Bacterium That Produces Antifouling Agents

International Journal of Systematic Bacteriology (1998), 48, 1205-1 21 2 Printed in Great Britain ~-

Pseudoalteromonas tunicata sp. now, a bacterium that produces antifouling agents

Carola Holmstromfl Sally James,’ Brett A. Neilan,’ David C. White’ and Staffan Kjellebergl

Author for correspondence : Carola Holmstrom. Tel: + 61 2 9385 260 1. Fax: + 6 1 2 9385 159 1. e-mail: c.holmstrom(cx unsw.edu.au

1 School of Microbiology A dark-green-pigmented marine bacterium, previously designated D2, which and Immunology, The produces components that are inhibitory to common marine fouling organisms University of New South Wales, Sydney 2052, has been characterized and assessed for taxonomic assignment. Based on Australia direct double-stranded sequencing of the 16s rRNA gene, D2Twas found to show the highest similarity to members of the genus 2 Center for Environmental (93%) B iotechnology, Un iversity Pseudoalteromonas. The G+C content of DZT is 42 molo/o, and it is a of Tennessee, 10515 facultatively anaerobic rod and oxidase-positive. DZT is motile by a sheathed research Drive, Suite 300, Knoxville, TN 37932, USA polar flagellum, exhibited non-fermentative metabolism and required sodium ions for growth. The strain was not capable of using citrate, fructose, sucrose, sorbitol and glycerol but it utilizes mannose and maltose and hydrolyses gelatin. The molecular evidence, together with phenotypic characteristics, showed that this bacterium which produces an antifouling agent constitutes a new species of the genus Pseudoalteromonas.The name Pseudoalteromonas tunicata is proposed for this bacterium, and the type strain is DZT (= CCUG 2 6 7 5 7T).

1 Kevwords: PseudoalttJvomonas tunicata, pigment, antifouling bacterium, marine, 16s I rRNA sequence .__ ,

INTRODUCTION results suggested that the genus Alteromonas should be separated into two genera. The new genus was de- Heterotrophic bacteria which are Gram-negative and signated Pseudoalteromonas. Originally the genus Al- motile through the use of flagella are commonly teromonas contained four species and was created for isolated from marine environments. These culturable marine bacteria that are Pseudomonas-like but which bacteria can be divided into two groups based on their have a lower G+C content (Baumann et al., 1972). capacity to ferment carbohydrates. The fermentative The new genus, Pseudoalteromonas, was recommended strains belong to the genera Vibrio,Listonella, Photo- to contain 11 species previously belonging to species of bacterium. Colivellia and Aeromonas and the non- Alteromonas and one species that previously was fermentative strains belong to Alteromorzas, Pseudo- placed in the genus Pseudomonas, Pseudoalteromonas monas, Alc digenes, Halomonas, Deleya, Marinomonas, piscicida comb. nov. (Gauthier et al., 1995). The genus Shewmelltr and Flavohacterium (Kita-Tsukamoto et Alteromonas is thereby restricted to a single species, al., 1993). The two new genera Listonella and Colwellia Alteromonas mucleodii. have been suggested for species formerly considered to The strain in this study, a pigmented Gram-negative be members of the genus Vibrio (Deming et a/., 1988; marine bacterium, designated D2T (T = type strain), MacDonell & Colwell, 1985) while most members of was isolated from an adult tunicate, Ciona intestinalis. the genus Alteromonas have been reclassified within The bacterium was shown to produce extracellular the new genus Pseudoalteromonas (Gauthier et al., components that kill the larvae of Balanus amphitrite 1995). and Ciona intestinalis (Holmstrom et al., 1992). In Gauthier et al. (1995) made a comparison of the addition, recent studies have demonstrated that the phylogenetic relationships among 17 isolates of isolate produces at least four different extracellular the genera Alteromonas, Slzewanella and Moritella. The compounds with various degrees of specific inhibitory

, ...... , . . . , ...... , .. . . . , ...... , . . ., . . , ...... , .. . . ., ...... , . , ,...... activity against a variety of organisms including The GenBank accession number for the 165 rDNA sequence of strain DZT is bacteria, algae, diatoms and fungi (Egan, 1995 ; Holm- 225522. strom et al., 1992, 1996, 1997; Holmstrom &

