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Rhizobial isolates that were obtained from both surface and deep soil samples in the Sahelian and Sudano-Guinean areas of Senegal (West Africa) under Acacia albida trees were compared with representative strains of known rhizobial species and genera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was used to determine the taxonomic positions of these organisms and the relationships between isolates obtained from the surface and isolates obtained from deep soil. Most of the isolates belonged to eight electrophoretic clusters containing representative strains of Bradyrhizobium japonicum, Bradyrhizobium elkanii, and Bradyrhizobium sp. Isolates were also characterized by the Biolog system, and the results were compared with the results obtained by SDS-PAGE of total proteins; the level of correlation was very low. DNA-rRNA hybridizations with 16s or 23s rRNA from Bradyrhizobium japonicum LMG 6138T (T = type strain) confirmed that most of the protein electrophoretic clusters belong in the Bradyrhizobium-Rhodopseudomonas rRNA complex. Sequencing of 16s rRNA genes showed that some of the A. ulbida-nodulating isolates belong to a separate lineage together with representatives of other protein electrophoretic clusters. Other isolates that belong to the same electrophoretic cluster as the type strain of Bradyrhizobium japonicum are considered members of the lineage represented by this type strain. The first lineage is as far removed from Bradyrhizobium japonicum as it is from the genus Afipia, Bhstobacter denitnpcans, and the genus Rhodopseudomonas. The possible relationship among electrophoretic group, geographic origin, and depth of isolation at a particular site is discussed.Jordan (15, 16) divided the rhizobia into two genera, Rhizobium and Bradyrhizobium, containing fast-growing and slowly growing strains, respectively.At one time the genus Rhizobium was divided into three species, Rhizobium leguminosarum (with Rhizobium leguminosarum bv. viciae, Rhizobium leguminosarum bv. phaseoli, and Rhizobium leguminosarum bv. trifolii), Rhizobium meliloti, and Rhizobium loti. In 1984 Scholla and Elkan (27) proposed a new species, Rhizobium fredii; later, Chen et al. (3) transferred this taxon on phenotypic grounds to the new genus Sinorhizobium and described a second species, Sinorhizobium xinjiangensis. Recently, however, on the basis of the results of partial 16s rRNA sequence analyses Jarvis et al. (13) concluded that the name Sinorhizobium is a synonym of the name Rhizobium, that all fast-growing soybean-nodulating strains belong to a single species, Rhizobium fredii, and that additional studies are needed to confirm the status of Sinorhizobium xinjiangensis as a separate taxon. Since that time the following four new species have been proposed: Rhizobium galegae (21), Rhizobium huakuii (2), Rhizobium tropici (for the former Rhizobium leguminomrum bv. phaseoli type 11) (22), and Rhizobium etli (for Rhizobium leguminosarum bv. phaseoli type I strains) (28).According to Jordan (16), the genus B...

Members of the genus Methanosarcina are recognized as major aceticlastic methanogens, and several species which thrive in low-salt, pH-neutral culture medium at mesophilic temperatures have been described. However, the environmental conditions which support the fastest growth of these species (Methanosarcina barkeri MST [T = type strain] and 227, Methanosarcina mazei S-6T, and Methanosarcina vacuolata Z-761T) have not been reported previously. Although the members of the genus Methanosarcina are widely assumed to grow best at pH values near neutrality, we found that some strains prefer acidic pH values. M. vacuolata and the two strains of M. barkeri which we tested were acidophilic when they were grown on H, plus methanol, growing most rapidly at pH 5 and growing at pH values as low as 4.3. M. mazei grew best at pH values near neutrality. We found that all of the strains tested grew most rapidly at 37 to 42°C on all of the growth substrates which we tested. None of the strains was strongly halophilic, although the growth of some strains was slightly stimulated by small amounts of added NaCI. The catabolic substrates which supported most rapid growth were H, plus methanol; this combination sometimes allowed growth of a strain under extreme environmental conditions which prevented growth on other substrates. The cell morphology of all strains was affected by growth conditions.Although Methanosarcina species have been the subjects of extensive metabolic studies, other aspects of the characterization of these organisms do not meet even the minimal standards for description of methanogenic taxa (9). In this study we examined the physiology of the following three mesophilic, aceticlastic species: Methanosarcina barkeri, Methanosarcina mazei, and Methanosarcina vacuolata. Methanosarcina acetivorans, the only other mesophilic and aceticlastic Methanosarcina species, was not included in this study because it has been well characterized (35).The original type strain of M. barkeri, isolated by C. G. T. P. Schnellen (32a), has been lost. In 1966, Bryant isolated strain MS (lo), which has been adopted as the type strain (4). Although this strain is one of the most extensively studied methanogens, previous studies have not included definitive determinations of its responses to environmental conditions such as pH, temperature, and salinity. Strain 227 (25) is another extensively studied strain which DNA hybridization studies (37) indicate is a member of M. barkeri. Methanogenic cocci were first described by Groenewege (12) in 1920 and were later observed by Kluyver and van Niel (17). These cocci were physiologically similar to M . barkeri, but because of morphological differences between the cocci and M . barkeri, Kluyver and van Niel proposed a new genus (Methanococcus) for the cocci. Later Barker (6) studied a purified, nonaxenic culture and named it Methanococcus mazei. Pure cultures of this species were not available until more than 40 years later. Zhilina and Zavarzin (44) also studied an aceticlastic enrichment culture w...

