Rhizobacterium

J Bacteriol, 1983 Dec, 156(3), 1236 - 42
Regulation of hydrogenase in Rhizobium japonicum: analysis of mutants altered in regulation by carbon substrates and oxygen; Merberg D et al.; The synthesis of the H2 uptake system in free-living Rhizobium japonicum SR is repressed both by oxygen and by carbon substrates . Mutants selected for the ability to express hydrogenase in 10.0% partial pressure O2 were also less sensitive than the wild type to repression by carbon substrates such as arabinose, glycerol, gluconate, and succinate . The H2 uptake system in another class of mutants, previously shown to be hypersensitive to repression by O2, is also more sensitive to repression by carbon substrates . The oxygen- and carbon-insensitive mutants express the hydrogen uptake system during heterotrophic growth in the absence of hydrogen and thus can be considered constitutive (Hupc) . The amount of cytochromes in the Hupc mutants is similar to that in the wild-type strain; however, the Hupc mutants contain greater methylene blue-dependent and O2-dependent hydrogenase activity, both as free-living cells and as bacteroids . Two-dimensional polyacrylamide gel electrophoresis revealed that during heterotrophic growth the Hupc mutant strain SR470 synthesized at least six peptides not found in the wild-type strain . The concentrations of cyclic AMP and guanosine tetraphosphate were similar in strain SR and the Hupc mutants during heterotrophic growth.

J Bacteriol, 1983 Dec, 156(3), 1035 - 45
Sym plasmid transfer to various symbiotic mutants of Rhizobium trifolii, R . leguminosarum, and R . meliloti; Djordjevic MA et al.; Two self-transmissible Sym(biosis) plasmids, one encoding pea-specific nodulation and nitrogen-fixation functions (plasmid pJB5JI) and the other encoding clover-specific nodulation and nitrogen-fixation functions (plasmid pBR1AN) were used to determine whether the symbiotic genes encoded on these plasmids are expressed in various members of the Rhizobiaceae . The host specificity of Rhizobium trifolii and R . leguminosarum Sym plasmid-cured strains could be directly determined by the transfer to these strains of the appropriate Sym plasmid . The nodulation of white clovers was restored by either plasmid pJB5JI or pBR1AN when these plasmids were transferred to two transposon Tn5-induced hair-curling (Hac-) R . trifolii mutants . In addition, lucerne nodulation was restored to a Hac- R . meliloti mutant when either plasmid pBR1AN or pJB5JI was transferred to this strain . The phenotype of nonmucoid (Muc-) Rhizobium mutants, which had altered cell surfaces, was not influenced by the transfer to these strains of plasmid pBR1AN or plasmid pJB5JI.

J Bacteriol, 1983 Dec, 156(3), 1025 - 34
Molecular characterization of Tn5-induced symbiotic (Fix-) mutants of Rhizobium meliloti; Zimmerman JL et al.; To investigate the expression of specific symbiotic genes during the development of nitrogen-fixing root nodules, we conducted a systematic analysis of nodule-specific proteins and RNAs produced after the inoculation of alfalfa roots with a series of Rhizobium meliloti mutants generated by site-directed transposon Tn5 mutagenesis . The mutagenized region of the Rhizobium genome covered approximately 10 kilobases and included the region encoding the nitrogenase polypeptides . All mutant strains that were analyzed produced nodules, but with several strains the nodules failed to fix nitrogen (Nod+ Fix- phenotype) . All Fix- nodules accumulated reduced levels of the host plant protein leghemoglobin . In addition, Tn5 insertions in the nitrogenase operon (nifHDK genes) eliminated some or all of the nitrogenase polypeptides and nifHDK RNA transcripts, depending on the site of insertion . Finally, mutation of a region approximately 5 kilobases upstream from the nitrogenase operon resulted in the absence of all three nitrogenase polypeptides and their corresponding RNAs, suggesting that this region may serve a regulatory function during nitrogen fixation . The studies presented here indicate that site-directed mutagenesis coupled with biochemical analysis of nodule proteins and RNAs allows the identification of products of specific gene regions as well as the assignment of specific functions to previously unidentified regions of the R . meliloti genome.

J Bacteriol, 1983 Nov, 156(2), 937 - 40
High-frequency induction of nodulation and nitrogen fixation mutants of Rhizobium japonicum; Skogen-Hagenson MJ et al.; More than 50 symbiotic mutants of Rhizobium japonicum were isolated by purported plasmid-curing techniques . Wild-type R . japonicum strains were grown in liquid culture at 28 or 36 degrees C in different concentrations of acridine orange, ethidium bromide, or sodium dodecyl sulfate for selection of mutants . The symbiotic traits of 133 isolates from nine treatment groups were determined . Forty-two isolates were Nod- Nif+, seven were Nod+ Nif-, and two were Nod- Nif- . The nifDH genes were deleted in three mutants and consequently showed no hybridization to a nifDH probe . None of these mutants showed any detectable loss of plasmid DNA.

