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1 CEA/Cadarache, DSV-DEVM, Laboratoire d'Ecologie Microbienne de la Rhizosphère UMR 163 CNRS-CEA–Univ. Méditerranée, F-13108 Saint-Paul-lez-Durance, France
2 UMR 6078 CNRS–Université de Nice Sophia-Antipolis, Bât. Jean Maetz, F-06230 Villefranche-sur-mer, France
3 Service Commun de Microscopie Electronique, CNRS & Université d'Angers, rue Haute de Reculée, F-49045 Angers, France
4 UMR 077 de Pathologie Végétale, INRA-INH-Université d'Angers, 42 rue Georges-Morel, BP 57, F-49071 Beaucouzé Cedex, France
Correspondence
Thierry Heulin
thierry.heulin{at}cea.fr
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-Proteobacteria. They belong to two different species, as verified by DNA–DNA hybridization (23·5 % reassociation). The type species of the genus is Ramlibacter tataouinensis sp. nov., with the type strain TTB310T (=DSM 14655T =ATCC BAA-407T =LMG 21543T). The second species is Ramlibacter henchirensis sp. nov., with the type strain TMB834T (=DSM 14656T =ATCC BAA-408T =LMG 21542T). The G+C contents of R. tataouinensis and R. henchirensis are 69·6 and 66·6 mol%, respectively.
Published online ahead of print on 23 August 2002 as DOI 10.1099/ijs.0.02482-0.
The GenBank/EMBL accession numbers for the rrs sequences of Ramlibacter tataouinensis strain TTB310T and Ramlibacter henchirensis strain TMB834T are AF144383 and AF439400, respectively.
Deceased 16 December 2002; this paper is dedicated to his memory. 
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) and the largest fragments were collected immediately and sent to the Muséum National d'Histoire Naturelle in Paris. The strewn field was revisited in 1994 and several weathered fragments of the meteorite were recovered by sieving the first few centimetres of the sandy soil. Scanning electron microscopic observations of both pristine and weathered samples of this meteorite revealed numerous bacterium-like forms on the surface of the meteorite minerals (pyroxene and chromite), and also on the secondary calcite crystals resulting from terrestrial weathering (Barrat et al., 1998, 1999). We have described two types of bacterium-like forms present in the alteration zones of the meteorite (Barrat et al., 1999). One of these bacterium-like forms was characterized by a ‘nanometric’ size (80 nm in diameter) (Gillet et al., 2000). Chemical analyses and electron-diffraction patterns confirmed that the bacterium-like forms could not be magnetite or other iron oxides, iron hydroxides, silicates or carbonates, and showed them to contain C, O, N, Na, K and traces of P and S (Gillet et al., 2000). In an experiment designed to determine whether a soil bacterium was responsible for the colonization and alteration of the meteorite, we isolated a ‘nanometric’ bacterial strain from a meteorite fragment collected from Tataouine subdesert soil in 1994. Here we report the characteristics of two pleomorphic bacteria (including a ‘nanometric’ form), for which the names Ramlibacter tataouinensis gen. nov., sp. nov. and Ramlibacter henchirensis sp. nov. are proposed.
Isolation and morphological characteristics
We tested several procedures to isolate bacteria of unusually small size; the following one was successful. A fragment of weathered meteorite embedded in sandy soil (4·3 g) was sampled at the soil surface, close to boundary stones at the top of a hill (1 km east of the town of Tataouine in Tunisia; coordinates 32° 57' 22'' N, 10° 29' 03'' E). This sample was crushed with a mortar in 10 ml sterile water and stored for 11 days at 4 °C. One hundred microlitres of this suspension was diluted in 100 ml tenfold-diluted tryptic soy broth (TSB/10; 3 g l-1) supplemented with 100 mM CaCO3 (TTB medium) to mimic natural selective pressure (carbonated sandy soil). After 2 days incubation at 30 °C, the resulting culture was filtered using a 0·45 µm filter (Millipore) to select the smallest bacterial forms; 24 tubes containing the filtrate (diluted tenfold in TSB/10) were prepared and kept for 5 days at 30 °C. Associations of rod-shaped (0·2 µm diameter) and spherical (0·8 µm diameter) bacteria were observed by using an optical microscope in eight of the 24 tubes.
The contents of the positive tubes were streaked on TTA agar medium (TSB, 3 g l-1; granulated agar, 15 g l-1; CaCO3 100 mM) and stored at 30 °C for 1 month. After this incubation, colonies remained small (0·1–0·5 mm in diameter), were embedded in TTA medium and yellow-orange in colour. After ten purification steps consisting of serial streaking of single colonies, all purified colonies contained two bacterial forms (rods and coccoid cells). Strain TTB310T was selected for further characterization (Fig. 1).