Two strains of Methanogenium bourgense, strains MS2T (T = type strain) and LX1, were characterized, and, based in part on previously published DNA hybridization data, this species was transferred to a new genus, Methanoculleus, as Methanoculleus bourgense comb. nov. Methanogenium marisnigri JRIT and a new strain of Methanogenium marisnigri, strain ANS, were also characterized. This species was also transferred to the genus Methanoculleus as MethanocuUeus marisnigri comb. nov. et emend., and its description was emended to indicate that the species has a temperature optimum near 40°C, is halotolerant, and is slightly alkaliphilic; in contrast, the previous description of this organism indicates that it has a temperature optimum of 20 to 25"C, is halophilic, and is slightly acidophilic. We also propose the transfer of two other phylogenetically related species, Methanogenium thermophilicum and Methanogenium olentangyi, to the genus Methanoculleus as Methanoculleus thermophilicum and Methanoculleus olentangyi, respectively. Methanogenium cariaci JRIT was also further characterized, and its description is emended.

We examined 22 previously described and newly isolated Methanosarcina strains by performing denaturing gel electrophoresis of whole-cell proteins and assigned these strains to previously described species. Methanosarcina mazei S-6T (T = type strain) and Methanosarcinafi.isia C 16T were very similar in terms of the electrophoresis patterns of their proteins and in their DNA sequences (the results of reassociation experiments indicated that there was 77% sequence similarity). Thus, M. fish is a junior subjective synonym of M. mazei, and strain C 16 is a reference strain of M. mazei. M. muzei C 16 was similar to M. mazei in other characteristics that have not been reported previously, including the ability to catabolize acetate and a lack of halophily. All of the Methanosarcina strains examined, including the marine strains M. mazei C 16 (= M. jiisia C 16T) and Methunosarcina acetivorans C2AT, were slightly halotolerant (rather than halophilic, as originally described). Methunosarcina sp. strain FR-1, which has gas vesicles, was more similar to Methanosarcina barken' MST than to Methanosarcina vacuolata Z-761T in both its protein patterns and its DNA sequence (80% similarity to M. barken' MST and 38% similarity to M. vacuolata Z-761T). Thus, the presence of gas vesicles is not an adequate taxonomic characteristic for assigning Methunosarcina strains to M. vacuolata.The five commonly recognized mesophilic Methanosarcina species (Methanosarcina barkri, Methanosarcina mazei, Methanosarcina vacuolata , Methanosarcina fnsia , and Methanosarcina acetivorans) are found in marine and freshwater environments (24,33,34). The first three of these species are considered to be freshwater species, and their type strains were isolated from freshwater environments. However, early strains of M. barkri were isolated from marine or brackish mud (36,42), and the current type strains of M. barkeri and M. mazei thrive at marine salinities (40). It has been postulated that Methanosarcina species evolved in marine habitats and adapted to freshwater environments by developing a heteropolysaccharide matrix or capsule (41). When Methanosarcina strains are grown in the presence of marine concentrations of Na+ and Mg2+, they have a protein cell wall, are sensitive to lysis by detergents, and occur as individual cells (35,40,46). When these organisms are grown in the presence of low concentrations of these cations, a heteropolysaccharide matrix is often formed, and cells may occur as aggregates, "cysts," or lamina (27,40,46).The other two mesophilic Methanosarcina species, M. fnsia and M. acetivorans, are considered to be marine species because all of the known strains (M. fnsia C 16T [T = type strain] and M. acetivorans C2AT and C2E) were isolated from marine sediments (4, 39). The cells of these strains are sensitive to lysis by detergent, and their cell walls are protein (4, 39).In this paper we describe some of the physiological characteristics, especially halotolerance, of some Methanosarcina strains, the results of a phylogenetic analysis...