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1 Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie, Microbiologie, Faculteit Wetenschappen, Universiteit Gent, Ledeganckstraat 35, B-9000 Belgium
2 Grupo de Ecología Genética, Estación Experimental del Zaidin, CSIC, E-18008, Granada, Spain
3 Centro de Investigación sobre Fijación de Nitrógeno, Universidad Nacional Autónoma de México, Ap. P. 565-A, Cuernavaca, Mexico
Correspondence
Anne Willems
anne.willems{at}ugent.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-Proteobacteria, as well as in an analysis of partial recA gene sequences. They may therefore be regarded as a single genus. Because Sinorhizobium was proposed in 1988, according to the Bacteriological Code (1990 Revision) the older name, Ensifer, has priority. However, there are several reasons why a change from Sinorhizobium to Ensifer may not be the best solution and making an exception to Rule 38 may be more appropriate. We therefore propose the species Sinorhizobium adhaerens comb. nov. and put forward a Request for an Opinion to the Judicial Commission regarding the conservation of Sinorhizobium adhaerens over Ensifer adhaerens.
The GenBank/EMBL/DDBJ accession numbers for the new 16S rDNA sequences are AJ420773 (strain 5D19), AJ420774 (HAMBI 1631), AJ420775 (LMG 20582) and AJ420776 (LMG 20571) and for new recA gene sequences: AJ505595 (ATCC 33212T), AJ505596(LMG 20582), AJ505597 (5D19), AJ505598 (HAMBI 1631), AJ505599 (BR819), AJ505600 (BR8606) and AJ505601 (LMG 21331T).
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Sivakumaran et al., 1997; Laguerre et al., 1993). These strains can often become effective symbionts again when provided with a suitable symbiotic plasmid, as reported for strains from the rhizosphere of bean plants (Segovia et al., 1991) and white clover (Sivakumaran et al., 1997). The symbiotic phenotype can also be conferred by transfer of chromosomal symbiotic genes as demonstrated for Mesorhizobium loti ICMP 3153 and Lotus corniculatus rhizobia (Sullivan et al., 1995). It was proposed that non-symbiotic rhizobia persist in soils in the absence of legume plants, and upon introduction of legumes may acquire symbiotic genes from inoculant strains (Sullivan et al., 1996).
Ensifer adhaerens is a soil bacterium that is capable of adhering to and lysing other soil bacteria, although it is not an obligate predator and is not nutritionally fastidious (Casida, 1982). Because of this unusual predatory activity and other mostly morphological characteristics, the organism was considered to be most similar to the budding and appendaged bacteria sensu Bergey's Manual of Determinative Bacteriology, 8th edition (Buchanan & Gibbons, 1974). Because it differed significantly from all known genera in that group, it was placed in a new genus and species (Casida, 1982).
Recently the 16S rDNA sequence of Ensifer adhaerens has become available (Balkwill, 2003) and comparative analysis places this organism inside the genus Sinorhizobium. Together with the observation that non-symbiotic Ensifer adhaerens could effectively nodulate Phaseolus vulgaris and Leucaena leucocephala when provided with symbiotic plasmids of Rhizobium tropici CFN 299 (Rogel et al., 2001), this evidence suggests that Ensifer adhaerens may represent a non-symbiotic rhizobial group. Its remarkable predatory activity and the fact that phylogenetic methods were not widely used at the time have contributed to the fact that it was not readily recognized as a rhizobium when first isolated (Casida, 1982).
We report the characterization of a group of diverse rhizobial and soil isolates that are highly related to Ensifer adhaerens. In view of the phylogenetic position of the genus Ensifer, it should be merged with the genus Sinorhizobium which, according to the Bacteriological Code (1990 Revision) (Lapage et al., 1992), should then be called Ensifer. However, we believe that in this case there are several arguments against such a change and we therefore request the opinion of the Judicial Commission on a proposal to transfer Ensifer adhaerens to Sinorhizobium as Sinorhizobium adhaerens.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. They were grown on yeast extract mannitol agar (YMA) or, for the preparation of protein extracts, on tryptone yeast extract (TY) agar (de Lajudie et al., 1998).