~~ - 00710 0 1998 IUMS 1205 C. Holmstrom and others

Kjelleberg, 1994; James et al., 1996). In this study, we agent 0/129 was tested at a concentration of 150 pg ml-I. carried out a phenotypic and genetic characterization Disks (6.0 mm in diameter) impregnated with antibiotics of the isolate for taxonomic classification. The non- were placed on VNSS agar plates which had been surface- fermentative nature of this strain precludes its in- inoculated with D2' suspension. clusion in the genus Vibrio, and the G + C content of Some of the biochemical properties of the isolate were the genome is too low for the inclusion in the genera determined using the API 20E system (Analytab Products) Pseudomonas and Marinomonas. Isolate D2T revealed and exponential-phase bacterial cells which were washed 93.4% 16s rDNA sequence similarity to the next three times in minimal medium (MMM) before being closest related bacterial strain (Pseudoalteromonas inoculated into the test cupules. citrea) in current DNA sequence databases (GenBank, The motility of the cells was determined with a 100 x oil- EMBL and RDP). This relationship is well within the immersion objective. range of % similarities of the genus Pseudoalteromonas Negative staining and electron microscopy. Cell morphology (> 91 YO).We therefore propose isolate D2T as a new and size and the presence and type of flagellum were species of the genus Pseudoalteromonas, P. tunicata on examined by using cells from an 18 h bacterial growth the basis of its physiological, biochemical and genetic culture. The cells were suspended in a fixative (2.5% features. glutaraldehyde in 0.1 M sodium cacodylate buffer), fixed for 30 min, and washed three times in sterile Milli-Q water. A drop of the cell suspension was mixed with one drop of METHODS sodium phosphotungstate stain (2 % aqueous) for 10 s using a Formvar grid (300-square copper grid). The grid was Bacterial strain and isolation. Strain D2T was isolated from thereafter blotted with filter paper and dried for 10 min an adult tunicate Ciona intestinalis which was collected from before examination using a Hitachi H7000 electron micro- a depth of 10 m in Gullmarsfjorden on the western coast of scope operated at 75 kV. Sweden. A platinum loop was used to obtain samples from the surface of tunicates and loopfuls of the samples were Fatty acid analysis. D2T was grown on VNSS medium at diluted in sterile nine salt solution (NSS, which consists of, 25 "C for 24 h. The cells were harvested, washed, and freeze- per litre, 17.6 g NaCl, 1.47 g Na,SO,, 0.08 g NaHCO,, 0.25 g dried. The supernatant was collected and filtered through a KC1,0.04 g KBr, 1.87 g MgC1,. 6 H,O, 0.4 1 g CaC1,. 2 H,O, 0-22 pm filter and thereafter freeze-dried. A modified Bligh- 0.008 g SrCl. 6 H,O, 0.008 g H,BO,, pH 7). Aliquots of the Dyer lipid extraction (Bligh & Dyer, 1959; White et a/.. diluted samples were spread on VNSS agar plates (VNSS 1979) was performed on the extracts followed by silicic acid agar consists of per litre NSS, 1.0 g peptone, 0.5 g yeast chromatography to fractionate the lipids. Finally mild extract, 0.5 g glucose, 0.01 g FeSO, .7 H,O, 0.01 g Na,HPO, alkaline methanolysis was performed (White et al., 1979). and 15 g agar) and incubated for 3-5 d at 20 "C. Isolates The samples were then separated, quantified and identified