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Phylogenetic analysis
To confirm that the rods, coccoid and intermediary cells were pleomorphic forms of the same bacterial strain and were of clonal origin, the rrs genes (encoding 16S rRNA) of two different bacterial cultures of strain TTB310T were sequenced. The first contained 100 % coccoid cells (20-day-old culture) and the second 90 % rods (2-day-old culture, from a subculture of the first one). The rrs sequence of strain TMB834T was determined from a bacterial culture containing 90 % coccoid cells. Amplification of the entire rrs genes from strains TTB310T (two samples) and TMB834T (one sample) was performed using bacterial primers (fD1, rD1) as described by Achouak et al. (1999). The PCR product was purified using the QIAquick PCR purification kit (Qiagen). Sequencing reactions were performed using the ABI PRISM Dye Terminator Ready Reaction kit as specified by the manufacturer (Perkin-Elmer). Sequences were obtained with an ABI PRISM 310 DNA sequencer (Perkin-Elmer) using primers S6, S10, S12 and S17 (Achouak et al., 1999).
The rrs sequences from strains TTB310T and TMB834T were aligned, first automatically and then manually, by comparison with a database of 35 000 bacterial rrs sequences. The two rrs sequences of strain TTB310T were 100 % identical, proving that the rods and coccoid cells were of clonal origin. The rrs sequence of strain TMB834T was slightly different from that of TTB310T (98·0 % similarity); the genotypic difference between these two strains was also confirmed by their distinct REP-PCR patterns (data not shown).
Comparison of the complete rrs sequences of strains TTB310T and TMB834T with the GenBank database using the BLASTN 2.2.1 program (Altschul et al., 1997) showed that the closest species was Acidovorax avenae: both TTB310T and TMB834T share 95·0 % similarity with the rrs sequence of Acidovorax avenae strain ATCC 19307 (AB021421) at positions 43–1406. The closest rrs sequence, recently deposited in GenBank (AY102310, 97·4 % similarity with strain TTB310T), corresponded to an uncultured bacterial clone (a13103) isolated from heavy-metal-contaminated soil (R. J. Ellis, P. Morgan, A. J. Weightman and J. C. Fry, unpublished data).
Three methods were used to assess the phylogenetic positions of the rrs sequences of strains TTB310T and TMB834T: neighbour-joining (bioNJ), maximum-likelihood and maximum-parsimony. The domains used to construct the phylogenetic tree were regions of the rrs sequences available for all bacteria and excluding positions likely to show homoplasy: for the tree in this paper, positions 76–180, 194–428, 439–812, 824–1001, 1017–1103 and 1108–1419 of Ramlibacter tataouinensis were used. The robustness of the analysis was evaluated by bootstrapping with 500 replications (Kimura two-parameter correction and bioNJ); bioNJ was performed according to the method of Gascuel (1997); maximum-likelihood and maximum-parsimony data were from PHYLIP (Phylogeny Inference Package, version 3.573c, distributed by J. Felsenstein, Department of Genetics, UW, Seattle, WA, USA). The phylogenetic trees were drawn using the NJPLOT (Perrière & Gouy, 1996) and MacDRAW software packages for Apple Macintosh.
Preliminary large-scale analyses and BLAST searches indicated that the rrs sequences of these two strains were closely related to those of the genus Acidovorax. The final analysis included all rrs sequences found to be closely similar to those of TTB310T and TMB834T, and a set of rrs sequences from other species belonging to the -Proteobacteria, included as outgroups for the phylogenetic analysis (strain designation followed by accession number of their rrs sequence). Sequences belonging to uncultured organisms were then removed, the final list being: Kingella denitrificans ATCC 33394T, M22516; Bordetella holmesii CDC F5101T, U04820; Burkholderia gladioli ATCC 10248T, X67038; Chromobacterium violaceum ATCC 12472T, M22510; Dechloromonas agitata CKBT, AF047462; Formivibrio citricus DSM 6150T, Y17602; Hydrogenophilus thermoluteolus IFO 14978T, AB009828; Iodobacter fluviatilis ATCC 33051T, M22511; Methylobacillus flagellatus KTT, M95651; Methylophilus methylotrophus ATCC 53528T, L15475; Microvirgula aerodenitrificans SGLY2T, U89333; Ralstonia solanacearum strain ACH0158, U28224; Rhodocyclus purpureus strain 6770, M34132; Spirillum volutans ATCC 19554T, M34131; Sutterella wadsworthensis strain 9054, L37786; Thiobacillus thioparus strain LV43, AF005628; Thiomonas cuprina DSM 5495T, U67162; Thiomonas thermosulfata ATCC 51520T, U27839; Vogesella indigofera ATCC 19706T, U45995; Zoogloea ramigera ATCC 19544T, X74913.
