Accumulation of Amino Acids in Rhizobium sp. Strain WR1001 in Response to Sodium Chloride Salinity

Rhizobium sp. strain WR1001, isolated from the Sonoran Desert by Eskew and Ting, was found to be able to grow in defined medium containing NaCl up to 500 mM, a concentration approaching that of sea water. Therefore, it is a valuable strain for studying the biochemical basis of salt tolerance. Intracellular free glutamate was found to increase rapidly in response to osmotic stress by NaCl. It accounted for 88% of the amino acid pool when the bacterium was grown in 500 mM NaCl. The role of glutamate dehydrogenase in glutamate biosynthesis was examined in several Rhizobium strains. Both NADH- and NADPH-dependent glutamate dehydrogenase activities in various Rhizobium strains were observed. The range of activity differed considerably depending on the particular strain. KCl (500 mM) did not stimulate glutamate dehydrogenase activity, as reported in a number of bacterial strains by Measures. The low activity of glutamate dehydrogenase in Rhizobium sp. strain WR1001 apparently cannot fulfill a biosynthetic function of glutamate formation in response to medium NaCl concentrations.     

Increase in Internode Length of Phaseolus lunatus L. Caused by Inoculation with a Nitrate Reductase-deficient Strain of Rhizobium sp

Dramatic differences in the height of lima beans (Phaseolus lunatus L.) treated with two different Rhizobium strains were studied. Lima beans were grown in Perlite in the greenhouse or in a minus-N culture solution in the growth chamber. The plants were inoculated with either Rhizobium sp. (lima bean) strain 127E15, which contains the constitutive nitrate reductase activity, or strain 127E14, which lacks that activity. For up to 3 weeks, no growth differences were observed in the plants inoculated with either strain. Five weeks after inoculation, however, those plants inoculated with strain 127E14 were significantly taller and had a larger number of leaves than those inoculated with strain 127E15. The difference in plant height was the result of increased internode elongation caused by inoculation with Rhizobium sp. 127E14. This response was observed with all lima bean cultivars tested, including Henderson, Fordhook, Allgreen, and Early Thorogreen. The growth difference occurred in plants cultured in the greenhouse or in the growth chamber.     

Inoculation of Rhizobium (VR-1 and VA-1) induces an increasing growth and metal accumulation potential in Vigna radiata and Vigna angularis L. growing under fly-ash

Fly-ash-tolerant Rhizobium strains were isolated from plants grown in fly-ash-contaminated soil, axenically under laboratory conditions. Saplings of both plants were raised in N₂-free Jenson medium and inoculated with 2.6 × 10⁸ cell ml⁻¹ and 5.2 × 10⁸ cell ml⁻¹ of culture after 10 d of growth. Plants were transferred into 100% fly-ash under natural condition. Rhizobium-inoculated plants grown on 100% fly-ash showed marked increase in relation to root-shoot length, biomass yield, photosynthetic pigment, protein content and nodulation frequency compared to uninoculated plant grown in control (100% fly-ash). Inoculation of fly-ash-tolerant Rhizobium increased the accumulation of Fe, Zn, Cu Cd and Cr in different tissues vis-à-vis enhanced translocation of metals to the aboveground part of plant. Although inoculation of fly-ash-tolerant Rhizobium strains (VR-1 and VA-1) enhanced the translocation of more Fe to shoot parts, nevertheless, the amount of Rhizobium inoculants supplied to the plant was found to be very important since it has a positive role in increasing plant growth through increased N₂ supply via nitrogenase activity. Results suggest that an integrated approach employing biotechnological means and inoculation of plants with host-specific fly-ash-tolerant Rhizobium strain may prove a stimulus to a fly-ash management programme.     

