Matthew Hitchings

Paul Dyson

Cronfa at Swansea University

 Cronfa -  Swansea University Open Access Repository   _____________________________________________________________   This is an author produced version of a paper published in :Journal of Biotechnology                                        Cronfa URL for this paper:http://cronfa.swan.ac.uk/Record/cronfa26369_____________________________________________________________ Paper:Sun, H., Gao, T., Chen, X., Hitchings, M., Li, S., Chen, T., Zhang, H., An, L. & Dyson, P. (2016).  Complete genomesequence of a psychotrophic Arthrobacter strain A3 (CGMCC 1.8987), a novel long-chain hydrocarbons producer.Journal of Biotechnology, 222, 23-24.http://dx.doi.org/10.1016/j.jbiotec.2016.02.010        _____________________________________________________________  This article is brought to you by Swansea University. Any person downloading material is agreeing to abide by theterms of the repository licence. Authors are personally responsible for adhering to publisher restrictions or conditions.When uploading content they are required to comply with their publisher agreement and the SHERPA RoMEOdatabase to judge whether or not it is copyright safe to add this version of the paper to this repository. http://www.swansea.ac.uk/iss/researchsupport/cronfa-support/  Accepted ManuscriptTitle: Complete genome sequence of a psychotrophicArthrobacter strain A3 (CGMCC 1.8987), a novel long-chainhydrocarbons producerAuthor: Haili Sun Tianpeng Gao Ximing Chen Matthew D.Hitchings Shuyan Li Tao Chen Hua Zhang Lizhe An PaulDysonPII: S0168-1656(16)30055-4DOI: http://dx.doi.org/doi:10.1016/j.jbiotec.2016.02.010Reference: BIOTEC 7404To appear in: Journal of BiotechnologyReceived date: 2-2-2016Accepted date: 3-2-2016Please cite this article as: Sun, Haili, Gao, Tianpeng, Chen, Ximing, Hitchings,Matthew D., Li, Shuyan, Chen, Tao, Zhang, Hua, An, Lizhe, Dyson, Paul,Complete genome sequence of a psychotrophic Arthrobacter strain A3 (CGMCC1.8987), a novel long-chain hydrocarbons producer.Journal of Biotechnologyhttp://dx.doi.org/10.1016/j.jbiotec.2016.02.010This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.Complete genome sequence of a psychotrophic Arthrobacter strain A3 (CGMCC 1.8987), a novel Long-Chain Hydrocarbons producer Haili Suna,f,1, Tianpeng Gaof,1, Ximing Chenc,d, Matthew D. Hitchingsb, Shuyan Lie, Tao Chena, Hua Zhanga*, Lizhe Ana*, Paul Dysonb Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciencesa, State Key Laboratory of Applied Organic Chemistry, College of Chemistry & Chemical Engineeringe, Lanzhou University; Centre of Urban Ecology and Environmental Biotechnology; Lanzhou City Universityf; Key Laboratory of Extreme Environmental Microbial Resources and Engineering of Gansu Provincec; Key Laboratory of Desert and Desertification, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciencesd; Lanzhou 730000, China, Institute of Life Science; College of Medicine, Swansea Universityb, Singleton Park, Swansea SA2 8PP, UK * Corresponding authors  1 These authors contributed equally to this work. To whom correspondence should be addressed.  Email: lizhean@lzu.edu.cn                 Highlights	 	This	article	is	a	genome	announcement,	which	has	report	a	complete	genome	sequence	 of	 a	 psychotrophic	Arthrobacter	 strain	 A3.	Arthrobacter strain A3, a psychotrophic bacterium isolated from the Tian Shan Mountain of China, can degrade the cellulose and synthesis the long-chain hydrocarbons efficiently in low temperature.	 The availability of this genome sequence allows us to investigate the genetic basis of adaptation to growth in a nutrient-poor permafrost environment and to evaluate of the biofuel-synthetic potential of this species. The complete genome chromosome and plasmid sequences have been deposited in GenBank database with accession number CP013745.1 and CP013746.1 respectively. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC 1.8987).                   