Marine Genomics 38 (2018) 1–4

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Marine Genomics

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Complete genome sequence of Alcanivorax xenomutans P40, analkane-degrading bacterium isolated from deep seawater

Xiaoteng Fu a,b,c,d,e, Qiliang Lai a,c,d,e, Chunming Dong a,c,d,e, Wanpeng Wang a,c,d,e, Zongze Shao a,c,d,e,⁎a Key Laboratory of Marine Genetic Resources, Third Institute of Oceanography, SOA, Xiamen 361005, Chinab School of Life Sciences, Xiamen University, Xiamen 361005, Chinac State Key Laboratory Breeding Base of Marine Genetic Resources, Xiamen 361005, Chinad Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Xiamen 361005, Chinae Key Laboratory of Marine Genetic Resources of Fujian Province, Xiamen 361005, China

⁎ Corresponding author at: Key Laboratory of Marine Gof Oceanography, SOA, Xiamen 361005, China.

E-mail addresses: fuxiaoteng0129@163.com (X. Fu), ladongchunming@tio.org.cn (C. Dong), wangwanpeng2008shaozz@163.com (Z. Shao).

http://dx.doi.org/10.1016/j.margen.2017.05.0101874-7787/© 2017 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 12 April 2017Received in revised form 16 May 2017Accepted 16 May 2017Available online 23 May 2017

Strain P40 (MCCC 1A01128) is a member of Alcanivorax xenomutans, in the family Alcanivoracaceae ofGammaproteobacteria. Since Alcanivorax species play a pivotal role in bioremediation of oil spills in marine envi-ronments, further studies on these hydrocarbonoclastic marine bacteria will facilitate a better understanding oftheir alkane metabolic capacities. Previous study shows strain P40 has obvious ability of alkane degradation.Here, we describe the complete genome sequence and annotation of strain P40, which is the first strain withthe complete genome sequence of the species A. xenomutans. Strain P40 contains a 4,733,951 bp chromosomewithout any plasmids, and encodes 4148 protein-coding genes and 45 RNA-only encoding genes. With genes in-volving in alkane degradation, heavy-metal resistance, stress response and so on, A. xenomutans P40 may have apotential use in the bioremediation of oil polluted and heavy metal-contaminated environments.

© 2017 Elsevier B.V. All rights reserved.

Keywords:AlcanivoraxGenome sequencingAlkane degradationIndian Ocean

1. Introduction

Nowadays, oil spills are still a central source of ocean pollution,threatingmarine ecosystems. Fortunately, the activities of naturally mi-crobes have the ability of eliminating those considerable amounts of pe-troleum entering the sea (Timmis, 2010). Since Alcanivorax species canuse alkanes more effectively than other hydrocarbon-degrading bacte-ria (Hara et al., 2003; Liu et al., 2011), thus becoming the most impor-tant alkane-degrading bacteria in marine environments. The genusAlcanivorax belongs to family Alcanivoracaceae of Gammaproteobacteria,and currently comprises eleven species including A. borkumensis(Yakimov et al., 1998), A. jadensis (Bruns and Berthe-Corti, 1999), A.venustensis (Fernández-Martínez et al., 2003), A. dieselolei (Liu andShao, 2005), A. balearicus (Rivas et al., 2007), A. hongdengensis (Wu etal., 2009), A. pacificus (Lai et al., 2011), A. marinus (Lai et al., 2013), A.xenomutans (Rahul et al., 2014) and A. gelatiniphagus (Kwon et al.,2015), A. nanhaiticus (Lai et al., 2016).