J Bacteriol, 1983 Nov, 156(2), 888 - 97
Initial stages in the morphogenesis of nitrogen-fixing stem nodules of Sesbania rostrata; Tsien HC et al.; Morphogenesis of stem nodules in Sesbania rostrata was studied over a period of 6 days after inoculation with an appropriate species of Rhizobium . Nodulation sites were initially slightly raised, circular areas 0.3 to 0.6 mm in diameter and 4 to 5 mm apart in vertical rows along the length of the stem . Each site was underlaid by an adventitious root primordium . A site became susceptible to infection by a specific Rhizobium sp . when the root primordium broke through the epidermis, leaving a fissure . Rhizobia multiplied within this fissure and colonized the exposed intercellular spaces . The infection extended inward as narrow, branched intercellular threads moved into a cortical meristematic zone, where cell division was initiated, and invagination of infection thread branches into adjacent plant cells followed . Rhizobia were released into the plant cells and surrounded immediately by plant membrane . Intracellular rhizobia divided actively, leading to bacteroid-filled cells . Infected areas enlarged and coalesced as the nodule matured.

J Bacteriol, 1983 Nov, 156(2), 592 - 9
Identification of a Rhizobium trifolii plasmid coding for nitrogen fixation and nodulation genes and its interaction with pJB5JI, a Rhizobium leguminosarum plasmid; Christensen AH et al.; Rhizobium trifolii T37 contains at least three plasmids with sizes of greater than 250 megadaltons . Southern blots of agarose gels of these plasmids probed with Rhizobium meliloti nif DNA indicated that the smallest plasmid, pRtT37a, contains the nif genes . Transfer of the Rhizobium leguminosarum plasmid pJB5JI, which codes for pea nodulation and the nif genes and is genetically marked with Tn5, into R . trifolii T37 generated transconjugants containing a variety of plasmid profiles . The plasmid profiles and symbiotic properties of all of the transconjugants were stably maintained even after reisolation from nodules . The transconjugant strains were placed into three groups based on their plasmid profiles and symbiotic properties . The first group harbored a plasmid similar in size to pJB5JI (130 megadaltons) and lacked a plasmid corresponding to pRtT37a . These strains formed effective nodules on peas but were unable to nodulate clover and lacked the R . trifolii nif genes . This suggests that genes essential for clover nodulation as well as the R . trifolii nif genes are located on pRtT37a and have been deleted . The second group harbored hybrid plasmids formed from pRtT37a and pJB5JI which ranged in size from 140 to ca . 250 megadaltons . These transconjugants had lost the R . leguminosarum nif genes but retained the R . trifolii nif genes . Strains in this group nodulated both peas and clover but formed effective nodules only on clover . The third group of transconjugants contained a hybrid plasmid similar in size to pRtT37b . These strains contained the R . trifolii and R . leguminosarum nif genes and formed N2-fixing nodules on both peas and clover.

J Bacteriol, 1983 Oct, 156(1), 168 - 76
Cloning of the glutamine synthetase I gene from Rhizobium meliloti; Somerville JE et al.; Glutamine synthetase is a major enzyme in the assimilation of ammonia by members of the genus Rhizobium . Two forms of glutamine synthetase are found in members of the genus Rhizobium, a heat-stable glutamine synthetase I (GSI) and a heat-labile GSII . As a step toward clarifying the role of these enzymes in symbiotic nitrogen fixation, we have cloned the structural gene for GSI from Rhizobium meliloti 104A14 . A gene bank of R . meliloti was constructed by using the bacteriophage P4 cosmid pMK318 . Cosmids that contain the structural gene for GSI were isolated by selecting for plasmids that permit ET8051, an Escherichia coli glutamine autotroph, to grow with ammonia as the sole nitrogen source . One of the cosmids, pJS36, contains an insert of 11.9 kilobases . ET8051(pJS36) grows slowly on minimal media . When a 3.7-kilobase HindIII fragment derived from this DNA is cloned into the HindIII site of pACYC177 and the plasmids are transformed into ET8051, rapid growth is observed when the insert is in one orientation (pJS44) but not the other (pJS45) . Glutamine synthetase activity can be detected in ET8051(pJS44); most of this activity is heat stable . pJS36 hybridizes with the glnA structural gene from Escherichia coli . Insertion of a 2.7-kilobase Tetr determinant into a BglII site located within pJS44 abolishes all glutamine synthetase activity . This interrupted version of a glutamine synthetase gene was substituted for the normal R . meliloti sequence by homologous recombination in R . meliloti . Recombinants lose GSI activity, but retain GSII activity and grow well with ammonia as the sole nitrogen source . These mutants are unaffected in nodulation and nitrogen fixation.

