Deoxyribonucleate Binding and Transformation in Rhizobium japonicum

Rhizobium japonicum, capable of binding high-molecular-weight donor (32)P-labeled deoxyribonucleic acid (DNA) during late log phase in a competence medium, was transformed for streptomycin resistance with a frequency of transformation ranging between 0.02 and 0.08%. Eight to 10% of the homologous native (32)P-labeled input DNA was bound irreversibly in a temperature-dependent manner. Homologous denatured (32)P-labeled DNA was incapable of binding to the recipient under similar conditions. CsCl density gradient banding of the donor and recipient DNA indicated homology. The low frequency of transformation could be due to one or more steps that occur between DNA uptake and integration.     

L-Arabinose metabolism in Rhizobium japonicum

l-Arabinose was metabolized through an oxidative pathway by extracts of a strain of Rhizobium japonicum. The findings showed that l-arabinose is converted into 2-keto-3-deoxy-l-arabonate, which is cleaved into glycoaldehyde and pyruvate.     

Gluconate Catabolism in Rhizobium japonicum

Gluconate catabolism in Rhizobium japonicum ATCC 10324 was investigated by the radiorespirometric method and by assaying for key enzymes of the major energy-yielding pathways. Specifically labeled gluconate gave the following results for growing cells, with values expressed as per cent (14)CO(2) evolution: C-1 = 93%, C-2 = 57%, C-3 = 30%, C-4 = 70%, C-6 = 39%. The preferential release of (14)CO(2) from C-1 and C-4 indicate that gluconate is degraded primarily by the Entner-Doudoroff pathway but the inequalities between C-1 and C-4 and between C-3 and C-6 indicate that another pathway(s) also participates. The presence of gluconokinase and a system for converting 6-phosphogluconate to pyruvate also indicate a role for the Entner-Doudoroff pathway. The extraordinarily high yield of (14)CO(2) from C-1 labeled gluconate suggests that the other participating pathway is a C-1 decarboxylative pathway. The key enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase, could not be demonstrated. Specifically labeled 2-ketogluconate and 2,5-diketogluconate were oxidized by gluconate grown cells and gave ratios of C-1 to C-6 of 2.73 and 2.61, respectively. These compare with a ratio of 2.39 obtained with specifically labeled gluconate. Gluconate dehydrogenase, the first enzyme in the ketogluconate pathway found in acetic acid bacteria, was found. Oxidation of specifically labeled pyruvate, acetate, succinate, and glutamate by gluconate-grown cells yielded the preferential rates of (14)CO(2) evolution expected from the operation of the tricarboxylic acid cycle. These data are consistent with the operation of the Entner-Doudoroff pathway and tricarboxylic acid cycle as the primary pathways of gluconate oxidation in R. japonicum. An ancillary pathway for the initial breakdown of gluconate would appear to be the ketogluconate pathway which enters the tricarboxylic acid cycle at alpha-ketoglutarate.     

Streptomycin resistance in Rhizobium japonicum

Mutants resistant to varying concentrations of streptomycin were recovered from two streptomycin-sensitive, effective nitrogen-fixing strains of Rhizobium japonicum. To determine if there were an upper limit of resistible antibiotic concentration, 3 mutants which were resistant to 10000 μg/ml were challenged by higher concentrations of streptomycin. Only one grew well at 25000 μg/ml, and none grew at 50000 μg/ml. All mutants maintained a smooth colonial morphology, and none exhibited streptomycin-dependence. Streptomycin-resistant mutants of both strains were examined for properties of infectivity and effectiveness. All mutants tested retained the symbiotic properties of the parental strains. The retention of these parental properties by the streptomycin-resistant mutants of R. japonicum is different from the properties described for phenotypically similar mutants in certain other rhizobial species.     

Glucose Catabolism in Rhizobium japonicum

Glucose catabolism in Rhizobium japonicum ATCC 10324 was investigated by the radiorespirometric method and by assaying for key enzymes of the major energy-yielding pathways. Specifically labeled glucose gave the following results for resting cells, with values expressed as per cent (14)CO(2) evolution: C-1=59%, C-2=51%, C-3=45%, C-4=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%, C-4=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 (14)CO(2) 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 (14)CO(2) evolution to be C-1 > C-2=C-5 > C-3, 4, whereas a higher yield of (14)CO(2) 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.     

Protoporphyrin formation in Rhizobium japonicum.

The obligately aerobic soybean root nodule bacterium Rhizobium japonicum produces large amounts of heme (iron protoporphyrin) only under low oxygen tensions, such as exist in the symbiotic root nodule. Aerobically incubated suspensions of both laboratory-cultured and symbiotic bacteria (bacteroids) metabolize delta-aminolevulinic acid to uroporphyrin, coproporphyrin, and protoporphyrin. Under anaerobic conditions, suspensions of laboratory-cultured bacteria form greatly reduced amounts of protoporphyrin from delta-aminolevulinic acid, whereas protoporphyrin formation by bacteroid suspensions is unaffected by anaerobiosis, suggesting that bacteroids form protoporphyrin under anaerobic conditions more readily than do free-living bacteria. Oxygen is the major terminal electron acceptor for coproporphyrinogen oxidation in cell-free extracts of both bacteroids and free-living bacteria. In the absence of oxygen, ATP, NADP, Mg2+, and L-methionine are required for protoporphyrin formation in vitro. In the presence of these supplements, coproporphyrinogenase activity under anaerobic conditions is 5 to 10% of that observed under aerobic conditions. Two mechanisms for coproporphyrinogen oxidation exist in R. japonicum: an oxygen-dependent process and an anaerobic oxidation in which electrons are transferred to NADP. The significance of these findings with regard to heme biosynthesis in the microaerophilic soybean root nodule is discussed.     

