In Bradyrhizobium japonicum the common nodulation genes, nodABC, are linked to nifA and fixA

Summary Using cloned Rhizobium phaseoli nodulation (nod) genes as hybridization probes homologous restriction fragments were detected in the genome of the slow-growing soybean symbiont, Bradyrhizobium japonicum strain 110. These fragments were isolated from a cosmid library, and were shown to lie 10 kilobasepairs (kb) upstream from the nifA and fixA genes. Specific nod probes from Rhizobium leguminosarum were used to identify nodA-, nodB-, and nodC-like sequences clustered within a 4.5 kb PstI fragment. A mutant was constructed in which the kanamycin resistance gene from Tn5 was inserted into the nodA homologous B. japonicum region. This insertion was precisely located, by DNA sequencing, to near the middle of the nodA gene. B. japonicum mutants carrying this insertion were completely nodulation deficient (Nod^-).     

Bradyrhizobium japonicum mutants defective in root-nodule bacteroid development and nitrogen fixation

The genome of the slow-growing Bradyrhizobium japonicum (strain 110) was mutagenized with transposon Tn5. A total of 1623 kanamycin/streptomycin resistant derivatives were screened in soybean infection tests for nodulation (Nod) and symbiotic nitrogen fixation (Fix). In this report we describe 14 strains possessing a stable, reproducible Nod⁺Fix^- phenotype. These strains were also grown under microaerobic culture conditions to test them for free-living nitrogen fixation activity (Nif). In addition to strains having reduced Fix and Nif activities, there were also strains that had reduced symbiotic Fix activity but were Nif⁺ ex planta.Analysis of the genomic structure revealed that the majority of the strains had a single Tn5 insertion without any further apparent physical alteration. A few strains had additional insertions (by Tn5 or IS50), or a deletion, or had cointegrated part of the vector used for Tn5 mutagenesis. One of the insertions was found in a known nif gene (nifD) whereas all other mutations seem to affect different, hitherto unknown genes or operons.Several mutant strains had an altered nodulation phenotype, inducing numerous, small, widely distributed nodules. Light and electron microscopy revealed that most of these mutants were defective in different stages of bacteroid development and/or bacteroid persistence. The protein patterns of the mutants were inspected by two-dimensional gel electrophoresis after labelling microaerobic cultures with l-(³⁵S)methionine. Of particular interest were mutants lacking a group of proteins the synthesis of which was known to be under oxygen control. Such strains can be regarded as potential regulatory mutants.     

Isolation of adenylate cyclase mutants from Rhizobium meliloti deficient in nodulation

Six Rhizobium meliloti mutants were isolated after Tn5-mediated mutagenesis as resistant to inhibition by a mixture of amino acids (serine, methionine, glycine and leucine). All were defective in adenylate cyclase activity and failed to form nodules in infected roots of Medicago sativa. Furthermore, like other nodulation mutants, they showed altered motility and increased secretion of exopolysaccharides; addition of cAMP to the growth medium abolished some of these phenotypic defects. The possibility that adenylate cyclase participates in the transduction of signals inducing nodulation is discussed.     

Effect of glutamate transport and catabolism on symbiotic effectiveness in Rhizobium leguminosarum bv. phaseoli

Seven Tn5 induced mutants unable to use glutamate as sole carbon and nitrogen source were isolated from the effective Rhizobium leguminosarum bv. phaseoli strain P121-R. As indicated by restriction and hybridisation analysis, all the mutants arose from a single Tn5 insertion in the chromosome. The ¹⁴C-glutamate uptake rate of the mutants was 76 to 88% lower than that of strain P121-R. Inoculation of Phaseolus vulgaris cv. Labrador with these mutants significantly decreased shoot dry matter yield and the total nitrogen content respectively, as compared to inoculation with the parental strain P121-R. All the mutants formed nodules, however they were smaller, white to greenish and approximately 30% less numerous than those formed by strain P121-R. These observations suggest that glutamate transport and catabolism in R. leguminosarum bv. phaseoli P121-R may play an important role in the establishment of an effective symbiosis in field bean. None of the mutants isolated was an auxotroph. All mutants were unable to grow on aspartate suggesting that glutamate and aspartate, probably have the same transporter as indicated in Rhizobium meliloti and in Bacillus subtilis. All mutants readily used glutamine, proline, arginine as sole carbon and nitrogen source, but grew more slowly than the wild type strain. On the other hand, all the mutants were impaired in growth on histidine and -aminobutyrate as sole carbon and nitrogen source. As the catabolism of these amino acids occurs predominantly through glutamate, our results indicate that mutants are also impaired in their ability to use histidine and -aminobutyrate as a nitrogen source. Our results also suggest that other amino acids catabolized through the glutamate pathways may be an additional important carbon source for bacteroids in nodules.     

