Transfer of R factors to and between genetically marked sublines of Rhizobium japonicum.

Plasmids R1822 and pRD1 of the P-1 incompatibility group, for which Rhizobium japonicum had not previously been shown to serve as host, were introduced into a strain of R. japonicum. Acquisition of R68 and R68.45 plasmids by this Rhizobium was equivocal. Transfer of R1822 from Pseudomonas aeruginosa and of pRD1 from Escherichia coli to R. japonicum was unambiguous, because the exconjugants subsequently cotransferred the three R-factor resistance determinants (kanamycin, tetracycline, and penicillin) between genetically marked sublines of strain I-110. Under optimal conditions the transfer of R1822 and pRD1 occurred at frequencies of approximately 10(-3) in plate matings of strains bearing as many as five dissimilar genetic markers. In matings with R1822 on membrane filters, recombinants were formed at incidences as high as 4%.     

Transfer of RP4 and R68.45 factors to Rhizobium.

Two R factor were introduced by conjugation into Rhizobium trifolii and Rhizobium meliloti strains at a frequency of 10(-5) to 10(-6). Plasmids RP4 from Escherichia coli J53 and R68.45 from Pseudomonas aeruginosa PAO.25 were maintained stably in Rhizobium hosts and could be retransferred to other Rhizobium recipients. Some of the transconjugants were able to mobilize chromosome and transfer his or met genes in intra-, and interspecies matings.     

Genetically marked Rhizobium identifiable as inoculum strain in nodules of soybean plants grown in fields populated with Rhizobium japonicum.

The fate of an inoculum strain of Rhizobium japonicum was studied using a genetically marked strain I-11O subline carrying resistance markers for azide, rifampin, and streptomycin (I-110 ARS). At the time of planting into a field populated with R. japonicum, seeds of soybean cultivars Kent and Peking were inoculated with varying cell densities of strain I-110 ARS. At various times during the growing season, surface-sterilized root nodules were examined for the presence of the inoculum strain by plating onto selective media. The recovery of the inoculum strain was unambiguous, varying, in the case of Kent cultivar, from about 5% with plants (sampled at 51 days) that had been inoculated with 3 X 10(8) cells per cm of row to about 20% with plants (sampled at 90 days) that had been inoculated with 3 X 10(9) cells per cm. The symbiotically incompatible interaction of Peking and strain 110 in Rhizobium-populated field soil was confirmed by the finding that at 60 days after planting, only one nodule in 360 sampled contained strain I-110 ARS. The use of genetically marked Rhizobium bacteria was found to provide for precise identification of the inoculum strain in nodules of field-grown soybeans.     

Transfer from Rhizobium japonicum to Azotobacter vinelandii of genes required for nodulation

A mutant strain of Azotobacter vinelandii that is unable to fix N2 (Nif-) was transformed to Nif+ with DNA from Rhizobium japonicum. Of 50 Nif+ transformants tested, 3 contained the O antigen-related polysaccharide that is present on the cell surface of a nodulating R. japonicum strain, but is absent from a non-nodulating mutant strain.     

Genetically marked Rhizobium identifiable as inoculum strain in nodules of soybean plants grown in fields populated with Rhizobium japonicum

The fate of an inoculum strain of Rhizobium japonicum was studied using a genetically marked strain I-11O subline carrying resistance markers for azide, rifampin, and streptomycin (I-110 ARS). At the time of planting into a field populated with R. japonicum, seeds of soybean cultivars Kent and Peking were inoculated with varying cell densities of strain I-110 ARS. At various times during the growing season, surface-sterilized root nodules were examined for the presence of the inoculum strain by plating onto selective media. The recovery of the inoculum strain was unambiguous, varying, in the case of Kent cultivar, from about 5% with plants (sampled at 51 days) that had been inoculated with 3 X 10(8) cells per cm of row to about 20% with plants (sampled at 90 days) that had been inoculated with 3 X 10(9) cells per cm. The symbiotically incompatible interaction of Peking and strain 110 in Rhizobium-populated field soil was confirmed by the finding that at 60 days after planting, only one nodule in 360 sampled contained strain I-110 ARS. The use of genetically marked Rhizobium bacteria was found to provide for precise identification of the inoculum strain in nodules of field-grown soybeans.     

