Construction and characterization of Bordetella pertussis RecA− mutants

Abstract Antibiotic drug-resistance cassettes (DRCs) were used to insertionally inactivate the wild-type Bordetella pertussis recA gene cloned into a suicide vector. The mutant allele was mobilized by conjugal gene transfer from Escherichia coli strain SM10 into different genetic backgrounds of B. pertussis . Southern hybridization studies of one of these mutants showed that it contained a DRC integrated within a recA gene situated within a Cla I genomic DNA fragment. Selected mutants were assayed to quantify recombinational and DNA repair deficiencies. These mutants were shown to be highly sensitive to both chemically and physically induced DNA damage. Gene transfer studies of another RecA⁻ mutant also indicated that it was defective in intergenic recombination. No difference in hemolytic activity or production of capsule was detected between the RecA⁻ mutants and their corresponding wild-type strains. The results of this investigation corroborate previous studies with the cloned B. pertussis recA gene, and demonstrate that the expression of the B. pertussis recA gene in the original host promotes both DNA repair and recombination.     

The pha gene cluster of Rhizobium meliloti involved in pH adaptation and symbiosis encodes a novel type of K+ efflux system

The fix-2 mutant of Rhizobium meliloti affected in the invasion of alfalfa root nodules (Inf⁻/Fix⁻) is K⁺ sensitive and unable to adapt to alkaline pH in the presence of K⁺. Using directed Tn 5 mutagenesis, we delimited a 6 kb genomic region in which mutations resulted in both Inf⁻/Fix⁻ and K⁺-sensitive phenotypes. In this DNA region, seven open reading frames (ORFs) were identified and the corresponding genes were designated phaA , B , C , D , E , F and G . The putative PhaABC proteins exhibit homology to the subunits of a Na⁺/H⁺ antiporter from an alkalophilic Bacillus strain. Moreover, PhaA and PhaD also show similarity to the ND5 and ND4 subunits of the proton-pumping NADH:ubiquinone oxidoreductase respectively. Computer analysis suggests that all seven proteins are highly hydrophobic with several possible transmembrane domains. Some of these domains were confirmed by generating active alkaline phosphatase fusions. Ion transport studies on phaA mutant cells revealed a defect in K⁺ efflux at alkaline pH after the addition of a membrane-permeable amine. These results suggest that the pha genes of R. meliloti encode for a novel type of K⁺ efflux system that is involved in pH adaptation and is required for the adaptation to the altered environment inside the plant.     

Toxicity of Sodium and Chloride Ions to Rhizobium spp. in Broth and Peat Culture

The growth of a strain of Rhizobium trifolii and of R. meliloti was studied in broth and peat cultures to determine the relative toxicity of Na⁺ and Cl^-. The following salts were added in a range of concentrations: Na₂HPO₄ as a source of Na⁺, CaCl₂.2H₂O as a source of Cl^-, and NaCl. Disodium hydrogen orthophosphate affected the growth rate of both strains in broth culture but not in peat culture. Unexpectedly, calcium chloride was more toxic than NaCl in broth and peat culture. The toxicity of NaCl can be ascribed to the Cl^-. Rhizobium meliloti strains grew on 3·5% NaCl after adaptation during a long period. Rhizobia for soya bean and cowpea grew at 0·5% NaCl and those for clover and pea, at 1·0% NaCl.     

Phosphodiesterase and phosphotriesterase in Rhizobium and Bradyrhizobium strains and their roles in the degradation of organophosphorus pesticides

Of 13 Rhizobium and Bradyrhizobium strains investigated for the production of cellular and extracellular phosphodiesterase and phosphotriesterase, all were found to produce both enzymes. Phosphodiesterase was produced at a much higher level than phosphotriesterase. Rhizobium meliloti TAL 1373 was the most productive. The extracellular enzymes were activated by inclusion in the assay mixture of Ca²⁺ or Mg²⁺. The enzymes were inhibited by Zn²⁺ but not significantly affected by Cu²⁺, Co²⁺ and Mn²⁺. Both hydrolases were inhibited by dithiothreitol but not by thiol-directed inhibitors, suggesting that sulphydryl groups are not directly involved in catalysis. The enzymes have the ability to hydrolyse some organophosphorus compounds, suggesting that Rhizobium and Bradyrhizobium strains play an important role in the degradation of organophosphorus pesticides.     

