See-Too et al. Standards in Genomic Sciences (2017) 12:52 DOI 10.1186/s40793-017-0264-0 SHORT GENOME REPORT Open Access Complete genome of Arthrobacter alpinus strain R3.8, bioremediation potential unraveled with genomic analysis Wah-Seng See-Too1,2, Robson Ee1, Yan-Lue Lim1, Peter Convey2,3, David A. Pearce2,3,4, Taznim Begam Mohd Mohidin5, Wai-Fong Yin1 and Kok Gan Chan1,6,7* Abstract Arthrobacter alpinus R3.8 is a psychrotolerant bacterial strain isolated from a soil sample obtained at Rothera Point, Adelaide Island, close to the Antarctic Peninsula. Strain R3.8 was sequenced in order to help discover potential cold active enzymes with biotechnological applications. Genome analysis identified various cold adaptation genes including some coding for anti-freeze proteins and cold-shock proteins, genes involved in bioremediation of xenobiotic compounds including naphthalene, and genes with chitinolytic and N-acetylglucosamine utilization properties and also plant-growth-influencing properties. In this genome report, we present a complete genome sequence of A. alpinus strain R3.8 and its annotation data, which will facilitate exploitation of potential novel cold-active enzymes. Keywords: Cold active enzymes, Anti-freeze protein, Chitinase, Pyschrotolerant bacteria, Plant growth promoting bacteria Introduction Adelaide Island, maritime Antarctica. The optimum The production of cold-adapted enzymes by psychroto- growth temperature range of this bacterium is 10–16 °C, lerant bacteria has important scientific and industrial which rendered it a promising source for discovery of interest due to their highly specific activity and catalytic novel cold-adapted enzymes. The complete genome efficiency at low and moderate temperatures [1]. The sequence of A. alpinus strain R3.8 was generated using use of cold-adapted enzymes offers various advantages Single Molecule Real Time sequencing technology to such as the reduction of undesirable chemical reactions provide a rapid and complete insight into its biotechno- that take place at high temperature, rapid enzymatic in- logical potential. Here, we highlight various genome activation through thermal treatment, and reduction in features that indicate the potential biotechnological energy demand required to fuel industrial processes at value of A. alpinus strain R3.8 in the context of xeno- higher temperatures [2–4]. These beneficial traits are biotic biodegradation and metabolism, chitin utilization, particularly useful in the development of sequential mo- and as a potential component in bio-fertilizers. lecular biology processes, low temperature detergents, food and industrial bio-catalytic enzymes, and for bio- Organism information remediation agents applicable during cold seasons and Classification and features in cold regions. In this study, we perform complete A. alpinus strain R3.8, is a psychrotolerant soil bacter- genome sequencing on a psychrotolerant bacterium, Arthrobacter alpinus ium originally isolated from a soil sample collected at strain R3.8 (=DSM 100969), origin- Rothera Research Station, close to Antarctic Special ally isolated from soil collected from Rothera Point, Protected Area No.129 (68°07′S, 67°34′W). Strain R3.8 was isolated using basal medium supplied with C6-HSL * Correspondence: [email protected] as sole carbon source. An isolation temperature of 4 °C 1 Division of Genetics and Molecular Biology, Institute of Biological Sciences, was used to select for psychrophilic or psychrotolerant Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia 6UM Omics Centre, University of Malaya, Kuala Lumpur, Malaysia bacteria maintained on Luria Bertani (LB) agar [5, 6]. Full list of author information is available at the end of the article The strain exhibited a 98.6% 16S rRNA nucleotide © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. See-Too et al. Standards in Genomic Sciences (2017) 12:52 Page 2 of 7 sequence similarity with A. alpinus, the most phylogen- etically closely related Arthrobacter species with standing in nomenclature (Fig. 1). The cells are Gram-positive, coccoid, and approximately 2.0 μM in width and 1.8 μM in length (Fig. 2). This pairwise 16S rRNA gene sequence similarity value suggested that strain R3.8 is A. alpinus, following the species delineation threshold recom- mended by Stackebrandt and Ebers [7]. API test strips (API 20 E, API 20 E and API ZYM) incubated at 20 °C were used according to the manufacturer’s instructions to determine the physiological