Complete genome sequence of Halogeometricum borinquense type strain (PR3T)

Halogeometricum borinquense Montalvo-Rodríguez et al. 1998 is the type species of the genus, and is of phylogenetic interest because of its distinct location between the halobacterial genera Haloquadratum and Halosarcina. H. borinquense requires extremely high salt (NaCl) concentrations for growth. It can not only grow aerobically but also anaerobically using nitrate as electron acceptor. The strain described in this report is a free-living, motile, pleomorphic, euryarchaeon, which was originally isolated from the solar salterns of Cabo Rojo, Puerto Rico. 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 halobacterial genus Halogeometricum, and this 3,944,467 bp long six replicon genome with its 3937 protein-coding and 57 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain PR3 T (= DSM 11551 = ATCC 700274 = JCM 10706) is the type strain of Halogeometricum borinquense, representing the sole species of the genus Halogeometricum [1]. Strain PR3 T was first described by Montalvo-Rodríguez et al. in 1998 [1] as Gram-stain negative and motile. The organism is of interest because of its position in the tree of life, where it is located between members of the Haloferax/Halorubrum cluster within the large euryarchaeal family Halobacteraceae (Figure 1). Here we present a summary classification and a set of features for H. geometricum PR3 T together with the description of the complete genomic sequencing and annotation.

Classification and features
In addition to the solar salterns of Cabo Rojo, Puerto Rico, where the type strain PR3 T and two accompanying strains (PR7 and PR9) were initially isolated [1], the occurrence of strains or phylotypes closely related or belonging to H. borinquense have so far only been reported from high salt environments such as an Australian crystallizer pond [6], Maras Salterns in the Peruvian Andes [7], a salt field at Nie, Ishikawa Prefecture, Japan [8], the salterns of Tamilnadu, India (Kannan et al. unpublished), Exportadora del Sal, Guerro Negro, Mexico (FJ609942), a Taiwanese saltern soil (FJ348412), and a low-salt, sulfide-and sulfur-rich spring in southwestern Oklahoma, USA [9].
H. geometricum PR3 T cells are highly pleomorphic (short and long rods, squares, triangles and ovals) and motile by peritrichous flagella (Table 1 and Figure 2). Cells lyse in distilled water. Gas vesicles are present and are responsible for modifying the color of colonies or cell suspensions from red to pink. H. geometricum PR3 T is aerobic, but also capable of anaerobic growth with nitrate. No anaerobic growth on arginine (arginine dihydrolase is not present). At least 8% NaCl (w/v) is required for growth, reflecting the primary characteristic requirement for high salt concentrations of the Halobacteriaceae [18]. The optimal NaCl concentration range is 20-25% NaCl (w/v) at 40°C (optimal growth temperature). Nitrate is reduced to nitrite with the production of gas [1]. Spores or other resting stages have not been reported [1]. H. geometricum PR3 T is capable of degrading gelatin, but starch is not hydrolysed. A number of sugars and polyols are used as carbon sources, and acid is produced from some sugars [1]. Figure 1 shows the phylogenetic neighborhood of H. borinquense strain PR3 T in a 16S rRNA based tree. Analysis of the two 16S rRNA gene sequences in the genome of strain PR3 T indicated that the two genes differ by five nucleotides (nts) from each other, and by 3-5 nts from the previously published 16S rRNA sequence generated from DSM 11551 (AF002984). The slight differences between the genome data and the reported 16S rRNA gene sequence are most likely the result of sequencing errors in the previously reported sequence data.

Chemotaxonomy
The quinone composition of H. borinquense strain PR3 T has not been recorded, but based on reports from other members of the family Halobacteriaceae menaquinones with eight isoprenoid units are likely to be present. Typically both MK-8 and MK-8 (VIII-H2) are predicted. The lipids are based on isoprenoid diether lipids, but the exact nature of the isoprenoid side chains remains to be investigated. The major phospholipids are the diether, isoprenoid analogs of phosphatidylglycerol and methyl-phosphatidylglycerophosphate (typical of all members of the family Halobacteriaceae), the diether analog of phosphatidyl-glycerol sulfate is absent [1]. A single glycolipid has been reported with an Rf value similar to that of the triglycosyl diether from Haloarcula marismortui, but its structure has not been determined [1]. The pigments responsible for the red color of the cells have not been determined, but it may be predicted that they are carotenoids, probably bacterioruberins. Outer cell layers are probably proteinaceous. The presence of peptidoglycan has not been investigated, but is generally absent from members of this family Halobacteriaceae.  [2] under the maximum likelihood criterion [3,4]. The tree was rooted with Natronomonas pharaonsis, the deepest branching member of the family Halobacteriaceae. 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. Strains with a genome sequencing project registered in GOLD [5] are printed in blue; published genomes in bold.  , 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 [17]. If the evidence code is IDA then the property was directly observed for a living isolate 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 each phylogenetic position, and is part of the Genomic Encyclopedia of Bacteria and Archaea project. The genome project is deposited in the Genome OnLine Database [5]. The complete genome sequence has not yet been released from 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. . DNA was isolated from 1-1.5 g of cell paste using a Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with a modified protocol for cell lysis, LALMP procedure according to Wu et al. [20]..

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 http://www.jgi.doe.gov/. 454 Pyrosequencing reads were assembled using the Newbler assembler version v 2.0.0 (Roche). Large Newbler contigs were broken into 4,435 overlapping fragments of 1,000 bp and entered into assembly as pseudoreads. 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 as-sembly was made using the PGA assembler. Possible mis-assemblies were corrected and gaps between contigs were closed by custom primer walks from sub-clones or PCR products. A total of 2,826 Sanger finishing reads were produced. The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequence types provided 31.5× coverage of the genome.

Genome annotation
Genes were identified using GeneMark [21] as part of the genome annotation pipeline in the Integrated Microbial Genomes Expert Review (IMG-ER) system [22], followed by a round of manual curation using the JGI GenePRIMP pipeline [23]. 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. The tRNAScanSE tool [24] was used to find tRNA genes, whereas ribosomal RNAs were found by using the tool RNAmmer [25]. Other non coding RNAs were identified by searching the ge-nome for the Rfam profiles using INFERNAL (v0.81) [26]. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG) platform [27].

Metabolic network analysis
The metabolic Pathway/Genome Database (PGDB) was computationally generated using Pathway Tools software version 12.5 [28] and MetaCyc version 12.5 [29], based on annotated EC numbers and a customized enzyme name mapping file. It has undergone no subsequent manual curation and may contain errors, similar to a Tier 3 BioCyc PGDB [30].

Genome properties
The genome is 3,944,467 bp long and comprises one main circular chromosome with a 60% GC content and five plasmids. Of the 3,994 genes predicted, 3,937 were protein coding genes, and 57 RNAs. Thirty seven pseudogenes were also identified. A total of 62% of the genes were assigned a putative function while the remaining ones are annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 3. The distribution of genes into COGs functional categories is presented in Figure 3 and Table 4. A cellular overview diagram is presented in Figure 4, followed by a summary of metabolic network statistics shown in Table 5.