Legume-Rhizobium Interactions: Cowpea Root Exudate Elicits Faster Nodulation Response by Rhizobium Species

Preinfection events in legume-Rhizobium symbiosis were analyzed by studying the different nodulation behaviors of two rhizobial strains in cowpeas (Vigna sinensis). Log-phase cultures of Rhizobium sp. strain 1001, an isolate from the plant nodule, initiated host responses leading to infection within 2 h after inoculation, whereas log-phase cultures of Rhizobium sp. strain 32H1 took at least 7 h to trigger a discernible response. The delay observed with strain 32H1 could be eliminated by incubating the rhizobial suspension, before inoculation, for 4.5 h either in the cowpea rhizosphere/rhizoplane condition or in the root exudate of cowpea plants, grown without NH₄⁺ in the rooting medium. The delay could not be eliminated by incubating the rhizobial suspension in the rooting medium of plants grown in the presence of 5 mM NH₄⁺, indicating that there is a regulatory role of combined nitrogen in triggering preinfection events by the legume. The substance(s) in the root exudate which elicited the faster nodulation response by Rhizobium sp. strain 32H1 could be separated into a high-molecular-weight fraction by Sephadex G-100 gel filtration. The data support the notion that legume roots release substances that favor the development of rhizobial features essential for infection and nodulation.     

Inhibition of Soybean Cell Growth by the Adsorption of Rhizobium japonicum

Soybean cells in suspension culture were inhibited in their growth by mixed culture with Rhizobium japonicum 5033. Rhizobium cells had the ability to adsorb on the surface of soybean cells. Cell envelope prepared from Rhizobium by sonic oscillation inhibited the growth of soybean cells. The growth-inhibiting activity of the cell envelope was depressed by β-glucosidase, KIO₄, urea, sodium cholate, and Triton X-100, but was stable on heating at 120 C for 15 minutes. Adsorption of the cell envelope on soybean cells was depressed by only β-glucosidase. The sodium cholate-soluble fraction of the cell envelope had the growth-inhibiting activity. Results in this paper suggest that components of the Rhizobium cell surface cause the inhibition of soybean cell growth after the adsorption of the Rhizobium cell to the soybean cell.     

Biodegradation of nonylphenol by two alphaproteobacterial strains in liquid culture and sediment microcosm

Nonylphenol (NP) has been a contaminant of great environmental concern due to its ubiquity, toxicity and endocrine activity. Biodegradation is an ideal way to clean up NP pollution. In this study, two NP degraders were isolated from river sediment. Their ability to degrade NP was tested in both liquid culture and sediment microcosm. Phylogenetic analysis indicated that one isolate belonged to genus Rhizobium, while another was a Sphingobium species. The Rhizobium strain contained ALK gene, while the Sphingobium strain harbored ALK and C23O genes. Both of the two strains showed strong NP degradation ability in liquid culture. However, only the Rhizobium strain demonstrated a potential of bioremediating NP-contaminated sediment. This study can provide some new insights towards NP biodegradation and bioremediation.     

Procedure for Obtaining Efficient Root Nodulation of a Pea Cultivar by a Desired Rhizobium Strain and Preempting Nodulation by Other Strains

The specificity between the sym-2 gene bred into certain cultivars of pea (Pisum sativum L.) and the nodX gene, present only rarely in isolates of Rhizobium leguminosarum, can be exploited to preempt competition or nodulation blocking by a Rhizobium strain indigenous to a soil environment. The principle is to isolate an R. leguminosarum strain prevalent in a locale, convert it into a strain that will nodulate a desirable pea cultivar carrying sym-2 by establishing nodX in it, and then use the resulting Rhizobium strain with the pea cultivar carrying sym-2. To accomplish this, we first constructed a transposon Tn5 derivative called Tn5nodX and an efficient delivery vehicle that is suicidal in R. leguminosarum. We tested the potential utility of the system in greenhouse experiments. The results are encouraging enough to warrant extensive experiments under field conditions.     

Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium

The Rhizobium strain ORS571, which is associated with the tropical legume Sesbania rostrata, has the property of growing in the free-living state at the expense of ammonia or N₂ as sole nitrogen source. Five mutants, isolated as unable to form colonies on plates under conditions of nitrogen fixation, were studied. All of them, which appear as Fix^- in planta, are nif mutants. With mutant 5740, nitrogenase activity of the crude extract was restored by addition of pure Mo-Fe protein of Klebsiella pneumoniae. A 13-kb BamHI DNA fragment from the wild-type strain, which hybridized with a probe carrying the nifHDK genes of K. pneumoniae, was cloned in vector pRK290 to yield plasmid pRS1. The extent of homology between the probe and the BamHI fragment was estimated at 4 kb and hybridization with K. pneumoniae nifH, nifK, and possibly nifD was detected. The pRS1 plasmid was introduced into the sesbania rhizobium nif mutants. Genetic complementation was observed with strain 5740(pRS1) both in the free-living state and in planta. It thus appears that biochemistry and genetics of nitrogen fixation in this particular Rhizobium strain can be performed with bacteria grown under non-symbiotic conditions.     