were maintained on VNSS agar plates at 4 "C. Bacteria were by capillary gas-liquid chromatography/electron impact also stored at - 70 "C in 30 YO(v/v) glycerol. Isolate D2T(= mass spectrometry (Guckert et al., 1985). The results were CCUG 26757T)has been deposited in the Culture Collection based on three replicates. of the University of Goteborg. All media used in this study Genome analysis. The G+C content was determined by were sterilized at 121 "C for 20 min, unless otherwise stated. using a thermal denaturation procedure (Johnson, 1994). Phenotypic characterization of strain D2T.The bacterium was The DNA samples used for the analysis were extracted and routinely grown on the complex marine medium, VNSS, and purified as described by Wilson (1992) and the results were incubated at 20 "C to provide inocula for the biochemical based on four replicates. Pseudoalteromonas haloplanktis tests. ACAM 547 was used as a reference strain (G+C = 43.0 mol YO). Oxidative or fermentative utilization of glucose was determined by the method of Hugh & Leifson (1953). Catalase 165 rDNA amplification and sequencing. Bacterial cells for activity was determined by the methods of Skerman (1967) the DNA extraction were grown in a complex marine and oxidase activity was tested by the Kovaks method medium VNSS and harvested (1.5 ml bacterial suspension) (Kovaks, 1956). in late-exponential phase of growth. The DNA extraction, PCR-mediated amplification of the 16s rDNA and puri- The salinity tests were performed using the medium VNSS fication of PCR products were carried out as described with NaCl concentrations ranging from 0 to 10 YO(w/v). We previously by Hallbeck et al. (1993). Both strands were also tested for growth on Tryptone soy broth (TSB) (Oxoid) sequenced with the Sequenase version 2.0 DNA sequencing and Luria broth (LB 20) supplemented with 2% salt (w/v) kit (USB). The PCR product was sequenced directly, using (Ostling 1991). A marine minimal medium (MMM) et al., the primers as described by Hallbeck et al. (1993). (Ostling et a/., 1991) was used during tests of growth of D2' on different substrates as sole carbon and energy sources at Nucleotide sequence accession numbers. EMBL/GenBank/ the concentration of 4 g 1-I. RDP accession numbers (in parentheses) for small-subunit rDNA sequences of strains other than D2' used in this study Swarming (Givskov 1996) was tested on four different et al., are as follows : Alteromonas macleodii IAM 12920' media, VNSS, TSB, LB 20 and AB (Clark & Maaloe, 1967). (X82 145), ATCC 271 19' (X67025), The last medium was supplemented with 0.4% glucose, Marinomonas vaga Moritella marinus NCIMB 1 144' (X82 142), Pseudoultero- 0.5 % Casamino acids, and all four media were supplemented CECT4664' (X98 3 6), with 0.6% Bacto agar. monas antarctica 3 Pseudoalteromouiirs atlantica IAM 12927T (X82 134), PseudoalteronPonas aurantia The susceptibility of strain D2T to the antibiotics (Bauer et ATCC 33046' (X82 139, Pseudoalteronionas cnrrageenovorcr al., 1966) erythromycin, rifampicin, gentamicin, tetracycline, IAM 12662' (X82136), Pseudoalteromonas citrea NCIMB ampicillin, neomycin, kanamycin and nalidixic acid was 1889T (X82 137), Pseudoalteronionas denitrificcms ATCC tested at 100 yg ml-l and its sensitivity to the vibriostatic 43337T (X82138), Pseudoalteromonns espejiana NCIMB