Abstract  Arthrobacter strain A3, a psychotrophic bacterium isolated from the Tian Shan Mountain of China, can degrade the cellulose and synthesis the long-chain hydrocarbons efficiently in low temperature. Here we report the complete genome sequence of this bacterium. The complete genome sequence of Arthrobacter strain A3, consisting of a cycle chromosome with a size of 4.26 Mbp and a cycle plasmid with a size of 194kbp. In this genome, a hydrocarbon biosynthesis gene cluster (oleA, oleB/oleC and oleD) was identified. To resistant the extreme environment, this strain contains a unique mycothiol-biosynthetic pathway (mshA-D), which has not been found in other Arthrobacter species before. The availability of this genome sequence allows us to investigate the genetic basis of adaptation to growth in a nutrient-poor permafrost environment and to evaluate of the biofuel-synthetic potential of this species.                 Arthrobacter are most frequently isolated in soils and play important roles in biogeochemical cycles and decontamination (Unell et al., 2008). Additionally, members of the genus Arthrobacter, Shewanella and Micrococcus have the ability to produce long-chain hydrocarbons as biofuel (Beller et al., 2010; Frias et al., 2009; Sukovich et al., 2010). Arthrobacter Strain A3 was isolated from permafrost of the Tian Shan Mountain in NW China, is a psychotrophic bacterium that can grow at temperatures between -4 oC to 25 oC, with an optimum growth temperature of 20oC (Chen et al., 2011). Gas chromatography-mass spectrometry (GC-MS) has indicated that this strain can produce hydrocarbons as biofuel at low temperatures. Here we present the complete genome sequence of Arthrobacter strain A3 (previous name is Arthrobacter xinjiangense TSBY90). The raw sequence was generated using a shotgun approach employing the PacBio® RS II system and whole-genome shotgun technology with the Illumina HiSeq 2000 system yielding ∼430× coverage (Roberts et al., 2013). Genomic DNA was sheared to an average fragment size of approximately 20 kb, and subsequently constructed to a SMRTbellTM library with a recovery of 40%. To generate the longest reads possible for this genome, the 20 kb SMRTbell library was size-selected using the BluePippinTM size selection system to remove SMRTbells less than 15 kb. Approximately, 14% of the final SMRTbell library was recovered with an average insert size of approximately 20 kb. The size-selected SMRTbell library was bound with P6 polymerase and sequenced with C4 chemistry and loaded using the one-cell-per-well protocol in the RS II instrument. Three SMRT Cells with 1.45 Gb post-filtering data were used for assembly. De novo assembly was carried out using the Hierarchical Genome Assembly Process (HGAP) version 3 (PacBio DevNet; Pacific Biosciences) workflow, including consensus polishing with Quiver as available in the SMRT® Analysis v 2.3.  The annotation for Arthrobacter strain A3 was conducted with NCBI Prokaryotic Genome Annotation Pipeline (Pruitt et al., 2012). The chromosome of A3 consists of 4,264,061 bp, the G+C content is 60.78%, 3735 coding sequence were identified, and 52 tRNA genes. The plasmid of A3 consists of 194,226 bp, the G+C content is 57.11% and 175 coding sequence were identified (Table1). Over 58% (2267) of the predicted open reading frames (ORFs) present in the A3 genome could not be assigned a putative function. Previous studies identified the oleABCD genes involved in head-to-head olefinic hydrocarbon biosynthesis (Sukovich et al., 2010). In the A3 genome, one ole gene cluster (MB46_09735- MB46_09745) was identified. Like related hydrocarbon biosynthesis clusters from other sequenced Arthrobacter species, this gene cluster also contains one copie of oleA (MB46_09735), a copy of oleB/oleC fusion gene (MB46_09740) and oleD (MB46_09745) gene.  