Isolation of Insertion Sequence ISRLdTAL1145-1 from a Rhizobium sp. (Leucaena diversifolia) and Distribution of Homologous Sequences Identifying Cross-Inoculation Group Relationships

Insertion sequence (IS) element ISRLdTAL1145-1 from Rhizobium sp. (Leucaena diversifolia) strain TAL 1145 was entrapped in the sacB gene of the positive selection vector pUCD800 by insertional inactivation. A hybridization probe prepared from the whole 2.5-kb element was used to determine the distribution of homologous sequences in a diverse collection of 135 Rhizobium and Bradyrhizobium strains. The IS probe hybridized strongly to Southern blots of genomic DNAs from 10 rhizobial strains that nodulate both Phaseolus vulgaris (beans) and Leucaena leucocephala (leguminous trees), 1 Rhizobium sp. that nodulates Leucaena spp., 9 R. meliloti (alfalfa) strains, 4 Rhizobium spp. that nodulate Sophora chrysophylla (leguminous trees), and 1 nonnodulating bacterium associated with the nodules of Pithecellobium dulce from the Leucaena cross-inoculation group, producing distinguishing IS patterns for each strain. Hybridization analysis revealed that ISRLdTAL1145-1 was strongly homologous with and closely related to a previously isolated element, ISRm USDA1024-1 from R. meliloti, while restriction enzyme analysis found structural similarities and differences between the two IS homologs. Two internal segments of these IS elements were used to construct hybridization probes of 1.2 kb and 380 bp that delineate a structural similarity and a difference, respectively, of the two IS homologs. The internal segment probes were used to analyze the structures of homologous IS elements in other strains. Five types of structural variation in homolog IS elements were found. The predominate IS structural type naturally occurring in a strain can reasonably identify the strain's cross-inoculation group relationships. Three IS structural types were found in Rhizobium species that nodulate beans and Leucaena species, one of which included the designated type IIB strain of R. tropici (CIAT 899). Weak homology to the whole IS probe, but not with the internal segments, was found with two Bradyrhizobium japonicum strains. The taxonomic and ecological implications of the distribution of ISRLdTAL1145-1 are discussed.     

Two rhizobacterial strains,individually and in interactions with Rhizobium sp., enhance fusarial wilt control,growth, and yield in pigeon pea

A Pseudomonas aeruginosa strain, RRLJ 04, and a Bacillus cereus strain, BS 03, were tested both individually and in combination with a Rhizobium strain, RH 2, for their ability to enhance plant growth and nodulation in pigeon pea (Cajanus cajan L.) under gnotobiotic, greenhouse and field conditions. Both of the rhizobacterial strains exhibited a positive effect on growth in terms of shoot height, root length, fresh and dry weight, nodulation and yield over the non-treated control. Co-inoculation of seeds with these strains and Rhizobium RH 2 also reduced the number of wilted plants, when grown in soil infested with Fusarium udum. Gnotobiotic studies confirmed that the suppression of wilt disease was due to the presence of the respective PGPR strains. Seed bacterization with drug-marked mutants of RRLJ 04 and BS 03 confirmed their ability to colonize and multiply along the roots. The results suggest that co-inoculation of these strains with Rhizobium strain RH 2 can be further exploited for enhanced growth, nodulation and yield in addition to control of fusarial wilt in pigeon pea.     