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). SDS-PAGE, digitization, normalization and numerical analyses of the protein patterns were performed with the GelCompar 4.0 software (Applied Maths) as described by Pot et al. (1994).
Nodulation assays.
Seeds of Sesbania rostrata, Vigna unguiculata UC46 and Leucaena gigante were surface-sterilized and germinated for 2 days on paper plates according to Fernández-López et al. (1998). Thereafter, the seedlings were transferred to sterile Leonard jars filled with vermiculite, inoculated with 1 ml of a 24-h culture of the appropriate strain and covered with sterile perlite. Two replicas with four plants each, per strain, were performed. Plants were grown on nitrogen-free medium (Rigaud & Puppo, 1975). Growth conditions were 16 h day and 8 h night with a temperature of 25 and 20 °C, respectively. The plants were harvested 25 days after inoculation and checked for nodulation.
DNA hybridization with nod gene probes.
Total DNA was extracted as previously described (Laguerre et al., 1992), digested with HindIII and separated on 0·8 % agarose gel. The DNA was transferred to nylon filters (Roche) using a VacuGene XL blotting apparatus according to the manufacturer's instructions (Pharmacia Biotech). Probes were obtained by PCR amplification of internal fragments of nodC and nodD genes of Sinorhizobium meliloti GR4 and Rhizobium sp. LPU83 (Del Papa et al., 1999). Oligonucleotides for the nodD gene were: RD1 (5'-TCATCTGCGATATGGATGC-3') and RD2 (5'-GTCTCGAACGAGATGTTCC-3') (Van Dillewijn, 2000); and for nodC: C1 (5' ACATGGAGTATTGGCTTGCC-3') and C2 (5'-AGTTGTTGGCGCAGATATGG-3'). Amplified fragments were isolated from agarose gels and labelled with dioxigenin-11-dUTP (Roche Molecular Biochemicals) by PCR. PCR was performed using 1 cycle at 95 °C for 5 min; 30 cycles at 95 °C for 1 min, at 60 °C for 1 min, at 72 °C for 1 min; followed by a final extension at 72 °C for 5 min. Hybridizations were carried out under high-stringency conditions [formamide 50 %, 5x SSC (1x SSC is 0·15 M NaCl plus 0·015 M trisodium citrate, pH 7·0 ± 0·2), 42 °C]. Washing was carried out twice, 5 min each, in 2x SSC, 0·1 % SDS at room temperature and twice, 15 min each, in 0·1x SSC, 0·1 % SDS at 68 °C. Alternatively, the last step of the washing was carried out at 50 °C and 1x SSC. Detection of the hybridization signals was performed by using as substrate CSPD (disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenylphosphate) from Roche Molecular Biochemicals with an exposure of 5 h and 18 h to an X-ray film.
DNA–DNA hybridization.
DNA was prepared according to a slightly modified procedure of Marmur (1961) as described previously (Willems et al., 2001). Hybridizations were carried out using a microplate method in which unlabelled DNA, non-covalently bound to a microplate, is hybridized with biotinylated probe DNA (Ezaki et al., 1989; Willems et al., 2001). Hybridizations were performed at 45 °C in 2x SSC in the presence of 50 % formamide.
Determination of the DNA base composition.
DNA was degraded enzymically into nucleosides as described by Mesbah et al. (1989). The resulting nucleoside mixtures were separated by HPLC using a Waters SymmetryShield C8 column at 37 °C. The solvent was 0·02 M NH4H2PO4 (pH 4·0) with 1·5 % acetonitrile. Non-methylated lambda phage DNA (Sigma) was used as the calibration reference.
Sequencing and phylogenetic analysis of the 16S rDNA and part of the recA gene.
