Abstract
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain LW36T is DQ512861.
A scanning electron micrograph of cells of strain LW36T and a table showing its cellular fatty acid profile are available as supplementary material in IJSEM Online.
The genus Paracoccus was first erected by Davis et al. (1969) and the description was emended by Katayama et al. (1995). At the time of writing, the genus comprises 19 recognized species (see Fig. 1). Members of this genus are metabolically versatile and are widely distributed in soil, sediment and activated sludge environments. They take part in bioconversion of organic and inorganic compounds. For example, Paracoccus denitrificans utilizes organic compounds for growth and can use nitrate for respiration (Rainey et al., 1999, and references therein). Some other Paracoccus species, such as Paracoccus pantotrophus (Robertson & Kuenen, 1983; Rainey et al., 1999), Paracoccus thiocyanatus and Paracoccus versutus, can use reduced sulfur as an energy source (Harrison, 1983; Katayama et al., 1995). In this study, a sulfur-oxidizing, heterotrophic bacterial strain, designated LW36T, was isolated from activated sludge. Based on phenotypic and genotypic studies it is concluded that this isolate represents a novel species of the genus Paracoccus.
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Bacterial strain LW36T was isolated from activated sludge of a sequential batch reactor treating mixed wastewater containing various nitroaromatic compounds (nitrobenzene, nitrophenol, 2,4-dinitrophenol) and aniline. The sludge sample was suspended in sterile saline solution by using vigorous vortexing, and a portion of the suspension was spread directly on LB agar plates. The plates were incubated at 30 °C for about a week. Single colonies on the plates were picked up and bacterial strain LW36T was obtained by repeatedly streaking cultures on new plates from a single colony.
Routine cultivation was conducted at 30 °C with LB media. Gram reactions were determined according to the method described by Gerhardt et al. (1994). Presence of cell flagella and cell morphology were examined by transmission electron microscopy (H600; Hitachi) and scanning electron microscopy (FEI QUANTA 200). Growth temperature range was determined with a TN3F temperature-gradient incubator (Advantec). Catalase and oxidase activities, the VogesProskauer reaction, carbon source utilization and other biochemical characterization methods were performed according to Dong & Cai (2001). For testing growth on reduced sulfur and elemental sulfur, strain LW36T was inoculated into Allen's medium (Allen, 1959), the pH of which was adjusted to 6.5, supplemented with elemental sulfur (1 %), sulfide (1 %), thiosulfate (1 %) or sulfite (1 %). Cell growth was estimated either by monitoring the increase in optical density at 600 nm or by determining the increase in protein content of cultures.
Cells of strain LW36T were Gram-negative, coccoid to short rods with a size range of 1.01.2x0.5 µm (see Supplementary Fig. S1 in IJSEM Online). Colonies were cream-coloured, smooth, circular and 0.42.0 mm in diameter. Growth was observed over a temperature range of 2536 °C and a pH range of 510. Strain LW36T grew chemolithotrophically on thiosulfate, sulfide and elemental sulfur but growth was not observed with sulfite. Other physiological and biochemical characteristics are given in Table 1 and in the species description below.
Table 1. Phenotypic characteristics that differentiate strain LW36T from related species of the genus Paracoccus Taxa: 1, strain LW36T; 2, P. denitrificans (data from Doronina et al., 1998; Dworkin, 2001); 3, P. methylutens (Doronina et al., 1998; Dworkin, 2001); 4, P. versutus (Katayama et al., 1995; Dworkin, 2001); 5, P. pantotrophus (Robertson & Kuenen, 1983; Dworkin, 2001). +, Positive; , negative; NA, no data available.
Biomass for chemotaxonomic analyses was harvested from LB cultures at 30 °C after incubation for 24 h. Cellular fatty acids were extracted and analysed as described by Hu et al. (2004). Quinones were extracted and purified according to the method of Collins (1985) and were analysed by HPLC (Wu et al., 1989), with a commercial ubiquinone-10 (Q-10) sample (China Pharmacy Corp.) as reference. Results showed that the major cellular fatty acids (>1 % of the total) of strain LW36T were C18 : 1ω7c (64.2 %), C16 : 0 (16.4 %), C18 : 0 (3.7 %), C14 : 0 3-OH (3.0 %), C16 : 1ω7c (2.3 %), C14 : 0 (1.9 %), iso-C15 : 1 (1.8 %) and C19cyc (1.4 %). Detailed information on the cellular fatty acid composition of strain LW36T is provided in Supplementary Table S1 in IJSEM Online. Strain LW36T had Q-10 as the sole respiratory quinone. DNA base composition was determined by thermal denaturation (Marmur & Doty, 1962), with Escherichia coli K-12 as reference. The DNA G+C content of strain LW36T was 61.3 mol%.
The nearly complete 16S rRNA gene of strain LW36T (1343 bp) was amplified and sequenced as described by Zhang et al. (2003). Alignments of 16S rRNA gene sequences were performed with the CLUSTAL_X program, version 1.64b (Thompson et al., 1997). A neighbour-joining (Saitou & Nei, 1987) phylogenetic tree (Fig. 1) was constructed based on evolutionary distances calculated with the Kimura two-parameter model. When aligned, positions with insertions or deletions were excluded from the calculations. The 16S rRNA gene sequence analysis indicated that strain LW36T was phylogenetically related to members of the genus Paracoccus, with similarities ranging from 92.4 to 94.9 %.
Based on our phenotypic and phylogenetic studies, it is clear that strain LW36T represents a member of the genus Paracoccus. Strain LW36T showed a range of phenotypic characteristics that differentiated it from recognized species of the genus (as listed in Table 1). Its phylogenetic divergence (Fig. 1) also differentiates strain LW36T from recognized Paracoccus species. Therefore, we conclude that strain LW36T represents a novel species of the genus Paracoccus, for which the name Paracoccus sulfuroxidans sp. nov. is proposed.
Description of Paracoccus sulfuroxidans sp. nov.
Paracoccus sulfuroxidans (sul.fur.ox'i.dans. L. n. sulfur sulfur; N.L. part. adj. oxidans oxidizing; N.L. part. adj. sulfuroxidans pertaining to the ability to oxidize sulfur).
Cells are Gram-negative, coccoid to short rods, 1.01.2x0.5 µm in size. Colonies are cream-coloured, smooth, circular and 0.42.0 mm in diameter. Growth occurs over a temperature range of 2536 °C (optimum growth at 31.533.5 °C) and a pH range of 510. Positive for catalase and oxidase activities. Negative for VogesProskauer reaction and lipase activity. Does not hydrolyse gelatin, starch or casein. Utilizes citric acid, nitrilosides, L-arabinose, D-glucose and maltose, but not D-arabitol, fructose, mannitol, rhamnose, melibiose, xylitol, malic acid, L-glutamic acid, L-lactic acid, cellobiose, D-lactose, inositol, D-mannose, D-melibiose, raffinose, D-ribose, salicin, sorbitol, glycerol, trehalose or dextrin. Grows chemolithotrophically on thiosulfate, sulfide and elemental sulfur but not on sulfite. The sole respiratory quinone is Q-10. The predominant cellular fatty acid is C18 : 1ω7c (64.2 %). The G+C content of the DNA is 61.3 mol% (Tm).
The type strain, LW36T (=CGMCC 1.5364T=JCM 14013T), was isolated from activated sludge of a wastewater-treatment bioreactor.