Complete genome sequence of Thermocrinis albus type strain (HI 11/12).

Thermocrinis albus Eder and Huber 2002 is one of three species in the genus Thermocrinis in the family Aquificaceae. Members of this family have become of significant interest because of their involvement in global biogeochemical cycles in high-temperature ecosystems. This interest had already spurred several genome sequencing projects for members of the family. We here report the first completed genome sequence a member of the genus Thermocrinis and the first type strain genome from a member of the family Aquificaceae. The 1,500,577 bp long genome with its 1,603 protein-coding and 47 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Introduction

Strain HI 11/12T (= DSM 14484 = JCM 11386) is the type strain of the species Thermocrinis albus [1]. Officially, the genus Thermocrinis currently contains three species [2], however, it should be noted that at the time of writing, the 16S rDNA sequence of the type strain of Thermocrinis ruber held in the DSMZ open collection as DSM 12173 does not correspond with that published under AJ005640. The generic name derives from the Greek word ‘therme’, meaning ‘heat’, and the Latin word ‘crinis’, hair, meaning ‘hot hair’, referring to the long hair-like filamentous cell structures found in the high-temperature environments, such as hot-spring outlets [3]. These long filaments are formed under conditions where there is a continuous flow of medium. The species name is derived from the Greek word ‘alphos’, white, referring to the cell color [1]. Strain HI 11/12T has been isolated from whitish streamers in Hveragerthi, Iceland [3]. Other strains of the species have been isolated from further high-temperature habitats in Iceland, but also in Kamchatka, Russia [1]. Members of the genus Thermocrinis appear to play a major ecological role in global biochemical cycles in such high-temperature habitats [4-7]. As currently defined the genus does not appear to form a monophyletic group, suggesting that further taxonomic work is necessary.

The large interest in the involvement of members of the family Aquificaceae in global biogeochemical cycles in high-temperature ecosystems made them attractive targets for early genome sequencing, e.g. ‘Aquifex aeolicus’ [8], the third hyperthermophile whose genome was already decoded in 1998 [9]. Like ‘A. aeolicus’ (a name that was never validly published) strain VF5 [10], Hydrogenobaculum sp. Y04AAS1 (CP001130, JGI unpublished) and Hydrogenivirga sp. 128-5-R1-1 (draft, Moore Foundation) do not represent type strains. Here we present a summary classification and a set of features for T. albus HI 11/12T, together with the description of the complete genomic sequencing and annotation.

Classification and features

Only four cultivated strains are reported for the species T. albus in addition to HI 11/12T: Strains H7L1B and G3L1B from the same team that isolated HI 11/12T [1], and SRI-48 (AF255599) from hot spring microbial mats [11]. All three strains originate from Iceland and share 98.9-99.7% 16S rRNA sequence identity with HI 11/12T. The only non-Icelandic isolate, UZ23L3A (99.2%), originates from Kamchatka (Russia) [1]. Almost all uncultured clones also originate from Iceland: clones KF6 and HV-7 (GU233821 and GU233840, >99%) from water-saturated sediment in the Krafla and Hveragerdi geothermal systems, respectively. Clones GY1-1 and GY1-2 (GU233809, GU233812, >99%) from water-saturated sediment Geysir hot springs; clone SUBT-1 (AF361217, 99.2%) from subterranean hot springs [12], and clone PIce1 (AF301907, 99.3%) as the dominant clone from a blue filament community of a thermal spring. Only clone PNG_TB_4A2.5H2_B11 (EF100635, 95.9%) originated from a non-Icelandic source: a heated, arsenic-rich sediment of a shallow submarine hydrothermal system on Ambitle Island, Papua New Guinea. According to the original publication the 16S rRNA of the type strain of the closest related species within the genus, T. ruber [3], shares 95.2% sequence identity, whereas the type strains from the closest related genus, Hydrogenobacter, share 94.7-95.0% sequence identity, as determined with EzTaxon [13]. However, as noted above the 16S rDNA sequence of the T. ruber strain held in the DSMZ (DSM 12173) does not correspond with the sequence deposited (AJ005640). Environmental samples and metagenomic surveys featured in the NCBI database contain not a single sequence with >88% sequence identity (as of February 2010), indicating that the species T. albus might play a rather limited and regional role in the environment.

Figure 1 shows the phylogenetic neighborhood of T. albus HI 11/12T in a 16S rRNA based tree. The sequence of the single 16S rRNA gene copy in the genome differs by seven nucleotides from the previously published 16S rRNA sequence generated from DSM 14484 (AJ278895), which contains two ambiguous base calls.

Figure 1

Phylogenetic tree highlighting the position of T. albus HI 11/12T relative to the other type strains within the family Aquificaceae. The tree was inferred from 1,439 aligned characters [14,15] of the 16S rRNA gene sequence under the maximum likelihood criterion [16] 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 250 bootstrap replicates [17] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [18] are shown in blue, published genomes in bold. Note that the sequence AJ005640 does not correspond with that from the type strain of T. ruber deposited as DSM 12173.

When grown in the laboratory in a continuous flow of medium, for example in a glass chamber [1], strain HI 11/12T exhibits filamentous growth with a length of 10-60 µm [1]. When grown in static culture, the strain grows singly or in pairs [1]. The cells are short rods with 0.5-0.6 µm in width by 1-3 µm in length and motile by means of a monopolar monotrichous flagellum [1] (Figure 2 and Table 1). However, no flagella are visible in Figure 2. A regularly arrayed surface layer protein was not observed [1].

Figure 2

Scanning electron micrograph of T. albus HI 11/12T

Table 1

Classification and general features of T. albus HI 11/12T according to the MIGS recommendations [19]

MIGS ID	Property	Term	Evidence code
	Current classification	Domain Bacteria	TAS [20]
		Phylum Aquificae	TAS [21]
		Class Aquificae	TAS [21]
		Order Aquificales	TAS [22]
		Family Aquificaceae	TAS [23]
		Genus Thermocrinis	TAS [3]
		Species Thermocrinis albus	TAS [1]
		Type strain HI 11/12	TAS [1]
	Gram stain	Gram negative	TAS [1]
	Cell shape	both filament and rod	TAS [1]
	Motility	monopolar monotrichous flagellation	TAS [1]
	Sporulation	non-sporulating	TAS [1]
	Temperature range	55–89°C	TAS [1]
	Optimum temperature	not determined	TAS [1]
	Salinity	≤ 0.7%	TAS [1]
MIGS-22	Oxygen requirement	aerobic	TAS [1]
	Carbon source	CO2, no organic carbon source reported	TAS [1,24]
	Energy source	chemolithoautotrophic	TAS [1]
MIGS-6	Habitat	hot spring	TAS [1]
MIGS-15	Biotic relationship	free living	TAS [1]
MIGS-14	Pathogenicity	not reported	TAS [3,25]
	Biosafety level	1	TAS [25]
	Isolation	hot streamlet	TAS [1]
MIGS-4	Geographic location	Hverageroi, Iceland	TAS [1]
MIGS-5	Sample collection time	1998 or before	TAS [3]
MIGS-4.1MIGS-4.2	Latitude      Longitude	64,      -21.2	NAS
MIGS-4.3	Depth	unknown
MIGS-4.4	Altitude	30 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 for a live isolate by one of the authors or an expert mentioned in the acknowledgements.

Strain 11/12T is microaerophilic with oxygen as electron acceptor [1]. Strain 11/12T appears to be strictly chemolithoautotrophic [1]. This differentiates T. albus from its two sister species T. ruber and T. minervae, which both can also grow chemoorganoheterotrophically [3,24]. Strain 11/12T grows optimally under microaerophilic conditions when hydrogen and sulfur are present simultaneously as electron donors [1], however, no growth is observed on nitrate. Physiological characteristics such as the wide temperature preference are reported in Table 1.

Chemotaxonomy

The cell wall of strain 11/12T contains meso-diaminopimelic acid [1]. There are no reports on the presence of a lipopolysaccharide in the typical Gram-negative cell wall, although there are reports of an LPS in Aquifex pyrophilus [27,28]. Cellular polyamines are important to stabilize cellular nucleic acid structure as a major function, and may function in thermophilic eubacteria as important chemotaxonomic markers [29]. In the genus Thermocrinis, the major polyamines are spermidine and a quaternary branched penta-amine, N4-bis(aminopropyl)-norspermidine [29].

The major fatty acids in strain HI 11/12T are cyclo-C21:0 (42%, 2 isomers), C18:0 (14%), C20:1 cΔ11 (10.7%), C20:1 cΔ13 (8.2%), C20:1 tΔ11 (5.4%), and C20:1 tΔ13 (3.5%) [30]. All other fatty acids are below 2.9%) [30]. The polar lipids are based on ester linked fatty acids (diacyl glycerols) and monoether (probably in the form of monoester-monoether glycerols), in which the ether linked side chain includes C18:0 (78.5%), C20:0 (2.0%), C20:1 (17.6%) and C21:0 (1.9%) side chains [30].

T. albus belongs to a group of organisms where characteristic sulfur containing napthoquinones, menathioquinones (2-methylthio-1,4-naphthoquinone) are present [31-35]. The polar lipids reported in members of the genera Aquifex, Hydrogenobaculum, Hydrogenothermus and Thermocrinis are also characteristic, with an unusual aminopentanetetrol phospholipid derivative being present in all strains examined [36,37]. Where detailed analyses have been carried out phosphatidylinositol has also been reported [37]. Stöhr et al. labeled these lipids PNL and PL1 respectively [31].

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position, and is part of the enomic ncyclopedia of acteria and rchaea project [38]. The genome project is deposited in the Genome OnLine Database [18] 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 (17 kb insert size)
MIGS-29	Sequencing platforms	454 Titanium, Illumina GAii
MIGS-31.2	Sequencing coverage	52.9× 454 Titanim; 298× Illumina
MIGS-30	Assemblers	Newbler, Velvet, phrap
MIGS-32	Gene calling method	Prodigal, GenePRIMP
	INSDC ID	CP001931
	Genbank Date of Release	February 19, 2010
	GOLD ID	Gc01206
	NCBI project ID	37275
	Database: IMG-GEBA	2502082116
MIGS-13	Source material identifier	DSM 14484
	Project relevance	Tree of Life, GEBA

Growth conditions and DNA isolation

T. albus HI 11/12T, DSM 14484, was grown in DSMZ medium 887 (OS Medium) [39] at 80°C. DNA was isolated from 1-1.5 g of cell paste using MasterPure Gram-positive Kit (Epicentre MGP04100) with a modified protocol for cell lysis, using an additional 5 µl mutanolysin to standard lysis solution, and one hour incubation on ice after the MPC-step.

Genome sequencing and assembly

The genome of strain HI 11/12T was sequenced using a combination of Illumina [40] and 454. An Illumina GAii shotgun library with reads of 447 Mb, a 454 Titanium draft library with average read length of 287 bases, and a paired end 454 library with average insert size of 17 Kb were generated for this genome. All general aspects of library construction and sequencing can be found at http://www.jgi.doe.gov/. Illumina sequencing data were assembled with VELVET [41], and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. Draft assemblies were based on 79 Mb 454 draft data. Newbler parameters were -consed -a 50 -l 350 -g -m -ml 20. The initial assembly contained six contigs in one scaffold. We converted the initial 454 assembly into a phrap assembly by making fake reads from the consensus, collecting the read pairs in the 454 paired end library. The Phred/Phrap/Consed software package (www.phrap.com) was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap. Possible mis-assemblies were corrected with gapResolution (unpublished; http://www.jgi.doe.gov/), Dupfinisher or sequencing cloned bridging PCR fragments with subcloning or transposon bombing [42]. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J-F. Chan, unpublished). A total of 68 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. The completed genome sequence had an error rate of less than 1 in 100,000 bp

Genome annotation

Genes were identified using Prodigal [43] as part of the Oak Ridge National Laboratory genome an-notation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [44]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform [45].

Genome properties

The genome consists of a 1,500,577 bp long chromosome with a 46.9% GC content (Table 3 and Figure 3). Of the 1,650 genes predicted, 1,593 were protein coding genes, and 47 RNAs; 10 pseudogenes were identified. The majority of the protein-coding genes (75.2%) were assigned with 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)	1,500,577	100.00%
DNA coding region (bp)	1,459,457	97.26%
DNA G+C content (bp)	704,229	46.93%
Number of replicons	1
Extrachromosomal elements	0
Total genes	1,650	100.00%
RNA genes	47	2.85%
rRNA operons	1
Protein-coding genes	1,603	97.15%
Pseudo genes	10	0.61%
Genes with function prediction	1,241	75.21%
Genes in paralog clusters	124	7.52%
Genes assigned to COGs	1,316	79.76%
Genes assigned Pfam domains	1,333	80.79%
Genes with signal peptides	243	14.73%
Genes with transmembrane helices	322	19.52%
CRISPR repeats	4

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	131	8.2	Translation, ribosomal structure and biogenesis
A	0	0.0	RNA processing and modification
K	45	2.8	Transcription
L	80	5.0	Replication, recombination and repair
B	2	0.1	Chromatin structure and dynamics
D	0	0.0	Cell cycle control, mitosis and meiosis
Y	0	0.0	Nuclear structure
V	11	0.7	Defense mechanisms
T	47	2.9	Signal transduction mechanisms
M	111	6.9	Cell wall/membrane biogenesis
N	57	3.6	Cell motility
Z	0	0.0	Cytoskeleton
W	0	0.0	Extracellular structures
U	58	3.6	Intracellular trafficking and secretion
O	78	4.9	Posttranslational modification, protein turnover, chaperones
C	140	8.7	Energy production and conversion
G	48	3.0	Carbohydrate transport and metabolism
E	118	7.4	Amino acid transport and metabolism
F	49	3.1	Nucleotide transport and metabolism
H	96	6.0	Coenzyme transport and metabolism
I	39	2.4	Lipid transport and metabolism
P	74	4.6	Inorganic ion transport and metabolism
Q	16	1.0	Secondary metabolites biosynthesis, transport and catabolism
R	148	9.2	General function prediction only
S	79	4.9	Function unknown
-	334	20.8	Not in COGs

Insights from the genome sequence

With only very few papers published on the organism [1,2], and only one gene sequence (16S rRNA) available in GenBank from strain HI 11/12T, a comparison of already known sequences to the here presented novel genomic data is rather meager for T. albus. As shown in Figure 1, there are presently no other type strain genomes from the Aquificaceae available either to allow a meaningful comparative genomics analysis. This might change when other type/neotype strains of species within the genus Thermocrinis which are also part of the enomic ncyclopedia of acteria and rchaea project [38] will become available in the near future.