Classification and features
Figure 1 shows the phylogenetic neighborhood of L. hongkongensis DSM 17492T in a 16S rRNA gene based tree. The sequence of the single 16S rRNA gene copy in the genome does not differ from the previously published 16S rRNA gene sequence (AY600300).
The single genomic 16S rRNA gene sequence of
L. hong-kongensis
DSM 17492T was compared with the Greengenes database for determining the weighted relative frequencies of taxa and (truncated) keywords as previously described [6]. The most frequently occurring genera were
Loktanella
(46.2 %),
Ketogulonicigenium
(14.9 %),
Methylarcula
(10.3 %),
Silicibacter
(10.0 %) and
Ruegeria
(8.5 %) (65 hits in total). Regarding the five hits to sequences from representatives of the species, the average identity within high-scoring segment pairs was 99.6 %, whereas the average coverage by HSPs was 98.0 %. Regarding the 13 hits to sequences from other representatives of the genus, the average identity within HSPs was 95.6 %, whereas the average coverage by HSPs was 97.6 %. Among all other species, the one yielding the highest score was
Loktanella vestfoldensis
(NR_029021), which corresponded to an identity of 95.8 % and a HSP coverage of 99.4 %. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification). The highest-scoring environmental sequence was FJ869048 (Greengenes short name ‘
Roseobacter
isolates Chesapeake Bay water 2 m depth isolate CB1079Rhodobacterales str. CB1079’), which showed an identity of 99.2 % and an HSP coverage of 99.9 %. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were ‘lake’ (8.6 %), ‘tin’ (7.1 %), ‘qinghai’ (6.4 %), ‘microbi’ (3.2 %) and ‘sea’ (3.1 %) (185 hits in total). The most frequently occurring keywords within the labels of those environmental samples which yielded hits of a higher score than the highest scoring species were ‘sea’ (15.4 %), ‘water’ (7.7 %), ‘bloom, chl, concentr, contrast, diatom, dure, filter, non-bloom, spring, station, success, surfac, yel’ (5.1 %) and ‘bai, chesapeak, depth, roseobact’ (2.6 %) (3 hits in total). These keywords fit well to the isolation site of strain UST950107-009PT.
L. hongkongensis
UST950107-009PT is Gram-negative and non-spore forming (Table 1). Cells are short rods and non-motile (Fig. 2). When grown on Marine Agar 2216 (Difco) at 30 ˚C in the absence of light, colonies are pink in color, convex with entire margin, and have smooth and shiny surface; brown diffusible pigment is produced. However, whitish colonies would emerge from every culture upon aging (3 days or beyond). The colonies of the white morphovar, with otherwise identical morphological properties, can be maintained as separate cultures (UST950701-009 W) without turning pink.
L. hongkongensis
UST950107-009PT cannot grow on nutrient agar or trypticase-soy agar (both from Oxoid).
The growth of
L. hongkongensis
UST950701-009PT is strictly aerobic and requires at least 2 % NaCl (up to 14 %). The ranges of temperature and pH where its growth can occur are 8–44 ˚C and 5.0–10.0, respectively.
L. hongkongensis
UST950107-009PT can utilize a wide range of mono-, di-, tri- and polysaccharides, and sugar alcohols. Citrate is not utilized. Catalase, oxidase and beta-galactosidase activities are positive whereas arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, urease, tryptophane deaminase and gelatinase are negative.
L. hongkongensis
UST950701-009PT does not produce bacteriochlorophyll a, indole, acetoin or H2S. It cannot hydrolysis casein or tween 80. Streptomycin, penicillin, chloramphenicol, amplicilin and tetracycline can inhibit the growth of
L. hongkongensis
UST950107-009PT but kanamycin cannot (all data from [1]).
