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PubMed2006

Identification of a cowpea gamma-thionin with bactericidal activity.

Antimicrobial peptides are an abundant group of proteinaceous compounds widely produced in the plant kingdom. Among them, the gamma-thionin family, also known as plant defensins, represents one typical family and comprises low molecular mass cysteine-rich proteins, usually cationic and distributed in different plant tissues. Here, we report the purification and characterization of a novel gamma-thionin from cowpea seeds (Vigna unguiculata), named Cp-thionin II, with bactericidal activity against Gram-positive and Gram-negative bacteria. Once the primary structure was elucidated, molecular modelling experiments were used to investigate the multimerization and mechanism of action of plant gamma-thionins. Furthermore, Cp-thionin II was also localized in different tissues in cowpea seedlings during germination in contrasting conditions, to better understand the plant protection processes. The use of plant defensins in the construction of transgenic plants and also in the production of novel drugs with activity against human pathogens is discussed.

Amino Acid Sequence↗
PubMed1978

Toxicity of Leucaena leucocephala. The effect of iodine and mineral supplements on penned steers fed a sole diet of Leucaena.

Steers fed a sole diet of Leucaena leucocephala cv. Peru (leucaena), rapidly developed a hypothyroid condition. Total serum thyroxine (T4) levels declined from c 120 n mol litre-1 to 13 n mol litre-1 within six weeks of full leucaena feeding. Associated with the hypothyroidism was a declined in feed intake, poor weight gain, hair loss, excessive salivation and the development of lesions on the oesophagus of some animals. Light steers (119 kg) were more severely affected than heavy steers (220 kg) and two light steers died. During the eight week period of full leucaena feeding animals gained only 0.18 kg head-1 day-1 compared with 0.73 kg head-1 day -1 for steers fed cowpea (Vigna sinensis) hay. Supplementation with minerals (Fe, Cu, Zn) significantly increased mean daily intake (85.4 v 68.9 g/kg0.75), daily liveweight gain, (334 v 14 g), and serum T3 resin uptake (42.5 v 39.6%); decreased hair loss and skin lesions but did not alleviate the low serum T4 levels. Iodine supplementation did not affect intake, bodyweight gain, serum T4 levels, serum T3 uptake or toxicity scores compared with unsupplemented leucaena fed steers. When the leucaena fed animals were subsequently fed cowpea hay, appetite improved and the serum T4 levels returned to normal within two weeks. For the heavy steers over this period, weight gain was similar to the controls; however, the light steers continued to gain poorly.

Animals↗
PubMed1976

Insecticidal action of the phytohemagglutinin in black beans on a bruchid beetle.

Black bean (Phaseolus vulgaris) phytohemagglutinin added to the normal diet of a bruchid beetle (Callosobruchus maculatus) that can eat phytohemagglutinin-free cowpeas (Vigna unguiculata) but not P. vulgaris seeds kills the bruchid larvae. Trypsin inhibitors, found in both P. vulgaris and V. unguiculata seeds, have virtually no effect on the bruchid larvae. The conclusion is that a major part of the adaptive significance of phytohemagglutinins in black bean and other legume seeds is to protect them from attack by insect seed predators.

Adaptation, Biological↗
PubMed1982

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 NH(4) 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 NH(4), 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.

Journal Article↗
PubMed1986

Comparison of hup trait and intrinsic antibiotic resistance for assessing rhizobial competitiveness axenically and in soil.

