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1 Korea Research Institute of Bioscience and Biotechnology, 52 Oeundong, Yusong, Daejeon 305-333, Republic of Korea
2 Department of Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
Correspondence
Yong-Ha Park
yhpark{at}kribb.re.kr
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ABSTRACT |
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7c as the dominant cellular fatty acid. Analysis of the 16S rRNA gene sequence revealed that this strain is affiliated with a cluster within the Alphaproteobacteria. Strain mano18T synthesized bacteriochlorophyll under aerobic conditions. The 16S rRNA gene sequence similarity between strain mano18T and the most closely related species, Stappia aggregata DSM 13394T, was 98·5 %. Levels of DNA–DNA relatedness between strain mano18T and the type strains of S. aggregata and Stappia stellulata were respectively 6·2–11·2 and 3·3–7·6 %. Strain mano18T, like other Stappia strains, possesses carbon monoxide dehydrogenase genes. The results of DNA–DNA hybridization and the polyphasic data confirmed that strain mano18T can be considered to represent a novel taxon in the genus Stappia. The name Stappia marina sp. nov. is proposed for the tidal flat isolate; the type strain is strain mano18T (=KCTC 12288T=DSM 17023T).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and form I and form II coxL gene sequences of strain mano18T are respectively AY628423, AY753548 and AY753549.
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; Stapp & Knösel, 1954). In a study of marine star-shaped-aggregate-forming bacteria, Rüger & Höfle (1992) concluded that ‘Agrobacterium aggregatum’ (Ahrens, 1968) was a later heterotypic synonym of Agrobacterium stellulatum (Stapp & Knösel, 1954), which had nomenclatural priority. Later phylogenetic studies based on 16S rRNA gene sequences suggested that the marine species of the genus Agrobacterium have no relation to the terrestrial Agrobacterium species; therefore, the taxonomic position of the marine subdivision of Agrobacterium was reassessed and a proposal was made to transfer two species belonging to Agrobacterium to a new genus as Stappia aggregata and Stappia stellulata (Uchino et al., 1998). All known strains of the genus Stappia have been shown to oxidize carbon monoxide (CO) and to possess the gene for CO dehydrogenase (coxL) in a survey of the diversity of aerobic CO oxidizers (King, 2003).
Recently, a number of bacterial strains have been isolated in a tidal flat area as part of a study aimed at understanding the diversity of micro-organisms and their function in a tidal flat ecosystem. Many of them have been identified as phylogenetically novel micro-organisms (Yoon et al., 2003a, b, c). In this study, we describe a Stappia-like strain, mano18T, which was isolated from a tidal flat area of the Yellow Sea, Korea. The organism was considered to be Stappia-like on the basis of 16S rRNA gene sequence comparison. Accordingly, the aim of present work was to elucidate the taxonomic position of strain mano18T by means of phenotypic, genetic and chemotaxonomic analyses.
Strain mano18T was isolated from a sample of a tidal flat obtained in Dae-Chun, Chung-Nam, Korea (36° 17' 45·2'' N 126° 31' 9·5'' E) by using the dilution plating technique on marine agar 2216 (MA; Difco). The strain was routinely grown at 25 °C for 3 days. S. aggregata DSM 13394T and S. stellulata DSM 5886T, obtained from the DSMZ, were grown under the same conditions and used as reference strains. Morphology of live cells and the presence of flagella were investigated by using light microscopy (Nikon E600) and transmission electron microscopy (TEM). For TEM observation, cells from exponentially grown culture were negatively stained with 1 % (w/v) phosphotungstic acid. After air drying, the grid was examined by using a model H-7600 transmission electron microscope (Hitachi). Anaerobic growth was tested by using a BBL GasPak Pouch (Becton Dickinson) on MA supplemented with nitrate. Sodium ion requirements were determined by comparing growth on BM medium (Baumann et al., 1971) and modified BM medium containing potassium ions in place of sodium ions and by investigating growth in trypticase soy broth without NaCl. Growth at various NaCl concentrations was investigated in marine broth 2216 (MB; Difco) or trypticase soy broth (Difco). API 20 NE test strips (bioMérieux) were used for analysing biochemical and physiological traits of bacterial strains and standard microbiological methods were used for studying Gram staining, motility and catalase and oxidase activities (Smibert & Krieg, 1994).
