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Int J Syst Evol Microbiol 55 (2005), 1593-1596; DOI  10.1099/ijs.0.63638-0
© 2005 International Union of Microbiological Societies

Pseudoalteromonas spongiae sp. nov., a novel member of the {gamma}-Proteobacteria isolated from the sponge Mycale adhaerens in Hong Kong waters

Stanley C. K. Lau1, Mandy M. Y. Tsoi1, Xiancui Li1, Sergey Dobretsov1, Yulia Plakhotnikova1, Po-Keung Wong2 and Pei-Yuan Qian1

1 Coastal Marine Laboratory/Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong
2 Department of Biology, Chinese University of Hong Kong, Shatin, N.T., Hong Kong

Correspondence
Pei-Yuan Qian
boqianpy{at}ust.hk


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A Gram-negative, non-spore-forming, short rod-shaped bacterium (UST010723-006T) was isolated from the surface of the sponge Mycale adhaerens in Hong Kong waters. Cells of UST010723-006T did not have flagella and were non-motile. Colonies were pale orange in colour, 2–4 mm in diameter, convex with a smooth surface and an entire translucent margin. Gas bubbles were observed in the colonies and also in the agar matrix underneath and adjacent to the colonies. UST010723-006T was heterotrophic, strictly aerobic and required NaCl for growth (2·0–6·0 %). It grew at pH 5·0–10·0 and between 12 and 44 °C. Phylogenetic analysis of the 16S rRNA gene sequence placed UST010723-006T within the genus Pseudoalteromonas of the {gamma}-subclass of the Proteobacteria. The DNA G+C content is 40·6 mol% and the dominant fatty acids were 12 : 0 3-OH, 14 : 0, 15 : 0 iso 2-OH, 16 : 0, 16 : 1{omega}7, 17 : 1{omega}8 and 18 : 1{omega}7 (altogether representing 75·9 % of the total).These data supported the affiliation of UST010723-006T to the genus Pseudoalteromonas. The closest relatives were Pseudoalteromonas luteviolacea, P. phenolica, P. rubra and P. ruthenica with similarity values ranging from 95·4 to 96·8 %. UST010723-006T differed from these closest relatives by 9–19 traits. Molecular evidence, together with phenotypic characteristics, suggests that UST010723-006T constitutes a novel species within the genus Pseudoalteromonas. The name Pseudoalteromonas spongiae sp. nov. is proposed for this bacterium. The type strain is UST010723-006T (=NRRL B-41100T=JCM 12884T).

Published online ahead of print on 4 March 2005 as DOI 10.1099/ijs.0.63638-0.

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain UST010723-006T is AY769918.

Supplementary material available in IJSEM Online comprises a table showing primers used for the construction of 16S rRNA gene sequences, TEM and SEM images and a table detailing the carbohydrate metabolism of strain UST010723-006T.


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The genus Pseudoalteromonas currently comprises 32 described members; many isolated from marine macroorganisms, such as Pseudoalteromonas ruthenica from mussels and scallops (Ivanova et al., 2002aGo), Pseudoalteromonas maricaloris from sponges (Ivanova et al., 2002bGo), Pseudoalteromonas tunicata from tunicates (Holmström et al., 1998Go) and Pseudoalteromonas ulvae from algae (Egan et al., 2001Go). They are often suggested to have important ecological implications for the survival and fitness of the host organism (Holmström et al., 2002Go). In this study, we describe a novel member of Pseudoalteromonas isolated from the surface of a sponge in subtropical water.

During the characterization of bacteria isolated from the surface of the sponge Mycale adhaerens in Hong Kong waters, the strain UST010723-006T was isolated on nutrient agar (Oxoid) supplemented with 0·22-µm-filtered sea water (referred to as marine agar) at 30 °C. Unless otherwise specified, all characteristics described hereafter were based on cultures grown on marine agar for 48 h at 30 °C. UST010723-006T appeared as pale orange, convex colonies with an entire translucent margin and a smooth surface. No diffusible pigment was observed. Gas bubbles were observed in the colonies as well as in the agar matrix underneath and adjacent to the colonies; this property has not been reported for any of the already described members of Pseudoalteromonas.