1206 International Journal of Systematic Bacteriology 48 Pseudoalteromonas tunicata sp. nov.

2 127T (X82143), Pseudoalteromonas haloplanktis subsp. Table 7. Phenotypic characteristics of ha1oplankti.v ATCC 14393"' (X67024), Pseudoalteromonas Pseudoalteromonas tunicata Iuteovio1rc.cn NCIMB 1893' (X82 144), Pseudoalteromonas nigrifacim\ NCIMB 8614' (X82135), Pseudoalteromonas -, Negative; +, positive. piscicida ATCC 15057T (X822 15), Pseudoalteromonas rubra ATCC 29 570' (X82 147), Pseudoalteromonas haloplanktis Characteristic P. tunicata subsp. tetraodonis IAM 14 160' (X82 139), Pseudoaltero- nzonas undina NCIMB 2 1 28' (X82 140), Pseudoalteromonas sp. IC006 (U85856), Pseudoalteromonas sp. IC013 (U85859), Swarming Pseudorltc~ronionas sp. M B6-05 (U85860), Pseudoaltero- Growth at 4 "C monas sp. MB6-03 (U85857), Pseudoalteromonas sp. MB8- Growth at 37 "C 1 1 (U85855), Pseudoalteromonas sp. SW08 (U85861), Optimal NaCl concentration (YO) Pseudoaltcronionas sp. SW29 (U85862), Pseudoalteromonas Optimal pH sp. S9 (U80834), Pseudoalteromonas sp. Y (AF03038 l), Hugh-Leifson test Pseudoaltoronionas sp. ANG.RO2 (AF022407), Pseudo- Production of: alteromoncis peptidysin F 12-50-A 1' (AF007286), Shewanella /3-Galactosidase activity benthica ATCC 43992' (X82 13 l), Shewanella hanedai CIP Arginine dihydrolase activity* 103207" (X82132), Shewanella putrefaciens ATCC 807 1 Lysine decarboxylase activity* (X82 133), Aeromonas allosaccharophila CECT 4 199T (S39232), Photobacterium sp. SS9 (U91586), VibrioJischeri Ornithine decarboxylase activity* ATCC 7744" (X74702) and Escherichia coli (501859). Cul- Hydrolysis of: ture collection designations are : ATCC, American Type Urease Culture ('ollection, Rockville, MD, USA; NCIMB, Gelatin National Collection of Industrial and Marine Bacteria, Utilization of: Aberdeen, UK ; IAM, Institute of Applied Microbiology, Glucose, maltose, trehalose Tokyo, Japan; CIP, Collection of Institute Pasteur, Paris, Mannose France. Galactose, fructose, xylose, sucrose, Phylogenetic analysis. DNA sequences were aligned using lactose, raffinose, melibiose, rhamnose, the programs PILEUP, GCG (Wisconsin package, 1994) and arabinose, glycerol, mannitol, sorbitol, the multiple sequence alignment tool from CLUSTAL w inositol, dulcitol, melizitose, xylitol, (Thompson et al., 1994). Manual confirmation of the erythritol, glycine, citrate sequence alignment was performed and checked against DL-Threonine both primary and secondary structure considerations of the L- Asparagine 16s rRNA molecule. The aligned sequences were applied to genetic dibtance and maximum parsimony methods for L-Proline phylogeneiic inference. For all multiple sequence alignments L-Histidine and phylogenetic inference programs the input order of taxa Tween 20 was randoinized. Genetic distances were calculated using the Oxidase formula of Jukes & Cantor (1969), where D = -3/4 Catalase In( 1 -4/34 and dis the sequence dissimilarity. Phylogenetic Sheathed polar flagellum inference protocols, DNADIST, NEIGHBOR, DNAPARS, CONSENSE G + C content (mol YO) and SEQBOOT were supplied by the PHYLIP package (version 3.57~)(Felsenstein, 1989). All sequence manipulation and * Tests using the API 20E system. phylogeny programs were made available through the ? Growth after 48 h. Australian National Genome Information Service (ANGIS, Sydney, Australia). galactosidase, arginine dihydrolase, lysine-ornithine RESULTS decarboxylase and urease activities were not detected. Phenotypic characterization of the marine isolate Did not show any H,S or NO, production. Growth on different carbon and energy sources showed that the Strain D2T grew at 4 "C but not at 37 "C, with optimal bacterium utilizes trehalose, glucose, maltose and growth at approximately 28 "C. Growth at the lower Tween 20 after 24 h incubation and mannose and temperatures of 4 and 10 "C was very slow compared proline after 48 h. We have not been able to dem- with that at 28 "C. The water temperature surrounding onstrate growth on additional carbohydrates or amino the tunicate specimen, at the time of the isolation, was acids. It did not ferment sugar in the Hugh-Leifson approximately 10 "C. D2T tolerated a pH range of test and the cells were sensitive to erythromycin, 5-5-9 (optimum pH 7-8) while the low-molecular-mass rifampicin, gentamicin, tetracycline, ampicillin, neo- component, which is active against invertebrate larvae, mycin, kanamycin and nalidixic acid at concentrations tolerated a pH range of 2-11 without losing its of 100 pg ml-l. The isolate is sensitive to the vibrio- inhibitor) activity (Holmstrom, 1993). D2T required static agent O/ 129 at a concentration of 150 pg and is Na+ at a concentration of 0.3% (w/v) NaCl, and oxidase and catalase-positive. Strain D2T cells are optimal salinity for growth of the strain was found to 2.0-3-4 pm in length, motile and rod-shaped, and be 1-2 O/O (w/v) NaCl concentration. possess a single sheathed polar flagellum. Swarming of The isolate exhibited gelatinase activity while p- the bacteria was not detected (Table 1).