Isoprenoid quinones of strain mano18T was extracted from 100 mg freeze-dried cells according to Collins & Jones (1981) and purified by preparative TLC (silica gel F254; Merck). The ubiquinone fraction was analysed by HPLC (Hitachi L-5000) equipped with a reversed-phase column (YMC pack ODS-AM; YMC Co.) as described by Shin et al. (1996). Bacterial strains grown on MA plates at 25 °C for 5 days were used for fatty acid methyl ester (FAME) analysis. FAMEs were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). The DNA G+C content was determined by the method of Tamaoka & Komagata (1984). Chromosomal DNA was extracted and purified according to the method described by Sambrook et al. (1989). DNA was hydrolysed and the resultant nucleotides were analysed by HPLC using a reversed-phase column (Supelcosil LC-18-S; Supelco). The 16S rRNA gene was amplified by PCR using two universal primers as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA gene and phylogenetic analysis were performed as described by Yoon et al. (2003a). DNA–DNA hybridization was based on the method described by Kusuda et al. (1991) and Willcox (1996). DNA was transferred to a nylon membrane (Hybond-N+; Amersham). The membrane was incubated for 1 h at 40 °C for prehybridization and then for 12 h at 40 °C for hybridization. A DIG High Prime DNA Labelling and Detection starter kit II (Roche Molecular Biochemicals) was used for the detection of DNA. After washing, the membrane was exposed to autoradiography film (Hyperfilm-ECL; Amersham) for 10 min and signal intensities were determined using the TINA 2.0 program (Lee et al., 2003). The signal produced by self-hybridization was taken as 100 %, and relative intensities of genomic DNAs of other strains were determined to be the percentage similarity.
The coxL gene of strain mano18T was amplified according to the method of King (2003). Two forward primers, OMPf [5'-GGCGGCTT(C/T)GG(C/G)AA(C/G)AAGGT-3'] and BMSf [5'-GGCGGCTT(C/T)GG(C/G)TC(C/G)AAGAT-3'], and a reverse primer, O/Br [5'-(C/T)TCGA(T/C)GATCATCGG(A/G)TTGA-3'], were designed on the basis of conserved motifs of gene sequences for the large subunit of authentic CO dehydrogenases (King, 2003). Primer pairs OMPf and O/Br or BMSf and O/Br were used for amplification of the form I and form II large subunit genes of CO dehydrogenase, respectively.
The production of bacteriochlorophyll a (BChl a) by strain mano18T was investigated by PCR amplification of a phototrophism-related gene (pufM). Primers pufMF (5'-CGCACCTGGACTGGAC-3'; Achenbach et al., 2001) and pufMR [5'-CCAT(G/C)GTCCAGCGCCAGAA-3'; Beja et al., 2002] were used for PCR amplification of the pufLM gene. Extracted genomic DNA of strain mano18T was used as the PCR template. The PCR mixture contained 1·5 mM MgCl2, 0·25 µM each dNTP, 5 U Taq DNA polymerase, 0·5 µM each primer and 10 ng template DNA in a total volume of 20 µl (Bioneer). PCR was performed in a thermocycler (iCycler; Bio-Rad) with an initial denaturation step at 95 °C for 4 min followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 47 °C for 40 s and extension at 72 °C for 1·5 min and a final extension at 72 °C for 7 min. The amplified DNA was separated by agarose gel electrophoresis (1 % agarose in 1x TAE), stained with ethidium bromide, viewed by UV illumination and photographed.
Cells of strain mano18T were Gram-negative, regular and club shaped. They occurred singly, in irregular clusters or in star-like aggregates or rosette-like aggregates (Fig. 1). However, star-shaped aggregate formation could not always be observed. It seems to be dependent on the physiological state of the cells (Suzuki et al., 2000). Strain mano18T has been grown at 25–30 °C and needed sodium ions for growth. Growth occurs in the presence of 3–6 % (w/v) NaCl. Optimum growth of strain mano18T occurred at 25 °C and in 3 % (w/v) NaCl. The physiological and biochemical properties of strain mano18T are summarized in Table 1. A partial 16S rRNA gene sequence (1355 bp) was determined from strain mano18T. The result of a BLAST search indicated that the isolate was a member of the Alphaproteobacteria and was closely related to several marine bacteria. The 16S rRNA gene sequence of strain mano18T showed the highest similarity to that of S. aggregata DSM 13394T (98·5 %); the next highest similarity was observed to members of the genus Roseibium (97 %) and S. stellulata DSM 5886T (95 %). 16S rRNA gene sequence alignment and phylogenetic tree construction (Fig. 2) were conducted by using CLUSTAL X software (Thompson et al., 1997). DNA–DNA hybridization studies were carried out between strain mano18T and closely related strains selected on the basis of their 16S rRNA gene sequence similarities and phylogenetic positions. The low DNA–DNA relatedness between strain mano18T and S. aggregata DSM 13394T (6·2–11·2 %) and S. stellulata DSM 5886T (3·3–7·6 %) confirmed that strain mano18T represents a novel species.