The competitiveness of dual-strain inocula of cowpea rhizobia for nodulation of Vigna unguiculata (L.) Walp. was studied axenically between one slow-growing strain (P132, HP147, 401, or 22A1) and one fast-growing strain (176A26 or 176A28) at logarithmic inoculum ratios ranging from 10 to 10. Nodule infectivity was determined by multiple intrinsic antibiotic resistance, since both fast-growing strains were sensitive. Different hydrogen uptake (Hup) efficiencies of dual-strain inocula allowed for the comparison of an indirect rapid method. Infectivity data based on antibiotic resistance and Hup efficiency were fit to linearized fractile plots of log-normal distributions to determine C(AB) (percent infectivity at a 1:1 inoculum density) or I(50) (inoculum ratio at 50% infectivity). The slow growers were always better competitors and had I(50) values which ranged from 7 to 160,000 and C(AB) values which ranged from 62 to 97%. P132 was the best competitor of all those tested. Antibiotic resistance and Hup efficiency methods were in agreement with 401 (Hup) and 176A26 (Hup), but the Hup efficiency method overestimated the I(50) index with 22A1 (Hup) and 176A28 (Hup). The competition of each of the four slow-growing strains with indigenous rhizobia was examined in Cajanus cajan from three tropical soils. Nodule infectivity for all strains ranged from 42 to 96%, and P132 was the best competitor in all the soils. Hup efficiency overestimated infectivity by about 2-fold when Hup inocula (P132 and HP147) were used but underestimated infectivity by more than 100-fold when Hup inocula (401 and 22A1) were used. Although the Hup trait has limited quantitative usage axenically, it is only qualitative in soil competition studies and can only be used with Hup inocula.

Journal Article↗
PubMed1986

Inoculation response of legumes in relation to the number and effectiveness of indigenous Rhizobium populations.

The response of legumes to inoculation with rhizobia can be affected by many factors. Little work has been undertaken to examine how indigenous populations or rhizobia affect this response. We conducted a series of inoculation trials in four Hawaiian soils with six legume species (Glycine max, Vigna unguiculata, Phaseolus lunatus, Leucaena leucocephala, Arachis hypogaea, and Phaseolus vulgaris) and characterized the native rhizobial populations for each species in terms of the number and effectiveness of the population for a particular host. Inoculated plants had, on average, 76% of the nodules formed by the inoculum strain, which effectively eliminated competition from native strains as a variable between soils. Rhizobia populations ranged from less than 6 x 10/g of soil to 1 x 10/g of soil. The concentration of nitrogen in shoots of inoculated plants was not higher than that in uninoculated controls when the most probable number MPN counts of rhizobia were at or above 2 x 10/g of soil unless the native population was completely ineffective. Tests of random isolates from nodules of uninoculated plants revealed that within most soil populations there was a wide range of effectiveness for N(2) fixation. All populations had isolates that were ineffective in fixing N(2). The inoculum strains generally did not fix more N(2) than the average isolate from the soil population in single-isolate tests. Even when the inoculum strain proved to be a better symbiont than the soil rhizobia, there was no response to inoculation. Enhanced N(2) fixation after inoculation was related to increased nodule dry weights. Although inoculation generally increased nodule number when there were less than 1 x 10 rhizobia per g of soil, there was no corresponding increase in nodule dry weight when native populations were effective. Most species compensated for reduced nodulation in soils with few rhizobia by increasing the size of nodules and therefore maintaining a nodule dry weight similar to that of inoculated plants with more nodules. Even when competition by native soil strains was overcome with a selected inoculum strain, it was not always possible to enhance N(2) fixation when soil populations were above a threshold number and had some effective strains.

Journal Article↗
PubMed1986

Fermentation of aqueous plant seed extracts by lactic Acid bacteria.

The effects of lactic acid bacterial fermentation on chemical and physical changes in aqueous extracts of cowpea (Vigna unguiculata), peanut (Arachis hypogea), soybean (Glycine max), and sorghum (Sorghum vulgare) were studied. The bacteria investigated were Lactobacillus helveticus, L. delbrueckii, L. casei, L. bulgaricus, L. acidophilus, and Streptococcus thermophilus. Organisms were inoculated individually into all of the seed extracts; L. bulgaricus and S. thermophilus were also evaluated together as inocula for fermenting the legume extracts. During fermentation, bacterial population and changes in titratable acidity, pH, viscosity, and color were measured over a 72-h period at 37 degrees C. Maximum bacterial populations, titratable acidity, pH, and viscosity varied depending upon the type of extract and bacterial strain. The maximum population of each organism was influenced by fermentable carbohydrates, which, in turn, influenced acid production and change in pH. Change in viscosity was correlated with the amount of protein and titratable acidity of products. Color was affected by pasteurization treatment and fermentation as well as the source of extract. In the extracts inoculated simultaneously with L. bulgaricus and S. thermophilus, a synergistic effect resulted in increased bacterial populations, titratable acidity, and viscosity, and decreased pH in all the legume extracts when compared to the extracts fermented with either of these organisms individually. Fermented extracts offer potential as substitutes for cultured dairy products.