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.     

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.     

Nif- Hup- mutants of Rhizobium japonicum.

Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity.     

Relationship between Raman spectroscopic lines and growth of Rhizobium japonicum

The Raman spectroscopic lines of liquid cultures of Rhizobium japonicum have been compared with electron microscopic examinations and growth measurements of these cells. The results showed that the significant Raman lines are related to the reproduction activities of the procaryotic cells.     

Competitiveness of Rhizobium sp. [Leucaena leucocephala (Lam.) Dewit] strains and their genetically marked derivatives

D.M. SWELIM, L.D. KUYKENDALL, F.M. HASHEM, S.M. ABDEL-WAHAB AND N.I. HEGAZI. 1996. The competitiveness of wild-type strains of Rhizobium sp. ( Leucaena ) and their genetically marked double mutants was examined in mixed infection experiments in the greenhouse. Antibiotic resistance markers were selected for use in strain identification, but these genetic markers apparently lowered both competitiveness and effectiveness, except in the case of strain DS 144/2 where the genetically marked derivative was evidently superior to the wild-type parent strain in effectiveness. Four wild-type strains and their genetically marked derivatives were carefully evaluated using double reciprocal pairs, the results of which nevertheless allowed the formulation of some conclusions. Strains DS 65 and DS 78 were more competitive than strain DS 144/2; only strain DS 78 was more competitive than DS 158; and strains DS 158 and DS 65 were equally competitive. There was no correlation between nodule number and competitiveness. Shoot dry weight and nitrogen mass, as well as nitrogenase activity, decreased with some strain mixtures indicating that relatively ineffective symbioses had formed, as compared with single-strain inoculations using symbiotically competent 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.     

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.     

Bacteriophage that can distinguish between wild-type Rhizobium japonicum and a non-nodulating mutant.

A bacteriophage (phage TN1) that lyses Rhizobium japonicum 3I1b110 was isolated from Tennessee soil. Structurally, this phage resembles the Escherichia coli phage T4, having an icosahedral head (47 by 60 nm) and a contractile tail (17 by 80 nm). An interesting feature of this phage is that it lyses all of the symbiotic defective mutants derived from R. japonicum 3I1b110 that were tested, except one, mutant strain HS123. Mutant strain HS123 is a non-nodulating mutant that is defective in attachment to soybean roots. Since Rhizobium attachment to host roots is thought to be mediated by a specific cell surface interaction, it is likely that mutant strain HS123 is defective in some way in its cell surface. Mutant strain HS123 bound soybean lectin to the same extent as the wild type as measured by the binding of tritium-labeled lectin. Phage TN1 did not attach to the surface of strain HS123, nor did cells of strain HS123 inactivate phage TN1. A hot phenol-water cell extract from the wild-type inactivated phage TN1, whereas a similar cell extract from mutant HS123 did not. Capsular polysaccharide isolated from mutant or wild type did not inactivate the phage. Capsular polysaccharide and exopolysaccharide from the mutant and wild type do not differ in sugar composition. These results indicate that capsular polysaccharide may not play a role in attachment to the plant root surface and that other cell wall components may be important.     

Interaction between Rhizobium japonicum phage M-1 and its receptor

The receptor for phage M-1 was present in the exopolysaccharide (EPS) of Rhizobium japonicum D211. The EPS was a heteropolysaccharide consisting of glucose, galactose, glucuronic acid, and glucosamine units. These monosaccharides prevented phage-cell attachment indicating that they may mimick the receptor. Phage-cell attachment was either stimulated or inhibited by Mg2+ and Ca2+ depending upon their concentration. An enzyme which depolymerized the exopolysaccharide releasing oligosaccharides was detected in the phage-infected cell lysates. A comparison of the properties of adsorption and those of the depolymerase enzyme indicated that the latter was a component of the phage and appeared to be involved in the phage-receptor interaction.     

Ineffective and non-nodulating mutant strains of Rhizobium japonicum.

