| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 GBF – Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D38124 Braunschweig, Germany
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany
Correspondence
Irene Wagner-Döbler
iwd{at}gbf.de
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
Published online ahead of print on 8 July 2005 as DOI 10.1099/ijs.0.63832-0.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DFL-24T is AJ534215.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). The 16 strains that were found to be positive for these genes were characterized based on 16S rRNA gene sequence analysis and found to be phylogenetically diverse, forming five groups within the ‘Alphaproteobacteria’. Only one group exhibited well-pigmented cultures under appropriate growth conditions and was shown to contain bacteriochlorophyll a (bchl a) (Allgaier et al., 2003; Biebl et al., 2005). The other groups appeared beige, slightly pink or yellow; bchl a was not detected initially, but was found later in small amounts or under specific conditions. Two strains were related closely to Roseovarius tolerans (Labrenz et al., 1999) according to their 16S rRNA gene sequence. One of these strains, DFL-24T, is morphologically, physiologically and biochemically characterized here, and the results suggest that it should be classified as a novel species of the genus Roseovarius, a member of the Roseobacter–Sulfitobacter–Silicibacter group. In addition, a hitherto-unidentified marine strain, which was isolated by Hold et al. (2001), was also shown to be a member of this species. All three strains of the novel species were obtained from dinoflagellates. DFL-24T was isolated from a culture of Alexandrium ostenfeldii KO287, which was maintained in the collection of the Biological Institute, Helgoland. The algal cells were isolated and washed, and the attached bacteria were spread on an agar medium containing 0·25 g peptone, 0·25 g yeast extract, 0·25 g glucose and 10 g sea salts l–1. After 2 weeks incubation at 18 °C, two faintly pink colonies were purified and maintained as strains DFL-24T and DFL-35, which appeared to be very similar. DFL-24T was further investigated.
Strain DFL-24T grew well on marine agar 2216 (Difco). Colonies were whitish to faintly pink, circular, convex and had an entire margin and a glistening surface. A slimy consistency was often observed. By using a dissecting microscope and light from underneath, two stable colony morphotypes were discriminated: a rough, slightly slimy type and a glistening, somewhat darker type. The 16S rRNA gene sequences of the two types were found to be identical. For further characterization, cultures of the rough type were used. Cells were ovoid to rod-shaped and 0·5–0·7x1·3–3·0 µm in size, often with unequal ends (Fig. 1a), suggesting a budding mode of division. The cells in the rough colony type were distributed evenly, whereas they formed star-shaped aggregates in the glistening colony type (Fig. 1b). Real motility was not observed, but occasionally cells showed a turning motion on the spot, which was therefore not caused by Brownian motion. In negatively stained preparations (Biebl et al., 2005), the electron micrographs revealed extensions of the outer membrane at the cell poles (Fig. 2a), as has been described for Roseovarius nubinhibens (González et al., 2003). Flagella were not observed. In ultrathin sections, the periplasmatic space appeared rather translucent (Fig. 2b, inset); the peptidoglucan sacculus was not visible. The outer membrane outlined the cell as an intensely stained layer with an undulating contour (Fig. 2b).
|
|
), growth was observed throughout, with maximum growth observed at 31 °C. Growth was equally good at initial pH values between 6·5 and 8·8. Below pH 6·5, growth was increasingly slower, and was lacking above pH 9·1.
Utilization of carbon sources was tested in a medium containing 20 g sea salts, 0·3 g (NH4)2SO4, 0·1 g KH2PO4, 0·1 g yeast extract and 1 ml trace-element solution (Pfennig & Trüper, 1992) l–1 at a pH of 7·5. The strain was able to use acetate and butyrate, intermediates of the tricarboxylic acid cycle, lactate, glutamate and glycerol, but not glucose, fructose, ethanol or methanol (see Table 1 and the species description for further details). Interestingly, the inability to grow on glucose was also found for R. tolerans (Labrenz et al., 1999). DFL-24T did not grow in a medium without yeast extract, but yeast extract could be replaced by a mixture of seven vitamins. Tests with mixtures in which one of these vitamins was omitted revealed that only biotin, thiamine and nicotinic acid were essential for growth. Strain DFL-24T was able to degrade gelatin, but not starch, alginate or Tween 80. Dissimilatory nitrate reduction was not found; neither nitrite (sulfanilic acid/N,N-dimethylnaphthylamine test) nor nitrogen (Durham tubes) was formed.