A large part of the 16S rRNA gene (corresponding to positions 39–1521 of the Escherichia coli rDNA) was amplified by PCR using conserved primers at the 5' and 3' ends of the gene. Approximately 550 bases of the recA gene (corresponding to positions 6–555 of the recA gene of Rhizobium leguminosarum, GenBank no. X59956) were amplified as described by Gaunt et al. (2001). PCR products were purified using a Qiaquick PCR purification kit (Qiagen). The 16S rDNA PCR products were sequenced using conserved primers, a BigDye DideoxyTerminator Cycle Sequencing kit (Perkin Elmer) and an ABI 310 Genetic Analyzer (Perkin Elmer) according to the manufacturer's instructions. The recA PCR products were sequenced using primers described by Gaunt et al. (2001) on an ABI 3100 Genetic Analyzer. Consensus sequences were constructed using the AutoAssembler software (Perkin Elmer). The new 16S rDNA sequence data were aligned with those of related strains from the EMBL database with the program PILEUP of the GCG software (Devereux et al., 1984) and phylogenetic analyses were performed using the TREECON package (Van de Peer & De Wachter, 1994). The phylogenetic analysis of recA sequences was performed using the program Bionumerics (Applied Maths). Distances were calculated using the Kimura correction and clustering was performed with the neighbour-joining algorithm. Bootstrap analysis was performed using 500 (16S rDNA) or 1000 (recA) replications. For comparison maximum-likelihood and maximum-parsimony trees were also calculated using the program Bionumerics (Applied Maths).
Phenotypic characterization.
Strains were studied using API 20NE systems (bioMérieux) according to the manufacturer's instructions. We also used API 50CH systems (bioMérieux) to study growth on different carbon sources following a previously described protocol (Kersters et al., 1984). Growth was scored after 1, 2, 4 and 7 days incubation at 28 °C. Each strain, except for LMG 20582, was tested in API 50CH on two separate occasions and agreement was very good. The only differences observed occasionally were quantitative (e.g. a later onset of a positive reaction) and not qualitative.
Antibiotic sensitivities.
We used the disk-diffusion method to study antibiotic sensitivities. Because the presence or concentrations of certain ions (Mg2+, Ca2+), sulphates, pyruvate and some trace elements can influence resistance patterns (Phillips, 1991), we modified the composition of YMA by using, per litre, 5 instead of 10 g mannitol and 0·0747 g MgCl2.6H2O instead of 0·1 g MgSO4.7H2O. Care was taken to produce agar plates of uniform thickness. For each strain, a cell suspension in physiological water (0·86 % NaCl in distilled water, sterilized) was prepared with a density of 0·5 on the McFarland scale. Using sterile swabs, plates were then uniformly inoculated. Antibiotic disks were applied with a dispenser and plates were incubated for 2 days at 28 °C. Inhibition zones were determined using a digital caliper.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), in order to analyse and characterize the diversity within a natural bacterial population. Sinorhizobium meliloti GR4 exhibits a highly competitive phenotype linked to the nfe (nodule formation efficiency) locus (Soto et al., 1993) and belongs to the major infective groups of the indigenous Sinorhizobium meliloti population at EEZ (Villadas et al., 1995). Strain 5D19 was isolated from an alfalfa nodule, which was also occupied by a Sinorhizobium meliloti GR4-type isolate. Strain 5D19 is unable to elicit nodules on alfalfa roots by itself (Nod-), but it can occupy nodules when co-inoculated with Sinorhizobium meliloti strain GR4. It is of particular interest because it contains a group II intron closely related to Sinorhizobium meliloti intron RmInt1, which will be reported on separately. In a first step towards its identification, we determined the 16S rDNA sequence of strain 5D19 and found that the isolate belongs to Sinorhizobium, where it formed a separate line.
To search among our collection of unnamed rhizobial isolates for similar strains, we prepared a protein extract from strain 5D19 and compared the SDS-PAGE protein profile with our database. This way we found three additional strains with a similar protein profile: BR819, BR8606 and HAMBI 1631. The first two of these strains formed SDS-PAGE cluster 3 in the study of Moreira et al. (1993). The third strain is an unclassified fast-growing rhizobial isolate from Sesbania grandiflora in Sri Lanka (Lindström & Lehtomäki, 1988). The 16S rDNA of a representative of these three, HAMBI 1631, was sequenced. A sequence analysis (Fig. 1) was performed with more organisms that had meanwhile become available from the EMBL database and from a study of soil isolates in our own laboratory (Goris et al., 2002). This revealed that strains 5D19 and HAMBI 1631 are closely related to the new species Sinorhizobium morelense (Wang et al., 2002), to Ensifer adhaerens, a soil bacterium capable of lysing other bacteria (Casida, 1982), to two other soil isolates, LMG 20571 and LMG 20582 (Dejonghe et al., 2000), and to an isolate from the gut of the termite Nasutitermes nigriceps (EMBL/GenBank accession no. AJ298869). All these strains form a relatively tight cluster among Sinorhizobium species. Although this cluster was strongly supported in the bootstrap analysis, the internal branching is poorly resolved because of the high internal sequence similarity (Fig. 1, 99–100 %). Sinorhizobium morelense and strain LMG 20571 form a small subgroup inside this cluster, with sequence similarities of 99·0–99·6 % with the other members of the cluster. The maximum-likelihood and parsimony trees (data not shown) gave essentially the same groupings as the neighbour-joining tree (Fig. 1).
