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-Proteobacteria originating from marine biofilms in Hong Kong waters
1 Coastal Marine Laboratory/Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR
2 Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR
Correspondence
Pei-Yuan Qian
boqianpy{at}ust.hk
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ABSTRACT |
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7c) contents. Phylogenetic analysis of the 16S rRNA gene sequence places UST950701-009PT within the Rhodobacter group of the 
-subclass of the Proteobacteria. The nearest neighbours belong to the genus Loktanella, with similarity values ranging from 94·5 to 95·5 %. Data on G+C and fatty acid contents support the affiliation to the genus Loktanella. UST950701-009PT and -009W are heterotrophic, strictly aerobic and require NaCl for growth (2·0–14·0 %). Both grow in pH 5·0–10·0 and at 8–44 °C. Both are positive in oxidase, catalase and 
-galactosidase tests, but they differ in the pattern of carbohydrate oxidation and assimilation. Molecular evidence together with phenotypic characteristics shows that UST950701-009PT constitutes a novel species within the genus Loktanella. The name Loktanella hongkongensis sp. nov. is proposed; the type strain is UST950701-009PT (=NRRL B-41039T=JCM 12479T) and a morphovar is UST950701-009W (=NRRL B-41040=JCM 12480).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains UST950701-009PT and UST950701-009W are AY600300 and AY600301.
An expanded phylogenetic tree, scanning electron micrographs and details of sequencing primers and API test results are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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-subclass of the Proteobacteria comprises one of the largest fractions of heterotrophic bacteria in the marine environment (Cottrell & Kirchman, 2000
; Hagström et al., 2002). Among the three major groups of -Proteobacteria (the SAR11, SAR116 and Roseobacter clades), only the Roseobacter clade contains culturable members. Amongst the 13 recognized genera of the Roseobacter clade, Loktanella Van Trappen et al. 2004 is the most recently established. Loktanella already has three described species, Loktanella fryxellensis, Loktanella vestfoldensis and Loktanella salsilacus (Van Trappen et al., 2004); all have been recovered from microbial mats in Antarctic lakes. In this study, we propose a novel species of Loktanella originating from a marine biofilm in subtropical water. During the characterization of bacteria isolated from a 7-day-old marine biofilm in Hong Kong waters, strain UST950701-009PT was isolated on marine agar 2216 (Difco) at 30 °C. UST950701-009PT grows as pink, convex colonies with an entire margin and a smooth and shiny surface. Unless otherwise specified, all characteristics described hereafter were based on cultures grown on marine agar 2216 for 48 h at 30 °C. Brown diffusible pigment is produced under all culture conditions. Whitish colonies, with otherwise identical colony morphological characteristics, emerge from every culture upon ageing (3 days or beyond). The whitish colonies can be maintained as separate cultures without returning to the pink colour and are therefore regarded as a morphovar (UST950701-009W) of UST950701-009PT.
The nearly complete 16S rRNA gene sequences of UST950701-009PT and -009W (1407 and 1336 bp, respectively) were resolved on a capillary genetic analyser (Megabace) using a dye terminator method (Rosenblum et al., 1997). Primers used in the sequencing reactions are detailed in Supplementary Table A available in IJSEM Online. Fragments of DNA sequences obtained from individual primers with replications (n=3) were assembled using the Sequencher software package (Gene Codes). Sequence alignment using the bl2seq function at the NCBI server indicated full identity in the 1333 overlapping nucleotide positions of the UST950701-009PT and -009W 16S rRNA gene sequences with the exception of an ambiguous nucleotide position in UST950701-009W. A phylogenetic tree (Fig. 1; see also Supplementary Fig. A for a fuller tree) was constructed using neighbour-joining in the ARB software package (Ludwig et al., 2004). The stability of grouping was estimated by the bootstrap analysis (500 replications). The closest relatives of UST950701-009PT were L. vestfoldensis (95·5 %), L. salsilacus (95·0 %) and L. fryxellensis (94·5 %). A tree based on maximum-parsimony method showed the same topology (data not shown).
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were respectively 65·9±0·4 and 66·2±0·4 mol%. These values are within the range of the DNA G+C contents described for the genus Loktanella (Van Trappen et al., 2004). UST950701-009PT and -009W showed highly similar cellular fatty acid profiles, as determined using the Sherlock Microbial Identification Systems according to the manufacturer's protocols (Table 1). 18 : 17c is the predominant fatty acid in both strains, comprising 84·5–87·0 % of the total fatty acid, which is congruent with the characteristics of the genus Loktanella. On the other hand, the presence of 12 : 0 3-OH and the absence of summed features 2 and 7 distinguish the two strains from the already described members of the genus Loktanella (Table 1).