Cytokinin Production by Rhizobium japonicum

Possible hormonal interactions between soybean roots and the Rhizobium initiating nodule proliferation in this genus were studied. A cytokinin has been isolated by column and paper chromatography from an effective strain of Rhizobium japonicum grown in pure culture. The substance promotes cell proliferation in a cytokinin-requiring soybean callus tissue. The bacteria are capable of conditioning a cylokinin-free soybean culture medium so that it is able to support the cytokinin-requiring tissue. It is concluded that the substance is a product of the bacteria rather than an artifact of purification. This unidentified cytokinin or a substance moving to a similar Rf value on the paper chromalogram produces polyploid divisions when tested on cultured pea root segments. Some of the division figures exhibit the diploclirornosomes typical of the root nodule primordium in pea.     

Tryptophan auxotrophs of Rhizobium japonicum

Eleven tryptophan-requiring mutants of Rhizobium japonicum I-110 ARS were isolated after nitrous acid mutagenesis and fell into five groups based on characterization by supplementation with intermediates and enzyme assays.     

Induced plasmid-genome rearrangements in Rhizobium japonicum.

The P group resistance plasmids RP1 and RP4 were introduced into Rhizobium japonicum by polyethylene-glycol-induced transformation of spheroplasts. After cell wall regeneration, transformants were recovered by selecting for plasmid determinants. Plant nodulation, nitrogen fixation, serological, and bacterial genetics studies revealed that the transformants were derived from the parental strains and possessed the introduced plasmid genetic markers. Agarose gel electrophoresis, restriction enzyme analysis, and DNA hybridization studies showed that many of the transformant strains had undergone genome rearrangements. In the RP1 transformants, chromosomal DNA was found to have transposed into a large indigenous plasmid of R. japonicum, producing an even larger plasmid, and the introduced R plasmid DNA was found to be chromosomally integrated rather than replicating autonomously or integrated into the endogenous plasmid. Seemingly, a similar section of chromosomal DNA was involved in all the genomic rearrangements observed in the R. japonicum RP1 and RP4 transformant strains.     

Multiple antibiotic resistance in Rhizobium japonicum.

A total of 48 strains of the soil bacterium Rhizobium japonicum were screened for their response to several widely used antibiotics. Over 60% of the strains were resistant to chloramphenicol, polymyxin B, and erythromycin, and 47% or more of the strains were resistant to neomycin and penicillin G, when tested by disk assay procedures. The most common grouping of resistances in strains was simultaneous resistance to tetracycline, penicillin G, neomycin, chloramphenicol, and streptomycin (25% of all strains tested). The occurrence of multiple drug resistance in a soil bacterium that is not a vertebrate pathogen suggests that chemotherapeutic use of antibiotics is not required for the development of multiple drug resistance.     

Multiple antibiotic resistance in Rhizobium japonicum.

A total of 48 strains of the soil bacterium Rhizobium japonicum were screened for their response to several widely used antibiotics. Over 60% of the strains were resistant to chloramphenicol, polymyxin B, and erythromycin, and 47% or more of the strains were resistant to neomycin and penicillin G, when tested by disk assay procedures. The most common grouping of resistances in strains was simultaneous resistance to tetracycline, penicillin G, neomycin, chloramphenicol, and streptomycin (25% of all strains tested). The occurrence of multiple drug resistance in a soil bacterium that is not a vertebrate pathogen suggests that chemotherapeutic use of antibiotics is not required for the development of multiple drug resistance.     

Regulation of hydrogenase in Rhizobium japonicum.

Factors that regulate the expression of an H2 uptake system in free-living cultures of Rhizobium japonicum have been investigated. Rapid rates of H2 uptake by R. japonicum were obtained by incubation of cell suspensions in a Mg-phosphate buffer under a gas phase of 86.7% N2, 8.3% H2, 4.2% CO2, and 0.8% O2. Cultures incubated under conditions comparable with those above, with the exception that Ar replaced H2, showed no hydrogenase activity. When H2 was removed after initiation of hydrogenase derepression, further increase in hydrogenase activity ceased. Nitrogenase activity was not essential for expression of hydrogenase activity. All usable carbon substrates tested repressed hydrogenase formation, but none of them inhibited hydrogenase activity. No effect on hydrogenase formation was observed from the addition of KNO3 or NH4Cl at 10 mM. Oxygen repressed hydrogenase formation, but did not inhibit activity of the enzyme in whole cells. The addition of rifampin or chloramphenicol to derepressed cultures resulted in inhibition of enzyme formation similar to that observed by O2 repression. The removal of CO2 during derepression caused a decrease in the rate of hydrogenase formation. No direct effect of CO2 on hydrogenase activity was observed.     

Mutants of Rhizobium japonicum defective in nodulation

Several mutants defective in nodulation were isolated from Rhizobium japonicum strains 3I1b110 and 61A 76. Mutants of class I do not form nodules after incubation with soybean [Glycine max (L.) Merrill] for 17 days, but will do so by 28 days. When host plants other than G. max are infected with several of these strains, there is no detectable difference in the time of nodulation or size of nodules as compared to the wild type. Two mutants of class I (i. e., SM1 and SM2) have been shown previously to be altered in the lipopolysaccharide portion of their cell wall. Mutants of class II are not slow to nodulate but form fewer nodules than the wild type on all the host plants tested. Mutants of class III are unable to form nodules. Some bacteriophage-resistant mutants, altered in cell surface structure, fall into this class. Two mutants of class III do not bind to soybean roots.