Involvement of Genes on a Megaplasmid in the Acid-Tolerant Phenotype of Rhizobium leguminosarum Biovar Trifolii

The acid-tolerant Rhizobium leguminosarum biovar trifolii strain ANU1173 exhibited several new phenotypes when cured of its symbiotic (Sym) plasmid and the second largest megaplasmid. Strain P22, which has lost these two plasmids, had reduced exopolysaccharide production and cell mobility on TY medium. The parent strain ANU1173 was able to grow easily in laboratory media at pH 4.5, whereas the derivative strain P22 was unable to grow in media at a pH of <4.7. The intracellular pH of strain ANU1173 was 6.8 when the external pH was 4.5. In contrast, strain P22 had an acidic intracellular pH of <6.4 when the external pH was <5.5. Strain P22 had a dramatically increased membrane permeability to protons and decreased proton extrusion activity. Analysis with sodium dodecyl sulfate-polyacrylamide gels showed that strain P22 lacked a slow-migrating lipopolysaccharide (LPS) banding group which was present in the parent strain. Mobilization of the second largest megaplasmid of strain ANU1173 back into strain P22 restored the altered LPS structure and physiological characteristics of strain P22. Mobilization of the Sym plasmid of strain ANU1173 into strain P22 showed that the second largest megaplasmid of strain ANU1173 was required for the establishment of nitrogen-fixing nodules on Trifolium repens and Trifolium subterraneum. Furthermore, an examination of a large number of specific exopolysaccharide- or LPS-deficient Rhizobium mutants did not show a positive correlation between exopolysaccharide or LPS synthesis and acid tolerance.     

Rhizobium leguminosarum Biovar viciae 1-Aminocyclopropane-1-Carboxylate Deaminase Promotes Nodulation of Pea Plants

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.     

Construction of a Symbiotically Effective Strain of Rhizobium leguminosarum bv. trifolii with Increased Nodulation Competitiveness

Genes involved in nodulation competitiveness (tfx) were inserted by marker exchange into the genome of the effective strain Rhizobium leguminosarum bv. trifolii TA1. Isogenic strains of TA1 were constructed which differed only in their ability to produce trifolitoxin, an antirhizobial peptide. Trifolitoxin production by the ineffective strain R. leguminosarum bv. trifolii T24 limited nodulation of clover roots by trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii. The trifolitoxin-producing exconjugant TA1::10-15 was very competitive for nodulation on clover roots when coinoculated with a trifolitoxin-sensitive reference strain. The nonproducing exconjugant TA1::12-10 was not competitive for nodule occupancy when coinoculated with the reference strain. Tetracycline sensitivity and Southern analysis confirmed the loss of vector DNA in the exconjugants. Trifolitoxin production by TA1::10-15 was stable in the absence of selection pressure. Transfer of tfx to TA1 did not affect nodule number or nitrogenase activity. These experiments represent the first stable genetic transfer of genes involved in nodulation competitiveness to a symbiotically effective Rhizobium strain.     

Higher Diversity of Rhizobium leguminosarum Biovar viciae Populations in Arable Soils than in Grass Soils

The bacterial genetic diversity after long-term arable cultivation was compared with that under permanent grassland using replicated paired contrasts. Pea-nodulating Rhizobium leguminosarum populations were sampled from pairs of arable and grass sites at four locations in Yorkshire, United Kingdom. Isolates were characterized using both chromosomal (16S-23S ribosomal DNA internal transcribed spacer PCR-restriction fragment length polymorphism) and plasmid (group-specific repC PCR amplification) markers. The diversities of chromosomal types, repC profiles, and combined genotypes were calculated using richness in types (adjusted to equal sample sizes by rarefaction), Shannon-Wiener index, and Simpson's index. The relative differences in diversity within each pair of sites were similar for all three diversity measures. Chromosomal types, repC profiles, and combined genotypes were each more diverse in arable soils than in grass soils at two of the four locations. The other comparisons showed no significant differences. We conclude that rhizobial diversity can be affected by differences between these two management regimens. Multiple regression analyses indicated that lower diversity was associated with high potential nitrogen and phosphate levels or with acidity.     