Nitrogen fixation (C2H2 reduction) by Medicago nodules and bacteroids under sodium chloride stress

Medicago ciliaris (L.) All., a salt-tolerant legume, was not nodulated by Rhizobium meliloti (2011), a strain commonly used for field inoculation of alfalfas. A strain of Rhizobium meliloti (ABS7) was isolated from saline Algerian soils. It is generally more salt-resistant than strain 2011, exhibits a higher rate of growth and induces the formation of nodules on M. ciliaris . C₂H₂ reduction activity of M. ciliaris nodules was inhibited by 50% in the presence of 200 m M NaCl in the culture medium. whereas 100 m M NaCl was sufficient to inhibit the activity of nodules of M. sativa (L. cv. Europe). C₂H₂ reduction by bacteroids, isolated from nodules of the two species of alfalfa, was directly inhibited by the presence of NaCl in the incubation medium. In both cases, glucose could support bacteroid nitrogen fixation, but only in a narrow range of O₂ tensions. Bacteriods from M. ciliaris were more tolerant to salt than M. sativa ones. The salt resistance of bacteroids from nodules of plants watered with NaCl solutions was not improved in either species. Salt directly added to the incubation mixture of bacteroids or to the culture medium of plants inhibited O₂ uptake of bacteroids isolated from nodules of both M. ciliaris and M. sativa . The depressive effect of NaCl on bacteroid C₂H₂ reduction could be directly related to the drop in bacteroid respiration. The nitrogen fixation capacity of the M. ciliaris-Rhizobium meliloti (ABS7) symbiosis under saline conditions leads us to recommend the introduction of this association in salt-troubled areas.     

Carbon and nitrogen partitioning in young nodulated pea (wild type and nitrate reductase-deficient mutant) plants exposed to NH4NO3

Carbon and nitrogen partitioning was examined in a wild-type and a nitrate reductase-deficient mutant (A317) of Pisum sativum L. (ev. Juneau), effectively inoculated with two strains of Rhizobium leguminosarum (128C23 and 128C54) and grown hydroponically in medium without nitrogen for 21 days, followed by a further 7 days in medium without and with 5 mM NH₄NO₃. In wild-type symbioses the application of NH₄NO₃ significantly reduced nodule growth, nitrogenase (EC 1.7.99.2) activity, nodule carbohydrates (soluble sugars and starch) and allocation of [¹⁴C]-labelled (NO₃⁻, NH₄⁺, amino acids) in roots. In nodules, there was a decline in amino acids together with an increase in inorganic nitrogen concentration. In contrast, symbioses involving A317 exhibited no change in nitrogenase activity or nodule carbohydrates, and the concentrations of all nitrogenous solutes measured (including asparagine) in roots and nodules were enhanced. Photosynthate allocation to the nodule was reduced in the 128C23 symbiosis. Nitrite accumulation was not detected in any case. These data cannot be wholly explained by either the carbohydrate deprivation hypothesis or the nitrite hypothesis for the inhibition of symbiotic nitrogen fixation by combined nitrogen. Our result with A317 also provided evidence against the hypothesis that NO₃⁻ and NH₄⁺ or its assimilation products exert a direct effect on nitrogenase activity. It is concluded that more than one legume host and Rhizobium strain must be studied before generalizations about Rhizobium /legume interactions are made.     