The rrs sequences of strains TTB310T and TMB834T formed a clade identified by all the methods with a 100 % bootstrap, but did not constitute a robust clade at the genus level with any other rrs sequence of previously-described bacteria (Fig. 2), suggesting that strains TTB310T and TMB834T belong to a new genus. The grouping of the two rrs sequences within a single clade and their 98·0 % similarity also suggested that the two strains represented a single genus of the family Comamonadaceae (Willems et al., 1991).
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. Native DNA from strain TTB310T was labelled in vitro by random priming (Feinberg & Vogelstein, 1983) using the Megaprime DNA-labelling system (RPN 1604; Amersham). DNA–DNA hybridization experiments were duplicated. The percentage reassociation with strain TTB310T was 23·5 % for strain TMB834T, suggesting that strains TTB310T and TMB834T did not belong to the same species. The percentage reassociation with strain TTB310T was 10·5 % for Acidovorax avenae subsp. avenae ATCC 19860T.
The DNA base composition of strains TTB310T and TMB834T was determined by the thermal denaturing temperature method (Marmur & Doty, 1962) and was calculated using the equation of Owen & Lapage (1976). Escherichia coli K-12 CIP 54-117 (DNA G+C content 50·6 mol%) was used as a control. The DNA G+C content of strains TTB310T and TMB834T was 69·6 mol% and 66·6 mol%, respectively. These DNA G+C contents were similar to those of the closest genera in the family Comamonadaceae (Table 1).
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-Proteobacteria. This unusual phenotype was the coexistence of rods and coccoid cells at different growth stages of strains TTB310T and TMB834T. The rods are the ‘nanometric’ forms (diameter of 0·2 µm) we were initially seeking. They are the vegetative cells, whereas the coccoid cells have all the classical traits of cysts: cell encased by a thick capsular material, presence of white spherical granules in the cytoplasm and long-term resistance to desiccation (Fig. 3, top). Spherical granules were observed within both cysts and rods (Fig. 3, top), and their chemical composition (polyhydroxyalkanoate) was determined by Nile Red labelling (James et al., 1999) and observation under a confocal laser scanning microscope (Olympus Fluoview, excitation at 568 nm). After 6 months dehydration at room temperature under vacuum conditions, the coccoid cells were still able to germinate. Similar desiccation-resistant bacterial species have already been described in the genera Azotobacter (Pal et al., 1997; Núñez et al., 1999) and Azospirillum (Sadasivan & Neyra, 1985). Thin-walled coccoid bodies (termed ‘microcysts’) observed in old cultures of Prolinoborus (formerly Aquaspirillum) fasciculus seem to be a degenerative form rather than part of the life cycle of this bacterium (Koechlein & Krieg, 1998).
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). Despite the absence of polar and lateral flagella, motility of rods was observed on solid media. The mechanism involved is probably the gliding motility observed in some other bacterial species belonging to the genera Myxococcus, Flavobacterium and Flexibacter (Spormann, 1999). On solid agar medium (TSA/10, 1·5 % agar, 24 °C), the velocity of single cells was approximately 0·1 µm min-1 (videomicroscope observations; Olympus Fluoview).
Classical biochemical tests were done with API 20NE strips (bioMérieux). Both the TTB310T and TMB834T strains were positive for aesculin hydrolysis (-glucosidase) and reduction of nitrate to nitrite, and negative for denitrification, indole production, glucose fermentation, arginine dihydrolase, urease, assimilation of arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenylacetate. Gelatin hydrolysis, -galactosidase and glucose-assimilation tests were negative with TTB310T and positive with TMB834T.
Using Biolog GN and GP microplates, only the following carbon sources were metabolized by strain TTB310T: acetate, pyruvate, -hydroxybutyrate, 
-hydroxybutyrate, DL-lactate and propionate. Additional carbon sources were utilized by strain TMB834T: Tween 40, Tween 80, 
-ketovalerate and -ketobutyrate. For both strains, the growth factors present in 0·01 % yeast extract (Difco) were required for growth in minimal medium (M9), in the presence of 0·4 % sodium acetate as a carbon source.