|
Allgaier et al. (2003) were not able to detect bchl a in the cells of strain DFL-24T. However, when the pellet of the 25 ml culture grown in the above peptone medium was extracted with 3 ml acetone/methanol (7 : 2), a flat hump was seen in the absorption spectrum around 770 nm, the IR absorption range of bchl a. Faint IR absorption was also detected in a succinate-grown continuous culture of the strain, particularly during dark growth following an illumination period (H. Biebl, unpublished observations).
Very small levels of bchl a have previously been reported for aerobic anoxygenic phototrophs (Sato, 1978; Yurkov et al., 1993; Hiraishi et al., 2000; Hiraishi & Shimada, 2001). In visibly pigmented cultures, a specific bchl a content of 1–4 nmol (mg protein)–1 has been measured, whereas <0·1 nmol (mg protein)–1 was found generally in colourless or beige-coloured cultures. The lowest value, 
0·004 nmol (mg protein)–1, was reported for Acidiphilium acidophilum (Hiraishi & Shimada, 2001). For strain DFL-24T, our estimated bchl a content was 0·05 nmol (mg protein)–1. In Blastomonas natatoria, which was originally regarded as a pigment-free close relative of Erythromonas ursincola (Yurkov et al., 1997), bchl a was identified by HPLC and photodiode-array detection and found to be present in very small amounts. Interestingly, in the genome of this species, the pufL/M genes were also shown to be present (Hiraishi et al., 2000), as in strain DFL-24T. For R. tolerans, no quantitative data on bchl a content are available, but it is noticeable that some strains belonging to this species formed bchl a and others did not, and two formed photosynthetic pigments only after several years of cultivation (Labrenz et al., 1999). However, the pufL/M genes were present in all eight existing Roseovarius strains (Allgaier et al., 2003). González et al. (2003) described a second species of Roseovarius, R. nubinhibens, which was free of photosynthetic pigments. Boettcher et al. (2005) showed that neither R. nubinhibens nor a newly proposed species, Roseovarius crassostreae, contains the pufL/M genes. The significance of very small amounts of bchl a for aerobic metabolism is unknown at present. Possibly, there are hitherto undetected conditions under which the bchl a content is increased to an effective quantity, or the bchl a found is merely a remnant of a formerly expressed physiological activity.
Polar lipids were extracted with chloroform/methanol/0·3 % NaCl and were separated by TLC according to the method of Tindall (1990). Phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine were found, as previously shown in R. tolerans. By contrast, the lipid composition of other Roseobacter group members, such as Antarctobacter heliothermus, Ruegeria algicola, Ruegeria denitrificans and Ruegeria litoralis, differs considerably from this pattern (Labrenz et al., 1999). The predominant lipoquinone was ubiquinone-10. The fatty acid profile of strain DFL-24T was also similar to that of R. tolerans (Table 2). As in most members of the ‘Alphaproteobacteria’, 18 : 1
7c is the predominant component, amounting to 70 % of the total fatty acid content (76 % as determined by González et al., 2003), followed by 16 : 1 (about 10 %). Labrenz et al. (1999) also reported 10 % 18 : 2, which may have arisen from the low cultivation temperature used by these authors. The occurrence of the hydroxylated acids 12 : 0 2-OH, 12 : 0 3-OH and 12 : 1 3-OH might be more characteristic of the genus Roseovarius. 12 : 1 3-OH was present in both strain DFL-24T and R. tolerans, whereas R. nubinhibens contained 12 : 0 3-OH instead. Detection of 12 : 0 2-OH requires acid hydrolysis, which was not applied by González et al. (2003).