Nodulation of Pole Bean (Phaseolus vulgaris L.) by Rhizobium Species of Two Cross-Inoculation Groups

Physiology and morphology of pole bean (Phaseolus vulgaris L. cv Kentucky Wonder) root nodules induced by two Rhizobium species of different cross-inoculation groups have been compared. Root nodules induced by Rhizobium sp. 127E15, which is a strain of the cowpea group Rhizobium, were pinkish, had irregular shapes, and were only partially effective. Their peak acetylene reduction activity was 4.36 μmol of C₂H₄ formed per g of fresh nodules per h at 30 days after inoculation. The effective nodules induced by Rhizobium phaseoli 127K14, which is a strain of the bean group Rhizobium, were dark red, spherical, and showed peak acetylene reduction activity of 15.95 μmol of C₂H₄ formed per g of fresh nodules per h at 15 days after inoculation. The partial effectiveness of 127E15-induced nodules was associated with fewer infected cells, a delay in the increase of bacteroid population within the host cells, abundance of cytoplasmic vesicles in the host cells, more bacteroids within a membrane envelope (peribacteroid membrane), and the inability of bacteroids to completely fill up the host cytoplasmic space. The 127K14-induced nodules were fully mature, with host cells filled with bacteroids by 12 days after inoculation. In contrast, the 127E15-induced nodules did not reach a similar developmental stage even 30 days after inoculation.     

Nitrate Effect on Nitrogen Fixation (Acetylene Reduction): ACTIVITIES OF LEGUME ROOT NODULES INDUCED BY RHIZOBIA WITH VARIED NITRATE REDUCTASE ACTIVITIES

The effect of nitrate on symbiotic nitrogen fixation by root nodules of cowpea (Vigna unguiculata L., Walp., cv. California Blackeye) and lupine (Lupinus augustifolius L., cv. Frost) plants inoculated with nitrate reductase-expressing and nitrate reductase-nonexpressing Rhizobium strains were examined. Nitrate reductase of Rhizobium bacteroids in the nodules of cowpea and lupine reduced nitrate to nitrite. Both cowpea and lupine nodules accumulated nitrite when grown in the presence of 15 millimolar nitrate and induced by Rhizobium strains which express nitrate reductase activity (Rhizobium sp. 32H1 and 127E15). The nitrogen fixation (acetylene reduction) activities of cowpea and lupine nodules were inhibited by nitrate whether the nodules were induced by Rhizobium strains that express (Rhizobium sp. 32H1 and 127E15) or do not express (Rhizobium sp. 127E14 and R. lupini ATCC 10318) nitrate reductase activity. These findings indicate that nitrite, the product of bacteroid nitrate reductase, may not play a role in the inhibitory effect of nitrate on nitrogen fixation activities of legume root nodules. However, the degree of inhibition on the fixation activity by nitrate varied in different legume-Rhizobium combinations.     

Genome Sequence of Rhizobium sp. Strain CCGE510, a Symbiont Isolated from Nodules of the Endangered Wild Bean Phaseolus albescens

We present the genome sequence of Rhizobium sp. strain CCGE510, a nitrogen fixing bacterium taxonomically affiliated with the R. leguminosarum-R. etli group, isolated from wild Phaseolus albescens nodules grown in native pine forests in western Mexico. P. albescens is an endangered bean species phylogenetically related to P. vulgaris. In spite of the close host relatedness, Rhizobium sp. CCGE510 does not establish an efficient symbiosis with P. vulgaris. This is the first genome of a Rhizobium symbiont from a Phaseolus species other than P. vulgaris, and it will provide valuable new insights about symbiont-host specificity.     

Production of Proteasome Inhibitor Syringolin A by the Endophyte Rhizobium sp. Strain AP16

Syringolin A, the product of a mixed nonribosomal peptide synthetase/polyketide synthase encoded by the syl gene cluster, is a virulence factor secreted by certain Pseudomonas syringae strains. Together with the glidobactins produced by a number of beta- and gammaproteobacterial human and animal pathogens, it belongs to the syrbactins, a structurally novel class of proteasome inhibitors. In plants, proteasome inhibition by syringolin A-producing P. syringae strains leads to the suppression of host defense pathways requiring proteasome activity, such as the ones mediated by salicylic acid and jasmonic acid. Here we report the discovery of a syl-like gene cluster with some unusual features in the alphaproteobacterial endophyte Rhizobium sp. strain AP16 that encodes a putative syringolin A-like synthetase whose components share 55% to 65% sequence identity (72% to 79% similarity) at the amino acid level. As revealed by average nucleotide identity (ANI) calculations, this strain likely belongs to the same species as biocontrol strain R. rhizogenes K84 (formely known as Agrobacterium radiobacter K84), which, however, carries a nonfunctional deletion remnant of the syl-like gene cluster. Here we present a functional analysis of the syl-like gene cluster of Rhizobium sp. strain AP16 and demonstrate that this endophyte synthesizes syringolin A and some related minor variants, suggesting that proteasome inhibition by syrbactin production can be important not only for pathogens but also for endophytic bacteria in the interaction with their hosts.     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

Nucleotide Sequence and Genetic Structure of a Novel Carbaryl Hydrolase Gene (cehA) from Rhizobium sp. Strain AC100

Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene (cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA. No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the cehA gene. Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon.     