International Journal of Systernatic Bacteriology 48 1207 C. Holmstrom and others

Table 2. Fatty acid profiles of cell and supernatant four major components accounted for almost 90 YOof extracts of P. tunicata the total cellular fatty acids. The balance of the profile of D2T cells consisted of saturated and unsaturated Results are expressed as mean mol '%I ( f. SD). fatty acids and a trace of terminally branched fatty acids. There was one unknown 18 carbon fatty acid, Fatty acid Extract however, this constituted less than 1 % of the profile. The supernatant also contained normal and mono- Cell Supernatant enoic fatty acids but the biomass of the supernatant - __- was four orders of magnitude less than that of the cells 12:o 0.03 f0.0 1 0.00 themselves. The saturated fatty acid 16:O was the 13:O 0.06 f. 0.02 0.00 major fatty acid (46.99 & 7.53) in the supernatant while 14: la 0-44f 0.0 1 0.00 16: lw7c and 16: lc05c were the second and third most 14: lb 0.03 f. 0.00 0.00 common fatty acids (23.47 & 0.53 and 19.51 & 6.1 3 YO). 14: 0 2.10 * 0.02 0.00 respectively (Table 2). 15: la 0.45 f. 0.02 0.00 15: lb 0.08 f0.0 1 0.00 15:O 0.90 * 0.0 1 0.00 DNA base composition 16:Oi 0.13 fO.01 0.00 The G + C content of the DNA of Pseudoalteron-zoncis 16: lw9c 0.58 f0.04 0.00 tunicata was 42.2 & 0.7 mol YO.The accepted range for 16: lw7c 47.47 f0.76 23.47 f0.53 members of the genus Pseudoalteromonas is 38- 16: ls7t 6.69 f0.06 0.00 50 mol YO. 16 : 105c 0.35f0.13 1951 f.6.13 16:O 18.31 f0.38 46.99 & 7.53 17:Oi 0-0I & 0.00 0.00 165 rDNA sequence and phylogenetic 17:Oa 3.54 f. 0.03 0.00 characterization 17: la 0.20 0.00 0.00 f The sequencing strategy used in the current inves- 17: lb 0.23 0.0 1 0.00 f. tigation generated 1370 bases of the 16s rRNA gene 17:O 1.30 & 0.07 2.84 4.02 f between positions 34 and 1404 corresponding to the 18 unknown 0.04 0.00 0.00 f numbering of nucleotides in the coli 16s rRNA 18: 1~9~ 0.36 0.0 1 0.00 E. f molecule (Brosius et 1981). The resulting sequence 18: lw7c 14.46 0.29 3.1 4.45 al., f 5 f was aligned to currently available 16s rRNA gene 18: 1~7t 0.29 0.0 1 0.00 f sequences which showed significant identity from the 18: Iw5c 0.08 0.03 0.00 f EMBL, GenBank and RDP databases. The derived 18:O 1 a50 0.09 4.04 5.72 f f. multiple sequence alignment, including both conserved 18: 1br 0.02 0.0 1 0.00 f and variable domains of the 16s rRNA primary 19: 1 0.17 0.01 0.00 f. structure, was used as the basis to generate pairwise 20 : 109c 0.1 6 0.02 0.00 f similarities and genetic distances between D2T and 20:o 0.04 0.0 I 0.00 f. related bacteria. Several phylogenetic trees were con- structed using different methods to analyse the sequence data, including genetic distance matrices and The colony and cellular morphology on different maximum parsimony. The input order of sequences growth media showed that young colonies grow in into the various phylogenetic programs and the selec- punctiform with a diameter around 2 mm on TSB and tion of outgroups were also varied during this study. LB 20 agar plates or 1 to 2 mm on VNSS plates. The Statistical evaluation of the derived genetic divergences colonies were smooth and convex with regular edges. was performed by bootsrap resampling of the sequence However, the occurrence of pigmentation varied. After data. 24 h on VNSS medium, the colonies turned dark The tree topology shown in Fig. 1 was identical to green, while on LB 20 and TSB the colonies remained other statistical representations of the sequence data, white. Streaking white colonies from plates on VNSS including parsimony and consensus trees. Based on plates gave dark green colonies after 24 h incubation phylogenetic characterization, the strain D2T belongs and vice versa. to the gamma-3 subclass of Proteobacteria and was most closely branched to the monophyletic cluster Fatty acid analysis containing members of the genus Pseudoalteromor-las. However, in relation to the Pseudoalteromonas clade, The unsaturated fatty acid 16: lc07c was shown to be this strain comprised a distinct ancestral lineage, as the predominant fatty acid of D2T cells (mean level, seen in both parsimony (data not shown) and genetic 47.47 & 0.76 %), whereas the fatty acids 16 : 0, 18 : lc07c distance trees (Fig. 1). The isolate shared 93 YO 16s and 16: lw7t were the second, third and fourth most rDNA sequence similarity with P. citrea and P. common fatty acids (relative percentages, 18.31 0.38, aurantia. Within this genus, D2T had a range of 14.46 & 0.29 and 6.69 0.06 YO),respectively. These sequence identities between 91-3-93.4 Yo, for the