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demonstrated the phylogeny of recA supports that based on 16S rRNA genes among the rhizobia. We took this study as a starting point and extended it by determining the partial recA gene sequences for our unnamed strains and for Ensifer and Sinorhizobium morelense strains. The resulting neighbour-joining dendrogram (Fig. 2) confirms that Ensifer and Sinorhizobium form a single well-supported cluster (bootstrap value 93). The partial recA sequences within the Sinorhizobium–Ensifer cluster had similarity levels ranging from 85·4 to 100 %. Within this cluster a small well-supported subcluster (bootstrap value 98) was formed by the newly sequenced strains. Furthermore, partial recA sequences for strains HAMBI 1631, BR819 and BR8606 were identical, as were the sequences for strains LMG 20582 and ATCC 33212T. Parsimony and maximum-likelihood analyses showed essentially the same grouping (data not shown).
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). This shows that strains BR 819, BR 8606 and HAMBI 1631 had a highly similar protein profile. The protein profile of strain 5D19 is similar to that of these strains, but overall shows more similarity to the pattern of strains ATCC 33212T, ATCC 33499, 4FB6 and LMG 20582. The protein pattern of strain LMG 20571 is distinct from all these strains and most similar to that of Sinorhizobium morelense Lc04T.
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). These data demonstrate the presence of five groups among the strains, all different from Sinorhizobium fredii and Sinorhizobium xinjiangense. The first group (group A) consists of strains BR 819, BR 8606 and HAMBI 1631. Strain 5D19 shows hybridization values of 62–67 % with group A and is therefore highly related. In view of its distinct SDS-PAGE protein profile (Fig. 3
), it can be regarded as representing a separate group (group B). The third DNA group (group C) contains two Ensifer adhaerens strains, Ensifer sp. strain 4FB6 and soil isolate LMG 20582. With hybridization values above 92 % with Ensifer adhaerens strains, these latter two strains can be identified as Ensifer adhaerens. Hybridization values of DNA groups A and B with C were rather high: 43–62 % and 46–57 %, respectively. The type strain of Sinorhizobium morelense (Lco4T) shows a mean hybridization value of 59 % with Sinorhizobium strain LMG 20571. If we use 70 % as a cut-off value, both can be regarded as representing two further DNA groups D and E. It is striking that the grouping found by DNA–DNA hybridizations is neatly corroborated by the recA grouping (Fig. 2).
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) show that DNA groups A and B cannot be differentiated from Ensifer adhaerens (DNA group C) on the basis of these tests. In view of the relatively high DNA–DNA hybridization between the groups (43–62 %, Table 2), we conclude that, for now, they have to be regarded as a single species, Ensifer adhaerens, in line with international recommendations (Wayne et al., 1987). However, in view of the genomic diversity that clearly exists in this species and that is also reflected in the protein profiles (Fig. 3) and partial recA sequences, we propose to create three highly related genomovars (A, B and C) inside Ensifer adhaerens. This terminology was proposed for phenotypically similar genomic groups (Ursing et al., 1995) and a comparable genomovar system exists in Burkholderia cepacia (Vandamme et al., 1997), where genomovars are gradually being named as differentiating data become available (Vandamme et al., 2000; Coenye et al., 2001).
