International Journal of Systematic and Evolutionary Microbiology (2002), 52, 1017–1021 DOI: 10.1099/ijs.0.02131-0 Arthrobacter roseus sp. nov., a psychrophilic NOTE bacterium isolated from an Antarctic cyanobacterial mat sample 1 Centre for Cellular and G. S. N. Reddy,1 J. S. S. Prakash,1 G. I. Matsumoto,2 E. Stackebrandt3 Molecular Biology, Uppal 1 Road, Hyderabad 500007, and S. Shivaji India 2 Department of Author for correspondence: S. Shivaji. Tel: j91 40 7172241. Fax: j91 40 7171195. Environmental and e-mail: shivas!ccmb.ap.nic.in Information Science, Otsuma Women’s University, Tamashi, T Tokyo 206, Japan Strain CMS 90r , a red-pigmented bacterium, was isolated from a 3 cyanobacterial mat sample from a pond located in McMurdo, Antarctica. Based Deutsche Sammlung von T Mikroorganismen und on its chemotaxonomic and phylogenetic properties, strain CMS 90r was Zellkulturen GmbH, 38124 identified as a member of group I of Arthrobacter. It shared 16S rDNA Braunschweig, Germany similarity of 98% with Arthrobacter oxydans ATCC 14358T and Arthrobacter polychromogenes ATCC 15216T, while DNA–DNA similarities determined for these three organisms were less than 70%. It also differed from all 17 reported Arthrobacter species with A3α-variant peptidoglycan in that it possessed a unique peptidoglycan (Lys–Gly–Ala3) and contained galactose, glucose, ribose and rhamnose as cell-wall sugars. These data and the presence of diagnostic phenotypic traits support the description of CMS 90rT as a novel species of Arthrobacter, for which the name Arthrobacter roseus sp. nov. is proposed. The type strain is strain CMS 90rT (l MTCC 3712T l DSM 14508T). Keywords: Arthrobacter, psychrophile, Antarctica, cyanobacterial mat Prokaryotes are the predominant biomass in Antarc- 160m 45h E) located in the Wright valley, McMurdo, tica (Wynn-Williams, 1996; Franzmann, 1996) and Antarctica (Matsumoto, 1993; Matsumoto et al., they influence the food chain and the biogeochemical 1993). The mat sample was found to contain an cycles (Friedmann et al., 1993; Matsumoto et al., Aphanothece sp. and coccoid green algae in low 1989). Among the prokaryotes, cyanobacteria belong- densities (Matsumoto et al., 1993). About 200 mg of ing to the genera Phormidium, Anabaena, Calothrix, the sample was suspended in 1 ml sterile saline Nostoc, Lyngbya and Synechococcus are the most (150 mM NaCl) and an aliquot of the suspension abundant and they form mats in the polar lakes (100 µl) was plated on Antarctic bacterial medium (Parker & Wharton, 1985; Vincent et al., 1993). These (ABM) plates containing 0n5% (w\v) peptone, 0n2% mats contain a large biomass of bacteria that contri- (w\v) yeast extract and 1n5% (w\v) agar (pH 6n9) and butes to the mineral and nutrient cycling in the lakes. incubated at 5 mC (Shivaji et al., 1988, 1989, 1992; However, these mat-associated, heterotrophic bacteria Reddy et al., 2000). The appearance of colonies was have rarely been identified. In an attempt to establish monitored on a regular basis and pure cultures of the the microbial biodiversity of the cyanobacterial mat bacteria were established. Following incubation for 15 communities of the Antarctic lakes, a red-pigmented days, colonies appeared that were predominantly white bacterium (CMS 90rT), isolated from a cyanobacterial in colour, with the exception of CMS 90rT, which was mat sample, was studied in detail with respect to its red-pigmented (indicated by the suffix ‘r’). phenotypic characteristics and phylogenetic relation- ships. Phylogenetic analysis Source and isolation Amplification and sequence analysis of the 16S rRNA T gene were done as published previously (Reddy et al., Strain CMS 90r was isolated from a cyanobacterial 2000; Shivaji et al., 2000). The almost complete mat sample collected from pond L4 (77m 32h 18d S, sequence of strain CMS 90rT (1470 nucleotides) was ................................................................................................................................................. aligned with those deposited in EMBL using The EMBL accession number for the 16S rDNA sequence of strain CMS 90rT (Higgins et al., 1992). Following an initial crude is AJ278870. taxonomic affiliation, a more-detailed analysis was 02131 # 2002 IUMS Printed in Great Britain 1017 G. S. N. Reddy and others A. roseus CMS 90rT (AJ278870) the membrane-filter method (Shivaji et al., 1992; T 100 A. polychromogenes DSM 20136 (X80741) Reddy et al., 2000). The results indicated that CMS T T A. oxydans DSM 20119 (X83408) 90r shared 69n8 and 65n46% DNA similarity, re- A. chlorophenolicus strain unknown (AF102267) 52 spectively, with the type strains of A. oxydans and A. A. citreus DSM 20133T (X80737) polychromogenes. 