The utilization of carbon compounds by
L. hongkongensis
DSM 17492T grown at 28 °C was also determined for this study using Generation-III microplates in an OmniLog phenotyping device (BIOLOG Inc., Hayward, CA, USA). The microplates were inoculated at 28 °C with dye IF-A and a cell suspension at a cell density of 95–96 % turbidity. Further additives were vitamin, micronutrient and sea-salt solutions [14]. The plates were sealed with parafilm to avoid a loss of fluid. The exported measurement data were further analyzed with the opm package for R [15, 16], using its functionality for statistically estimating parameters from the respiration curves such as the maximum height, and automatically translating these values into negative, ambiguous, and positive reactions. The reactions were recorded in three individual biological replicates. Positive results were received for the following substrates: positive control, pH 6, 1 % NaCl, 4 % NaCl, 8 % NaCl, D-galactose, 3-O-methyl-D-glucose, D-fucose, L-fucose, L-rhamnose, inosine, 1 % sodium lactate, myo-inositol, rifamycin SV, L-aspartic acid, L-glutamic acid, L-histidine, L-serine, D-glucuronic acid, glucuronamide, quinic acid, L-lactic acid, citric acid, α-keto-glutaric acid, D-malic acid, L-malic acid, nalidixic acid, acetic acid and sodium formate.
According to Generation-III plates the strain is negative for dextrin, D-maltose, D-trehalose, D-cellobiose, β-gentiobiose, sucrose, D-turanose, stachyose, pH 5, D-raffinose, α-D-lactose, D-melibiose, β-methyl-D-galactoside, D-salicin, N-acetyl-D-glucosamine, N-acetyl-β-D-mannosamine, N-acetyl-D-galactosamine, N-acetyl-neuraminic acid, D-glucose, D-mannose, D-fructose, fusidic acid, D-serine, D-sorbitol, D-mannitol, D-arabitol, glycerol, D-glucose-6-phosphate, D-fructose-6-phosphate, D-aspartic acid, D-serine, troleandomycin, minocycline, gelatin, glycyl-L-proline, L-alanine, L-arginine, L-pyroglutamic acid, lincomycin, guanidine hydrochloride, niaproof, pectin, D-galacturonic acid, L-galactonic acid-γ-lactone, D-gluconic acid, mucic acid, D-saccharic acid, vancomycin, tetrazolium violet, tetrazolium blue, p-hydroxyphenylacetic acid, methyl pyruvate, D-lactic acid methyl ester, bromo-succinic acid, lithium chloride, potassium tellurite, tween 40, γ-amino-n-butyric acid, α-hydroxy-butyric acid, β-hydroxybutyric acid, α-keto-butyric acid, acetoacetic acid, propionic acid, aztreonam, butyric acid and sodium bromate and the negative control.
The phenotype of the strain was described as well as the assimilation of a wide range of sugars was tested by Lau et al. [1] with the API50CH system, which is based on the detection of biochemical reactions. Using the API50CH system positive reactions were found for more than 20 carbon sources. None of these results could be confirmed by the OmniLog measurement.
L. hongkongensis
was positive for only five sugars, as well as for a number of carboxylic acids (e.g. malate and citrate) and amino acids. This observation agrees with the finding of Van Trappen et al. [6], who determined the phenotype of three Loktanella strains using API20NE, except for the difference that no positive reaction was found for the carbon sources given in [6]. Positive reactions found in the OmniLog measurements but not in growth experiments might be due to the higher sensitivity of the former [17].
Chemotaxonomy
The predominant fatty acids of
L. hongkongensis
UST950107-009PT are C18:1 Ω7C (84.5 %), C16:0 (5.8 %), C18:0 (3.5 %), C10:0 3-OH (2.0 %) and C12:0 3-OH (1.9 %), making up to 97.7 % of the total [1]. The remaining fatty acids are C12:1 3-OH, C17:O, C18:1 Ω7C 11-methyl, summed feature 3 (comprising C16:1 Ω7c and C15 iso 2-OH), and an unknown peak with an expected chain length equivalent to 11.799.