The genetic basis for resistance to a number of antibiotics was examined in Rhizobium japonicum . Resistance to penicillin G, neomycin, and chloramphenicol appears to be mediated by an extrachromosomal element similar to that found in the Enterobacteriaceae . Resistance to these antibiotics was eliminated from cells by treatment with acridine orange, and resistance to all three antibiotics could be transferred en bloc to Agrobacterium tumefaciens under conditions excluding transformation or transduction as possible genetic mechanisms.

The symbiotic nitrogen-fixing bacteria associated with soybeans (Glycine max) have been classified routinely as R hizobium japonicum. DNA : DNA hybridization studies were conducted to estimate the genetic relationships of strains labelled as R. japonicum to clarify their taxonomic status. Reassociation reactions were carried out using DNA from various R hizobium and Agrobacterium species. Relationships of DNA sequences were also estimated by comparing the thermal stability of heterologous DNA duplexes with that of the homologous DNA duplex. The results indicated that strains labelled as R. japonicum can be separated into at least three DNA homology groups. Reference strains of other Rhizobium species, with the exception of R. Zupini, were not closely related to these homology groups.

A new species, Rhizobium fredii, is proposed for fast-growing root nodule bacteria isolated from soybeans. The type strain was isolated from a root nodule of Glycine mnx growing in Honan Province, China, and is designated strain USDA 205 (= ATCC 35423 = PRC 205). This new species is differentiated from currently recognized Rhizobium and Bradyrhizobium species by deoxyribonucleic acid hybridization comparisons, plant specificity, generation times, antibiotic resistance, and serology. The strains of R. fredii are differentiated into two proposed chemovars, R. fredii chemovar fredii chemovar nov. and R . fredii chemovar siensis chemovar nov., by deoxyribonucleic acid hybridization tests, growth in the presence of erythromycin (20 kg/ml), final pH of yeast extract-mannitol medium, and serology.Bacteria which form effective nitrogen-fixing nodules on the roots of leguminous plants are presently classified into two genera, Rhizobium and Bradyrhizobium (6, 8). Differentiation of these genera is based upon the cross-inoculation group concept (6,8). The distinguishing features between the two genera are differences in physiology when the organisms are grown on medium containing yeast extract and mannitol and the plant host groups which are nodulated. These differences have been reviewed elsewhere (3, 4, 6, 8). Rhizobium spp. are considered to be fast growing, with generation times of less than 6 h, whereas Bradyrhizobium strains have generation times in excess of 6 h (6, 8). The genus Rhizobium presently includes three species, Rhizobium meliloti, Rhizobium loti (3, and Rhizobium leguminosarum (8), which includes the former species Rhizobium trifolii and Rhizobium phaseoli (7). Bradyrhizobium presently has one species, Brpdyrhizobium japonicum, and includes strains that are capable of effectively nodulating lupines and soybeafis (6). Also included within the bradyrhizobia is the cowpea miscellany (6).Recently, a new group of fast-growing rhizobia which nodulate soybeans was isolated from mainland China (9). A list of these strains and appropriate references are shown in Table 1. Comparative studies of growth rate and acid production on yeast extract-mdnnitol medium (YEM) (9, 13, 15), plant cross-inoculation (9, 15), plasmid profiles (10, 12), location of nitrogen fixation ( n i f ) genes (lo), location of nodulation genes (12), susceptibility to ahtibiotics (15), physiological characteristics (9, 13, 15), vitamin requirements (15), tolerance to NaCl (13, 15), diffentiation on litmus milk (13), and deoxyribonucleic acid (DNA) hybridization (14) indicate that this group has cultural characteristics of species in the fast-growing genus Rhizobium and symbiotic characteristics of species in the slow-growing genus Bradyrhizobium. DNA homology indicates that these fast-growing, soybean-nodulating strains are not genetically related to the type strain of any of the previously recognized species, but the strains in each proposed chemovar are related among themselves (14). Several investigators have indicated that this group of ba...

Symbiotic gene diversity and other measures of genetic diversity were examined in Bradyrhizobium isolates that form an effective symbiosis with peanut (Arachis hypogaea). Initially, restriction fragment length polymorphism (RFLP) analysis using a nitrogenase (nif) gene probe was performed on 33 isolates along with one Bradyrhizobium elkanii and two Bradyrhizobium japonicum strains. Considerable diversity was observed among the RFLP patterns of many of the isolates, especially those from South America. Some isolates, however, were found to have similar nif and subsequent nod (nodulation) gene RFLP patterns, indicating symbiotic gene relatedness. With some noted exceptions, symbiotic gene relatedness correlated with relatedness based on total DNA homology and ribotyping analyses. Symbiotic gene relatedness also correlated with symbiotic effectiveness. The RFLP and DNA homology analyses indicate that bradyrhizobia effective with peanut are genetically diverse and consist of at least three different species. This diversity, however, was not particularly evident with partial 16S rRNA gene sequencing. Sequences obtained from the isolates were very similar to each other as well as to sequences previously reported for other Bradyrhizobium strains.

Glucose catabolism in Rhizobium japonicum ATCC 10324 was investigated by the radiorespirometric method and by assaying for key enzymes of the major energyyielding pathways. Specifically labeled glucose gave the following results for resting cells, with values expressed as per cent 14CO2 evolution: C-1 = 59%, C-2 = 51%, C-3 =45%, C4 = 59%, and C-6 = 43%. These values indicate that glucose was degraded by the Entner-Doudoroff pathway alone. Cells which grew in glucose-yeast extract-salts medium gave essentially the same pattern except for retardation of the C-6 carbon. The rates were: C-1 = 54%, C-2 = 42%, C-3 = 51%, C4 = 59%, and C-6 =32%. Hexokinase, glucose-6-phosphate dehydrogenase, transketolase, and an enzyme system which produces pyruvate from 6-phosphogluconate were found to be present in these cells. No 6-phosphogluconate dehydrogenase was detected. Oxidation of specifically labeled pyruvate gave the following 14CO2 evolution pattern: C-1 =78%, C-2 =48%, and C-3 = 37%; the pattern from acetate was C-1 = 73%; and C-2 = 56%. Oxidation of glutamate showed the preferential rate of "4CO2 evolution to be C-1 > C-2 =C-5 > C-3, 4, whereas a higher yield of "(CO2 was obtained from the C-1 and C-4 carbons of succinate than from the C-2 and C-3 carbons. These data are consistent with the operation of the Entner-Doudoroff pathway and tricarboxylic acid cycle as the catabolic pathways of glucose oxidation in R. japonicum.