High quality draft genome sequence of the heavy metal resistant bacterium Halomonas zincidurans type strain B6T

Halomonas zincidurans strain B6T was isolated from a deep-sea heavy metal rich sediment from the South Atlantic Mid-Ocean Ridge. The strain showed significant resistance to heavy metals, especially to zinc. Here we describe the genome sequence and annotation, as well as the features, of the organism. The genome contains 3,325 protein-coding genes (2,848 with predicted functions), 61 tRNA genes and 6 rRNA genes. H. zincidurans strain B6T encodes 31 genes related to heavy metal resistance. And HGT may play an important role in its adaption to the heavy metal rich environment. H. zincidurans strain B6T may have potential applications in the bioremediation of heavy metal-contaminated environments.


Introduction
Heavy metals, either essential (e.g. Mn, Zn, Cu, Co, Ni and Mo) or toxic (e.g. Hg, Ag and Cd), are generally harmful to microbial cells even at low concentrations, as to other living organisms [1,2]. However, some microorganisms are able to resist to certain kinds and concentrations of heavy metals through several mechanisms, such as incorporating or precipitating heavy metals into complexes, oxidizing or reducing metals to less toxic valence states, and direct transporting metals out of the cell [3,4]. These heavy metal resistant microorganisms have been attracting great interests because of their potential biotechnological applications in bio-mining of expensive heavy metals and bioremediation of heavy metal-contaminated environment [2].
Halomonas, the largest genus of the family Halomonadaceae, can be found in most saline environments, including marine environments, salterns, saline lakes and soils, as well as salty foods, etc. [5,6]. Halomonas zincidurans strain B6 T , a moderately halophilic bacterium, was isolated from a deep-sea sediment from the South Atlantic Mid-Ocean Ridge [5]. The strain was able to grow in medium containing high concentrations of heavy metals, especially Zn 2+ ion, which is not detected in the reference strains and other moderately halophiles [5,7]. Therefore, the novel isolate was named as H. zincidurans due to its particular resistance to zinc ion [5]. Here, we present a summary classification and a set of features of H. zincidurans strain B6 T , together with the description of the genomic sequencing and annotation.

Organism information
A deep-sea sediment sample, TVG10, was collected from the South Atlantic Mid-Ocean Ridge (Table 1). There were many small hard orange red-colored lumps mixed in the sediment sample, which might be the particles containing ferric oxide and diffusing with hydrothermal plumes [8]. Not surprisingly, the concentrations of heavy metals in sample TVG10 were much higher than those in the samples collected from deep-sea seamount sediment [9], offshore sediment [10] and continental crust [11] (Additional file 1: Table S1), including Fe (98.99 mg/g), Mn (42.48 mg/g), Cu (0.839 mg/g), Ni (0.338 mg/g), Zn (0.285 mg/g), Cr (0.195 mg/g) and Co (0.064 mg/g). With consideration of the heavy metal rich environment, marine broth 2216 medium (MB, BD) containing 20 mM Mn 2+ was used to isolate heavy metal resistant strains. Subsequently a strain named B6 T was obtained [5].
H. zincidurans strain B6 T is a Gram-stained negative, rod-shaped (Figure 1), moderately halophilic bacterium growing at 0.5-15% (w/v) NaCl (Table 1). Strain B6 T exhibited the highest 16S rRNA gene sequence similarity with H. xinjiangensis (96.1%). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B6 T and H. xinjiangensis clustered together in a distinct branch within the genus Halomonas with a high bootstrap value ( Figure 2). Strain B6 T was able to resist high concentrations of heavy metals in liquid HM medium, including Mn 2+ (200 mM), Co 2+ (1.0 mM), Cu 2+ (2.5 mM) and Zn 2+ (14 mM). Its resistance to Zn 2+ could be much higher (30 mM) when incubated on marine agar 2216 medium (MA, BD) [5], comparing to only 1 mM Zn 2+ resisted by H. xinjiangensis TRM0175 T . And the maximum zinc resistance concentration for 250 moderately halophilic bacteria, reported by Nieto et al., was only 2.5 mM [7]. Therefore, H. zincidurans strain B6 T is of significant interest due to its prominent resistance to zinc.
(BGI, Shenzhen). The gap closure and annotation processes were performed by the authors. The Whole Genome Shotgun project of H. zincidurans strain B6 T has been deposited at DDBJ/EMBL/GenBank under the accession JNCK00000000. The version described in this paper is version JNCK01000000. Table 2 presents the project information and its association with MIGS version 2.0 compliance [12].

Growth conditions and DNA isolation
H. zincidurans strain B6 T was aerobically cultivated in MB medium at 30°C. Total genomic DNA was extracted using the method described by Marmur [32]. The quality and quantity of the genomic DNA was determined by 0.6% agarose gel electrophoresis with λ-Hind III digest DNA marker (TaKaRa, Dalian, China) and by a Qubit® fluorometer (Invitrogen, CA, USA) with Qubit dsDNA BR Assay kit (Invitrogen, CA, USA). About 350 μg DNA with a concentration of 450 ng/μl was obtained.