The	 potential	 of	 Arthrobacter strain A3	 to	 produce	 secondary	metabolites	 was	 analyzed	 with	 the	 secondary	 metabolites	 search	 tool	antiSMASH(Weber	 et	 al.,	 2015),	 which	 indicated	 that	 5	 putative	 gene	clusters	involved	in	the	biosynthesis	of	different	natural	products	(1	NRPS,	1	 typeI	 PKS,	 1	 Siderophore,	 1	 Terpene	 and	 1	 unspecified	 cluster)	 are	located	in the chromosome and 1 bacteriocin putative gene cluster is located in the plasmid. Other features of the genome indicate that Arthrobacter strain A3 has evolved a metabolism adapted to this nutrient-poor environment, specifically by recruiting the potential to utilize a variety of carbon sources from plants. In contrast to other Arthrobacter species, A3 has a unique predicted beta-glucoside metabolism with potential to utilise plant-derived carbohydrates that include salicin, arbutin, cellobiose and aesculin. In addition, A3 has the potential to utilize fructooligosaccharides and raffinose. The genome analysis of Arthrobacter strain A3 indicates considerable investment, greater than in other sequenced Arthrobacter species, in iron acquisition capability. Iron-uptake systems include a unique low-pH-induced iron transporter system and utilization systems. Siderophores are small, high-affinity iron chelating compounds secreted by many microorganisms as a means to acquire sufficient iron for their growth. A3 contains a gene cluster to direct synthesis of Desferrioxamine E, a pathway it shares with other Arthrobacter species.  The ability to cope with oxidative, osmotic and low temperature stresses is likely a key factor for life in the permafrost. Previous studies have indicated that many Actinobacteria can synthesis a mycothiol (1D-myo-inosityl 2-(N-acetylcysteinyl) amido-2-deoxy-a-D-glucopyranoside) to provide protection against oxidative stress (Fahey, 2001; Newton et al., 2008). However, until now, the mycothiol-biosynthetic pathway has not been found in representatives of the Arthrobacter, Agromyces, or Actinomyces genera (Newton et al., 1996). Here we report the first example of a potential mycothiol-biosynthetic pathway in an Arthrobacter; the A3 genome contains genes for myo-Inositol-1-phosphate synthase (MB46_13210), mshA (MB46_13160), mshB (MB46_12525), mshC (MB46_05370) and mshD (MB46_13645). The A3 genome also contains two pathways for synthesis of the osmotic stress protector trehalose: the OtsA/B (MB46_00090, MB46_00085) and TreS (MB46_19105) pathways (Chen et al., 2011). Additionally, in contrast to other sequenced Arthrobacter species, A3 contains a predicted hyperosmotic potassium uptake system, consisting of trkA (MB46_07900, MB46_07905, MB46_17505) and trkH (MB46_17510). In terms of cold adaption, the genome of this psychrotrophic bacterium has two aquaporin Z genes (MB46_15885, MB46_10660). Although bacterial aquaporin proteins can theoretically contribute to osmoregulation, studies indicate that they likely function to improve freeze tolerance under rapid-freezing conditions (Tanghe et al., 2006). In addition, A3 encodes a type 1 antifreeze protein (AFP, MB46_12945). AFPs bind to small ice crystals to inhibit growth and recrystallization of ice that would otherwise be fatal (Daley et al., 2002).  The complete genome chromosome and plasmid sequences have been deposited in GenBank database with accession number CP013745.1 and CP013746.1 respectively. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC 1.8987). ACKNOWLEDGEMENTS This study was supported by the National Key Basic Research Program of China (973) (2013CB429904), the State Key Program of the National Natural Science Foundation of China (31230014), the Major Project of Cultivating New Varieties of Transgenic Organisms (2014ZX08009-003-002)， the Fundamental Research Funds for the Central Universities (lzujbky-2015-bt05 and lzujbky-2015-ct01), the National Natural Science Foundation of China (grant 31400437) and UK BBSRC China Partnering Grant (BB/J020419/1).                               References Beller,	H.R.,	Goh,	E.B.,	Keasling,	 J.D.,	 (2010)	Genes	 involved	 in	 long‐chain	alkene	biosynthesis	in	Micrococcus	luteus.	Applied	and	environmental	microbiology	76,	1212‐1223.	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Table 1 General genome features of Arthrobacter strain A3 Feature Chromosome Plasmid Length (bp) 4,264,061 194,226 G + C content (%) 60.78 57.11 Protein coding genes 3735 175 rRNA number 20 0 tRNA number 52 0 GenBank accession CP013745.1 CP013746.1   

Complete genome sequence of a psychotrophic Arthrobacter strain A3 (CGMCC 1.8987), a novel long-chain hydrocarbons producer

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