Poly(β-hydroxyalkanoate) biosynthesis and accumulation by different Rhizobium species

Abstract Synthesis and accumulation of poly(β-hydroxy-alkanoate) in Rhizobium leguminosarum , R. leguminosarum biovar. trifolii, Rhizobium 'galega', Rhizobium 'hedysarum' and R. meliloti were studied.
Poly(β-hydroxybutyrate) is accumulated up to 55% of the cell dry weight. At 30–50% air saturation R. meliloti accumulates 50% of its biomass (5.5 g·1⁻¹ dry weight) as PHB after 90 h of batch fermentation. At 90% air saturation maximum accumulation (37.5%) of PHB occurs after 70 h cultivation.
R. meliloti strain 41 is able to synthesize the copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) at concentrations up to 58% of the biomass dry weight, containing up to 22 mol%β-hydroxyvalerate. Different concentrations of both copolymer and hydroxyvalerate were obtained when the microorganism was grown, in batch culture, in the presence of propionate or valerate and with glucose, sucrose or succinate as main carbon source.     

Effect of Mg2+ on production and on O-acetylation of glucuronan excreted by the Rhizobium meliloti M5N1 CS strain during fermentation

The effect of Mg²⁺ on glucuronan O -acetylation and production by the Rhizobium meliloti M5N1 CS strain was studied. Magnesium ion induced the production of a more acetylated exopolysaccharide containing a greater molar ratio of 2,3-di- O -acetyl residues.     

Rhizobial siderophore as an iron source for clover

Iron uptake by clover plants ( Trifoliuin pratense L. cv. Hruszowska) was studied using radioactive ferric iron (⁵⁵FeCl₂). As shown by autoradiography of non-infected plants, purified rhizobial siderophore isolated from Rhizobium leguminosarum by, trifolii , stimulated the uptake and shoot transport of iron. Addition of rhizobial siderophore into the growth medium of nodulated clover did not affect the iron transport to the shoots. In the absence of the rhizobial siderophore, clover infected by either nitrogen-fixing (Nod⁻ Fix⁺) or nonfixing (Nod⁺ Fix⁻) R. L. trifolii strains took up and transported into the shoots more iron than the non-infected control plants. Nodulated clover reduced Fe(III) more efficiently than the non-infected control plants.     

The construction, detection and use of bioluminescent Rhizobium leguminosarum biovar trifolii strains

A. CRESSWELL, L. SKØT AND A.R. COOKSON. 1994. The gene encoding the firefly luciferase enzyme ( luc ) was introduced to Rhizobium leguminosarum biovar trifolii strains with a view to using the resulting bioluminescent strains to study the survival of genetically-engineered rhizobia in soil microcosms. The genetically-engineered micro-organisms (GEMs) behaved similarly to their parent strains with respect to growth rate in laboratory media and in their symbiotic performance with their host plants. No gene transfer could be detected in laboratory mating experiments. When inoculated onto a non-sterile soil the population of the GEM declined sharply from an initial cell density of 2 times 107⁷ g^-1 soil to approach a stable cell density of approximately 3 times 10² g^-1 after 150 d. Direct photography of bioluminescent rhizobia enabled the detection of colonies as small as 0.1 mm in diameter without the need for transferring colonies onto filter paper. When a Rhizobium strain carrying the luc marker on a plasmid was used as inoculant it was possible to visualize differences in colonization of the rhizosphere of white clover and ryegrass by contact print and colour transparency films. The photographic detection methods described here demonstrate the possibilities of using bioluminescent rhizobia for assessing their survival in soil, and for looking at rhizosphere populations which may be an important site for potential gene transfer.     

Location of nif genes on large plasmids in Rhizobium strains isolated from legume tree root nodules

Abstract The presence of plasmids in Rhizobium strains isolated from legume tree root nodules has been studied. Bacteria isolated from Acacia melanoxylon, A. cyanophylla, Prosopis chilensis and Sophora microphylla harbour 2 to 5 plasmids of an M _r higher than 115 · 10⁶. Hybridization experiments have shown DNA homology between plasmid pID1 ( R. meliloti 41 nifH and D genes) and one of the plasmids in each of the bacteria studied.     