Induction of Symbiotically Defective Auxotrophic Mutants of Rhizobium fredii HH303 by Transposon Mutagenesis

Symbiotically defective auxotrophic mutants were isolated by transposon Tn5 mutagenesis of Rhizobium fredii HH303, a fast-growing microsymbiont of North American commercial soybean cultivars such as Glycine max cv. Williams. Three different Tn5-carrying suicide vectors, pBLK1-2, pSUP1011, and pGS9, were used for mutagenesis with transposition frequencies of 4 x 10, 3 x 10, and 1 x 10, respectively, while the frequency of background mutation resistant to 500 mug of kanamycin per ml was 1 x 10. From 2,600 Tn5-induced mutants, 14 auxotrophic mutants were isolated and classified in seven groups including adenosine (four), aspartate (two), cysteine or methionine (two), isoleucine and valine (two), nicotinic acid (one), pantothenic acid (one), and uracil (two). All the auxotrophs induced nodulation on soybean, but the symbiotic effectiveness of each mutant was different. Three auxotrophs (two cysteine or methionine and one pantothenic acid) formed effective nodules similar to those of the wild type. Three auxotrophs (one nicotinic acid and two aspartate) produced mature nodules like those of the wild type, but the nodules lacked the characteristic pink color inside and were unable to fix nitrogen. Four auxotrophs (two adenosine and two uracil) induced pseudonodules unable to fix nitrogen. The other four auxotrophs repeatedly induced both effective and ineffective nodules, but bacteroids isolated from the effective nodules were prototrophic revertants. The symbiotic phenotype and the degree of effectiveness of the auxotrophic mutants varied with the type of mutation.     

Transposon-induced symbiotic mutants of Bradyrhizobium japonicum: isolation of two gene regions essential for nodulation

Summary Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod^-) or nitrogen fixation (Fix^-). Seven Nod^- mutants were isolated from strain USDA 110 and from strain 61 A101 C, 4 Nod^- mutants and 14 Fix^- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His^- Nod^- mutants of USDA 110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix^- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three nonauxotrophic Nod^- mutants and one His^-Nod^- mutant of USDA 110. Homogenotization of the cloned fragments into wild-type strain USDA 110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod^- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA 110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.     

Respiratory deficient mutants of Rhodopseudomonas capsulata

The facultative photosynthetic bacterium Rhodopseudomonas capsulata was mutagenized by transfer of the plasmid pSUP201::Tn5 from Escherichia coli to R. capsulata. Mutants defective in cytochrome oxidase and other respiratory functions were selected by replica plating, NADI-reaction and immunological methods. Among 20,000 mutants no clone was detected, which lacks the 65,000-protein of the cytochrome oxidase, but many mutants have been isolated which were cytochrome oxidase deficient (or inactive). Other mutants excrete heme and cytochrome c into the medium and lack cytochrome c ₂.Abbreviations Ap ampicillin - CIE crossed immunoelectrophoresis - cyt cytochrome - Cm chloramphenicol - Km kanamycin - SDS sodium dodecylsulfate - Tc tetracycline     

Phenylalanine and tyrosine metabolism in the facultative methylotroph Nocardia sp. 239