J Bacteriol, 1983 Sep, 155(3), 1429 - 33
Plasmid visualization and nif gene location in nitrogen-fixing Azospirillum strains; Plazinski J et al.; A modified gel electrophoresis technique provided a reproducible way of detecting and isolating plasmids with molecular weights ranging from 12 X 10(6) to 370 X 10(6) for Azospirillum species . Analysis with the nifHD region of Rhizobium trifolii showed that the Azospirillum nif genes were chromosomally located in all eight strains investigated and not on endogenous plasmids.

J Bacteriol, 1983 Aug, 155(2), 926 - 9
Nif- Hup- mutants of Rhizobium japonicum; Moshiri F et al.; Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity . One of the mutants formed green nodules and was deficient in heme . Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts . Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins . Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity.

J Bacteriol, 1983 Aug, 155(2), 481 - 7
Involvement of cytochromes and a flavoprotein in hydrogen oxidation in Rhizobium japonicum bacteroids; O'Brian MR et al.; Electron transport components involved in H2 oxidation were studied in membranes from Rhizobium japonicum bacteroids . Hydrogen oxidation in membranes was inhibited by antimycin A and 2-n-heptyl-4-hydroxyquinoline-N-oxide with Ki values of 39.4 and 5.6 microM, respectively . The inhibition of H2 uptake by cyanide was triphasic with Ki values of 0.8, 9.9, and 93.6 microM . This result suggested that three cyanide-reactive components were involved in H2 oxidation . H2-reduced minus O2-oxidized absorption difference spectra showed peaks at 551.5 and 560 nm, indicating the involvement of c- and b-type cytochromes, respectively . This spectrum also revealed a trough at 455 nm, showing that H2 oxidation involves a flavoprotein . This flavoprotein was not reduced by H2 in the presence of cyanide . The inhibition of H2 or cytochrome c oxidation by the flavoprotein inhibitor Atebrin was monophasic; the Ki values were similar for both substrates . A role for the flavoprotein as a terminal oxidase was implicated based on its high redox potential and its sensitivity to cyanide . Cytochromes o and c-552 were identified based on their ability to bind carbon monoxide and cyanide.

Arch Microbiol, 1983 Aug, 135(2), 103 - 9
RNA polymerase from Rhizobium japonicum; Regensburger B et al.; DNA-dependent RNA polymerase (EC 2.7.7.6) from Rhizobium japonicum was purified . The subunit structure was found to be beta beta' alpha 2 alpha, with the following apparent molecular weights determined by electrophoresis: Mr (beta and beta') 150,000 each, Mr (sigma) 96,000, Mr (alpha) 40,000, Mr (holoenzyme) 490,000, Mr (core enzyme) 380,000 . The recovery of sigma was 28% . RNA polymerase from aerobically grown R . japonicum cells and from nitrogen-fixing cells have the same electrophoretic properties suggesting that no chemical modification of the enzyme occurs when cells undergo this metabolic differentiation . The enzyme is Mg2+-dependent, rifampicin-sensitive, and has optimal activity at alkaline pH (8--10) and at 35--40 degrees C . It binds strongly to bacteriophage T7 promoters, weakly to antibiotic resistance genes, and not at all to cloned R . japonicum nif DNA . Preliminary in vitro transcription experiments, including nif DNA as template, revealed that additional factors may be required for selective transcription from promoters.

J Gen Microbiol, 1983 Aug, 129 (Pt 8), 2531 - 4
Transfer of nitrate reductase genes of the cyanobacterium Nostoc muscorum into Rhizobium japonicum; Singh RK et al.; Transformation of Rhizobium japonicum CB1809 was studied using DNA from the cyanobacterium Nostoc muscorum ATCC 27893 . A spontaneous nitrate reductase deficient (Nar-) mutant (NR-6) of R . japonicum CB1809 was isolated with a frequency of 8.4 X 10(-7) . Streptomycin (Sm) and Neomycin (Neo) resistance markers were introduced into strain NR-6, and the resulting strain was designated NR-6 SmR NeoR . Experiments with cyanobacterial DNA and live cells of strain NR-6 SmR NeoR indicated transformation of nitrate reductase (nar) genes of N . muscorum into this strain . This conclusion was supported by the reversion frequency of strain NR-6 SmR NeoR to Nar+ and the transformation frequency when recipient cells were exposed to N . muscorum DNA (with heat-treated DNA as control) . Comparisons of growth, nitrate uptake, assimilatory nitrate reductase activity and nodulation of parent CB1809, NR-6 SmR NeoR and five transformant clones (Nar+) suggest that there may be considerable homology between the nar genes of R . japonicum CB1809 and N . muscorum.