The nearly complete 16S rRNA gene sequence of UST010723-006T (1425 bp) was resolved on a MegaBACE capillary genetic analyser using a dye terminator method according to the manufacturer's protocol. Primers used in the sequencing reactions are detailed in Supplementary Table S1 available in IJSEM Online. Fragments of DNA sequence obtained from individual primers with at least six replicates were assembled using the Sequencher software package (Gene Codes). Comparison of the 16S rRNA gene sequence of UST010723-006T with those available from GenBank revealed that UST010723-006T was a member of the {gamma}-subclass of the Proteobacteria and shared 99·2 % sequence similarity with Pseudoalteromonas sp. S9 (Techkarnjanaruk et al., 1997Go). A neighbour-joining phylogenetic tree (Fig. 1Go) constructed using the ARB software package (Ludwig et al., 2004Go) showed that UST010723-006T and Pseudoalteromonas sp. S9 belonged to a clade (100 % bootstrap value, 500 replications), that did not cluster robustly with any recognized species of Pseudoalteromonas (63 % bootstrap value, 500 replications). Trees based on maximum-parsimony and maximum-likelihood methods showed essentially the same topology (data not shown). The results of phylogenetic analysis suggest that UST010723-006T represents a novel species within the genus Pseudoalteromonas. The closest relatives of UST010723-006T were Pseudoalteromonas luteviolacea (Gauthier, 1982Go), P. ruthenica (Ivanova et al., 2002aGo), P. rubra (Gauthier, 1976Go) and P. phenolica (Isnansetyo & Kamei, 2003Go) with 96·8, 96·6, 96·5 and 95·4 % sequence identity, respectively.



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  		Fig. 1. Neighbour-joining dendrogram showing the estimated phylogenetic relationships among UST010723-006T and related Pseudoalteromonas species on the basis of 16S rRNA gene sequences. Vibrio marinus ATCC 15381T was chosen as the outgroup. Scale bar represents 1 nucleotide substitution per 100 nucleotides. Bootstrap values of >50 % (500 replicates) are indicated at nodes. The GenBank accession number for each reference strain is shown in parentheses.

 
The dominant cellular fatty acids of UST010723-006T are 12 : 0 3-OH, 14 : 0, 15 : 0 iso 2-OH, 16 : 0, 16 : 1{omega}7, 17 : 1{omega}8 and 18 : 1{omega}7 (altogether representing 75·9 % of the total; three replications) as determined using the Sherlock Microbial Identification System. This fatty acid profile is similar to that of P. ruthenica (Table 1Go). No fatty acid profiles have been reported for P. phenolica, P. luteviolacea or P. rubra. The DNA G+C content of the strains UST010723-006T as determined by the HPLC method of Mesbah et al. (1989)Go was 40·6±0·2 mol%. This value was similar to those described for P. luteoviolacea and P. phenolica (Table 2Go).


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  		Table 1. Dominant cellular fatty acids of UST010723-006T and Pseudoalteromonas ruthenica

Numbers given are the mean percentages (±SD) of total fatty acids. Data for Pseudoalteromonas ruthenica are from Ivanova et al. (2002a)Go. NR, not reported by Ivanova et al. (2002a)Go.

 

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  		Table 2. Differentiation of UST010723-006T from other recognized members of Pseudoalteromonas

Data for P. ruthenica, P. luteoviolacea, P. rubra and P. phenolica are from Ivanova et al. (2002a)Go, Gauthier (1982)Go, Gauthier (1976)Go and Isnansetyo & Kamei (2003)Go, respectively. +, Positive; (+), weakly positive; –, negative; V, variable; ND, not described. All are oxidase-, DNase- and gelatinase-positive, but are arginine dihydrolase-negative; all utilize glucose, but not D-adonitol, D-arabinose, erythritol, D-galactose, glycerol, D-lactose, D-mannitol, L-rhamnose or D-sorbitol.