and biochemical char- acteristics as well as enzyme activities of strain R3.8. The results were compared with type strain of A. alpinus strain S6-3T.StrainR3.8showedaclosely similar biochemical profile with S6-3T in all the API tests. Both strains did not produce catalase and cytochrome oxidase and were able to hydrolyze aesculin. Both strains were positive for activities of acidic phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, α-glucosidase, ß-glucosi- Fig. 2 Transmission electron micrograph of Arthrobacter alpinus dase, α-galactosidase, ß-galactosidase, ß-glucuronidase strain R3.8. The image was taken under a scanning transmission α electron microscope (STEM, LIBRA 120; Carl Zeiss AG, Germany) at an and -mannosidase, and could utilizes D-glucose, lac- operation voltage of 80 kV. The scale bar represents 500 nm tose, L-arabinose, maltose, D-mannose, D-mannitol and N-acetylglucosamine as sole carbon source. Both strains were negative in indole production, H2S and α-fucosidase, and negative for the fermentation production and citrate utilization. Both were also of glucose, mannitol, sucrose, inositol, sorbitol, rham- negative for activities of arginine dihydrolase, lysine nose, melibiose, and amygdalin. However, strain R3.8 dihydrolase, ornithine dihydrolase, lipase (C14), N- was not able to hydrolyze urea, unlike strain S6-3T, acetyl-ß-glucosaminidase, trypsin, α-chymotrypsin, in both API 20 E and API 20 NE tests. In the API 100 A.defluvii 4C1-a(T) (AM409361) 84 A.niigatensis LC4(T) (AB248526) 88 A.equi IMMIB L-1606(T) (FN673551) 57 A.chlorophenolicus A6(T) (AF102267) 70 A.phenanthrenivorans Sphe3(T) (AM176541) A.scleromae YH-2001(T) (AF330692) 100 A.polychromogenes DSM20136(T) (X80741) 51 A.oxydans DSM20119(T) (X83408) A.livingstonensis LI2(T) (GQ406811) A.alpinus S6-3(T) (GQ227413) 63 100 73 A.alpinus strain R3.8 A.cryoconiti Cr6-08(T) (GU784867) 69 51 A.stackebrandtii CCM 2783(T) (AJ640198) A.roseus CMS90(T) (AJ278870) A.sulfonivorans ALL(T) (AF235091) A.humicola KV-653(T) (AB279890) 97 A.globiformis DSM20124(T) (X80736) 78 A.pascens DSM20545(T) (X80740) Nocardioides simplex DSM20130T (Z78212) 0.01 Fig. 1 Neighbour-joining phylogenetic tree based on complete 16S rRNA gene sequences of Arthrobacter alpinus strain R3.8 and closely related species of the genus Arthrobacter. Bootstrap values (expressed as percentages of 1000 replications) are shown at the branching points. Bar, 1 nt substitutions per 100 nt See-Too et al. Standards in Genomic Sciences (2017) 12:52 Page 3 of 7 20 NE test, strain R3.8 was positive for nitrate reduc- Table 1 Classification and general features of Arthrobacter tion, differing from strain S6-3T.IntheAPI20E alpinus R3.8 according to the MIGS recommendations [34] test, strain R3.8 was positive for fermentation of L- MIGS ID Property Term Evidence codea T arabinose but strain S6-3 was negative. In the API Current Domain Bacteria TAS [35] classification ZYM test, strain R3.8 did not produced alkaline Phylum Actinobacteria TAS [36] phosphatase and naphthol-AS-BI- phosphohydrolase Class Actinobacteria TAS [37] as produced by strain S6-3T. Minimum Information about the Genome Sequence of Subclass Actinobacteridae TAS [37, 38] A. alpinus strain R3.8 is summarized in Table 1. Order Actinomycetales TAS [37–40] Family Micrococcaceae TAS [37–39, 41] Genome sequencing information Genus Arthrobacter TAS [39, 42, 43] Genome project history Species Arthrobacter alpinus TAS [42] The genome of A. alpinus strain R3.8 was sequenced to Strain R3.8 IDA study its bioremediation properties, specifically focusing Gram stain Positive TAS [42] on naphthalene biodegradation. The assembled and an- Cell Shape irregular rods, coccoid IDA notated genome of A. alpinus strain R3.8 described in this paper has been deposited in GenBank (accession Motility Non-motile TAS [42] number of CP12677.1), the KEGG database (entry num- Sporulation Non-sporulating TAS [42] ber of T04095) and the JGI portal with GOLD ID of Temperature 4–30 °C IDA Gp0124186 and IMG taxon ID of 2645727552. Sequen- range cing, assembly and annotation of the complete genome Optimum 20–25 °C IDA were performed by the UM Omics Centre, University of temperature Malaya, Malaysia. A summary of the project information pH range; 6.0–9.0;7.0 IDA Optimum is shown in Table 2. Carbon Acyl-homoserine lactone IDA source (AHLs),Yeast extract Growth conditions and genomic DNA preparation MIGS-6 Habitat soil IDA A. alpinus strain R3.8 was grown aerobically in 5.0 ml MIGS-6.3