Journal Article↗
PubMed1987

Variability in Effectiveness of Rhizobia during Culture and in Nodules.

The ability of three strains of Bradyrhizobium sp. (Vigna) to fix dinitrogen in symbiotic association with siratro (Macropitilium atropurpureum) was measured after culture in broth and after isolation from nodules. Seven transfers were made between the initial broth culture and the final broth culture. A total of 40 single-colony isolates were obtained from cultures 1 and 7 to test effectiveness. Variation in dinitrogen-fixing effectiveness of the population of one strain did not change on culturing, whereas there was considerable variation in effectiveness of populations of the other two strains. Generally, single-colony isolates from individual nodules had similar levels of effectiveness, but some exceptions occurred. Isolates from different nodules formed by the same Bradyrhizobium strain often differed in their effectiveness.

Journal Article↗
PubMed1988

Nodulation, Nitrogen Fixation, and Hydrogen Oxidation by Pigeon Pea Bradyrhizobium spp. in Symbiotic Association with Pigeon Pea, Cowpea, and Soybean.

The pigeon pea strains of Bradyrhizobium CC-1, CC-8, UASGR(S), and F4 were evaluated for nodulation, effectiveness for N(2) fixation, and H(2) oxidation with homologous and nonhomologous host plants. Strain CC-1 nodulated Macroptilium atropurpureum, Vigna unguiculata, Glycine max, and G. soja but did not nodulate Pisum sativum, Phaseolus vulgaris, Trigonella foenum-graecum, and Trifolium repens. Strain F4 nodulated G. max cv. Peking and PI 434937 (Malayan), but the symbioses formed were poor. Similarly, G. max cv. Peking, cv. Bragg, PI 434937, PR 13-28-2-8-7, and HM-1 were nodulated by strain CC-1, and symbioses were also poor. G. max cv. Williams and cv. Clark were not nodulated. H(2) uptake activity was expressed with pigeon pea and cowpea, but not with soybean. G. max cv. Bragg grown in Bangalore, India, in local soil not previously exposed to Bradyrhizobium japonicum formed nodules with indigenous Bradyrhizobium spp. Six randomly chosen isolates, each originating from a different nodule, formed effective symbioses with pigeon pea host ICPL-407, nodulated PR 13-28-2-8-7 soybean forming moderately effective symbioses, and did not nodulate Williams soybean. These results indicate the six isolates to be pigeon pea strains although they originated from soybean nodules. Host-determined nodulation of soybean by pigeon pea Bradyrhizobium spp. may depend upon the ancestral backgrounds of the cultivars. The poor symbioses formed by the pigeon pea strains with soybean indicate that this crop should be inoculated with B. japonicum for its cultivation in soils containing only pigeon pea Bradyrhizobium spp.

Journal Article↗
PubMed1989

Survival of Azorhizobium caulinodans in the Soil and Rhizosphere of Wetland Rice under Sesbania rostrata-Rice Rotation.