1208 International Journal of Systematic Bacteriology 48 Pseudoaltero~~ionastunicata sp. nov.

Marinomonas VURU sheathed flagellum which may disallow inclusion in the 7-Morirella mrinus genus Pseu~oalteromonas. However, earlier studies I Alteromonas macleodii I D2 (Pseudoalteromonas tunicata) have suggested that similar bacteria with sheathed 1Pseudoalteromonas denirrificans flagella may be admitted to the genus Pseudoaltero- P.rruiioalteromonas sp. Y nzunus- (Novic & Tyler, 1985). Enger et a/. (I 987) also Pseudoalteromonus peptidysin classified an isolate, with a sheathed Pseudoalteromonas piscicida P. denitriJicans, flagellum to the genus Pseudoalteron~o?ias. A further Pseudoalteromonas iuteoviolacea distinctive difference to the generic description of the Pseudoalieromonas sp S9 genus Pseudoalteromonas is that D2T is a facultative Pseudoalteromonas citrea anaerobic bacterium, however, similar results have Pseudoalteromonas aurantia also been reported for P. denitrzjicans (Enger et al., Pseudoalteromonas sp. MB8- I1 pseudoa alter om on as sp. ANC.RO2 1987) and Pseudoalteromonas sp. S9 (Techkarnjanaruk Pseudoalteromonus nigrifaciens et al., 1997; Wrangstadh et a/., 1986). We therefore do Pseudoalteromonas haloplunktis not regard the presence of a flagellar sheath and the

Pseudoalteromonus antartica ability to grow anaerobically as decisive for exclusion Pseudoalteromonas sp. SWO8 of our strain from the genus Pseudoalteronionas.

Pseudoalteromonas sp. ICO13 The genus Pseudoalteromonas classification is some- Pseudoalteromonas sp. MB6-05 what unsatisfactory (Akagawa-Matsushita et al., 1993) Pseudoalteromonas sp. SW29 given that the present members of this taxonomic Pseudoalteromonas tetraodonis group are related by 16s rDNA sequence identities as Pseudoalteromonas undina Pseudoalteromonus carrageenovora low as 91 YO.The D2T isolate showed the highest Pseudomlteromonas arlantica similarities with P. citrea (93.4%) and P. aurantia Pseudoalteromonas espejianu (93.3 YO).These two species also have many charac- Shewanella putrefuc lens teristics in common with P. tunicata. When comparing 23 taxonomic parameters (Table 3) the proposed type Shewanella benthica Escherichia coli strain of P. tunicata (D2T) differed by only six + Arromonas allo~accharophilu characteristics from the profiles of P. citrea and P. Vibriofisheri aurantia. All three species are pigmented, can hy- -Phc,lobacterium rp. SS9 drolyse gelatin, and can utilize mannose. None of the 10 species can utilize sucrose, citrate, glycerol and sorbitol. In Table 3, the comparisons of characteristics ...... , ., ., . , , , , , . . . , . . , . . . ., . ., . , ., ., ...... , . . . . ., . . , , , , , ,...... , , , , ...... between P. tunicata and other Pseudoalteromonas Fig, 1. Phylogenetic affiliations between D2T (P. tunicata), strains show that most strains differ by between nine strains of the genus Pseudoalteromonas, and other closely related bacteria as indicated by near complete 165 rRNA gene and six characteristics except P. haloplmktis subsp. sequence alignment. An alignment of 1370 characters was used haloplanktis (12 characteristics) and Alteronwnas to calculate genetic distances according to the method of Jukes macleodii (1 3 characteristics). The latter species also & Cantor (1969). The phenogram was reconstructed from the showed less than 90 % similarities with D2T 16s rDNA pairwise distance matrix using the neighbour-joining method of Saitou & Nei (Satuito eta/., 1996). The scale represents 1 base sequence. With respect to evolutionary distances substitution per 10 nucleotide positions. Strain designations, among strains assigned to genus Pseudoalteron~onas source culture collections, and sequence database accession the closest phylogenetic relative to P. tunicata is numbers are listed in the text. P. denitrificaris. P. denitr~canshas been classified to ______~ ~ .~ have a sheathed flagellum and can grow anaerobically. These strains, like D2T cells, can show varied expres- Pseudocilt~~ro~zoiiasstrains included in this analysis. sion of pigment depending on the growth medium The next highest sequence similarities to this genus was that is employed (Enger et d.,1987). This is a 88 %, for members of Slieivanella and Moritella, and characteristic which is also expressed by Shewanella (Baumann 1984) and 87 and 85 % for the genera Alteromonas and Marino- hanedai et al., P. nigrifaciens monas, respectively. (Ivanova et a/., 1996). P. tunicata express at least one diffusable dark pigment when grown on the complex marine medium VNSS. DISCUSSION The identity of the pigment(s) are not yet known but it The strain that we investigated was isolated from an was observed that the pigment was produced and adult tunicate collected in Gullmarsfjorden on the released into the supernatant at the same time as the western coast of Sweden. The isolate is a pigmented, antilarval and antibacterial components. However, it motile and Gram-negative rod, with a G+C content has clearly been shown that the dark pigment produced of 42.2 & 0.7 mol %. Strain D2T is oxidase-positive, can by the bacterium is not a fouling-inhibitory component hydrolyse gelatin and requires Na+ ion for growth with (Holmstrom et al., 1992). However, it is possible that an optimal growth at 1-2% Na' ion concentration. the active components may be produced by the same D2Tcan grow at 4 "C but not at 37 "C and is susceptible pathway or by a branch of the pathway leading to the to all antibiotics tested. Strain D2T possesses a pigment production. The genus Pseudoalteromonas