Strain P40 was isolated from the deep seawater of the Indian Oceanduring Chinese Global Ocean Expedition of the “the R/V Dayang Yihao”

enetic Resources, Third Institute

i719@163.com (Q. Lai),@126.com (W. Wang),

in 2005 after enriched with petroleum and diesel as carbon source. The16S rRNA gene shared 99.8%, 99.4% and 99.4% similarities withAlcanivorax dieselolei B-5T, Alcanivorax xenomutans JC109T andAlcanivorax balearicus MACL04T, respectively. And the gyrB gene se-quence of strain P40 shared the highest similarity with Alcanivoraxxenomutans JC109T (99.7%), followed by Alcanivorax dieselolei B-5T

(84.5%) and Alcanivorax balearicusMACL04T (83.3%), and other speciesof genus Alcanivorax (70.9%–77.5%),thus indicating strain P40 belongedto the species Alcanivorax xenomutans. Based on its phylogenetic posi-tion and dominance position in the crude oil-degrading, we selectedthis organism for sequencing. In this report, we present the genome se-quence and annotation of A. xenomutans P40, which is the first strainwith the complete genome sequence of the species A. xenomutans. Itsgenome sequence and its curated annotation will provide significantdatasets to better understand the physiology and metabolic potentialof A. xenomutans and will greatly facilitate functional genomic studiesof A. xenomutans.

2. Data description

General features of strain P40 are summarized in Table 1. The strainis positive for catalase, oxidase (weak), nitrate reduction, and negativefor denitrification, arginine dihydrolase, indole production, D-glucosefermentation, urease, β-glucosidase, gelatin hydrolysis, β-galactosidase,

Table 1General features of Alcanivorax xenomutans P40 and MIGS mandatory information.

Items Description

General featuresClassification Domain Bacteria

Phylum ProteobacteriaClass GammaproteobacteriaOrder OceanospirillalesFamily OceanospirillalesGenus AlcanivoraxSpecies Alcanivorax xenomutans

Gram stain NegativeCell shape RodMotility MotilePigmentation No-pigmentedSporulation Non-sporulatingTemperature range 4–43 °COptimum temperature 28 °CCarbon source Sodium acetate, alkanesEnergy source ChemoorganotrophicTerminal electron receptor OxygenSalinity 0.5–15%Oxygen AerobicMIGS dataSubmitted to INSDC GenBank (ID: CP012331)Investigation type BacteriaProject name Alcanivorax xenomutans strain: P40 genome

sequencingGeographic location (latitude andlongitude)

25.32°S, 70.04°E

Geographic location (depth) −668 mGeographic location (country) Indian OceanCollection date 2005Environment (biome) OceanEnvironment (feature) WaterEnvironment (material) Sea waterEnvironmental package Deep sea water samples from Indian OceanBiotic relationship Free-livingPathogenicity NoneSequencing method Illumina Hiseq2000, 454 FLX+Assembly GS De Novo Assembler packageFinishing strategy Complete

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D-glucose, L-arabinose, D-mannose, D-mannitol, D-maltose, potassiumgluconate. API ZYM test strip results indicate that it is positive for alka-line phosphatase, acid phosphatase, esterase (C4), esterase lipase (C8),lipase (C14), leucine aminopeptidase, naphtol-AS-Bl-phosphoamidase,valine aminopeptidase (weak); negative for cystine aminopeptidase,trypsin, α-chymotrypsin,α-galactosidase, β-galactosidase, β-glucuroni-dase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase. The API 20NE test strip shows that strainP40 can utilize adipic acid, capric acid, phenylacetic acid, N-acetyl-glu-cosamine (weak), trisodium citrate (weak), cannot utilize malic acid.

