Draft genome sequence of Halomonas lutea strain YIM 91125T (DSM 23508T) isolated from the alkaline Lake Ebinur in Northwest China

Species of the genus Halomonas are halophilic and their flexible adaption to changes of salinity and temperature brings considerable potential biotechnology applications, such as degradation of organic pollutants and enzyme production. The type strain Halomonas lutea YIM 91125T was isolated from a hypersaline lake in China. The genome of strain YIM 91125T becomes the twelfth species sequenced in Halomonas, and the thirteenth species sequenced in Halomonadaceae. We described the features of H. lutea YIM 91125T, together with the high quality draft genome sequence and annotation of its type strain. The 4,533,090 bp long genome of strain YIM 91125T with its 4,284 protein-coding and 84 RNA genes is a part of Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG-I) project. From the viewpoint of comparative genomics, H. lutea has a larger genome size and more specific genes, which indicated acquisition of function bringing better adaption to its environment. DDH analysis demonstrated that H. lutea is a distinctive species, and halophilic features and nitrogen metabolism related genes were discovered in its genome.


Introduction
Strain YIM 91125 T (= DSM 23508 T = KCTC 12847 T = CCTCC AB 206093 T ) is the type strain of Halomonas lutea [1]. Currently, there are 83 validly named species in the genus Halomonas on the basis of most recent released from LPSN [2] and EzTaxon-e [3]. Halomonadaceae comprises the largest number of halophilic and halotolerant bacteria described to date, and Halomonas is the largest genus in this family. However, most of the taxa in Halomonadaceae have been reclassified in the past due to their heterogeneous features [4][5][6][7]. In Halomonas, a small group of species has been formally re-located to Chromohalobacter, Cobetia and Kushneria by further taxonomic studies. Members of the genus Halomonas were usually isolated from saline environments [8][9][10][11][12]. Strain YIM 91125 T was originally isolated from soil sample of Ebinur Lake, which has been a long-term target for the studies of element cycling and microbial biota under extremely high-saline conditions in Xinjiang, Northwest China. As a type strain, it's the original isolate used in species description, which exhibits the relevant phenotypic and genotypic properties cited in the original published taxonomic circumscriptions [13]. This organism grows well across a wide range of salinity and temperature and also participates in nitrogen reduction. In this context, strain YIM 91125 T has been sequenced as a halophilic representative, and becomes a part of Genomic Encylopedia of Type Strains, Phase I: the one thousand microbial genomes project.
Here, we present a summary classification and a set of features for H. lutea strain YIM 91125 T , together with the description of the genomic sequencing and annotation, and provide brief findings of its genome sequence as compared to genomes of other Halomonas species. The genomic data will provide insights into its new biotechnological applications, such as sewage treatment. The comprehensive genomes of this genus will facilitate our understanding of the ecological roles that Halomonas species play in those hypersaline habitats and their relationships with other halophilic and nonhalophilic microorganisms.
16S rRNA gene sequence of strain YIM 91125 T was compared with the newly released database from the Greengenes [26], using NCBI BLAST [27,28] under default settings (e.g., considering only HSPs from the best 250 hits) and the relative frequencies of taxa were determined, weighted by BLAST scores. The most frequently occurring genera were Halomonas (71.4%), Chromohalobacter (17.8%), Bacillus (3.6%), Haererehalobacter (3.6%) and Modicisalibacter (3.6%) (228 hits in total). Regarding 186 hits to sequences from members of the genus Halomonas, the average identity within HSPs was 95.5%, whereas the average coverage by HSPs was 98.3%. Among all other species, the one yielding the highest score was Halomonas xinjiangensis, which corresponded to identity of 99.9% and HSP coverage of 98.0%. (Note that the Greengenes database uses the INSDC (=EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest scoring environmental sequences were EF157249 and EF157230 (Greengenes short name 'tar pits clone 101-11 k' and 'tar pits clone 101-120 k'), which showed identity of 96.3% and an HSP coverage of 99.6%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were soil like 'soil', 'seafloor', 'drilling deep-earth'; water like 'groundwater', 'aquatic', 'lake', 'marine'; oil and plant. Environmental samples yielded hits of a higher score than the highest scoring species were not found.
Phylogenetic analyses were carried out with two different algorithms, i.e., neighbor-joining (NJ) and maximumlikelihood (ML). The phylogenetic tree was shown in Figure 2 and Additional file 1: Figure S1, which provides an interesting insight into the nomenclature and classification of members of the genus Halomonas, and also indicates the phylogenetic neighborhood of H. lutea. The phylogenetic relationships indicate that H. lutea YIM 91125 T is most closely to H. xianhensis A-1 T with 99% similarity and the sequence of the sole 16S rRNA gene in the genome differs by 10 nucleotides from the previously published 16S rRNA sequence (EF674852). Evidence codes -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 [25].  According to the most recent release of the EzTaxon-e database, all the 16S rRNA gene sequences of the type strains within genus Halomonas were retained. The tree was inferred from 1,383 aligned bases [29] under the neighbor-joining (NJ) [30] and maximum-likelihood (ML) [31] methods with 1,000 randomly selected bootstrap replicates using MEGA version 5.2 [32]. The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 1,000 NJ bootstrap (left) and from 1,000 ML bootstrap (right) replicates [33] if they are larger than 50%. Lineages with type strain genome sequencing projects registered in Genomes OnLine Database (GOLD) [34] are labeled with one asterisk, and those have available genomic data are labeled with two asterisks. Non-type strain LS21of H. campaniensis and H. elongata DSM 2581 T listed 'Complete and Published' are also labeled with two asterisks.

Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position and biological application importance [35,36], and for a better understand the mechanism of its halophilic adaptation. Sequencing of H. lutea YIM 91125 T is part of Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG-I) project [37], a follow-up of the GEBA project [38], which aims for increasing the sequencing coverage of key reference microbial genomes. The genome project is deposited in the Genomes OnLine Database (GOLD), and the high quality draft genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE JGI using state of the art sequencing technology [39]. A summary of the project information is shown in Table 2. It presents the project information and in compliance with MIGS version 2.0 compliance [18].

Growth conditions and DNA isolation
H. lutea strain YIM 91125 T (DSM 23508 T ), was grown in DSMZ medium 514b (Medium 514 plus additional salt) at 37°C [40]. DNA was isolated from 0.5-1.0 g of cell pasted using Jetflex Genomic DNA Purification Kit (Qiagen, Hilden, Germany), following the standard protocol as recommended by the manufacturer, but with an additional incubation (60 min, 37°C) with 50 μl proteinase K and finally adding 200 μl protein precipitation buffer (PPT). DNA is available through the DNA Bank Network [41].

Genome sequencing and assembly
The draft genome of strain YIM 91125 T was generated at JGI using Illumina technology [42]. An Illumina standard shotgun library was constructed and sequenced using the Illumina HiSeq 2000 platform which generated 9,251,032 reads totaling 1,387.7 Mb. All general aspects of library construction and sequencing performed at the JGI. All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts. The following steps were then performed for assembly: (1) filtered Illumina reads were assembled using Velvet version 1.1.04 [43]; (2) 1-3 Kb simulated paired end reads were created from Velvet contigs using Wgsim [44]; (3) Illumina reads were assembled with simulated read pairs using Allpaths-LG [45]. The final draft assembly contained 49 contigs in 42 scaffolds. The total size of the genome is 4.5 Mbp and the final assembly is based on 538.9 Mbp of Illumina data, which provides an average 119.0 × coverage of the genome.

Genome annotation
Genes were identified using Prodigal [46] as part of the DOE JGI genome annotation pipeline [47], following by a round of manual curation using the JGI GenePRIMP pipeline [48]. The predicted CDSs were translated and used to search the NCBI non-redundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro database. These data sources were combined to assert a product description for each predicted protein.
Additional gene prediction analysis and functional annotation were performed within the Integrated Microbial Genomes-Expert Review (IMG-ER) platform [49].

Genome properties
The assembly of the draft genome sequence consists of 42 scaffolds (Figure 3) amounting to 4,533,090 bp, and G+C content is 59.1%. The majority of the protein-coding genes (83.0%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. 3,325 protein coding genes belong to 422 paralogous families in this genome. 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.

Insights from the genome sequence
The genomic sequences of twelve Halomonas species are available, including H. lutea YIM 91125 T . Genome properties of those Halomonas species are shown in  (Figure 4). And H. lutea has more specific genes, since proteins with COG only account for 71.18% which is lower than other members. Compared with other Halomonas species, the proportions of genes with signal peptide and transmembrane helices of H. lutea are respectively 7.46% and 23.65%, close to the corresponding averages. The abundance of transmembrane   The total is based on the total number of protein-coding genes in the annotated genome.
helices related genes indicates the important role in metabolism process of Halomonas. DNA-DNA hybridization is considered as a goldstandard of distinguishing species [51]. Digital DDH similarities between genome of H. lutea and those of other Halomonas species were calculated using GGDC web server version 2.0 under recommend setting [52,53]. The probabilities of DDH value > 70% assessed via logistic regression under three formulae indicate that H. lutea is different from other species of the genus (   inherent uncertainty in estimating DDH values from intergenomic distances based on models derived from empirical test data sets. Given that the low degree of DNA-DNA similarity among Halomonas species, it appears justified to assume that these strains represent different species. For better understanding of the relationships between H. lutea and other Halomonas members, availability of more genome sequences of representatives are needed to implement phylogenomic inference. As a halophilic bacterium, the genome of H. lutea also shows properties related to solute and ion transport, 203 genes related ion transport and metablism, 60 genes related TRAP-type C4-dicarboxylate transport system which is a crucial family of solute transporters. Moreover, nitrate reduction was tested using API 20NE system and 57 genes were predicted to participate in the nitrogen metabolism. PTS IIA-like nitrogen-regulatory protein, nitrate and sulfonate transport systems related genes were also detected in its genome.

Conclusions
The genome sequence and annotation of H. lutea YIM 91125 T were presented. The genome comprises 42 scaffolds which together represent the organism of approximately 4.53 Mb. It encodes for key genes and pathways involved in the compatible solutes production and nitrogen degradation. This provides clues to discover novel genes and functions, and leads to an improved understanding of halophilic microbial evolution and function in the extremely salty conditions. YIM 91125 T participates in nitrogen cycling, although the process of reducing nitrogen needs further studies to fully understand the related pathways. The genome sequencing of H. lutea marks an important step toward a comprehensive genomic catalog and the metabolic diversity of halophilic bacteria. It may contribute to further studies on important process for Halomonas, such as quorum-sensing regulatory and osmoadaption. Combining with genomes of other members in Halomonas, will make an important advance in understanding of the ecological roles that Halomonas species play in those hypersaline environments and their relationships with other halophilic and nonhalophilic microorganisms.

Additional file
Additional file 1: Figure S1. Phylogenetic tree of the genus Halomonas.