~~ ~- InternationaI Journal of Systematic Bacteriology 48 1209 Table 3. Differential characteristics of Pseudoalteromonas species and Alteromonas macleodii

Data from references 4 and 12. +, Positive; -, negative; d, 11-89 Yo of the strains are positive; ND, not determined.

Characteristic P. runicutu P. huloplunkfis P. niucleodii P. rubru P. citriw P. uuruntiu subsp. halnplunh tic

Growth at 4 "C' + - Growth at 35 "C' + d t d Piginentat ion + - + Hydrolysis of gelatin + + + t Utlllzatlon of. Mannose + + t Sucrose - + t M;iltose + + + -

Sorhitol -

Fructose - d + t C'itrate - + Glycerol - + Galactow d + Laclose - + - Melihiose - +

Mannitol - d d

Xylose - d 'Trchdohe + d t +

Inosito1 - R hainnose - Glycine - d d \I> I.-Proline + d

- 1,-Histiciine - d Oxidase + + + + G+C content (mol%) 42-43 41 45 41 44 41 45 No. of characteristics I2 I3 0 differing from P. /iiiii(,~t(i

contains both pigmented and non-pigmented strains. not shown). This result is in agreement with the Many of the strains within this genus produce met- findings by Bozal et al. (1997) who performed a fatty abolites active against other organisms. Five pig- acid analysis on P.seudoalteron.lorzas antarctica species. mented species belonging to Pseudoalteromonas have They showed that the two fatty acids,l6: Iu7c and been found to produce inhibitory components. Four 16: 0, were the dominant components. Svetashev et d. of these species, P. rubra, P. luteoviolucea, P. citrea and (1995) determined cellular Fdtty acid composition of P. aurarztia have been shown to produce high-mol- seven type strains of Pseudoalteromonas species. They ecular-mass components which are autotoxic and also showed that the fatty acids 16: lu7c and 16:O active against both Gram-positive and Gram-negative were the main components. In addition, only low bacteria (Baumann et al., 1984). This is a characteristic amounts of branched-chain and hydroxy fatty acids which P. tunicata also possesses (James et al., 1996). were detected in the seven type strains (Svetashev et al., The fifth species, P. piscida, has been suggested 1995). These results are in agreement with the data on to cause fish death (Bein, 1954). Furthermore, the the fatty acid composition of P. tunicata cells. no n - pi g me n t ed P . h alop Ian k t is subs p . t e t r a o dorz is (Akagawa-Matsushita et a/., 1993 ; Simidu et al., 1990) has been found to produce tetrodotoxin (Simidu et nl., Description of Pseudoalteromonas tunicata 1990). P. denitry7cans can also produce autotoxic substances that kill cells and inhibit growth in dense Pseudoulteromonas tunicata (tu.ni.ca'ta L. fem. n. bacterial cultures (Enger et al., 1987). Specific bio- tur?icata shirt-like classical piece of Roman underwear, logical activity has furthermore been demonstrated for tunic; L. fem. adj. tunicata clothed with a tunic). other Pseudoalteromonas strains. Pseudoalteromonas Facultative anaerobic Gram-negative rod shaped cells sp. S9 and Pseudoalteromoms col\velliam have been that are 2.0-3.4 pm in length. Motile by means of a shown to contribute to larval settlement (Szewzyk et polar sheathed flagellum. Growth on VNSS medium al., 1991; Weiner et al., 1988). results in dark-green-pigmented colonies while growth P. tunicata cells are dominated by monoenoic fatty on TSB medium results in white colonies. Does not acids (> 70 YO)which is typical of Gram-negative ferment sugar in the Hugh-Leifson test. Sodium ions bacteria. The fatty acids 16: lw7c and 16:O were the are required for growth with the optimal NaCl dominant fatty acids in both the cell and the super- concentration of 1-2 %. Cannot grow at pH 5 or I1 : natant extracts. These two fatty acids were also the optimal pH range for growth is pH 7-8. Slow growth dominant molecules when D2T was grown in Tryptone occurs at 4 "C and no growth is detectable at 37 "C. soy broth complemented with 2% (w/v) NaCl (data Oxidase- and catalase-positive. Utilizes trehalose. glu-