For genome sequencing, strain P40 was then grown aerobically tomid logarithmic phase at 28 °C in 216 L medium (containing, per litreseawater: CH3COONa, 1.0 g; tryptone, 10.0 g; yeast extract, 2.0 g; sodi-um citrate, 0.5 g; NH4NO3, 0.2 g; pH7.5). The genomicDNAwas then ex-tracted, concentrated and purified using the AxyPrep bacterial genomicDNA mini-prep Kit (Axygen). The purity and quality of DNA (UV A260/A280) was assessed using a NanoDrop 2000 Spectrophotometer (Ther-mo Scientific, USA). The whole-genomic DNA sequencing was per-formed using a combination of Illumina and Roche454 platforms. Thepair-end 454 library with average insert size of 3 kb pyrosequencinggenerated 543,410 reads totaling 233.4 Mb. GS De Novo Assemblerpackage (v2.8) was then applied for assembly to obtain the draft ge-nome scaffolds. Then Illumina Hiseq 2000 pair-end reads with averageinsert size of both 300 bp and 500 bp were realigned to the scaffoldsto correct potential base errors and to fill the intra-scaffold gaps. Theinter-scaffold gaps were filled in by sequencing the PCR productsusing ABI 3730xl capillary sequencers. Finally, sequences were assem-bled and assessed quality using Phred/Phrap/Consed software packages.

Circos software was then used to get circularized map of the chro-mosome, setting calculation window as 2000 bp and steps as 500 bp(Krzywinski et al., 2009). Gene prediction and annotation were per-formed using National Center for Biotechnology Information (NCBI)prokaryotic genome annotation pipeline (Tatusova et al., 2016) andthe Rapid Annotation using Subsystem Technology (RAST) pipeline(http://rast.nmpdr.org/) (Overbeek et al., 2014). The functional annota-tion of predicted ORFs was used to search the KEGG and COG databasesby RPS-BLAST.

The complete genome consists of one chromosome with a totallength of 4,733,951 base pairs (bp) and a G + C content of 61.45%(Fig. 1). Of the 4341 genes predicted, 4148 were protein-coding genes(coding sequence, CDS), and 45 RNAs; 143 pseudogeneswere also iden-tified. Of the entire 4148 CDS, 2092 CDSwere identified to participate in223 pathways. In addition, 3618 could be assigned to cluster oforthologous groups (COGs), which were analyzed to understand howstrain P40 deploys its genes in the genome. These CDS could be assignedto 25 different categories (Table 2).

28 putative Genomic islands (GIs) were identified by IslandViewer(Langille and Brinkman, 2009) (Table S1). The size of the 28 putativeislands ranged from 3721 bp (GI 3) to 3,9969 bp (GI 21). The largestGI 21 contained 32 genes, whereas the smallest GI 3 had 6 genes. Inthese 28 GIs, 234 CDS were identified, including CDS encoding regula-tors, transmembrane proteins, heavymetal ion transport and resistancerelated proteins, CRISPR-associated protein for defense, oxidoreduc-tases for metabolism and so on. Among these GIs, eight contain mobilegenetic elements, such as integrase and transposase genes, suggestingthat these GIs can self-mobilize and could also support potential activehorizontal gene transfer in the strain.

To further understanding adaptive capacity of strain P40 to marineenvironment, metabolic features related to functional categories werethen analyzed (Table S2). 125 genes were found relating to “Virulence,Disease and Defense” which functioned as antibiotics and toxic com-pounds resistance, invasion and intracellular resistance and bacteriocinssynthesis. 72% genes belonged to the subcategory of resistance to anti-biotics and toxic compounds, andmajority involving in heavy-metal re-sistance, such as cobalt-zinc-cadmium resistance, mercuric resistance,arsenic resistance and copper tolerance, suggesting strain P40 hasevolved with metal-resistant genes as a means of adaptation. Withthese heavy metal-resistance genes, A. xenomutans P40 may have a po-tential use in the bioremediation of heavymetal-contaminated environ-ments. What's more, A. xenomutans P40 owned 136 genes related to“stress response”. These genes would facilitate strain P40 surviving inthe high-osmolality and cold environment. The high salt concentrationsresulted in high osmotic stress. One effective strategy to maintain os-motic balance across a membrane is accumulating compatible solutes(Boscari et al., 2004). The presence of the choline dehydrogenasegene, high-affinity choline uptake protein gene and several glycine be-taine transporters related genes indicated its strong tolerance to saltsto synthesize and transport of betaine and choline. 21 cold/heat shockproteins could protect the cell from extreme temperatures (WeberandMarahiel, 2003). A number of genes that respond to oxidative stresswere also identified such as genes coding for catalase, superoxide dis-mutase and so on.