The survival of indigenous and introduced strains of Azorhizobium caulinodans in flooded soil and in the rice rhizosphere, where in situ Sesbania rostrata was incorporated before the rice crop, is reported. The azorhizobia studied were both root and stem nodulating. In a pot experiment, two crop cycles each of inoculated and noninoculated Sesbania-rice were compared with two crop cycles of flooded fallow-rice. In a field experiment, the effect of repeated incorporation of in situ S. rostrata in the Sesbania-rice sequence was studied. Soils in which inoculated S. rostrata was incorporated contained about 3,000 times more azorhizobia than did soils in the flooded fallow treatment and about 50 times more azorhizobia than did soils in the noninoculated Sesbania treatment. Azorhizobial numbers in the inoculated Sesbania treatment declined toward rice harvest but remained much higher than in the flooded fallow-rice treatment. Repeated incorporation of S. rostrata increased the population density of indigenous soil azorhizobia, whereas the population of inoculated strain ORS571 (Str Spc) declined to an undetectable level; this finding suggested low competitiveness by the introduced strain. In the incorporated Sesbania treatment, the rice rhizosphere harbored significantly more A. caulinodans and supported higher nitrogenase activity per plant than did the rhizosphere of the flooded fallow-rice treatment. Sterile rice seedlings inoculated with A. caulinodans showed nitrogenase activity comparable to that of seedlings inoculated with Azospirillum lipoferum 34H, a rice root isolate. Rhizobia from Sesbania aculeata, Sesbania sesban, a Trifolium sp., and Vigna unguiculata did not support appreciable nitrogenase activity.

Journal Article↗
PubMed1991

Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field-grown legumes.

Indigenous rhizobia in soil present a competition barrier to the establishment of inoculant strains, possibly leading to inoculation failure. In this study, we used the natural diversity of rhizobial species and numbers in our fields to define, in quantitative terms, the relationship between indigenous rhizobial populations and inoculation response. Eight standardized inoculation trials were conducted at five well-characterized field sites on the island of Maui, Hawaii. Soil rhizobial populations ranged from 0 to over 3.5 x 10 g of soil for the different legumes used. At each site, no less than four but as many as seven legume species were planted from among the following: soybean (Glycine max), lima bean (Phaseolus lunatus), cowpea (Vigna unguiculata), bush bean (Phaseolus vulgaris), peanut (Arachis hypogaea), Leucaena leucocephala, tinga pea (Lathyrus tingeatus), alfalfa (Medicago sativa), and clover (Trifolium repens). Each legume was (i) inoculated with an equal mixture of three effective strains of homologous rhizobia, (ii) fertilized at high rates with urea, or (iii) left uninoculated. For soybeans, a nonnodulating isoline was used in all trials as the rhizobia-negative control. Inoculation increased economic yield for 22 of the 29 (76%) legume species-site combinations. While the yield increase was greater than 100 kg ha in all cases, in only 11 (38%) of the species-site combinations was the increase statistically significant (P </= 0.05). On average, inoculation increased yield by 62%. Soybean (G. max) responded to inoculation most frequently, while cowpea (V. unguiculata) failed to respond in all trials. Inoculation responses in the other legumes were site dependent. The response to inoculation and the competitive success of inoculant rhizobia were inversely related to numbers of indigenous rhizobia. As few as 50 rhizobia g of soil eliminated inoculation response. When fewer than 10 indigenous rhizobia g of soil were present, economic yield was significantly increased 85% of the time. Yield was significantly increased in only 6% of the observations when numbers of indigenous rhizobia were greater than 10 cells g of soil. A significant response to N application, significant increases in nodule parameters, and greater than 50% nodule occupancy by inoculant rhizobia did not necessarily coincide with significant inoculation responses. No less than a doubling of nodule mass and 66% nodule occupancy by inoculant rhizobia were required to significantly increase the yield of inoculated crops over that of uninoculated crops. However, lack of an inoculation response was common even when inoculum strains occupied the majority of nodules. In these trials, the symbiotic yield of crops was, on average, only 88% of the maximum yield potential, as defined by the fertilizer N treatment. The difference between the yield of N-fertilized crops and that of N(2)-fixing crops indicates a potential for improving inoculation technology, the N(2) fixation capacity of rhizobial strains, and the efficiency of symbiosis. In this study, we show that the probability of enhancing yield with existing inoculation technology decreases dramatically with increasing numbers of indigenous rhizobia.

Journal Article↗
PubMed1998

Genotypic characterization of Bradyrhizobium strains nodulating endemic woody legumes of the Canary Islands by PCR-restriction fragment length polymorphism analysis of genes encoding 16S rRNA (16S rDNA) and 16S-23S rDNA intergenic spacers, repetitive extragenic palindromic PCR genomic fingerprinting, and partial 16S rDNA sequencing.