Nocardia sp. 239 is able to use l-tyrosine and both d- and l-phenylalanine as carbon-, energy- and nitrogen sources for growth. The catabolism of these compounds is by way of (4-hydroxy)phenylpyruvate and (4-hydroxy)-phenylacetate as intermediates and the pathways merge at the level of homogentisate. The conversion of the amino acids into (4-hydroxy)phenylpyruvate is catalyzed by an inducible NAD-dependent phenylalanine dehydrogenase and l-tyrosine aminotransferase, respectively. Incubation of the organism in media with l-phenylalanine plus phenyl-pyruvate resulted in diauxic growth, with phenylpyruvate used first. Phenylalanine dehydrogenase activity cold only be detected after depletion of phenylpyruvate, in the ensuing second growth phase on l-phenylalanine. During growth on phenylalanine plus methanol, low levels of phenylalanine dehydrogenase were detected and this resulted in simultaneous utilization of the two substrates. Following diepoxyoctane treatment, mutants of Nocardia sp. 239 affected in phenylalanine and phenylpyruvate degradation were isolated. Double mutants blocked in both phenylalanine dehydrogenase and phenylpyruvate decarboxylase completely failed to catabolize phenylalanine. The absence of these enzymes did not affect growth on tyrosine.Abbreviations RuMP ribulose monophosphate - EMS ethylmethanesulphonate - NTG N-methyl-N-nitro-N-nitrosoguanidine     

Mutants of Rhizobium japonicum defective in nodulation

Several mutants defective in nodulation were isolated from Rhizobium japonicum strains 3I1b110 and 61A 76. Mutants of class I do not form nodules after incubation with soybean [Glycine max (L.) Merrill] for 17 days, but will do so by 28 days. When host plants other than G. max are infected with several of these strains, there is no detectable difference in the time of nodulation or size of nodules as compared to the wild type. Two mutants of class I (i. e., SM1 and SM2) have been shown previously to be altered in the lipopolysaccharide portion of their cell wall. Mutants of class II are not slow to nodulate but form fewer nodules than the wild type on all the host plants tested. Mutants of class III are unable to form nodules. Some bacteriophage-resistant mutants, altered in cell surface structure, fall into this class. Two mutants of class III do not bind to soybean roots.     

Enhanced nodule initiation on alfalfa by wild-typeRhizobium meliloti co-inoculated withnod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular signals that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.Abbreviations EH emergent root hairs - kb kilobase - RDU relative distance unit - RT root tip This is journal article No. 188-87 of the Ohio Agricultural Research and Development Center     

Analogue-resistant and auxotrophic mutants ofMethanobacterium thermoautotrophicum

The death rate ofMethanobacterium thermoautotrophicum strain Marburg upon exposure toN-methyl-N-nitro-N-nitrosoguanidine under anaerobic conditions was of the same order of magnitude as the death rates that have been reported forEscherichia coli. Cultures of the methanogenic bacterium, mutagenized by nitrosoguanidine-treatment and grown under non-selective conditions, yielded mutants resistant toDL-ethionine (30 mM) or to 2-bromoethane sulfonic acid (3.8 mM). No mutants were observed in untreated controls. Among 1500 clones obtained from nitrosoguanidine-treated cell suspensions there were 6 mutants requiring a single growth factor each, namelyl-leucine,l-phenylalanine, thiamine (2 mutants) or adenosine (2 mutants). Three mutant-strains were studied in more detail. They were genetically stable (no revertants among 10⁹ cells), and wild type growth rates were restored by 5 mml-leucine, 0.4 mM adenosine and 0.03 mM thiamine, respectively.Abbreviations 2-BES 2-bromoethanesulfonic acid - MIC minimum inhibitory concentration     

Establishment of Bradyrhizobium japonicum for soybean by inoculation of a preceding wheat crop

On fields with no history of soybean (Glycine max (L.) Merr.) production, inoculation alone is often inadequate to provide for adequate nodulation the first time this crop is grown. The objective of this study was to determine if inoculation of spring wheat (Triticum aestivum L.) seed with Bradyrhizobium japonicum would lead to an increase of B. japonicum numbers in the soil, and improve nodulation of a subsequent soybean crop. In the greenhouse, wheat seed inoculation increased B. japonicum numbers from undetectable numbers to >9000 g^–1 of soil, whereas the numbers of introduced B. japonicum declined in unseeded pots. In the field, inoculation of wheat seed increased B. japonicum numbers in the soil from undetectable levels to >4000 g^–1 the following year. When soybean seed was inoculated, but grown in soil devoid of B. japonicum, nodules formed only near the point of seed placement. The heaviest nodulation, and widest distribution of nodules in the topsoil were found whenB. japonicum was established the year before by wheat seed inoculation, plus soybean seed inoculation. Wheat seed inoculation the year before growing soybean, combined with proper soybean seed inoculation, should provide for abundant nodulation the first time soybean is grown on a field.     