Compatibility of Rhizobial Genotypes within Natural Populations of Rhizobium leguminosarum Biovar viciae for Nodulation of Host Legumes

Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.     

Uptake Hydrogenase Activity Determined by Plasmid pRL6JI in Rhizobium leguminosarum Does Not Increase Symbiotic Nitrogen Fixation

We examined three groups of wild baboons (Papio cynocephalus) in Amboseli National Park, Kenya, to determine the prevalence of aerobic antibiotic-resistant fecal bacteria in nonhuman primates with and without contact with human refuse. Using standard isolation and replica plating techniques, we found only low numbers of antibiotic-resistant gram-negative enteric bacteria in two groups of baboons leading an undisturbed existence in their natural habitat and having limited or no contact with humans. However, resistance was significantly higher among enteric bacteria from the third group of baboons living in close proximity to a tourist lodge and having daily contact with unprocessed human refuse. Conjugation studies and analysis of the cell DNA by gel electrophoresis showed that in many cases resistance was plasmid-borne and transferable. These data suggest that wild nonhuman primates in frequent contact with human debris have a higher proportion of antibiotic-resistant enteric bacteria than do conspecifics without this contact. The findings further suggest that such groups of wild animals may constitute a heretofore overlooked source of antibiotic resistance in the natural environment.     

Uptake Hydrogenase Activity Determined by Plasmid pRL6JI in Rhizobium leguminosarum Does Not Increase Symbiotic Nitrogen Fixation

We examined three groups of wild baboons (Papio cynocephalus) in Amboseli National Park, Kenya, to determine the prevalence of aerobic antibiotic-resistant fecal bacteria in nonhuman primates with and without contact with human refuse. Using standard isolation and replica plating techniques, we found only low numbers of antibiotic-resistant gram-negative enteric bacteria in two groups of baboons leading an undisturbed existence in their natural habitat and having limited or no contact with humans. However, resistance was significantly higher among enteric bacteria from the third group of baboons living in close proximity to a tourist lodge and having daily contact with unprocessed human refuse. Conjugation studies and analysis of the cell DNA by gel electrophoresis showed that in many cases resistance was plasmid-borne and transferable. These data suggest that wild nonhuman primates in frequent contact with human debris have a higher proportion of antibiotic-resistant enteric bacteria than do conspecifics without this contact. The findings further suggest that such groups of wild animals may constitute a heretofore overlooked source of antibiotic resistance in the natural environment.     

Rhizobium leguminosarum Biovar viciae Symbiotic Hydrogenase Activity and Processing Are Limited by the Level of Nickel in Agricultural Soils

Analysis of levels of hydrogenase processing and activity in Rhizobium leguminosarum biovar viciae bacteroids from pea (Pisum sativum) plants showed that the oxidation of nitrogenase-evolved hydrogen is limited by the availability of nickel in agricultural soils. This limitation was overcome by using an inoculant strain engineered for higher hydrogenase expression.     

Construction of a gene bank of Rhizobium leguminosarum Rld 164

The total genomic DNA of R. leguminosarum Rld164 (trp, sms, azi) was cloned in the EcoR1 site of the wide host and conjugally transferable cosmid vector pLAFR1. The average insert size in the gene clones of the bank was found to be 21.3 Kb. The strain R. leguminosarum Rld7 (leu-1) was employed as recepient to conjugally transfer and thus isolate the complementary leu+ allele carrying clones from the gene bank.     