~- -~ .~ 1210 In terna tionaI Journal of Systematic Bacteriology 48 Pseudoalteromonas tunicata sp. nov. cose, maltose and Tween 20 at a concentration of bacterium and description of a new genus, Cohvellia gen. nov. 4 g 1-1 but can not grow on galactose, fructose, xylose, Syst Appl Microbiol 10, 152-160. sucrose, lactose, raffinose, melibiose, glycerol, man- Egan, 5. (1995). Inhibition of crlgal spores germination by mcrrine nitol, rhamnose, sorbitol, inositol, dulcitol, melexitose, bacteria. Honors thesis, University of New South Wales. xylitol, erythritol, glycine, threonine, arabinose, L- Enger, O., Nygaard, H., Solberg, M., Schei, G., Nielsen, J. & aspargine and L-histidine. Positive for hydrolysis of Dundas, I. (1987). Characterization of Alteromonas denitrzj7cans gelatin. Sensitive to erythromycin, rifampicin, genta- sp. nov. hzt J Syst Bacteriol37, 416421. micin, tetracycline, ampicillin, neomycin, kanamycin Felsenstein, J. (1989). PHYLIP : phylogeny inference package. and nalidixic acid at a concentration of 100 pg ml-l Cladistics 5, 164166. and is sensitive to the vibriostatic agent 0/129 at a Gauthier, G., Gauthier, M. & Christen, R. (1995). Phylogenetic concentration of 150 pg rn1-l. The G + C content of analysis of the genera Alteromonas, She,vanella, and Moritella the DNA is 42.2 & 0.7 mol YO.The main cellular fatty using genes coding for small-subunit rRNA sequences and acids are 16:1co7c, 16:0, 18:lco7c and 16:lco7t1 division of the genus Altuomonas into two genera, Alteromonas respectively. Strain D2T is negative for H,S and NO, (emended) and Pseuc~oalteromoiiasgen. nov., and proposal of production and no P-galactosidase, arginine di- twelve new species combination. Int J Syst Bacteriol 45, hydrolase, lysine decarboxylase and ornithine decar- 755-761. boxylase xtivity has been detected. Isolated from an Givskov, M., De Nys, R., Manefield, M., Gram, L., Maximilien, R., adult tunicate, Ciona intestinalis, collected from water Eberl, L., Molin, S., Steinberg, P. & Kjelleberg, 5. (1996). off the wtstern coast of Sweden. The type strain has Eukaryotic interference with homoserine lactone mediated been deposited in the Culture Collection of University procaryotic signalling. J Bactcwbl 178, 66 18-6622. of Goteborg as strain CCUG 26757T. Guckert, J. B., Antworth, C. P., Nichols, P. D. & White, D. C. (1985). Phospholipid, ester-linked fattyacid profiles as re- producible assays for changes in prokaryotic community ACKNOWLEDGEMENTS structure of estuarine sediments. FEMS Microb Ecol 31, 58. This work was supported by the Australian Research 147-1 Hallbeck, L., St&hl, F. & Pedersen, K. (1993). Phylogeny and Council and Swedish National Board for Industrial and phenotypic characterization of stalk forming and iron-oxidizing Technical Development. 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