabolism
I	130	4.9	Lipid transport and metabolism
P	124	4.6	Inorganic ion transport and metabolism
Q	56	2.1	Secondary metabolites biosynthesis, transport and catabolism
R	312	11.5	General function prediction only
S	182	6.7	Function unknown
-	889	26.5	Not in COGs