Nodulation and Nitrogen Fixation Efficacy of Rhizobium fredii with Phaseolus vulgaris Genotypes

Phaseolus plant introduction (PI) genotypes (consisting of 684 P. vulgaris, 26 P. acutifolius, 39 P. lunatus, and 5 P. coccineus accessions) were evaluated for their ability to form effective symbioses with strains of six slow-growing (Bradyrhizobium) and four fast-growing (Rhizobium fredii) soybean rhizobia. Of the 684 P. vulgaris genotypes examined, three PIs were found to form effective nitrogen-fixing symbioses with the R. fredii strains. While none of the Bradyrhizobium strains nodulated any of the genotypes tested, some produced large numbers of undifferentiated root proliferations (hypertrophies). A symbiotic plasmid-cured R. fredii strain failed to nodulate the P. vulgaris PIs and cultivars, suggesting that P. vulgaris host range genes are Sym plasmid borne in the fast-growing soybean rhizobia.     

New Field Isolates of Rhizobium leguminosarum Biovar Viciae That Nodulate the Primitive Pea Cultivar Afghanistan in Addition to Modern Cultivars

A collection of 13 field isolates of Rhizobium leguminosarum bv. viciae that have the ability to nodulate the roots of current North American cultivars of peas as well as a “primitive” cultivar, Afghanistan, was examined. These isolates originated in diverse geographical regions of the world, which indicates that this phenotype is not restricted to isolates from any one region. When subclones of the nodulation region from one plasmid were used to examine EcoRI-fragment-length polymorphisms in this collection of strains as well as in a collection comprising strains that do not nodulate the primitive cultivar, polymorphism was found in both collections. With one exception, RisA6, all strains that nodulated cv. Afghanistan pea contained a region called nodX as an extension to the nodA BCIJ operon that has been observed in all R. leguminosarum bv. viciae strains, including those that do not nodulate cv. Afghanistan pea. RisA6 was also the only strain in which nodulating ability could not be associated with a conjugative plasmid.     

Effect of Agglutinins from Rhizobium leguminosarum Strain 252 on the Activity of Hydrolytic Enzymes

Cells of the nitrogen-fixing soil bacterium Rhizobium leguminosarum 252 and its hemagglutination-deficient mutant strain 252/7 were found to possess the activities of a variety of hydrolytic enzymes. The agglutinin proteins of rhizobia diminished β-glucosidase activity, pectinolytic activity, and acid and alkaline phosphatase activities while completely inhibiting proteolytic enzyme activity in the bacterial cell. The results here show that rhizobial agglutinins are capable of affecting enzyme functioning in Rhizobium. Received: 18 November 1999 / Accepted: 10 February 2000     

Adenine requirement for nodulation of pea by an auxotrophic mutant of Rhizobium leguminosarum

A non-nodulating auxotroph (L4-73) derived from an effective strain (L4) of Rhizobium leguminosarum has a growth requirement for adenine and thiamine. The auxotroph was able to infect the roots of the host plant Pisum sativum L. but formed root nodules (ineffective in nitrogen fixation) only when adenine and, to a lesser extent, thiamine were added to the plant substrate. Nodules formed in the presence of adenine were structurally abnormal, containing small cells in which infection threads appeared to have aborted. In the presence of thiamine the auxotroph produced a smaller number of nodules which were slightly more developed and were able to reduce trace amounts of acetylene to ethylene. The adenine effect predominated when both growth factors were added together or separately in different sequences. Adenine amendment was required during the first 6 days following inoculation to achieve the maximum number of nodules. The block in symbiosis could not be fully overcome by sequential addition or removal from the root medium of either compound or by addition of some other adeninecontaining compounds. Partial prototroph revertants requiring adenine but not thiamine induced a nodulation response similar to that of the original auxotroph in the presence of thiamine; partial prototrophs requiring thiamine only were almost fully effective. Bioassay of pea root tissue indicated the presence of significant amounts of both adenine and thiamine or related substances in the roots. The auxotroph was able to compete with the parent strain L4 in nodulation on roots of pea only in the presence of exogenous adenine.