97 A. flavus MTCC 3476T (AJ242532) A. agilis DSM 20550T (X80748) A. histidinolovorans DSM 20115T (X83406) 93 T Chemotaxonomic properties 59 A. nicotinovorans DSM 420 (X80743) T A. ureafaciens DSM 20126 (X80744) T Peptidoglycan was prepared according to Rosenthal & 53 A. aurescens DSM 20116 (X83405) T Dziarski (1994) and hydrolysed with 4 M HCl at A. ilicis DSM 20138 (X83407) T 120 C for 60 min and the composition of the main A. crystallopoietes DSM 20117 (X80738) m T chain was determined according to the method of A. globiformis DSM 20124 (X80736) T 98 A. ramosus DSM 20546 (X80742) Schleifer & Kandler (1972). T A. pascens DSM 20545 (X80740) T Cellular fatty acid methyl esters were prepared from A. woluwensis CUL 1808 (X93353) 50 T lyophilized bacterial cell pellets (Sato & Murata, 1988) A. protophormiae DSM 20168 (X80745) 99 T and separated by GC on a DB-23 capillary column 52 A. nicotianae DSM 20123 (X80739) 52 A. uratoxydans DSM 20647T (X83410) (30 mi0n25 mm) purchased from J & W Scientific. 100 A. sulfureus DSM 20167T (X83409) The fatty acids were identified by comparison with A. rhombi CCUG 38813T (Y15884) fatty acid standards run under similar GC conditions 100 A. psychrolactophilus ATCC 700733T (AF134182) and also by mass spectrometry (Shivaji et al., 1992; 100 75 A. creatinolyticus GIFU 12498T (D88211) Reddy et al., 2000). The carrier gas used for GC was N# −" A. cumminsii DMMZ 445T (X93354) (1 ml min ). The initial run temperature was main- A. atrocyaneus DSM 20127T (X80746) tained at 175 mC for 8 min and then raised to 200 mCat T −" Renibacterium salmoninarum ATCC 33209 (AB017538) a rate of 4 mCmin and held at 200 mC for 5 min. The A. siderocapsulatus NCIMB 11286T (AJ007910) injector port and FID detector temperatures were E. coli ATCC 11775T (X80725) respectively 200 and 240 mC. ................................................................................................................................................. Menaquinones were extracted as described by Collins Fig. 1. Phylogenetic relationship between A. roseus sp. nov. (CMS 90rT) and 26 reported species of Arthrobacter based on et al. (1977) and were separated by TLC using 16S rDNA sequence analysis using DNAPARS. Bootstrap values petroleum ether and diethyl ether (85:15, v\v) above 50% are given at the nodes. The branch lengths (Dunphy et al., 1971). The individual menaquinones indicated in the tree are not to scale. were also identified by mass spectrometry (Reddy et al., 2000). The method of Minnikin et al. (1975) was used for the extraction of polar lipids, using lyophilized cell pellets washed free of medium; polar lipids were done with members of Kocuria, Arthrobacter and resolved by thin-layer chromatography using chloro- related genera. Pairwise evolutionary distances were form\methanol\water (65:25:4, by vol.). computed using the program with Kimura’s two-parameter model (Kimura, 1980). To obtain Cell-wall sugars were prepared and analysed according confidence values for the rDNA sequence-based gen- to the method described by Komagata & Suzuki etic affiliations, the original sequence dataset was (1987). resampled 1000 times using and subjected to bootstrap analysis. Phylogenetic trees were obtained Pigments were extracted from lyophilized bacterial cell using parsimony analysis () and various dis- pellets with chloroform\methanol (1:2, v\v), centri- tance-matrix-based clustering algorithms such as fuged at 10000 g and the clear, pigmented supernatant , and as compiled in was flushed under a stream of N#. The dry pigment (Felsenstein, 1993). The different algorithms gave thus obtained was dissolved in a minimal volume of consistent results. chloroform\methanol (2:1, v\v) and separated by T TLC using chloroform\methanol\water (65:25:4) in Strain CMS 90r fell into the radiation of Arthrobacter the dark. The individual pigments were then scraped species (94n7–98% similarity), showing the highest from the plates, dissolved in methanol and their values to the sequences of the type strains of Arthro- absorption spectra were recorded in a Hitachi bacter oxydans and Arthrobacter polychromogenes. 2000 spectrophotometer (Chauhan & Shivaji, 1994; The topology of the consensus phylogenetic tree (Fig. Shivaji et al., 1992; Jagannadham et al., 1991). 1) displays the phylogenetic position of the two strains next to their closest phylogenetic neighbours. The The GjC content of the DNA was determined as statistical significance of these new lineages is high. described previously (Shivaji et al., 1992). In order to determine the overall genomic relatedness The phylogenetic affiliation of CMS 90rT to Arthro- between the two isolates and their phylogenetic neigh- bacter was supported by its chemotaxonomic proper- bours, DNA–DNA hybridization was performed by ties (Table 1). 1018 International Journal of Systematic and Evolutionary