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7c (58·47 %) together with C20 : 17c (7·80 %), 11-methyl C18 : 17c (9·08 %), C17 : 18c (0·55 %) and C18 : 19c (0·23 %) were the most abundant. Small amounts of saturated fatty acids C18 : 0 (8·03 %), C19 : 0 cyclo 8c (3·56 %) and C16 : 0 (1·65 %) were also detected. The fatty acid composition was most similar to that observed in the genus Stappia (Table 2) and clearly differentiated the isolate from other phylogenetically related genera (data not shown). Minor quantitative differences in amounts of C19 : 0 cyclo 8c and summed feature 7 were observed between our isolate, S. aggregata DSM 13394T and S. stellulata DSM 5886T. The presence of C18 : 17c as the predominant fatty acid and Q-10 as the dominant lipoquinone are characteristics of members of the Alphaproteobacteria (Uchino et al., 1998; Martínez-Cánovas et al., 2004). The coxL gene was amplified and then sequenced bidirectionally. Inferred amino acid sequences derived from partial sequence of CO dehydrogenase form I (GenBank accession no. AY753548) and form II (AY753549) large subunits of strain mano18T were compared with corresponding sequences from the GenBank database. coxL sequences of strain mano18T showed the highest identity (95–97 %) with S. aggregata DSM 13394T and S. stellulata DSM 5886T. Strain mano18T synthesizes BChl a to grow photosynthetically under aerobic conditions, as judged by employing PCR amplification of a phototrophism-related gene (pufM, encoding the M subunit of the photosynthetic reaction centre, universally distributed among aerobic phototrophic bacteria; Achenbach et al., 2001).
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showed that all known strains of Stappia oxidize CO and possess a gene for ribulose-1,5-bisphosphate carboxylase/oxygenase, which plays a central role in lithotrophic carbon fixation. Oxidization of CO by strain mano18T was not examined; however, the presence of coxL genes showing highest sequence similarity to those from members of the genus Stappia suggests that strain mano18T may oxidize CO, like other species in the genus Stappia. Therefore, on the basis of the data presented, strain mano18T should be placed in the genus Stappia as a representative of a novel species, for which the name Stappia marina sp. nov. is proposed.
Description of Stappia marina sp. nov.
Stappia marina (ma.ri'na. L. fem. adj. marina of the sea, marine).
Cells are Gram-negative, 1·3–2·0 µm in length and 0·6–0·8 µm in diameter, do not form spores and are motile by means of polar flagella. Cells occasionally form rosette-like aggregates. It is able to grow by nitrate reduction. Oxidase- and catalase-positive. Urease, aesculin hydrolysis and 
-galactosidase are positive. Acid production from glucose, indole production from tryptophan, arginine dihydrolase and gelatin hydrolysis are negative. Halophilic; requires sodium ions for growth. Optimal NaCl concentration for growth is 3 % (w/v). Optimal growth temperature is 25 °C on MA. Nitrate is reduced to nitrite, but is not reduced to nitrogen gas. Q-10 is the predominant respiratory quinone. The principal cellular fatty acids are C18 : 17c (58·4 %), 11-methyl C18 : 17c (9·0 %), C18 : 0 (8·0 %) and C20 : 17c (7·8 %). The G+C content of the DNA of the type strain is 59·7 mol%. Cells synthesize BChl under aerobic conditions.
The type strain, strain mano18T (=KCTC 12288T=DSM 17023T), was isolated from a sample of a tidal flat obtained in Dae-Chun, Chung-Nam, Korea.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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