 
The phenotypic characteristics of UST010723-006T are given in the species description. Anaerobic growth was examined in the Oxoid Anaerobic System. The requirement for NaCl was tested in a medium containing (per litre) 5 g MgCl2, 2 g MgSO4, 0·5 g CaCl2, 1 g KCl, 5 g peptone and various amounts of NaCl adjusted to pH 7·5 using KOH (Isnansetyo & Kamei, 2003Go). Cell morphology was examined using scanning electron microscopy (JEOL 7600F) according to Neu et al. (2001)Go. The presence of flagella was examined using light microscopy after Leifson staining according to Smibert & Krieg (1994)Go and using transmission electron microscopy (TEM) (JEOL 100 CX) (Grossart et al., 2000Go) (scanning electron microscopy and TEM pictures are available as Supplementary Fig. S1 in IJSEM Online). Reaction to Gram-stain was determined using light microscopy according to Smibert & Krieg (1994)Go. Cell motility was determined using both light microscopy and observation of growth in motility medium (Smibert & Krieg, 1994Go). Susceptibility to antibiotics was tested according to Acar (1980)Go. Agarase, amylase, catalase, DNase and oxidase activities were tested using the method of Smibert & Krieg (1994)Go. Substrate utilization patterns and other enzyme activities were tested with the commercial systems API 20E, API 20NE, API 50CH and API ZYM (bioMérieux). Cells for inoculation to the API systems were suspended in sterile solution of sea water mixture at 22 {per thousand} salinity (MacDonell et al., 1982Go).

UST010723-006T differs from all previously described members of Pseudoalteromonas by testing negative for lipase activity and by the formation of gas bubbles in the colonies (Table 1Go) (Gauthier et al., 1995Go). UST010723-006T can be distinguished from the four closest relatives (P. luteviolacea, P. ruthenica, P. rubra and P. phenolica) by 9–19 traits, particularly the lack of amylase activity, flagella and cellular motility (Table 2Go). Molecular evidence, together with phenotypic characteristics, suggests that UST010723-006T constitutes a novel species within the genus Pseudoalteromonas.

Description of Pseudoalteromonas spongiae sp. nov.
Pseudoalteromonas spongiae (spon'gi.ae. L. gen. n. spongiae of a sponge).

Cells are Gram-negative, short rods (1·2–1·5 µm in length and 0·5 µm in width) and do not form spores. Cells do not have flagella and are non-motile. Upon cultivation on marine agar, colonies are pale orange in colour, 2–4 mm in diameter, convex with a smooth surface and an entire translucent margin. Gas bubbles are observed in the colonies and also in the agar matrix underneath and adjacent to the colonies. Growth of UST010723-006T is strictly aerobic and occurs between 12 and 44 °C (but not at 4 or 52 °C) and between pH 5·0 and 10·0. UST010723-006T requires NaCl (2·0–6·0 %) for growth. The DNA G+C content is 40·6 mol% and the dominant fatty acids are 12 : 0 3-OH, 14 : 0, 15 : 0 iso 2-OH, 16 : 0, 16 : 1{omega}7, 17 : 1{omega}8 and 18 : 1{omega}7 (altogether representing 75·9 % of the total). Susceptible to benzylpenicillin (0·1 µg), chloramphenicol (0·1 µg), ampicillin (0·1 µg) and tetracycline (1 µg). Resistant to streptomycin and kanamycin (tested up to 100 µg). Acetoin, indole and H2S are not produced. Citrate is not utilized. Nitrate is not reduced. {alpha}-Chymotrypsin, acid phosphatase, alkaline phosphatase, catalase, DNase, esterase (C4), esterase lipase (C8), gelatinase, leucine arylamidase, oxidase, protease and trypsin activities are positive. N-Acetyl-{beta}-glucosaminidase, naphthol-AS-BI-phosphohydrolase and valine arylamidase activities are weakly positive. {alpha}-Fucosidase, {alpha}-galactosidase, {beta}-galactosidase, {alpha}-glucosidase, {beta}-glucosidase, {alpha}-mannosidase, {beta}-glucuronidase, agarase, amylase, arginine dihydrolase, cystine arylamidase, lipase (C14), lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase and urease activities are negative. Grows on D-glucose, D-fructose, inositol, D-mannose, D-maltose, inulin, N-acetylglucosamine and starch as sole carbon source. Acid is produced from D-maltose and N-acetylglucosamine. Carbohydrates not utilized by UST010723-006T are listed in Supplementary Table S2 available in IJSEM Online.

The type strain, UST010723-006T (=NRRL B-41100T=JCM 12884T), was isolated from the surface of the sponge Mycale adhaerens in Hong Kong waters.


   ACKNOWLEDGEMENTS
 
This work was supported by a Central Allocation Grant of Hong Kong Research Grant Council (CA04/05. Sc01) to P.-Y. Q.


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