|
and Biebl et al. (2005) with the following amendments. The sequences were aligned manually and compared with published sequences from the DSMZ 16S rRNA gene sequence database, including sequences available from the Ribosomal Data Project (Maidak et al., 1999) and GenBank/EMBL. Alignment was achieved with the BioEdit program (Hall, 1999) and was used for calculating the distance matrix corrected by Kimura's two-parameter method (Kimura, 1980) and CLUSTAL_X (Thompson et al., 1997). A phlyogenetic dendrogram was inferred by using the neighbour-joining method (Saitou & Nei, 1987). Bootstrap analysis was based on 1000 resamplings.
The strain was thus identified as a member of the Roseobacter group within the ‘Alphaproteobacteria’. Its closest phylogenetic neighbour was found to be R. tolerans EL-172T, showing a 16S rRNA gene sequence similarity value of 96·4 %, whilst R. nubinhibens showed only 93·9 % similarity. R. tolerans, R. nubinhibens and the novel species proposed herein, Roseovarius mucosus sp. nov., form a clearly separated group within the Roseobacter clade (Fig. 3). Furthermore, the 16S rRNA gene sequence from R. crassostreae (GenBank accession no. AF114484; Boettcher et al., 2005) is more closely related to R. nubinhibens and both species can clearly be differentiated from the R. tolerans/R. mucosus cluster by specific signature nucleotides at several positions of the secondary structure from the 16S rRNA gene. The pattern of 16S rRNA gene signatures consists of (A–U) for ‘Roseovarius crassostreae’ and Roseovarius nubinhibens. The following helical regions are characterized by base-pair exchanges and the nucleotides detected for the R. crassostreae/R. nubinhibens group are given in parentheses: helical region 240–242/284–286 UGG/CCA (UAG/CUA), position 113–115/312–314 GUG/CAC (CAA/GUU), position 577–580/764–767 GCAC/GUGC (GCGC/GUGC). Additionally, sequences of the strains differ in the loop region of the fifth variable region of the 16S rRNA gene sequence. 16S rRNA gene sequence similarity of R. mucosus and R. tolerans was determined to be 96·4 %, whereas that between R. nubinhibens and R. tolerans was 95·8 % (González et al., 2003), which is relatively low for species affiliation within a genus. However, if the pattern of the investigated characters is taken into consideration, the species designation is justified, whereby R. nubinhibens appears more distant from the other two species. The three species are morphologically very similar; they form short to medium-sized rods. For R. mucosus and R. tolerans, an uneven cell division was observed. Motility was almost absent in R. mucosus, tumbling in R. tolerans and present in R. nubinhibens, whilst flagella were not found in any of the strains. Substrate utilization agreed well in R. mucosus and R. tolerans, in particular the inability to use glucose, which is used readily by R. nubinhibens. The cellular fatty acid composition showed a good match for the three species; R. nubinhibens contained a different hydroxylated C12 acid. Polar lipids were almost identical in R. mucosus and R. tolerans. The DNA G+C content of the novel species is again closer to that of R. tolerans than to that of R. nubinhibens. Roseovarius species differ strongly in bacteriochlorophyll content, but because formation of photosynthetic pigments in obligately aerobic bacteria is strongly dependent on cultivation conditions, it cannot be used as a taxonomically relevant criterion, given the pigmented and colourless strains reported for R. tolerans and the trace amounts reported for R. mucosus. However, both species have the puf genes of the photosynthetic reaction centre. It is remarkable that in R. nubinhibens, as well as in R. crassostreae, the pufL/M genes could not be detected (Boettcher et al., 2005).