We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers. Cluster analysis of 16S rDNA restriction patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains, Bradyrhizobium japonicum USDA 110spc4 and Bradyrhizobium sp. strain (Centrosema) CIAT 3101, resolving three genotypes within these bradyrhizobia. In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR fingerprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints. Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with 16S rDNA RFLPs in computer-assisted cluster analysis of electrophoretic patterns. The DNA sequence analysis of a highly variable 264-bp segment of the 16S rRNA genes of these strains was found to be consistent with the fingerprint-based classification. Three different DNA sequences were obtained, one of which was not previously described, and all belonged to the B. japonicum/Rhodopseudomonas rDNA cluster. Nodulation assays revealed that none of the Canarian isolates nodulated Glycine max or Leucaena leucocephala, but all nodulated Acacia pendula, C. proliferus, Macroptilium atropurpureum, and Vigna unguiculata.

Atlantic Islands↗
PubMed2003

Enhanced production of alpha-galactosyl epitopes by metabolically engineered Pichia pastoris.

A metabolically engineered Pichia pastoris strain was constructed that harbored three heterologous enzymes: an S11E mutated sucrose synthase from Vigna radiata, a truncated UDP-glucose C4 epimerase from Saccharomyces cerevisiae, and a truncated bovine alpha-1,3-galactosyltransferase. Each gene has its own methanol-inducible alcohol oxidase 1 promoter and transcription terminator on the chromosomal DNA of P. pastoris strain GS115. The proteins were coexpressed intracellularly under the induction of methanol. After permeabilization, the whole P. pastoris cells were used to synthesize alpha-galactosyl (alpha-Gal) trisaccharide (Galalpha1,3Galbeta1,4Glc) with in situ regeneration of UDP-galactose. Up to 28 mM alpha-Gal was accumulated in a 200-ml reaction. The Pichia system described here is simple and flexible. This work demonstrates that recombinant P. pastoris is an excellent alternative to Escherichia coli transformants in large-scale synthesis of oligosaccharides.

Animals↗
PubMed2006

Exo-oligosaccharides of Rhizobium sp. strain NGR234 are required for symbiosis with various legumes.

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with beta-1,3, beta-1,4, beta-1,6, alpha-1,3, and alpha-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGROmegaexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGROmegaexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGROmegaexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, approximately 50 microg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-beta-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.

Bacterial Proteins↗
PubMed1977

Nitrogen fixation in nitrate reductase-deficient mutants of cultured rhizobia.

Forty-eight mutants unable to reduce nitrate were isolated from "cowpea" Rhizobium sp. strain 32Hl and examined for nitrogenase activity in culture. All but two of the mutants had nitrogenase activity comparable with the parental sttain and two nitrogenase-defective strains showed alterations in their symbiotic properties. One strain was unable to nodulate either Macroptilium atropurpureum or Vigna uguiculata and, with the other, nodules appeared promptly, but effective nitrogen fixation was delayed. These results, and the relatively low proportion of nitrate reductase mutants with impaired nitrogenase activity, do not support the proposed commanality between nitrogenase and nitrate reductase in cowpea rhizobia. Inhibition studies of the effect of nitrate and its reduction products on the nitrogenase activity in cultured strains 32Hl and the nitrate reductase-deficient, Nif+ strains, indicated that nitrogenase activity was sensitive to nitrite rather than to nitrate.

Anaerobiosis↗
PubMed1988

Two host-inducible genes of Rhizobium fredii and characterization of the inducing compound.