Microbial Production of l-Threonine

l-Threonine producing α-amino-β-hydroxyvaleric acid resistant mutants were derived from E. coli K-12 with 3 x 10^-5 frequency. One of mutants, strain β-101, accummulated maximum amount of l-threonine (1. 9 g/liter) in medium. Among isoleucine, methionine and lysine auxotrophs derived from E. coli K-12, only methionine auxotrophs produced l-threonine. In contrast, among isoleucine, methionine and lysine auxotrophs derived from β-101, l-threonine accumulation was generally enhanced in isoleucine auxotrophs. One of isoleucine auxotrophs, strain βI-67, produced maximum amount of l-threonine (4. 7 g/liter). Methionine auxotroph, βM-7, derived from β-101 produced 3.8 g/liter, and βIM-4, methionine auxotroph derived from β1-67, produced 6.1 g/liter, when it was cultured in 3% glucose medium supplemented with 100 μg/ml of l-isoleucine and l-methionine, respectively. These l-threonine productivities of E. coli mutants were discussed with respect to the regulatory mechanisms of threonine biosynthesis. A favourable fermentation medium for l-threonine production by E. coli mutants was established by using strain βM-4.     

Nodulation of soybean byRhizobium japonicum mutants with altered capsule synthesis

Spontaneous mutants with altered capsule synthesis were isolated from a marked strain of the symbiont,Rhizobium japonicum. Differential centrifugation was used to enrich serially for mutants incapable of forming capsules. The desired mutants were detected by altered colony morphology and altered ability to bind host plant lectin. Three mutants failed to form detectable capsules at any growth phase when cultured in vitro or in association with the host (soybean,Glycine max (L.) Merr.) roots. These mutants were all capable of nodulating and attaching to soybean roots, indicating that the presence of a capsule physically surrounding the bacterium is not required for attachment or for infection and nodulation. Nodulation by several of the mutants was linearly proportional to the amount of acidic exopolysaccharide that they released into the culture medium during the exponential growth phase, indicating that such polysaccharide synthesis is important and perhaps required for nodulation. Two of the mutants appeared to synthesize normal lectin-binding capsules when cultured in association with host roots, but not when cultured in vitro. Nodulation by these mutants appeared to depend on how rapidly after inoculation they synthesized capsular polysaccharide.Abbreviations CPS capsular polysaccharide - EPS exopolysaccharide - FITC fluorescein isothiocyanate Contribution No. 719 of the C.F. Kettering Research Laboratory     

Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K12

Summary Two new mutants of E. coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport. They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate(pCMB). The lesion was mapped at the putP gene, which is located at min 23 on the revised E. coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order. The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product).Hybrid plasmids carrying the put region (Motojima et al. 1979; Wood et al. 1979) were used to construct the physical map of the put region. The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants.Abbreviations pCMB p-chloromercuribenzoate - DM Davis and Mingioli - Ap ampicillin - NTG N-methyl-N-nitro-N-nitrosoguanidine - EMS ethylmethane sulfonate - Str streptomycin - Tet tetracycline - Ac l-azetidine-2-carboxylic acid - DHP 3, 4-dehydro-d,l-proline - MTT 3-(4,5-dimethyl-2)2,5-diphenyl tetrazolium bromide - Tris tris(hydroxymethyl)aminomethane - EDTA ethylenediamine tetraacetic acid - Kan kanamycin - Spc spectinomycin     

The role of motility in the efficiency of nodulation by Rhizobium meliloti

Tn5 mutants of Rhizobium meliloti L5.30 defective in motility (Mot^-) were isolated and compared to the parent with respect to the nodulation activity. Each of the mutants was able to generate normal nodules on the alfalfa (Medicago sativa) but had slightly delayed nodule formation. Coinoculation of lucerne with wild type Mot⁺ and Mot^- cells in the wide range of ratios resulted in nodules occupied in the majority by a motile strain suggesting that motility is a factor involved in the competition for nodule formation.     