|
and was listed as Roseobacter 253-13. We obtained this strain from C. Strömpl (GBF, Braunschweig) together with some identification data and additionally checked for pigment content, morphology, substrate utilization, fatty acids and polar lipids. Surprisingly, we found bchl a to be present at about the same level as in DFL-24T, i.e. 0·05 nmol (mg protein)–1. Cells were similar under phase-contrast microscopy and were immotile, as were cells of strain DFL-24T. Two types of colonies were observed, as with DFL-24T. Substrate utilization was comparable, with the exception that lactate was not used. Strain 253-13 was unable to grow on glucose or fructose, as are DFL-24T and R. tolerans. Fatty acid composition and polar lipid pattern were almost identical to those of DFL-24T. Strain 253-13 differed from DFL-24T in being unable to liquefy gelatin and in being resistant to penicillin G. The DNA G+C content was slightly lower (60·9 mol% for strain 253-13 versus 62·9 mol% for DFL-24T). In view of the high 16S rRNA gene sequence similarity (99·7 %), the bchl a content and a number of other characters, strain 253-13 can be unreservedly classified as belonging to R. mucosus.
Description of Roseovarius mucosus sp. nov.
Roseovarius mucosus (mu.co'sus. L. adj. mucosus slimy, a property of the colonies).
Cells are rods of 0·5–0·7x1·3–3·0 µm with pointed cell poles, frequently uneven before and after division (as described for the genus). Motility is rarely observed. Colonies on marine agar 2216 (Difco) appear whitish to faintly pink, circular, convex with an entire margin and a glistening surface, occasionally slimy. Two stable types of colony morphologies can be discriminated by using a dissecting microscope: a brighter, slightly slimy and a darker, somewhat smaller and more glistening type. Optimum growth occurs at a salinity between 1 and 7 %, a temperature range between 20 and 40 °C and a pH range between 6·0 and 8·8. Requires biotin, thiamine and nicotinic acid for growth. Growth substrates are acetate, butyrate, succinate, fumarate, malate, lactate (not for strain 253-13), glutamate, pyruvate and glycerol, but not glucose, fructose, citrate, ethanol or methanol. Gelatin is liquefied, but starch, alginate and Tween 80 are not degraded. Nitrate is not reduced to nitrite or to nitrogen. Cells are positive for catalase and oxidase. Indole is not formed from tryptophan. The DNA G+C content of strain DFL-24T is 62·9 mol% and that of strain 253-13 is 60·9 mol%.
The type strain, DFL-24T (=DSM 17069T=NCIMB 14077T), was isolated from a culture of Alexandrium ostenfeldii (dinoflagellate).
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Biebl, H., Allgaier, M., Tindall, B. J., Koblizek, M., Lünsdorf, H., Pukall, R. & Wagner-Döbler, I. (2005). Dinoroseobacter shibae gen. nov., sp. nov., a new aerobic phototrophic bacterium isolated from dinoflagellates. Int J Syst Evol Microbiol 55, 1089–1096.
Boettcher, K. J., Geaghan, K. K., Maloy, A. P. & Barber, B. J. (2005). Roseovarius crassostreae sp. nov., a member of the Roseobacter clade and the apparent cause of juvenile oyster disease (JOD) in cultured Eastern oysters. Int J Syst Evol Microbiol 55, 1531–1537.
González, J. M., Covert, J. S., Whitman, W. B. & 8 other authors (2003). Silicibacter pomeroyi sp. nov. and Roseovarius nubinhibens sp. nov., dimethylsulfoniopropionate-demethylating bacteria from marine environments. Int J Syst Evol Microbiol 53, 1261–1269.
Hall, T. A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41, 95–98.
Hiraishi, A. & Shimada, H. (2001). Aerobic anoxygenic photosynthetic bacteria with zinc-bacteriochlorophyll. J Gen Appl Microbiol 47, 161–180.