Random transcription fusions with Mu d1(Kan lac) generated three mutants in Rhizobium fredii (strain USDA 201) which showed induction of beta-galactosidase when grown in root exudate of the host plants Glycine max, Phaseolus vulgaris, and Vigna ungliculata. Two genes were isolated from a library of total plasmid DNA of one of the mutants, 3F1. These genes, present in tandem on a 4.2-kilobase HindIII fragment, appear in one copy each on the symbiotic plasmid and do not hybridize to the Rhizobium meliloti common nodulation region. They comprise two separate transcriptional units coding for about 450 and 950 nucleotides, both of which are transcribed in the same direction. The two open reading frames are separated by 586 base pairs, and the 5H regions of the two genes show a common sequence. No similarity was found with the promoter areas of Rhizobium trifolii, R. meliloti, or Bradyrhizobium japonicum nif genes and with any known nodulation genes. Regions homologous to both sequences were detected in EcoRI digests of genomic DNAs from B. japonicum USDA 110, USDA 122, and 61A76, but not in genomic DNA from R. trifolii, Rhizobium leguminosarum, or Rhizobium phaseoli. Mass spectrometry and nuclear magnetic resonance analysis indicated that the inducing compound has properties of 4',7-dihydroxyisoflavone, daidzein. These results suggest that, in addition to common nodulation genes, several other genes appear to be specifically induced by compounds in the root exudate of the host plants.

Base Sequence↗
PubMed1990

Sequence and analysis of the rpoN sigma factor gene of rhizobium sp. strain NGR234, a primary coregulator of symbiosis.

We report the nucleotide sequence of the rpoN gene from broad-host-range Rhizobium sp. strain NGR234 and analyze the encoded RPON protein, a sigma factor. Comparative analysis of the deduced amino acid sequence of RPON from NGR234 with sequences from other gram-negative bacteria identified a perfectly conserved RPON box unique to RPON sigma factors. Symbiotic regulatory phenotypes were defined for a site-directed internal deletion within the coding sequence of the rpoN gene of Rhizobium strain NGR234: they included quantitative nodulation kinetics on Vigna unguiculata and microscopic analysis of the Fix- determinate nodules of V. unguiculata and Macroptilium atropurpureum. RPON was a primary coregulator of nodulation and was implicated in establishment or maintenance of the plant-synthesized peribacteroid membrane. Phenotypes of rpoN in Rhizobium strain NGR234 could be grouped as symbiosis related, rather than simply pleiotropically physiological as in free-living bacteria such as Klebsiella pneumoniae and Pseudomonas putida.

Amino Acid Sequence↗
PubMed1996

Bradyrhizobium (Arachis) sp. strain NC92 contains two nodD genes involved in the repression of nodA and a nolA gene required for the efficient nodulation of host plants.

The common nodulation locus and closely linked nodulation genes of Bradyrhizobium (Arachis) sp. strain NC92 have been isolated on an 11.0-kb EcoRI restriction fragment. The nucleotide sequence of a 7.0-kb EcoRV-EcoRI subclone was determined and found to contain open reading frames (ORFs) homologous to the nodA, nodB, nodD1, nodD2, and nolA genes of Bradyrhizobium japonicum and Bradyrhizobium elkanii. Nodulation assays of nodD1, nodD2, or nolA deletion mutants on the host plants Macroptilium atropurpureum (siratro) and Vigna unguiculata (cowpea) indicate that nolA is required for efficient nodulation, as nolA mutants exhibit a 6-day nodulation delay and reduced nodule numbers. The nolA phenotype was complemented by providing the nolA ORF in trans, indicating that the phenotype is due to the lack of the nolA ORF. nodD1 mutants displayed a 2-day nodulation delay, whereas nodD2 strains were indistinguishable from the wild type. Translational nodA-lacZ, nodD1-lacZ, nodD2-lacZ, and nolA-lacZ fusions were created. Expression of the nodA-lacZ fusion was induced by the addition of peanut, cowpea, and siratro seed exudates and by the addition of the isoflavonoids genistein and daidzein. In a nodD1 or nodD2 background, basal expression of the nodA-lacZ fusion increased two- to threefold. The level of expression of the nodD2-lacZ and nolA-lacZ fusions was low in the wild type but increased in nodD1, nodD2, and nodD1 nodD2 backgrounds independently of the addition of the inducer genistein. nolA was required for increased expression of the nodD2-lacZ fusion. These data suggest that a common factor is involved in the regulation of nodD2 and nolA, and they are also consistent with a model of nod gene expression in Bradyrhizobium (Arachis) sp. strain NC92 in which negative regulation is mediated by the products of the nodD1 and nodD2 genes.

Acyltransferases↗