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.     

Change of Carbon Metabolic Activity of Rhizobium Under Carbon Starvation

One fast growing strain of Rhizobium sp (Vigna mungo) VBS 1 was tested for its metabolic activities under carbon starvation. Specific activities of the catabolic enzymes like phosphofructokinase, fructose-1,6-bisphosphate aldolase, iso-citrate dehydrogenase and malate dehydrogenase decreased remarkably whereas, induction of two anapleurotic enzymes like fructose-1,6-bisphosphatase and iso-citrate lyase took place in the cell-free extract of the strain. Almost unchanged specific activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase indicated its key role in maintaining a balance between catabolic and anabolic activities under carbon starvation.     

Enrichment, isolation, and characterization of 4-chlorophenol-degrading bacterium Rhizobium sp. 4-CP-20

The objectives of this research were to monitor the variations of species in mixed cultures during the enrichment period, isolate species and identify and characterize the pure 4-chlorophenol (4-CP) degrading strains from enriched mixed cultures. Strain Rhizobium sp. 4-CP-20 was isolated from the acclimated mixed culture. The DGGE result indicated that strain Rhizobium sp. 4-CP-20 was undetectable at the beginning but detectable after 2 weeks of enrichment. The optimum growth temperatures for Rhizobium sp. 4-CP-20 were both 36°C using 350 mg l⁻¹ glucose or sodium acetate as the substrate. The optimum pH range for degrading 100 mg l⁻¹ 4-CP was between 6.89 and 8.20. Strain Rhizobium sp. 4-CP-20 could degrade 4-CP completely within 3.95 days, as the initial 4-CP concentration was 100 mg l⁻¹. If the initial 4-CP concentration was higher than 240 mg l⁻¹, the growth of bacterial cells and the activity of degrading 4-CP were both inhibited.     

Adsorption of slow- and fast-growing rhizobia to soybean and cowpea roots

Roots of soybean (Glycine max [L.] Merr. cv Hardee) and cowpea (Vigna unguiculata [L.] Walp. cv Pink Eye Purple Hull) were immersed in suspensions containing 10⁴Rhizobium cells per milliliter of a nitrogen-free solution. After 30 to 120 minutes the roots were rinsed, and the distal 2-centimeter segments excised and homogenized. Portions of the homogenates then were plated on a yeast-extract mannitol medium for bacterial cell counts. The adsorption capacities of four slow-growing rhizobia and a fast-growing R. meliloti strain varied considerably. Adsorption was independent of plant species and of the abilities of the Rhizobium strains to infect and nodulate. R. lupini 96B9 had the greatest adsorption capacity, and Rhizobium sp. 3G4b16 the least. Rhizobium sp. 229, R. japonicum 138, and R. meliloti 102F51 were intermediate, except on cowpea, where the adsorption of strain 102F51 was similar to that of strain 3G4b16. The initial adsorption rates of bacteria cultured in synthetic media and in the presence of soybean roots were about the same. Addition of soybean lectin to the bacterial inoculum failed to influence initial adsorption rates. Both treatments, however, reduced the numbers of bacteria that bound after incubation with roots for 120 minutes. The relationship between the logarithm of the number of strain 138 cells bound per soybean root segment and the logarithm of the density of bacteria in the inoculum was linear over five orders of magnitude. Binding of strain 138 to soybean roots was greatest at room temperature (27°C) and substantially attenuated at both 4 and 37°C. Although R. lupini 96B9 strongly rejected a model hydrophobic plastic surface, there were no simple correlations between bacterial binding to model hydrophobic and hydrophilic plastic surfaces and bacterial adsorption to roots.