In this work, we isolated two bacterial strains (TTB310T and TMB834T), phylogenetically distant from other genera of -Proteobacteria, and representing a new genus for which the name Ramlibacter is proposed. Strain TTB310T is proposed as the type strain of a new species, named Ramlibacter tataouinensis. Strain TMB834T is proposed as the type strain of a second species of the genus, named Ramlibacter henchirensis. The two species can be phenotypically differentiated by gelatin hydrolysis, -galactosidase activity and the use of glucose, -ketovalerate, -ketobutyrate, Tween 40 and Tween 80 as carbon sources. A notable phenotypic peculiarity of this new genus belonging to -Proteobacteria and to the family Comamonadaceae is a life cycle that includes both rods and cysts. Work on a complete description of this life cycle is in progress, in particular to determine whether the cyst is the only stage in this bacterial life cycle that is able to divide (Fig. 3, bottom). If so, this will constitute a distinct bacterial life cycle, different from those of Azotobacter and Azospirillum, which also include cyst formation (Sadoff, 1975).
Despite the very small diameter of the rods (200 nm), we cannot yet assert that they were the bacterial inhabitants (60–70 nm in diameter) observed earlier at the surface of the meteorite found in Tataouine (Gillet et al., 2000). We are growing strain TTB310T in vitro in the presence of sterile orthopyroxene to characterize the mineral alteration and the effects of desiccation on cell diameter.
Description of Ramlibacter gen. nov.
Ramlibacter (Ram.li.bac'ter. N.L. n. ramlis from Arabic raml sand; M.L. masc. n. bacter rod; N.L. masc. n. Ramlibacter rod isolated from sandy soil).
Small yellow-orange colonies (diameter 0·1–0·5 mm on TSA/10 plates after 15 days at 30 °C). Pleomorphic, ranging from rods (vegetative cells) to coccoid cells (cysts). Vegetative cells are long thin rods (mean dimensions: 3 µm in length and 0·2 µm in diameter) that form cysts (0·8–0·9 µm in diameter). Polyhydroxyalkanoate is present in cytoplasm of rods and cysts. Cell division occurs in cysts. Cysts are desiccation-resistant. Gram-negative. Catalase-positive and oxidase-positive. Vegetative cells motile by gliding. Flagella absent in vegetative and cyst cells. Aerobic chemo-organotrophic. Slow-growing in optimal conditions. Optimal pH and temperature for growth: 7·5 and 30 °C. Weak growth at 37 °C. Growth factors required. Positive for reduction of nitrate to nitrite and aesculin hydrolysis (-glucosidase) but negative for denitrification, indole production and arginine dihydrolase and urease activities. Arabinose, mannose, mannitol, N-acetylglucosamine, maltose, gluconate, caprate, adipate, malate, citrate and phenylacetate not assimilated. The type species of the genus is Ramlibacter tataouinensis.
Description of Ramlibacter tataouinensis sp. nov.
Ramlibacter tataouinensis (ta.ta.oui.nen'sis. N.L. masc. adj. tataouinensis pertaining to Tataouine, Tunisia).
Ramlibacter tataouinensis has all the characteristics of the genus Ramlibacter. Aerobic chemo-organotrophic growth by oxidizing acetate, pyruvate, -hydroxybutyrate, -hydroxybutyrate, DL-lactate and propionate. None of the other substrates in Biolog GN or GP microplates are oxidized. Glucose is not assimilated. Negative for gelatin hydrolysis and -galactosidase activity. Slow-growing in optimal conditions. Optimal pH and temperature for growth: 7·5 and 30 °C. Growth factors required. The DNA G+C content is 69·6 mol%.
The type strain is TTB310T (=DSM 14655T =ATCC BAA-407T =LMG 21543T).
Description of Ramlibacter henchirensis sp. nov.
Ramlibacter henchirensis (hen.chi.ren'sis. N.L. adj. masc. henchirensis from Arabic henchir boundary stones).
Ramlibacter henchirensis has all the characteristics of the genus Ramlibacter. Aerobic chemo-organotrophic growth by oxidizing acetate, pyruvate, -hydroxybutyrate, -hydroxybutyrate, DL-lactate, propionate, Tween 40, Tween 80, -ketovalerate and -ketobutyrate. None of the other substrates of Biolog GN or GP microplates are oxidized. Glucose is assimilated. Positive for gelatin hydrolysis and -galactosidase activity. Slow-growing in optimal conditions. Optimal pH and temperature for growth: 7·5 and 30 °C. Growth factors required. The DNA G+C content is 66·6 mol%.
The type strain is TMB834T (=DSM 14656T =ATCC BAA-408T =LMG 21542T).
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ACKNOWLEDGEMENTS |
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