Sulphation of Rhizobium sp. NGR234 Nod factors is dependent on noeE, a new host-specificity gene

Rhizobia secrete specific lipo-chitooligosaccharide signals (LCOs) called Nod factors that are required for infection and nodulation of legumes. In Rhizobium sp. NGR234, the reducing N -acetyl- d -glucosamine of LCOs is substituted at C₆ with 2- O -methyl- l -fucose which can be acetylated or sulphated. We identified a flavonoid-inducible locus on the symbiotic plasmid pNGR234 a that contains a new nodulation gene, noeE which is required for the sulphation of NGR234 Nod factors (NodNGR). noeE was identified by conjugation into the closely related Rhizobium fredii strain USDA257, which produces fucosylated but non-sulphated Nod factors (NodUSDA). R. fredii transconjugants producing sulphated LCOs acquire the capacity to nodulate Calopogonium caeruleum . Furthermore, mutation of noeE (NGRΔ noeE  ) abolishes the production of sulphated LCOs and prevents nodulation of Pachyrhizus tuberosus . The sulphotransferase activity linked to NoeE is specific for fucose. In contrast, the sulphotransferase NodH of Rhizobium meliloti seems to be less specific than NoeE, because its introduction into NGRΔ noeE leads to the production of a mixture of LCOs that are sulphated on C₆ of the reducing terminus and sulphated on the 2- O -methylfucose residue. Together, these findings show that noeE is a host-specificity gene which probably encodes a fucose-specific sulphotransferase.     

Low vapour pressure deficit reduces the beneficial effect of elevated CO2 on growth of N2-fixing alfalfa plants

Plant responses to elevated CO₂ can be modified by many environmental factors, but very little attention has been paid to the interaction between CO₂ and changes in vapour pressure deficit (VPD). Thirty-day-old alfalfa plants ( Medicago sativa L. cv. Aragón), which were inoculated with Sinorhizobium meliloti 102F78 strain, were grown for 1 month in controlled environment chambers at 25/15°C, 14 h photoperiod, and 600 µmol m⁻² s⁻¹ photosynthetic photon flux (PPF), using a factorial combination of CO₂ concentration (400 µmol mol⁻¹ or 700 µmol mol⁻¹) and vapour pressure deficit (0.48 kPa or 1.74 kPa, which corresponded to relative humidities of 85% and 45% at 25°C, respectively). Elevated CO₂ strongly stimulated plant growth under high VPD conditions, but this beneficial effect was not observed under low VPD. Under low VPD, elevated CO₂ also did not enhance plant photosynthesis, and plant water stress was greatest for plants grown at elevated CO₂ and low VPD. Moreover, plants grown under elevated CO₂ and low VPD had a lower leaf soluble protein and photosynthetic activity (photosynthetic rate and carboxylation efficiency) than plants grown under elevated CO₂ and high VPD. Elevated CO₂ significantly increased leaf adaxial and abaxial temperatures. Because the effects of elevated CO₂ were dependent on vapour pressure deficit, VPD needs to be controlled in experiments studying the effect of elevated CO₂ as well as considered in the extrapolations of results to a warmer, high-CO₂ world.     

Production of B-group vitamins by two Rhizobium strains in chemically defined media

Twenty-eight strains of Rhizobium spp. were tested for their ability to grow in chemically-defined medium lacking growth factors. Two strains, R. meliloti GR4B and Rhizobium spp. ( Acacia ) GRH28, were selected, on the basis of their good growth under the conditions imposed, for further quantification of the production of water-soluble vitamins (thiamine, niacin, riboflavin, pantothenic acid and biotin) in chemically defined media amended with different compounds (mannitol, glucose or sodium succinate) as sole carbon sources. Qualitative and quantitative production of vitamins in chemically-defined media was significantly affected by the use of C sources of a different nature and the age of the cultures. Strain GRH28 produced all the vitamins analysed, and high biological levels of biotin (14 ng ml^–1 culture) were detected after 6 d of culture in mineral medium amended with mannitol. Pantothenic acid was the vitamin detected in the highest amounts (up to 1 μg ml^–1 of culture) in culture supernatant fluids of strain GR4B grown for 6 d with succinate as sole carbon source.     

