Complete genome sequence of Halomicrobium mukohataei type strain (arg-2T)

Halomicrobium mukohataei (Ihara et al. 1997) Oren et al. 2002 is the type species of the genus Halomicrobium. It is of phylogenetic interest because of its isolated location within the large euryarchaeal family Halobacteriaceae. H. mukohataei is an extreme halophile that grows essentially aerobically, but can also grow anaerobically under a change of morphology and with nitrate as electron acceptor. The strain, whose genome is described in this report, is a free-living, motile, Gram-negative euryarchaeon, originally isolated from Salinas Grandes in Jujuy, Andes highlands, Argentina. Its genome contains three genes for the 16S rRNA that differ from each other by up to 9%. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence from the poorly populated genus Halomicrobium, and the 3,332,349 bp long genome (chromosome and one plasmid) with its 3416 protein-coding and 56 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain arg-2 T (= DSM 12286 = ATCC 700874 = JCM 9738) is the type strain of the species Halomicrobium mukohataei, and represents the type species of the genus Halomicrobium [1]. H. mukohataei was initially described as Haloarcula mukohataei (basonym) by Ihara et al. 1997 [2]. H. mukohataei is a motile, extremely halophilic euryarchaeon. The organism is of significant interest for its isolated position in the tree of life within the genus Halomicrobium in the family Halobacteriaceae. H. katesii [3] is currently the only other cultivated member of the genus Halomicrobium. Only two uncultivated archaeal clones related to the genus (>98% sequence identity) have been reported from diversity screenings: clone XCDLW-A62 from saline lakes on the Tibetan Plateau (FJ155620), and clone SA93 from an athalassohaline environment in the Tirez Lagoon in Spain (EU722674). No phylotypes from environmental samples or genomic surveys could be directly linked to H. mu-kohataei. Here we present a summary classification and a set of features for H. mukohataei arg-2 T , together with the description of the complete genomic sequencing and annotation. Figure 1 shows the phylogenetic neighborhood of H. mukohataei strain arg-2 T in a 16S rRNA based tree. Two of the three 16S rRNA gene copies in the H. mukohataei arg-2 T genome are identical, but differ by 131 nucleotides (9%) from the third copy (23S rRNA gene sequences differ by only 1-1.7%, this study). Studies on the ribosomes indicate that operons which differ significantly in their sequence are expressed under different environmental conditions [9], as has also been reported for members of the genus Haloarcula [10]. The symbols rrnA and rrnB used in Figure 1 for these distinct rRNA copies in Haloarcula and Halomicrobium are in accordance with the designations used by Cui et al. 2009 [9]. The two identical 16S rRNA genes differ in one nucleotide from the previously reported reference sequence of strain arg-2 T derived from JCM 9738 (EF645690).  [4,5] of the 16S rRNA gene using the neighbor-joining algorithm and K2P distances [6]. The tree was rooted with Natronomonas pharaonis, 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 if larger than 60%. Strains with a genome sequencing project registered in GOLD [7] are printed in blue; published genomes in bold, e.g. the GEBA genome from Halorhabdus utahensis [8].

Classification and features
H. mukohataei is rod shaped ( Table 1), but may produce pleomorphic cells in the stationary phase [1] (Figure 2). There are conflicting reports concerning the type of flagellation, which may be either polar or in tufts or peritrichous [1]. Gas vacuoles have not been reported and resting stages such as spores are not produced. Cells are Gramnegative, although peptidoglycan is probably absent [1]. Strain arg-2 T grows under aerobic conditions, but may also grow anaerobically in the presence of nitrate [1]. Arginine does no support anaerobic growth. Acids are produced from glucose, galactose, mannose, ribose, sucrose, maltose and glycerol [1]. Glucose, galactose, sucrose, maltose and glycerol support growth as single carbon and energy sources. Starch is hydrolyzed [1], however, gelatin, casein and Tween 80 are not hydrolyzed. Requires at least 2M NaCl to maintain cell shape, with optimal growth occurring at 3.0-3.5 M NaCl. Catalase and oxidase positive. Optimal growth temperature is 40-45°C [1].

Chemotaxonomy
The quinone composition of H. mukohataei arg-2 T has not been investigated, 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) may be predicted. The lipids are based on diphytanyl ether lipids. The major phospholipids are the diphytanyl ether analogues of phosphatidylglycerol and methylphosphatidylglycerophosphate (typical of all members of the family Halobacteriaceae), the diether analogue of phosphatidylglycerol sulfate is present [1]. Glycolipids have been reported, one of which has a molecular weight typical of a sulfate diglycosyl diphytanyl ether, the structure of which has not been determined [1]. A diglycosyl diphytanyl ether lipid is also present. The pigments responsible for the red color of the cells have not been recorded, 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.   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 the Gene Ontology project [18]. 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 its phylogenetic position, and is part of the Genomic Encyclopedia of Bacteria and Archaea project. The genome project is deposited in the Genome OnLine Database [7] and the complete genome sequence in GenBank Sequencing, finishing and annotation was performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.  [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 the JGI website (http://www.jgi.doe.gov/). 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 3,703 overlap-ping 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 mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones [21]. A total of 39 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. 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 44.4× coverage of the genome. The final assembly contains 48,917 Sanger reads and 443,713 pyrosequencing reads.

Genome annotation
Genes were identified using Prodigal [22] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline (http://geneprimp.jgi-psf.org/) [23]. 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 (http://img.jgi.doe.gov/er) [24].

Genome properties
The genome is 3,332,349 bp long and comprises one main circular chromosome of 3.11 Mbp and one 219 kbp megaplasmid with a 65.5% GC content ( Table 3, Figure 3a and Figure 3b). Of the 3,472 genes predicted, 3,416 were protein coding genes, and 56 RNAs. In addition, 66 pseudogenes were identified. The majority of the genes (59.4%) were assigned with a putative function while those remaining were 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 Table 4.