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). It can therefore be designated a member of this species. Sinorhizobium morelense is distinct from Ensifer adhaerens in that it is urease-negative and is sensitive to nalidixic acid (Table 3).
To explore the host range of Ensifer adhaerens and Sinorhizobium morelense, nodulation tests with different host plants were performed with strains 5D19, BR 819, BR 8606, HAMBI 1631, Lc04T, LMG 20571, LMG 20582, 4FB6, ATCC 33212T, ATCC 33499, Sinorhizobium meliloti GR4 and Rhizobium tropici CNFER90. On Leucaena gigante plants, all the strains tested were Nod-, including Sinorhizobium morelense Lc04T and Sinorhizobium sp. strain LMG 9954 that were isolated from nodules of Leucaena leucocephala. A similar Nod- phenotype was obtained with Vigna unguiculata UC46, a tropical crop, for all strains except Sinorhizobium sp. HAMBI 1631 and Rhizobium tropici CFNER90 that exhibited a Nod+ Fix- phenotype. On Sesbania rostrata, strains of DNA groups C, D and E (Sinorhizobium morelense) were Nod-, while the strains that belong to DNA groups A and B and Sinorhizobium meliloti strain GR4 elicited empty pseudonodules. Controls with Rhizobium tropici were Nod+, Fix+. To determine the presence of nod genes, DNA hybridizations were performed with PCR probes derived from nodC and nodD genes of Sinorhizobium meliloti strain GR4 and Rhizobium strain LPU83. The following strains were tested: 5D19, HAMBI 1631, BR819, BR8606, ATCC 33212T, ATCC 33499, LMG 20582, 4FB6, Lc04T and LMG 20571; Sinorhizobium meliloti GR4 was used as a positive control. The washing steps were performed at 68 and 50 °C. At 68 °C, only the positive control gave a hybridization signal. At 50 °C, a positive signal was observed for strain BR819, but its size (20 kb) was different from that of the positive control (500 bp; data not shown). No hybridization signals were observed for the other strains tested. Whether these strains carry nod genes will require further investigation, although strains HAMBI 1631, BR819 and BR8606 were isolated from nodules and must have a set of nodulation genes. Our results are in line with those of Rogel et al. (2001), who reported that strain ATCC 33499 did not form nodules on bean, Leucaena leucocephala, Vigna mungo, Macroptilium atropurpureum and alfalfa and that it did not carry nodC or nifH genes.
Casida (1980) described the ability of a streak of the type strain of Ensifer adhaerens to track an intersecting perpendicular streak of Micrococcus luteus. When we performed the same experiments with all Ensifer adhaerens and Sinorhizobium morelense strains in this study, we did observe clear tracking for strains ATCC 33212T, ATCC 33499 and HAMBI 1631 (2–5 mm after 7 days; in one instance >10 mm) and to a lesser extent for strains 5D19, Lc04T and LMG 20571 (1–2 mm). Escherichia coli LMG 2092T did not show tracking. However, tracking of 1–14 mm was observed for 8 of 11 strains tested when a perpendicular line was made with a sterile loop. Thus, the tracking experiment may not provide a reliable indication of predatory behaviour. To assess this direct microscopic observation may be needed.
Request for an Opinion
Comparative 16S rDNA sequence analysis (Fig. 1, and Balkwill, 2003) shows that Ensifer adhaerens is phylogenetically clearly a member of the Sinorhizobium subgroup of the -Proteobacteria, with sequence similarities of approximately 97·9–99·9 % with Sinorhizobium species. This observation is corroborated by recA gene sequence analysis (Fig. 2) and also by probe hybridization experiments with nolR, a regulatory gene involved in nodulation. A probe consisting of the 5' end of the Sinorhizobium meliloti nolR gene and its upstream region gave hybridization bands with different Sinorhizobium species as well as with Ensifer adhaerens, but not with eight Rhizobium species and Allorhizobium undicola. The same Rhizobium species did yield hybridization bands with a Rhizobium etli nolR probe (Toledo et al., 2003). Comparison of phenotypic data (our own API 20NE, API 50CH and antibiotic-sensitivity data, as well as the literature) indicates that Sinorhizobium and Ensifer are highly similar genera. Ensifer adhaerens strain ATCC 33499 did not nodulate Phaseolus vulgaris or L. leucocephala, but with symbiotic plasmids of Rhizobium tropici it did form nitrogen-fixing nodules on these hosts (Rogel et al., 2001). Our own finding of symbiotic strains very close to Ensifer adhaerens is further evidence of the similarities between Ensifer and Sinorhizobium. In view of all these data, it would seem logical to propose the merger of both genera.