Genome sequencing and assembly
Whole-genome shotgun DNA sequencing of H. zincidurans strain B6 T was performed using Solexa pairedend sequencing technology (HiSeq2000 system, Illumina, USA) [33]. Two libraries with insert size 494 bp and 2,586 bp were constructed and a total of 519 Mb and 416 Mb raw data were produced before filtering. After removing the adapter, duplicated reads and short inserts from the data of large library, there remained 433 Mb (~120-folds genome coverage) and 328 Mb (~90folds genome coverage) clean data from the small and large libraries for assembling, respectively. Then these sequences were assembled into 15 contigs using the SOAPdenovo v.1.05 [30], the contig N50 length of which was 1,864,365 bp. PCR primers for gap closure were designed by Primer Premier v.5. PCR reactions were performed with PrimeSTAR HS Polymerase (TaKaRa, Dalian, China) and the amplicons were sequenced using Sanger and primer walking technologies. The sequenced fragments were subsequently assembled with the contigs using SeqMan of the Lasergene package (DNAstar, Madison, WI) into 2 contigs.

Genome properties
The genome was assembled into 2 contigs, one with a size of 3,546,937 bp and the other with 7,823 bp ( Table 3). The G+C content determined based on the total 3,554,760 bp sequences was 66.41%. A total of 3,392 genes were predicted, including 3,325 proteincoding genes, 61 tRNA genes and two copies of 16S-23S-5S rRNA gene operons (Table 4 and Figure 2). Among the protein coding genes, 2,848 were assigned to putative functions, and the remaining was annotated as hypothetical proteins. In total, 1,938 and 442 protein coding genes were assigned to KEGG and subsystems, respectively. The detailed properties and the statistics of the genome as well as the distribution of genes into COG functional categories are summarized in Tables 3, 4 and 5, Figure 3 and Additional file 2: Table S2.

Insights into the genome
The genome of H. zincidurans strain B6 T contains 31 genes related to heavy metal resistance, especially to zinc resistance (Table 6). Zinc is an essential but also toxic metal for living being [2,43]. The concentration of zinc inside bacterial cells is maintained by importing limitation, efflux, accumulation and sequestration [44,45]. H. zincidurans strain B6 T possesses four heavy metal translocating P-type ATPases (HALZIN_733, HALZIN_1240, HALZIN_ 2196 and HALZIN_2262), which may participate in the transport of Zn 2+ , Mn 2+ , Cu 2+ , Cd 2+ , Pb 2+ , Ag + and Hg 2+ Figure 1 Micrograph of H. zincidurans strain B6 T obtained by scanning electron microscopy (S260; Cambridge).

Chromohalobacter israelensis ATCC 43985 T (AJ295144)
Chromohalobacter salexigens DSM 3043 T (AJ295146)  Figure 2 Phylogenetic tree highlighting the position of H. zincidurans strain B6 T relative to phylogenetically closely related type strains within the family Halomonadaceae. The sequences were aligned using Clustal W [26], and the neighbor-joining tree [27] was constructed based on kimura 2-parameter distance model [28] by using MEGA5 [29]. Bootstrap values above 60% are shown obtained from 1,000 bootstrap replications. Bar, 0.05 substitutions per nucleotide position. The corresponding GenBank accession numbers are displayed in parentheses.

HALZIN_922
). The other one encodes a transcriptional regulator (MerR, HALZIN_2469), two mercuric transport proteins (MerT and MerP, HALZIN_2470-2471) for inorganic mercury uptake [51] and a mercuric reductase (MerA, HALZIN_2472) catalyzing Hg 2+ to Hg 0 [53]. According to the genomic data, H. zincidurans strain B6 T is able to survive in both inorganic and organic mercury environments. Interestingly, the four ORFs of the inorganic mer-operon showed the highest sequence identities to those of Halomonas lutea. Nevertheless, all the six ORFs of the organic mer-operon did not show the highest sequence identities to those of the genus Halomonas, but to the genera Burkholderia, Pseudomonas, Gladiecola and Stenotrophomonas, which indicates that the organic mer-operon might be acquired by HGT. Of special interest are the three alkylmercury lyases (MerB, HALZIN_918-920), which had obvious differences between the G+C content (56.6%; 57.1, 56.6 and 56.0% for these three gene sequences, respectively) as well as the G+C content at the third-codon positions (60.3%; 60.4, 61.0 and 59.4% for these three gene sequences, respectively) and those of the total proteincoding genes (65.4 and 82.8%, respectively). Besides, the RSCUs of nearly half of the 59 codons used by the three genes (23, 27 and 26 codons for HALZIN_918-920, respectively) change more than 2 folds, compared with those used by total protein-coding genes. 13 of the 31

Conclusion
The draft genome sequence of the heavy metal resistant bacteria H. zincidurans strain B6 T isolated from the South Atlantic Mid-Ocean Ridge provide an insight into the genomic basis of its heavy metal resistance ability. And HGT may play an important role in its adaption to the heavy metal rich environment. On the basis of analysis and characterization of genome, H. zincidurans strain B6 T might be resistant more kinds of heavy metal than we tested, such as Hg 2+ , Cd 2+ , Pb 2+ , Ni 2+ and Ag + , etc. And it may have the potential for the bioremediation of multi-metal-contaminated environments.
In addition, further analysis will be performed to confirm its resistant ability to other heavy metals and determine the mechanism of heavy metal resistance that we don't know yet. Labeling from the outside to the inside circle: ORFs on the forward strand (colored by COG categories), ORFs on the reverse strand (colored by COG categories), RNA genes (tRNAs red, rRNAs blue), G+C content (peaks out/inside the circle indicate values higher or lower than the average G+C content, respectively), GC skew (calculated as (G-C)/(G+C), green/purple peaks out/inside the circle indicates values higher or lower than 1, respectively).