J Bacteriol, 1983 Aug, 155(2), 915 - 8
In Rhizobium japonicum the nitrogenase genes nifH and nifDK are separated; Kaluza K et al.; In contrast to Klebsiella pneumoniae or fast-growing Rhizobium species, such as R . meliloti, where the nitrogenase structural genes are clustered in one operon (nifHDK), in slow-growing Rhizobium japonicum 110, nifH and nifDK are on separate operons.

J Bacteriol, 1983 Jul, 155(1), 367 - 80
Ultrastructural analysis of ineffective alfalfa nodules formed by nif::Tn5 mutants of Rhizobium meliloti; Hirsch AM et al.; Ineffective alfalfa nodules formed by Rhizobium meliloti nif::Tn5 mutants were examined by light and electron microscopy . R . meliloti nifH::Tn5 mutants formed nodules that were similar in structure to wild-type nodules except that nifH- bacteroids accumulated a compact, electron-dense body . In contrast to nodules induced by wild type and nifH mutants, nifDK- R . meliloti mutants induced nodules which contained numerous starch grains and prematurely senescent bacteroids . In addition, meristematic activity in nifDK- nodules ceased significantly earlier than in nifH- nodules . All mutant nodules exhibited elevated levels of rough endoplasmic reticulum and Golgi membranes compared to wild-type nodule cells . These elevated levels may reflect either a response to nitrogen starvation in the ineffective nodules or an abnormal synthesis and export of nodule-specific proteins during later developmental stages.

Proc Natl Acad Sci U S A, 1983 Jul, 80(13), 4030 - 4
Activation of Klebsiella pneumoniae and Rhizobium meliloti nitrogenase promoters by gln (ntr) regulatory proteins; Sundaresan V et al.; We have studied the expression, in different Escherichia coli gln (ntr) mutants, of fusions (constructed in vitro) of the nifHDK (nitrogenase) promoters from Klebsiella pneumoniae and Rhizobium meliloti to E . coli lacZ . Derepression of the K . pneumoniae nifH::lacZ fusion requires the glnF (ntrA) gene product in addition to the K . pneumoniae nifA gene product, indicating that regulation of the K . pneumoniae nif genes is more closely integrated with the overall nitrogen control system than previously demonstrated . Derepression of the R . meliloti nifH::lacZ fusion in E . coli by the K . pneumoniae nifA gene product (which we had previously shown) exhibits the same requirement for glnF . Derepression of the R . meliloti nifH::lacZ fusion, but not the K . pneumoniae nifH::lacZ fusion, can be mediated by the glnG (ntrC) gene product, suggesting that the gln regulatory genes might directly regulate the symbiotic nitrogen fixation genes in Rhizobium.

J Bacteriol, 1983 Jul, 155(1), 311 - 6
Isolation and characterization of the recA gene of Rhizobium meliloti; Better M et al.; Interspecific complementation of an Escherichia coli recA mutant with plasmids containing a gene bank of Rhizobium meliloti DNA was used to identify a clone which contains the recA gene of R . meliloti . The R . meliloti recA protein can function in recombination and in response to DNA damage when expressed in an E . coli recA host, and hybridization studies have shown that DNA sequence homology exists between the recA gene of E . coli and that of R . meliloti . The isolated R . meliloti recA DNA was used to construct a recA R . meliloti, and this bacterium was not deficient in its ability to carry out symbiotic nitrogen fixation.