According to Rule 38 of the Bacteriological Code (1990 Revision), when two genera, including their respective type strains, are merged, the oldest genus name should be retained (Lapage et al., 1992). The genus Ensifer was proposed in 1982 (Casida, 1982) with a single species, Ensifer adhaerens, and only two strains. Sinorhizobium was proposed in 1988 (Chen et al., 1988) with Sinorhizobium fredii (the former Rhizobium fredii) as type species and Sinorhizobium xinjiangense as a second species. Therefore, Ensifer is the oldest valid name and should have priority over Sinorhizobium. However, several arguments can be raised against this change and do require consideration.
1. Sinorhizobium is a well-known genus of rhizobia. Its genetics are extensively studied and the genome sequence of Sinorhizobium meliloti has been determined (Capela et al., 2001; Finan et al., 2001; Barnett et al., 2001). Its is also widely studied for agronomic applications. Ensifer, on the other hand, is a rather less well-known organism.
2. Since its proposal, the genus Sinorhizobium was enlarged several times and it now contains nine species of symbiotic nitrogen-fixers, while Ensifer has only one species that until now contained just two strains (Nick et al., 1999; Casida, 1982).
3. Replacing the name Sinorhizobium with Ensifer would seem regrettable because the word ‘rhizobium’ – immediately clarifying the nature of the organisms – would be lost. The name Ensifer, meaning ‘sword-bearer’, does not have the same relevance for symbiotic, nitrogen-fixing bacteria. Replacing the name Sinorhizobium by the name Ensifer is likely to meet with resistance and misunderstanding or even derision by non-taxonomists.
4. Ensifer adhaerens was proposed in 1982, after an extensive morphological and phenotypic characterization of one strain. On this basis, the author placed this organism in part 4 (budding and appendaged bacteria) of division II (the bacteria) in Bergey's Manual of Determinative Bacteriology. However, because it differed significantly from all the genera in part 4, he proposed a new genus and species for this strain (Casida, 1982). More recent studies (Balkwill, 2003), determining the 16S rDNA sequence of Ensifer and Sinorhizobium strains, demonstrate clearly that Ensifer is phylogenetically a member of the Sinorhizobium cluster and we have now confirmed this finding using recA sequences. When Ensifer adhaerens was proposed, 16S rDNA sequence analysis was not yet widely used. Had it been used, the species would have been placed close to Rhizobium meliloti and therefore would probably have been described as a Rhizobium (and later Sinorhizobium) species since the diversity of rhizobia and their relatedness with other, non-rhizobial, genera was not well-known at the time. Therefore, renaming Ensifer adhaerens to Sinorhizobium adhaerens can be regarded as being in accordance with Principle 9 of the Bacteriological Code (1990 Revision), which states that ‘the name of a taxon should not be changed without sufficient reason based either on further taxonomic studies or on the necessity of giving up a nomenclature that is contrary to the Rules of this Code’. In this case, we propose that the above arguments represent ‘sufficient reason based on further taxonomic studies’.
In view of these arguments, we would prefer to preserve the genus name Sinorhizobium over the older name Ensifer for Ensifer adhaerens. This view was also expressed at a recent meeting of the Subcommittee on Rhizobium and Agrobacterium (Lindström & Martínez-Romero, 2002). Rule 38 also states ‘if however, this choice would lead to confusion in bacteriology, the author should refer this matter to the Judicial Commission’. We believe that replacing Sinorhizobium by Ensifer would cause considerable misunderstanding and confusion among rhizo-microbiologists and we therefore ask for the opinion of the Judicial Commission on this matter. We propose to transfer Ensifer adhaerens to the genus Sinorhizobium as Sinorhizobium adhaerens comb. nov. The species now includes symbiotic nitrogen-fixing strains and an emended description is provided below.
