Genetic mapping of symbiotic loci on the Rhizobium meliloti chromosome.

To facilitate genetic analyses of Rhizobium meliloti genes that are involved in symbiosis, we determined the map positions of 11 symbiotic loci on the R. meliloti chromosome by using a combination of the Tn5-Mob conjugational transfer method described by Klein et al. (S. Klein, K. Lohmann, G. C. Walker, and E. R. Signer. J. Bacteriol. 174:324-326, 1992) and co-transduction of genetic markers by bacteriophage phi M12. Loci involved in effective nodule formation (fix-379, fix-382, fix-383, fix-385, and fix-388), polysaccharide synthesis (exoR, exoS, exoC, and ndvB), nodule invasion (exoD), and nitrogen regulation (ntrA) were ordered with respect to previously mapped markers and each other. The positions of two other loci, degP and pho-1, were also determined.     

Genetic mapping of the chromosome of Rhizobium trifolii.

Cultures of the wild strain and auxotrophic mutants of Rhizobium trifolii T37 synchronized by means of phenylethanol have been mutagenized with nitrosoguanidine. Fifteen genetic markers were characterized in respect of their order and the time of replication based on the peaks of mutations of the genes. The time of R. trifolii chromosome replication was estimated using inhibitors of the initiation of DNA replication: rifampicin, chloramphenicol and phenylethanol. The replicative map of R. trifolii chromosome has been constructed. Taking into account the replicative map, linkages of the genes, and the bidirectional model of the Rhizobium chromosome replication, a circular genetic map of the chromosome of R. trifolii T37 was elaborated.     

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.     

Genetic mapping of isozyme loci in Secale cereale L.

Summary The genetics and linkage relationships of several isozymatic and morphological markers have been investigated in different cultivars of rye (Secale cereale L.). The inheritance and the variability among cultivars of three new isozymatic zones are described: GOT2 and LAP, each of them under the control of a two-allele single locus, namely Got2 and Lap, respectively; and 6PGD1 controlled by two loci, 6Pgd1a and 6Pgd1b, which have alleles in common. Four linkage groups have been found: Acp2-Acp3, Got3-Mdh2-Lper4, Mdh1-6Pgd2-Pgi2, and Pgm-Eper2-[Eper1-Eper3]. The assignment of these four groups to the chromosomes 7R, 3R, 1R, and 4R is discussed.     

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.     

Transformation in Rhizobium japonicum

Summary The transformation of streptomycin resistance in Rhizobium japonicum was studied. The susceptible strain 211 was selected from sixty strains and one step mutant resistant to streptomycin in concentration 1 mg per 1 ml was used as the donor. The peak of the competence curve appeared at the ninth hour of growth; the frequency, when the homologous strain had been used was 0.01 p.c. The transformed resistance was of the same level as in the donor strain.This investigation forms part of a contribution prepared by the Czechoslovak National Committee for the International Biological Programme (Section PP: Production Processes).     

Genetic locus in Rhizobium japonicum (fredii) affecting soybean root nodule differentiation.

A genetic locus in fast-growing Rhizobium japonicum (fredii) USDA 191 (Fix+ on several contemporary soybean cultivars) was identified by random Tn5 mutagenesis as affecting the development and differentiation of root nodules. This mutant (MU042) is prototrophic and shows no apparent alterations in its surface properties. It induces aberrant nodules, arrested at the same early level of differentiation, on all its host plants. An 8.1-kilobase EcoRI fragment containing Tn5 was cloned from MU042. In USDA 191 as well as another fast-growing strain, USDA 201, the affected locus was found to be unlinked to the large symbiotic plasmid and appears to be chromosomal. An analogous sequence has been shown to be present in Bradyrhizobium japonicum (J. Stanley, G.G. Brown, and D.P.S. Verma, J. Bacteriol. 163:148-154, 1985) as well as in R. trifolii and R. meliloti. MU042 was complemented for effective nodulation of soybean by a cosmid clone from USDA 201, and the complementing locus was delimited to a 6-kilobase EcoRI subfragment. An R. trifolii strain (MU225), whose indigenous symbiotic plasmid was replaced by that of strain USDA 191, induced more highly differentiated nodules on soybean than did MU042. This suggests that the mutation in MU042 can be functionally substituted by similar loci of other fast-growing rhizobia. Leghemoglobin and nodulin-35 (uricase II) were present in the differentiated Fix- nodules induced by MU225, whereas both were absent in MU042-induced pseudonodule structures.     

Genetic mapping of ripening and ethylene-related loci in tomato

 The regulation of tomato fruit development and ripening is influenced by a large number of loci as demonstrated by the number of existing non-allelic fruit development mutations and a multitude of genes showing ripening-related expression patterns. Furthermore, analysis of transgenic and naturally occurring tomato mutants confirms the pivotal role of the gaseous hormone ethylene in the regulation of climacteric ripening. Here we report RFLP mapping of 32 independent tomato loci corresponding to genes known or hypothesized to influence fruit ripening and/or ethylene response. Mapped ethylene-response sequences fall into the categories of genes involved in either hormone biosynthesis or perception, while additional ripening-related genes include those involved in cell-wall metabolism and pigment biosynthesis. The placement of ripening and ethylene-response loci on the tomato RFLP map will facilitate both the identification and exclusion of candidate gene sequences corresponding to identified single gene and quantitative trait loci contributing to fruit development and ethylene response. Received: 26 October 1998 / Accepted: 13 November 1998     

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.     

Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum.

Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.     

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.     

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.     

Genetic mapping of rust resistance loci in biomass willow

Rust diseases caused by Melampsora spp. represent a major threat to the productivity of short rotation coppice (SRC) willows grown for biomass, causing yield losses of up to 40%. The routine use of fungicide in SRC plantations is not a viable option because of economic and environmental considerations; thus, breeding for rust resistance is a major target for willow breeding programmes. To characterise the genetic basis of rust resistance in willow and provide targets for use in future marker-assisted selections, quantitative trait analyses were performed using a large full-sib mapping population (K8) which segregates for rust resistance and several other important agronomic traits. Rust resistance in field conditions was assessed in three consecutive years. For a more detailed genetic dissection, laboratory inoculation tests using isolates of two distinct and prevalent pathotypes (LET1 and LET5) were also performed. For field-based resistance, a major quantitative resistance locus, designated SRR1 (Salix Rust Resistance 1), was detected in addition to several quantitative trait loci (QTL) of more modest effect. Inoculation test data also supported an important role for SRR1. Specific interactions between particular rust isolates and different QTL were detected, and QTL that only influenced resistance in field conditions were identified. The QTL reported here represent an important basis for the future development of markers for use in willow breeding programmes. As the linkage map for the K8 population is anchored to the Populus trichocarpa genome sequence, a more efficient marker development for future fine-scale mapping and candidate gene identification is possible.