Reiko Hattori

Incubation time and media requirements of culturable bacteria from different phylogenetic groups

Previously, time courses of the colony formation process of soil bacteria on agar plates were simulated by colony-forming curves (CFCs). Bacteria were isolated from the colonies that appeared during an incubation period corresponding to each component curve of the CFC, respectively, and they were placed into groups I, II, III or IV, which were based on the order of colonization, so that their physiological characteristics could be studied (Gorlach, K. et al. (1994) J. Gen. Appl. Microbiol. 40, 507–517). We report here on the results of phylogenetic analysis of the isolates from each CFC group. A wide variety of eubacteria were included in the collection. Strains closely related to Arthrobacter were those from CFC groups I or II, groups of faster growers, and strains in Proteobacteria α-subdivision were those from groups III or IV, which grew more slowly. In contrast, some phylogenetic groups, such as Firmicutes low G+C, Cytophaga/Flexibacter/Bacteroides and Proteobacteria β-divisions exhibited heterogeneity regarding CFC grouping. As for their requirement for media, isolates belonging to the Cytophaga/Flexibacter/Bacteroides division, the Proteobacteria α-subdivision, β-subdivision and the Firmicutes high G+C division, except for Arthrobacter-related strains, were oligotrophic bacteria. In contrast, those belonging to the Firmicutes low G+C division, the Proteobacteria γ-subdivision and the A. globiformis-related group in the Firmicutes high G+C division were not oligotrophic.

Advances in soil microbial ecology and the biodiversity

Recent studies on the colony formation of soil bacteria opened the way to categorize soil bacteria into colony forming curve (CFC) groups of different growth rates. A bacterial culture collection comprising organisms from every CFC group is called an ecocollection. Outlines of ECs of paddy soil 1992 and grassland soil 1987 and 1992 were described. Phylogenetic studies by 16S rDNA sequencing showed a great diversity of culture strains of the ecocollections (EC). A set of alternative concepts was proposed; the active and the quiescent forms of bacterial cells in soil. The former is able to be cultivated and thus counted by the plate method, while the latter is not unless it transforms into the former. Based on the results several points required for extensive cataloguing of soil bacteria were noted.

Complete genome sequence of Bradyrhizobium sp. S23321: insights into symbiosis evolution in soil oligotrophs.

Bradyrhizobium sp. S23321 is an oligotrophic bacterium isolated from paddy field soil. Although S23321 is phylogenetically close to Bradyrhizobium japonicum USDA110, a legume symbiont, it is unable to induce root nodules in siratro, a legume often used for testing Nod factor-dependent nodulation. The genome of S23321 is a single circular chromosome, 7,231,841 bp in length, with an average GC content of 64.3%. The genome contains 6,898 potential protein-encoding genes, one set of rRNA genes, and 45 tRNA genes. Comparison of the genome structure between S23321 and USDA110 showed strong colinearity; however, the symbiosis islands present in USDA110 were absent in S23321, whose genome lacked a chaperonin gene cluster (groELS3) for symbiosis regulation found in USDA110. A comparison of sequences around the tRNA-Val gene strongly suggested that S23321 contains an ancestral-type genome that precedes the acquisition of a symbiosis island by horizontal gene transfer. Although S23321 contains a nif (nitrogen fixation) gene cluster, the organization, homology, and phylogeny of the genes in this cluster were more similar to those of photosynthetic bradyrhizobia ORS278 and BTAi1 than to those on the symbiosis island of USDA110. In addition, we found genes encoding a complete photosynthetic system, many ABC transporters for amino acids and oligopeptides, two types (polar and lateral) of flagella, multiple respiratory chains, and a system for lignin monomer catabolism in the S23321 genome. These features suggest that S23321 is able to adapt to a wide range of environments, probably including low-nutrient conditions, with multiple survival strategies in soil and rhizosphere.

Genome Analysis Suggests that the Soil Oligotrophic Bacterium Agromonas oligotrophica (Bradyrhizobium oligotrophicum) Is a Nitrogen-Fixing Symbiont of Aeschynomene indica

ABSTRACT Agromonas oligotrophica (Bradyrhizobium oligotrophicum) S58T is a nitrogen-fixing oligotrophic bacterium isolated from paddy field soil that is able to grow in extra-low-nutrient environments. Here, the complete genome sequence of S58 was determined. The S58 genome was found to comprise a circular chromosome of 8,264,165 bp with an average GC content of 65.1% lacking nodABC genes and the typical symbiosis island. The genome showed a high level of similarity to the genomes of Bradyrhizobium sp. ORS278 and Bradyrhizobium sp. BTAi1, including nitrogen fixation and photosynthesis gene clusters, which nodulate an aquatic legume plant, Aeschynomene indica, in a Nod factor-independent manner. Although nonsymbiotic (brady)rhizobia are significant components of rhizobial populations in soil, we found that most genes important for nodule development (ndv) and symbiotic nitrogen fixation (nif and fix) with A. indica were well conserved between the ORS278 and S58 genomes. Therefore, we performed inoculation experiments with five A. oligotrophica strains (S58, S42, S55, S72, and S80). Surprisingly, all five strains of A. oligotrophica formed effective nitrogen-fixing nodules on the roots and/or stems of A. indica, with differentiated bacteroids. Nonsymbiotic (brady)rhizobia are known to be significant components of rhizobial populations without a symbiosis island or symbiotic plasmids in soil, but the present results indicate that soil-dwelling A. oligotrophica generally possesses the ability to establish symbiosis with A. indica. Phylogenetic analyses suggest that Nod factor-independent symbiosis with A. indica is a common trait of nodABC- and symbiosis island-lacking strains within the members of the photosynthetic Bradyrhizobium clade, including A. oligotrophica.

Analysis of the Bacterial Community according to Colony Development on Solid Medium

Bacterial communities of paddy and grassland soils were studied by a plate count method. The colony formation processes were simulated by a superposition of the first order reaction (FOR) model curves. Isolates from colonies formed along each FOR curve were collected as a colony forming curve (CFC) group. Sequencing 16SrDNA base sequences revealed that isolates had diverse phylogenetic positions and those of each CFC group showed a trend to make clusters. Bacteria in microaggregate clumps (ca. 0.1 mg) also showed CFC groups similar to those obtained with Ig of soil and were phylogenetically diverse. Based on the results we propose to catalogue all the bacteria of the community in the microaggregates.