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(Supplementary Fig. B). Cell motility and reaction to Gram stain were determined using light microscopy at x1000 according to Smibert & Krieg (1994). Susceptibility to antibiotics was tested according to Acar (1980). The production of bacteriochlorophyll a was examined according to Lafay et al. (1995). Casein hydrolysis and catalase and oxidase activities were tested according to Smibert & Krieg (1994). The hydrolysis of Tween 80 was tested according to Baumann & Baumann (1988). Tests for indole, acetoin and H2S production, citrate utilization and -galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease, tryptophan deaminase and gelatinase activities were performed using the API 20E system (bioMérieux) according to the manufacturer's instructions. The metabolism of 49 different carbohydrates was tested using both the API 20E and API 50CH systems according to the manufacturer's instructions.
Strains UST950701-009PT and -009W, which have identical 16S rRNA gene sequences and highly similar phenotypic characteristics, can only be distinguished by colony colour, the metabolism of carbohydrates (detailed in Supplementary Table B) and the presence/absence of trace amounts of the fatty acid 14 : 0 (Table 1
). The two strains differ from the three already described species of Loktanella by their fatty acid profiles (Table 1) and a number of physiological characteristics (Table 2). Based on tests using the API 20NE and API 20E systems, Van Trappen et al. (2004) reported that the three already described members of Loktanella did not metabolize any of the carbohydrates included in the tests. We observed essentially the same negative result in our API 20E tests (see Supplementary Table B). However, strains UST950701-009PT and -009W appeared to be able to metabolize a wide range of carbohydrates in the API 50CH tests, including some of those that gave negative results in the API 20E tests such as L-arabinose, D-glucose and D-melibiose. Given the variability of results using different methods, the carbohydrate metabolism of the three already described members of Loktanella and the two strains in the present study is ambiguous.
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Cells of the type strain are Gram-negative, short rods, immotile and not forming spores. Colonies are pink in colour, convex with entire margins and have smooth and shiny surfaces. The colony size is 1–2 mm after 48 h of cultivation on marine agar 2216 at 30 °C. White colonies, with morphological characteristics otherwise identical to those of UST950701-009PT, emerge from every culture of UST950701-009PT after 3 days. The whitish colonies can be maintained as separate cultures without returning to the pink colour and are therefore regarded as a morphovar (UST950701-009W) of UST950701-009PT. The remaining characteristics refer to the type strain. Growth is strictly aerobic and occurs at 8–44 °C (but not at 4 or 48 °C) and at pH 5·0–10. Requires NaCl for growth and can grow on marine agar 2216 supplemented with NaCl up to 14 % final concentration. Cannot grow on nutrient agar or trypticase soy agar (both from Oxoid). The DNA G+C content is 65·9 mol% and the dominant fatty acid is 18 : 17c (84·5 % of total). Susceptible to streptomycin (50 µg), penicillin (0·1 µg), chloramphenicol (0·5 µg), ampicillin (0·1 µg) and tetracycline (0·1 µg) but not kanamycin (tested up to 100 µg). Bacteriochlorophyll a, indole, acetoin and H2S are not produced. Catalase, oxidase and -galactosidase activities are positive, whereas arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease, tryptophan deaminase and gelatinase are negative. Casein and Tween 80 are not hydrolysed. Citrate is not utilized. A wide range of carbohydrates are metabolized (acid production and/or assimilation) such as arabinose, fructose, glucose, mannitol, sucrose and xylose (see Supplementary Table B for details).
The type strain is UST950701-009PT (=NRRL B-41039T=JCM 12479T), which was isolated from a marine biofilm in Hong Kong waters. A morphovar of UST950701-009PT is UST950701-009W (=NRRL B-41040=JCM 12480).
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ACKNOWLEDGEMENTS |
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Baumann, P. & Baumann, L. (1988). The marine gram-negative eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas and Alcaligenes. In The Prokaryotes, vol. 1, pp. 1302–1331. Edited by M. P. Starr, H. Stolp, H. G. Trüper, A. Balows & H. Schlegel. Berlin: Springer.
Cottrell, M. T. & Kirchman, D. L. (2000). Community composition of marine bacterioplankton determined by 16S rRNA gene clone libraries and fluorescence in situ hybridization. Appl Environ Microbiol 66, 5116–5122.
Hagström, A., Pommier, T., Rohwer, F., Simu, K., Stolte, W., Svensson, D. & Zweifel, U. L. (2002). Use of 16S ribosomal DNA for delineation of marine bacterioplankton species. Appl Environ Microbiol 68, 3628–3633.
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