A Biochemical Characterization of Histidine Auxotrophs of Corynebacterium glutamicum

Two imidazoleglycerol (IG)-producing mutants and one imidazoleacetol (IA)-producing mutant were selected out of 14 histidine auxotrophs of C. glutamicum, by means of paper-chromatographic analysis of the culture broths of these mutants. Three of the histidine biosynthetic enzymes were determined for these mutants and a previously isolated histidinol-producing mutant of C. glutamicum. The IA-producing mutant and the l-histidinol-producing mutant had a defect in histidinol phosphate aminotransferase and histidinol dehydrogenase, respectively. IG-producers were not defective in these enzymes. These results were consistent with the histidine biosynthetic sequence known in other microorganisms.     

Genetic and functional analysis of the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI

Thirty Tn5- or Tn1831-induced nodulation (nod) mutants of Rhizobium leguminosarum were examined for their genetic and symbiotic properties. Thirteen mutants contained a deletion in Sym plasmid pRL1JI. These deletions cover the whole nod region and are 50 kb in size. All remaining seventeen mutations are located in a 6.6 kb EcoRI nod fragment of the Sym plasmid. Mutations in a 3.5 kb part on the right hand side of this 6.6 kb fragment completely prevent nodulation on Vicia sativa. All mutants in this 3.5 kb area are unable to induce marked root hair curling and thick and short roots.Mutations in a 1.5 kb area on the left hand side of the 6.6 kb nod fragment generate other symbiotic defects in that nodules are only rarely formed and only so after a delay of several days. Moreover, infection thread formation is delayed and root hair curling is more excessive than that caused by the parental strain. Their ability to induce thick and short roots is unaltered.Mutations in this 1.5 kb region are not complemented by pRmSL26, which carries nod genes of R. meliloti, whereas mutations in the 3.5 kb region are all complemented by pRmSL26.Abbreviations Rps repression of production of small bacteriocin - Mep medium bacteriocin production - Nod nodulation - Fix fixation - Tsr thick and short roots - Flac root hair curling - Hsp host specificity - Flad root hair deformation - Tc tetracycline - Km kanamycin - Cm chloramphenicol - Sp spectinomycin - Sm streptomycin - R resistant     

Chitinase and peroxidase in effective (fix+) and ineffective (fix−) soybean nodules

Chitinase and peroxidase, two enzymes thought to be involved in the defense of plants against pathogens, were measured in soybean (Glycine max L. Merr.) roots and in nodules colonized by Bradyrhizobium japonicum strains differing in their symbiotic potential. Activities of both enzymes were higher in nodules than in roots. In effective, nitrogen-fixing nodules, colonized by wild-type bacteria, chitinase and peroxidase activities had low levels in the central infected zone and were enhanced primarily in the nodule cortex. An ascorbate-specific peroxidase, possibly involved in radical scavenging, had similarly high activities in the infected zone and in the cortex. Ineffective nodules colonized by bacteria unable to fix nitrogen symbiotically showed a similar distribution of chitinase and peroxidase. In another type of ineffective nodule, colonized by a B. japonicum strain eliciting a hypersensitive response, activities of both enzymes were enhanced to a similar degree in the infected zone as well as in the cortex. Tissue prints using a direct assay for peroxidase and an antiserum against bean chitinase corroborated these results. The antiserum against bean chitinase cross-reacted with a nodule protein of M_r 32 000; it inhibited most of the chitinase activity in the nodules but barely affected the chitinase in uninfected roots. It is concluded that proteins characteristic of the defense reaction accumulate in the cortex of nodules independently of their ability to fix nitrogen, and in the entire body of hypersensitively reacting nodules.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate This work was supported by the Swiss National Science Foundation, Grants 31-25730.88 (to R.B. Mellor and T. Boller) and 31-27923.89 (to A. Wiemken).     

Peribacteroid membrane nodulin gene induction by Bradyrhizobium japonicum mutants

Seventeen translation products from Glycine max root mRNA precipitated with antiserum prepared against a peribacteroid membrane preparation from effective root nodules. Messenger RNA from fix ⁺ nodules coded for these 17 products plus 7 other nodule-specific polypeptides which bound to the antiserum. Of these 7 nodulins only 4 were present when nodules were infected with Bradyrhizobium japonicum 110 rif 15 2960, which induces the plant to produce empty peribacteroid membranes. In nodules infected with B. japonicum strains inducing either very short-lived or defective peribacteroid membrane, only 5 or 6, respectively, of these nodulins could be detected.From these results we hypothesize that the microsymbiont is responsible for the production of at least 4 different signals leading to peribacteriod membrane formation by the plant.