Description of Sinorhizobium adhaerens (Casida 1982) comb. nov.
Sinorhizobium adhaerens (ad'hae.rens. L. neut. adj. adhaerens adherent).
The original description of Ensifer adhaerens was based solely on the type strain (Casida, 1982). The data presented here include the type strain and seven other strains. Some of the characteristics we studied were also reported by Casida, (1982) and are in good agreement with our findings.
Colonies on TY medium (3 days, 30 °C) are smooth, shiny, creamy-white, convex, round with a diameter of 0·5–2 mm. In some strains, neighbouring colonies merge to form a single uniform area of growth because of exopolysaccharide production. Cells are motile rods, 0·3–0·6x1–3·5 µm, occurring singly or in pairs. It is not known if the predatory activity towards other bacteria reported for the type strain is also present in other strains of the species. Detailed phenotypic features for all strains are given in Table 3. The G+C content of the DNA, as determined by HPLC, ranges from 61·2 to 62·3 mol%. Using DNA–DNA hybridization, three highly related genomovar groups (43–62 % hybridization) were found. Strains of groups A and B were isolated from root-nodules on various host plants; strains of group C were isolated from soil. Group A and group B strains form empty pseudonodules on Sesbania rostrata; they do not nodulate Leucaena gigante or Vigna unguiculata, except for strain HAMBI 1631, which is Nod+ Fix- on the latter plant species. Group C strains do not nodulate Sesbania rostrata, Vigna unguiculata or Leucaena gigante. nodC or nodD genes are not detected by using hybridization probes derived by PCR from Sinorhizobium meliloti GR4 and Rhizobium sp. LPU83. No phenotypic differentiation of the three genomovars is currently possible, but they can be distinguished by their distinct protein profiles and recA gene sequences. The species has a very wide geographic distribution with strains so far recognized originating from the USA, Spain, Belgium, Sri Lanka and Brazil. The possible signature sequence for Ensifer adhaerens proposed by Balkwill (2003) is present in strains ATCC 33499, ATCC 33212T, LMG 20582 and 5D19, but has one mismatch in strain HAMBI 1631, two in the termite strain M3A and one in Sinorhizobium morelense. Of the signature positions reported for Ensifer adhaerens (Balkwill, 2003), only those at bases 658 (A), 659 (T), 746 (A), 747 (T) apply to Sinorhizobium adhaerens; they can differentiate this species from all other Sinorhizobium species except Sinorhizobium morelense.
The type strain is ATCC 33212T (=LMG 20216T). It was isolated from soil and is able to attach to Micrococcus luteus cells, causing their lysis. It is not, however, an obligate predator and is not nutritionally fastidious in the absence of prey cells. The DNA G+C content is 62·3 mol% (HPLC). The EMBL/GenBank accession number for the 16S rDNA sequence is AF191739. The type strain belongs to genomovar C.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Balkwill, D. L. (2003). The genus Ensifer. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 2 (in press). Edited by G. M. Garrity. New York: Springer Verlag.
Barnett, M. J., Fisher, R. F., Jones, T. & 23 other authors (2001). Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid. Proc Natl Acad Sci U S A 98, 9883–9888.
Buchanan, R. E. & Gibbons, N. E. (editors) (1974). Bergey's Manual of Determinative Bacteriology, 8th edn. Baltimore: Williams & Wilkins.
Capela, D., Barloy-Hubler, F., Gouzy, J. & 25 other authors (2001). Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021. Proc Natl Acad Sci U S A 98, 9877–9882.
Casida, L. E., Jr (1980). Bacterial predators of Micrococcus luteus in soil. Appl Environ Microbiol 39, 1035–1041.
Casida, L. E., Jr (1982). Ensifer adhaerens, gen. nov., sp. nov. a bacterial predator of bacteria in soil. Int J Syst Bacteriol 32, 339–345.
Chen, W. X., Yan, G. H. & Li, J. L. (1988). Numerical taxonomic study of fast-growing soybean rhizobia and proposal that Rhizobium fredii be assigned to Sinorhizobium gen. nov. Int J Syst Bacteriol 38, 392–397.
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-galactosidase, 116.