Hiraishi, A., Kuraishi, H. & Kawahara, K. (2000). Emendation of the description of Blastomonas natatoria (Sly 1985) Sly and Cahill 1997 as an aerobic photosynthetic bacterium and reclassification of Erythromonas ursincola Yurkov et al. 1997 as Blastomonas ursincola comb. nov. Int J Syst Evol Microbiol 50, 1113–1118.[Abstract]
Hold, G. L., Smith, E. A., Rappé, M. S. & 7 other authors (2001). Characterization of bacterial communities associated with toxic and non-toxic dinoflagellates: Alexandrium ssp. and Scrippsiella trochoidea. FEMS Microbiol Ecol 37, 161–173.
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Labrenz, M., Collins, M. D., Lawson, P. A., Tindall, B. J., Schumann, P. & Hirsch, P. (1999). Roseovarius tolerans gen. nov., sp. nov., a budding bacterium with variable bacteriochlorophyll a production from hypersaline Ekho Lake. Int J Syst Bacteriol 49, 137–147.
Maidak, B. L., Cole, J. R., Parker, C. T., Jr & 11 other authors (1999). A new version of the RDP (Ribosomal Database Project). Nucleic Acids Res 27, 171–173.
Pfennig, N. & Trüper, H. G. (1992). The family Chromatiaceae. In The Prokaryotes, 2nd edn, pp. 3200–3221. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. Berlin: Springer.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sato, K. (1978). Bacteriochlorophyll formation by facultative methylotrophs, Protaminobacter ruber and Pseudomonas AM 1. FEBS Lett 85, 207–210.[CrossRef][Medline]
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Tindall, B. J. (1990). Lipid composition of Halobacterium lacusprofundi. FEMS Microbiol Lett 66, 199–202.
Yurkov, V. V., Krasilnikova, E. N. & Gorlenko, V. M. (1993). Effect of light and oxygen on metabolism of the aerobic bacterium Erythromicrobium sibiricum. Microbiology (English translation of Mikrobiologiya) 62, 35–38.
Yurkov, V., Stackebrandt, E., Buss, O., Vermeglio, A., Gorlenko, V. M. & Beatty, J. T. (1997). Reorganization of the genus Erythromicrobium: description of "Erythromicrobium sibiricum" as Sandaracinobacter sibiricus gen. nov., sp. nov., and of "Erythromicrobium ursincola" as Erythromonas ursincola gen. nov., sp. nov. Int J Syst Bacteriol 47, 1172–1178.
This article has been cited by other articles:
|
|
 |
|
  C. Y. Hwang and B. C. Cho Ponticoccus litoralis gen. nov., sp. nov., a marine bacterium in the family Rhodobacteraceae Int J Syst Evol Microbiol, June 1, 2008; 58(6): 1332 - 1338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H. Yoon, S.-J. Kang, and T.-K. Oh Roseovarius aestuarii sp. nov., isolated from a tidal flat of the Yellow Sea in Korea Int J Syst Evol Microbiol, May 1, 2008; 58(5): 1198 - 1202. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Lee, Y.-J. Choo, S. J. Giovannoni, and J.-C. Cho Maritimibacter alkaliphilus gen. nov., sp. nov., a genome-sequenced marine bacterium of the Roseobacter clade in the order Rhodobacterales Int J Syst Evol Microbiol, July 1, 2007; 57(7): 1653 - 1658. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Eiler Evidence for the Ubiquity of Mixotrophic Bacteria in the Upper Ocean: Implications and Consequences Appl. Envir. Microbiol., December 1, 2006; 72(12): 7431 - 7437. [Full Text] [PDF]
|
|
|
|
|
|
T. Martens, T. Heidorn, R. Pukall, M. Simon, B. J. Tindall, and T. Brinkhoff Reclassification of Roseobacter gallaeciensis Ruiz-Ponte et al. 1998 as Phaeobacter gallaeciensis gen. nov., comb. nov., description of Phaeobacter inhibens sp. nov., reclassification of Ruegeria algicola (Lafay et al. 1995) Uchino et al. 1999 as Marinovum algicola gen. nov., comb. nov., and emended descriptions of the genera Roseobacter, Ruegeria and Leisingera Int J Syst Evol Microbiol, June 1, 2006; 56(6): 1293 - 1304. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
