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Complete genome sequence of Halorhabdus utahensis type strain (AX-2T)
Standards in Genomic Sciences volume 1, pages218–225(2009)
Halorhabdus utahensis Wainø et al. 2000 is the type species of the genus, which is of phylogenetic interest because of its location on one of the deepest branches within the very extensive euryarchaeal family Halobacteriaceae. H. utahensis is a free-living, motile, rod shaped to pleomorphic, Gram-negative archaeon, which was originally isolated from a sediment sample collected from the southern arm of Great Salt Lake, Utah, USA. When grown on appropriate media, H. utahensis can form polyhydroxybutyrate (PHB). 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 a member of halobacterial genus Halorhabdus, and the 3,116,795 bp long single replicon genome with its 3027 protein-coding and 48 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
Strain AX-2T (= DSM 12940 = JCM 11049) is the type strain of the species Halorhabdus utahensis, and represents one of only two species currently assigned to the genus Halorhabdus . Strain AX-2T was first described by Wainø et al. in 2000  as Gram-negative, motile and extremely pleomorphic organism. The organism is of interest because of its position in the tree of life, where the genera Halorhabdus and Halomicrobium constitute one of the deepest branches within the large euryarchaeal family Halobacteriaceae. Here we present a summary classification and a set of features for H. utahensis strain AX-2T together with the description of the complete genomic sequencing and annotation.
Classification and features
Only one other 16S rRNA encoding sequence has been deposited in the INSDC databases with a similarity of greater than 97% to that of strain AX-2T. That sequence belongs to the other species classified in the genus Halorhabdus, H. tiamatea, which was isolated from a sample of the brine-sediment interface of the Shaban Deep in the northern Red Sea . With 95% sequence identity, strain T4.2 (AJ270232), a halophilic archaeon that is neither validly published nor preserved in any collection  is the next cultivated neighbor of H. utahensis strain AX-2T. Screening of environmental genomic samples and surveys reported at the NCBI BLAST server indicated no closely related phylotypes (>91% sequence similarity) can be linked to the species or genus.
Figure 1 shows the phylogenetic neighborhood of H. utahensis strain AX-2T in a 16S rRNA based tree. The sequence of the unique 16S rRNA gene is identical with the previously published 16S rRNA sequence generated from DSM 12940 (AF071880).
H. utahensis strain AX-2T is rod shaped, but may also form pleomorphic cells (Table 1 and Figure 2). Cells are motile by a single flagellum. Strain AX-2T does not require amino acids for growth and will grow on defined medium containing a nitrogen source, using a single carbon source. Cells may grow anaerobically on glucose by fermentation. Polyhydoxybutyrate inclusions are formed on appropriate media. Spores or other resting stages are not produced. Oxidase and catalase are positive. Cells lyse in distilled water. Gelatin and starch were not hydrolyzed. Proteases not produced and urea was not hydrolyzed; aesculin is hydrolyzed. Esterase, lipase and glucosidase are produced. Arginine dihydrolase is not produced, and consequently arginine does not support anaerobic growth. Ornithine and lysine are not decarboxylated. Growth on glucose, xylose and fructose. Nitrate is reduced to nitrite, but does not support growth .
Menaquinones are the sole respiratory lipoquinones of H. utahensis strain AX-2T. Both MK-8 and MK-8 (VIII-H2) are present. The lipids are based on diphytanyl ether lipids. The major phospholipids are the C20 diphytanyl ether analogues of phosphatidylglycerol and methyl-phosphatidylglycerophosphate (typical of all members of the family Halobacteriaceae), the diether analogue of phosphatidylglycerol sulphate is absent . Two glycolipids have been reported with Rf values consistent with their identification as a triglycosyl diphytanyl ether and the sulfated derivative, sulfated triglycosyl diphytanyl. The structures of these two lipids have not been elucidated . 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.
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  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.
Growth conditions and DNA isolation
H. utahensis strain AX-2T, DSM 12940, was grown in DSMZ medium 927 (H. utahensis medium)  at 40°C. DNA was isolated from 1–1.5 g of cell paste using a Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) as described in Wu et al. .
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 on 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 3,474 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 to 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 . Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 212 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The final assembly consists of 26,545 Sanger and 382,722 pyrosequence (454) reads. Together all sequence types provided 29.5× coverage of the genome. The error rate of the completed genome sequence is less than 1 in 100,000.
Genes were identified using Prodigal  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/) . 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 functional annotation was performed within the Integrated Microbial Genomes Expert Review platform (http://img.jgi.doe.gov/er) .
The genome is 3,116,795 bp long and comprises one main circular chromosome with a 62.9% GC content (Table 3 and Figure 3). Of the 3,075 genes predicted, 3,027 were protein coding genes, and 48 RNAs; 29 pseudogenes were also identified. The majority of the protein-coding genes (60.5%) 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.
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We gratefully acknowledge the help of Susanne Schneider (DSMZ) for DNA extraction and quality analysis. This work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, as well as German Research Foundation (DFG) INST 599/1-1.
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Anderson, I., Tindall, B.J., Pomrenke, H. et al. Complete genome sequence of Halorhabdus utahensis type strain (AX-2T). Stand in Genomic Sci 1, 218–225 (2009). https://doi.org/10.4056/sigs.31864