Survival and infectivity of a Rhizobium meliloti strain maintained in water and buffer suspensions

Rhizobium meliloti B323 cells were suspended in deionized water, phosphate buffer pH 6.5 and 5.5 and these buffers supplemented with Ca²⁺, Mg²⁺ (1 mmol/l) and Fe³⁺ (0.1 mmol/l). Initial cell count was 1.10⁸ cells/ml. The viable count of rhizobia suspended in buffer at pH 6.5, with and without salts, remained constant or even increased during storage. Cells suspended in buffer at pH 5.5 with salts, decreased in numbers in the first 5 months, then, until the 10th month, the count remained at 10⁵ cells/ml. Rhizobia suspended in buffer at pH 5.5 and deionized water decreased in viability almost to zero by the 10th month. In those suspensions where viability was maintained, the symbiotic infectivity of cells was also maintained as compared with a control performed with fresh cultured rhizobia. In suspensions in deionized water and buffer at pH 5.5 where the viability diminished during the experiment, the rhizobia lost their ability to infect roots immediately after inoculation but maintained their capacity to form late nodules on the hosts.     

Isolation of an Asm− mutant of Rhizobium japonicum defective in symbiotic N2-fixation

Abstract A mutant strain of Rhizobium japonicum (CJ9) unable to assimilate ammonium (Asm⁻) was isolated following mutagenesis with N -methyl N -nitro-nitrosoguanidine (NTG). Glutamate synthase activity was not detectable in cell-free extracts of the mutant strain in contrast to the wild type and revertant strains. Although mutant CJ9 induced nitrogenase activity in an 'in vitro' assay system under microaerobic conditions, it failed to fix nitrogen (acetylene reduction) in soybean root nodules. These properties of mutant CJ9 constitute a new Asm⁻ mutant class in Rhizobium spp.     

Modulation of 45Ca2+ Influx into Cells Stably Expressing Recombinant Human NMDA Receptors by Ligands Acting at Distinct Recognition Sites

Abstract: A ⁴⁵Ca²⁺ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N -methyl- d -aspartate (NMDA) receptors. Inhibition of glutamate-stimulated ⁴⁵Ca²⁺ influx by six glycine-site antagonists and inhibition of glycine-stimulated ⁴⁵Ca²⁺ influx by five glutamate-site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated ⁴⁵Ca²⁺ influx for recombinant NMDA receptors, inhibiting and stimulating ⁴⁵Ca²⁺ influx into cells expressing NR1a/NR2A receptors (IC₅₀ = 408 µ M ) and NR1a/NR2B receptors (EC₅₀ = 37.3 µ M ), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC₅₀ = 0.099 µ M ) compared with NR1a/NR2A receptors (IC₅₀ = 164 µ M ). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg²⁺ and Zn²⁺ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of ⁴⁵Ca²⁺ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells.     

Incorporation of label from root-applied N6[8-14C]furfiuryIadenine into the guanine nucleotide fraction of pea bud ribonucleic acid

The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N ⁶[8-^I4C]furfuryladenine or of [8-¹⁴C]adenine to the root system of decapitated plants and to cultured excised buds. When N ⁶[8-¹⁴C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-¹⁴C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-¹⁴C]adenine or N ⁶[8-¹⁴C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N ⁶[8-^I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-¹⁴C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N ⁶ position of the adenine base.     

Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue

Genes of Rhizobium leguminosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK–fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRleVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain. An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R. leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr -like gene, designated fixK , whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix⁺ phenotype. A reduced nitrogen-fixation activity was found for a R. leguminosarum bv. viciae fnrN -deletion mutant, whereas no nitrogen-fixation activity was detectable for a fixK / fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified downstream of fixK–fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation.