Complete genome sequence of Haloterrigena turkmenica type strain (4kT)

Haloterrigena turkmenica (Zvyagintseva and Tarasov 1987) Ventosa et al. 1999, comb. nov. is the type species of the genus Haloterrigena in the euryarchaeal family Halobacteriaceae. It is of phylogenetic interest because of the yet unclear position of the genera Haloterrigena and Natrinema within the Halobacteriaceae, which created some taxonomic problems historically. H. turkmenica, was isolated from sulfate saline soil in Turkmenistan, is a relatively fast growing, chemoorganotrophic, carotenoid-containing, extreme halophile, requiring at least 2 M NaCl for growth. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the genus Haloterrigena, but the eighth genome sequence from a member of the family Halobacteriaceae. The 5,440,782 bp genome (including six plasmids) with its 5,287 protein-coding and 63 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain 4k T (= DSM 5511 = ATCC 51198 = VKM B-1734) is the type strain of the species Haloterrigena turkmenica, which is the type species of the genus Haloterrigena [1,2]. The strain was initially described in 1987 as Halococcus turkmenicus VKM B-1734 (basonym) by Zvyagintseva and Tarasov [3]. In 1999, Ventosa et al. proposed to transfer H. turkmenicus 4k as the type strain of the species H. turkmenica to the new genus Haloterrigena [1], whose name means salt, halos, (-requiring) and born from the earth, terrigena. Inconsistent data published on sequence similarity and DNA-DNA hybridization for some Haloterrigena and Natrinema strains created some confusion and taxonomic problems initially, but the problems were largely resolved in 2003 by Tindall [4], pointing to uncertainty about strain history. It has been suggested that the discrepancies may also be a result of 16S rDNA interoperon heterogeneity [5]. Published data appears to indicate that both strains GSL-11 and JCM 9743 (formally included in the species H. turkmenica by Ventosa et al. [1]) may be members of the genus Natrinema [4,6]. Those strains will not be considered further here.
There are no reliable reports of other strains of H. turkmenica having been isolated. 16S rRNA sequence identity with the other seven type strains in the genus, which were mainly isolated from salt lakes, range from 98.0% for H. salina [7] to 94.4% for H. longa [6]. The sequence similarity to the Natrinema type strains is somewhere in-between, 95.2-96.4% [8], underlining the taxonomic problems [4]. The sequence similarity to phylotypes in environmental metagenomic libraries was not above 87%, indicating a rather poor representation of closely related strains in the habitats analyzed (status January 2010). Here we present a summary classification and a set of features for H. turkmenica strain 4k T , together with the descrip-tion of the complete genome sequencing and annotation. Figure 1 shows the phylogenetic neighborhood of H. turkmenica strain 4k T in a 16S rRNA based tree. The three 16S rRNA gene sequences in the genome differ from each other by up to two nucleotides, and differ by up to six nucleotides from the previously published 16S rRNA sequence (AB004878) generated from DSM 5511. The difference between the genome data and the previously reported 16S rRNA gene sequences is most likely due to sequencing errors in the previously reported sequence data. As expected, Haloterrigena and Natrinema strains appear as intermixed in the tree, indicating a paraphyletic status of Haloterrigena (within which Natronorubrum and Natrinema branch off) and of Natrinema (within which H. longa is placed) [18].  [9,10] of the 16S rRNA sequence under the maximum likelihood criterion [11] and rooted with Natronomonas pharaonis [12]. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 800 bootstrap replicates [13] if larger than 60%. Strains with a genome sequencing project registered in GOLD [14] are printed in blue; published genomes in bold, e.g. the recently published GEBA genomes from Halogeometricum borinquense [15], Halorhabdus utahensis [16], and Halomicrobium mukohataei [17].

Classification and features
H. turkmenica cells occur mostly as single cells, rarely in pairs or tetrads [1]. They are described as Gram-negative, ovoid to coccoid, 1.5-2 μm in diameter [1], but can also be rod-shaped ( Figure 2 and Table 1) [1]. Neither spores, nor flagella, nor lipid granules were reported. Colonies are pigmented red or light pink due of the presence of C5O-carotenoids [1]. Stain 4k T is chemoorganotrophic and aerobic, and requires at least 2 M NaCl [1]. Detailed physiological characteristics were described by Zvyagintseva and Tarasov [3]. The G+C content of DNA was reported to be 59.2-60-2 mol % (Thermal denaturation method [1]), which is significantly less than the 64.3% found in the genome. At optimal growth temperatures, H. turkmenica is the fastest growing member of the Halobacteriaceae, with only 1.5 hours generation time [26]. Besides the chemical characterization of siderophores [29], there are no published reports on the molecular biology of H. turkmenica. Both diphytanyl moieties (C20, C20) and phytanylsesterterpanyl moieties (C20, C25) are present in polar lipids [1]. The presence of both phytanyl and esterterpanyl side chains implies the presence of three different prenyl transferases involved in lipid biosynthesis, which are probably chain length specific as well as stereospecific for the incorporation of the isoprenoid side chains into the glycerol backbone [30]. The presence of significant levels of both the diphytanyl moieties (C20, C20) and phytanyl-esterterpanyl moieties (C20, C25) is characteristic of all members examined of this evolutio-nary branch of the family Halobacteriaceae. Membrane polar lipids are glycerol-diether analogues of PG, PGP-Me and the disulfated digylcosyl diether lipid S2-DGD (mannose-2,6 disulfate 1→2 glucose-glycerol diether) [31], the characteristic glycolipid of Natrialba asiatica [32]. The presence of respiratory lipoquinones have not been reported, but it may be predicted that MK-8 and MK-8 (VIII-H2) should be present, since this is a feature of all members of the family Halobacteriaceae examined to date.

MIGS-4.4 Altitude unknown
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 [28]. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.

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 Genomic Encyclopedia of Bacteria and Archaea project [33]. The genome project is deposited in the Genomes OnLine Database [14] and the complete genome sequence 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.

Genome sequencing and assembly
The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing performed at the JGI can be found at the JGI website. 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.03.24 (Roche). Large Newbler contigs were broken into 6,060 overlapping fragments of 1,000 bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the parallel phrap assembler (High Performance Software, LLC). Possible misassemblies were corrected with Dupfinisher or transposon bombing of bridging clones [35]. A total of 1,183 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. Illumina reads were used to improve the final consensus quality using an in-house developed tool (the Polisher). The error rate of the completed genome sequence is less than 1 in 100,000.
Together, the combination of the Sanger and 454 sequencing platforms provided 26.8× coverage of the genome. The final assembly contains 33,433 Sanger reads and 394,632 pyrosequencing reads.

Genome annotation
Genes were identified using Prodigal [36] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [37]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, Uni-Prot, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and In-terPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review platform [38].

Genome properties
The genome is 5,440,782 bp long and comprises one main circular chromosome of 3,889,038 bp length and six circular plasmids of 15.8 to 698.5 kbp length, with an overall GC content of 64.3% (Table 3 and Figures 3 and 4). Of the 5,350 genes predicted, 5,287 were protein coding genes, and 63 RNAs; 174 pseudogenes were also identified. The majority of the protein-coding genes (60.1%) 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.