Mutant strains of Rhizobium japonicum that were unable to allow the Corsoy cultivar of soybean to reduce acetylene or fix N2 were isolated. These strains grow as well as the wild type in a variety of media. Mutant strains SM1 and SM2 did not form nodules on the host plant; however, they reduced acetylene in the nonsymbiotic assay. Strains SM3 and SM4 produced nodules that did not have the characteristic pink pigment caused by leghemoglobin. The nodules formed by these strains also were small. One mutant strain, SM5, produced large pink nodules. The lesion in this strain seems to be in the gene that specifies nitrogenase component II.     

Immunological homology between the membrane-bound uptake hydrogenases of Rhizobium japonicum and Escherichia coli.

Two polypeptides present in aerobic and anaerobic cultures of Escherichia coli HB101 were shown to cross-react with antibodies to the 30- and 60-kilodalton (kDa) subunits of the uptake hydrogenase of Rhizobium japonicum. The cross-reactive polypeptides in a series of different E. coli strains are of Mrs ca. 60,000 and 30,000, and both polypeptides are present in proportion to measurable hydrogen uptake (Hup) activity (r = 0.95). The 60-kDa polypeptide from E. coli HB101 comigrated on native gels with detectable Hup activity. The exact role of the 30-kDa polypeptide in E. coli is unclear. E. coli MBM7061, a natural Hup- variant, grown anaerobically or aerobically lacked detectable Hup activity and failed to cross-react with the antisera against the hydrogenase from R. japonicum. Anaerobically cultured E. coli MBM7061, however, did express formate hydrogenlyase activity, indicating that the hydrogenases involved in the oxygen-dependent activation of hydrogen and the formate-dependent evolution of hydrogen are biochemically distinct.     

Revertible hydrogen uptake-deficient mutants of Rhizobium japonicum.

We have developed mutants of Rhizobium japonicum which are deficient in H2 uptake capacity (Hup-) and which spontaneously revert to the parent type at a frequency consistent with that of a single-point mutation (ca. 1.0 x 10(-09)). The mutagenesis by nitrous acid and the selection of the Hup- phenotype by using penicillin and chemolithotrophy as enrichment for chemolithotrophy-deficient strains are described. Two mutants retain low but reproducible levels of ribulose bisphosphate-dependent CO2 fixation when grown on a low-carbon medium under an atmosphere of 1% O2, 4% H2, 5% CO2, and 90% N2. Neither O2 nor the artificial electron acceptors phenazine methosulfate or methylene blue supported detectable H2 uptake by the free-living Hup- mutants or by their bacteroids. Plant growth experiments under bacteriologically controlled conditions were conducted to assess the mutants' performance as inocula for soybean plants. Plants inoculated with Hup- strains had lower dry weights and contained less total N than did plants inoculated with the parent Hup+ strain. Use of either the Hup- mutants or the Hup+ parent strain as inocula, however, did not significantly affect the acetylene-reducing activity or the fresh weight of nodules. These results, obtained with apparently isogenic lines of H2 uptake-deficient R. japonicum, provide strong support for a beneficial role of the H2 uptake phenotype in legume symbiosis.     

Transfer of nitrate reductase genes of the cyanobacterium Nostoc muscorum into Rhizobium japonicum

Transformation of Rhizobium japonicum CB1809 was studied using DNA from the cyanobacterium Nostoc muscorum ATCC 27893. A spontaneous nitrate reductase deficient (Nar-) mutant (NR-6) of R. japonicum CB1809 was isolated with a frequency of 8.4 X 10(-7). Streptomycin (Sm) and Neomycin (Neo) resistance markers were introduced into strain NR-6, and the resulting strain was designated NR-6 SmR NeoR. Experiments with cyanobacterial DNA and live cells of strain NR-6 SmR NeoR indicated transformation of nitrate reductase (nar) genes of N. muscorum into this strain. This conclusion was supported by the reversion frequency of strain NR-6 SmR NeoR to Nar+ and the transformation frequency when recipient cells were exposed to N. muscorum DNA (with heat-treated DNA as control). Comparisons of growth, nitrate uptake, assimilatory nitrate reductase activity and nodulation of parent CB1809, NR-6 SmR NeoR and five transformant clones (Nar+) suggest that there may be considerable homology between the nar genes of R. japonicum CB1809 and N. muscorum.