Experiment showed that after 3 days culture of strain P40, 0.1% (w/v) hexadecane could be emulsification and degradation, thusconforming its good ability for alkane degradation. Genome analysis in-dicated that the genome of A. xenomutans P40 harbours three types ofgenes for alkane degrading, including three alkane hydroxylases(P40_00520, P40_03400, P40_21415), two P450 cytochrome(P40_09930, P40_11275) and one almA (P40_09820). Since the 16SrRNA gene shared 99.8%, the highest similarities with A. dieselolei B-5T,and the average percentage of nucleotide sequence identity (ANI)showed 93.52% between A. dieselolei B5T and strain P40, A. dieselolei B-5T served as the closest phylogenetic neighbor for strain P40. Then com-parative analysis was conducted between these two strains. Results

Fig. 1. Circular representation of Alcanivorax xenomutans P40 genome. From the outside to the center: label of genome size, CDSs on forward strand (colored by COG categories), CDSs onreverse strand (colored by COG categories), RNA genes (tRNAs and rRNA operons), G + C content and GC skew. CDSs are depicted in different colors according to COG categories. (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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indicated alkane monooxygenases in strain P40 were homologous tocorresponding genes in A. dieselole B-5T with at least 97% amino acididentity. The considerable high level of alkane monooxygenase genesequence conservation revealed similar function and regulation foralkane degrading between A. xenomutans P40 and A. dieselolei B-5T.Since the function of those genes have been conformed in A. dieseloleB-5T (Wang and Shao, 2014), P450 cytochrome in strain P40 wasthen predicted responsible for the initial oxidation of alkanes withchain lengths C5–C17; alkB from P40 would oxidize alkanes withchain lengths C12–C26; and almA might be essential for the degrada-tion of C32 and longer LC alkanes. These three types of alkanemonooxygenases could uptake distinctive length of alkane thusmaking the strain possessing wide range of substrate. Besides, pro-teins involved in alkane sensing, chemotaxis, signal transduction,uptake and pathway regulation, like outer membrane protein,

Table 2Genome statistics of Alcanivorax xenomutans P40.

Attribute Genome (total)

Value % of total

Genome size (bp) 4,733,951 100.00G + C content (bp) 2,909,013 61.45Coding region (bp) 4,168,815 88.06Total genes 4341 100.00RNA genes 45 1.04rRNA operons 2 0.05tRNA genes 43 1.00Protein-coding genes 4148 95.56Pseudo genes 143 3.29Genes in paralog clusters 802 18.48Genes assigned to COGs 3618 83.34Paralogous groups 55

chemotaxis complex, cytochrome-o ubiquinol oxidase, fatty acidtransporter protein and so on are all discovered in A. xenomutansP40 genome as well. The number of each protein is might differentbetween A. dieselole B-5T and A. xenomutans P40. These encodinggenes require further experiment validation to confirm their abilitiesand determine the mechanism of alkane degradation that we don'tknow yet, which might help in bioremediation of oil pollutedenvironments.

2.1. Nucleotide sequence accession number

The complete genome sequence of Alcanivorax xenomutans P40 hasbeen deposited at the GenBank database under number CP012331.The strain is available from the Marine Culture Collection of Chinaunder number MCCC 1A01128.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.margen.2017.05.010.

Author contributions

XF, QL, CD, WW performed the experiments and collected the data;XF analyzed data; XF and ZSwrote themanuscript. All authors approvedthe final manuscript.

Conflict of interest statement

The authors declare that the research was conducted in the absenceof any commercial or financial relationships that could be construed as apotential conflict of interest.

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Acknowledgments

This work was financially supported by COMRA program (No.DY125-15-R-01), National Natural Science Foundation of China(41276005) and China Postdoctoral Science Foundation (No.2015M571964).

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