J Bacteriol, 1983 Jun, 154(3), 1403 - 13
Symbiotic properties of C4-dicarboxylic acid transport mutants of Rhizobium leguminosarum; Finan TM et al.; The transport of succinate was studied in bacteroids of an effective, streptomycin-resistant strain (GF160) of Rhizobium leguminosarum . High levels of succinate transport occurred, and the kinetics, specificity, and sensitivity to metabolic inhibitors were similar to those previously described for free-living cells . The symbiotic properties of two transposon (Tn5)-mediated C4-dicarboxylate transport mutants (strains GF31 and GF252) were determined . Strain GF31 formed ineffective nodules, and bacteroids from these nodules showed no succinate transport activity . Strain GF252 formed partially effective nodules, and bacteroids from these nodules showed about 50% of the succinate transport activity of the parent bacteroids . Another dicarboxylic acid transport mutant (Dct-), strain GFS5, isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, formed ineffective nodules . The ability to form ineffective nodules in strains GF31 and GFS5 was shown to correlate with the Dct- phenotype . The data indicate that the presence of a functional C4-dicarboxylic acid transport system is essential for N2 fixation to occur in pea nodules.

Antibiotiki, 1983 May, 28(5), 323 - 5
{Induced resistance to kanamycin and tetracycline in Rhizobium japonicum}; Zlotnikov KM et al.; Spontaneous mutants Km-R and Tc-R of R . japonicum with various levels of resistance to kanamycin (0.8-20 mg/ml) and tetracycline (130-210 micrograms/ml) were isolated . No cross resistance in the mutants was observed . Plasmid R68.45 transferred to the wild strains resistance to 210 micrograms/ml of tetracycline and to 20 mg/ml of kanamycin . This plasmid did not practically increase the resistance to tetracycline in mutants Tc-R . At the same time it markedly increased the resistance to kanamycin in mutants Km-R.

J Bacteriol, 1983 May, 154(2), 838 - 45
Protoporphyrin formation in Rhizobium japonicum; Keithly JH et al.; The obligately aerobic soybean root nodule bacterium Rhizobium japonicum produces large amounts of heme (iron protoporphyrin) only under low oxygen tensions, such as exist in the symbiotic root nodule . Aerobically incubated suspensions of both laboratory-cultured and symbiotic bacteria (bacteroids) metabolize delta-aminolevulinic acid to uroporphyrin, coproporphyrin, and protoporphyrin . Under anaerobic conditions, suspensions of laboratory-cultured bacteria form greatly reduced amounts of protoporphyrin from delta-aminolevulinic acid, whereas protoporphyrin formation by bacteroid suspensions is unaffected by anaerobiosis, suggesting that bacteroids form protoporphyrin under anaerobic conditions more readily than do free-living bacteria . Oxygen is the major terminal electron acceptor for coproporphyrinogen oxidation in cell-free extracts of both bacteroids and free-living bacteria . In the absence of oxygen, ATP, NADP, Mg2+, and L-methionine are required for protoporphyrin formation in vitro . In the presence of these supplements, coproporphyrinogenase activity under anaerobic conditions is 5 to 10% of that observed under aerobic conditions . Two mechanisms for coproporphyrinogen oxidation exist in R . japonicum: an oxygen-dependent process and an anaerobic oxidation in which electrons are transferred to NADP . The significance of these findings with regard to heme biosynthesis in the microaerophilic soybean root nodule is discussed.

Proc Natl Acad Sci U S A, 1983 May, 80(9), 2524 - 8
Promoters regulated by the glnG (ntrC) and nifA gene products share a heptameric consensus sequence in the -15 region; Ow DW et al.; We have determined the nucleotide sequences of the Klebsiella pneumoniae nifL (regulation of N2 fixation genes) and the Escherichia coli glnA (glutamine synthetase) promoters . We compared these sequences with the published sequences of three other promoters that, like the nifL and glnA promoters, are activated by the general nitrogen regulators glnF (ntrA) and glnG (ntrC) . The three promoters are the argTr (arginine transport) and dhuA (histidine transport) promoters of Salmonella typhimurium and the nifH (nitrogenase) promoter of Rhizobium meliloti . All five sequences (with at most one mismatch) contain the heptameric consensus sequence T-T-T-T-G-C-A . In the R . meliloti nifH and K . pneumoniae nifL promoters, in which the transcription initiation sites have been determined, the consensus sequence is situated in the -15 region . We recently reported that the K . pneumoniae nifA product, which activates nif genes, can substitute for the glnG (ntrC) product in activating promoters of several genes involved in nitrogen assimilation, including the nifL, the glnA, and the R . meliloti nifH promoters . It is likely that nifA also activates the S . typhimurium argTr and dhuA promoters . In contrast, the glnG product cannot substitute for the nifA product in the activation of the K . pneumoniae nifH (nitrogenase) promoter . Consistent with this latter observation, and supporting the conclusion that the T-T-T-T-G-C-A sequence is a regulatory site for glnG product activation, the K . pneumoniae nifH promoter (C-C-C-T-G-C-A) has only partial similarity with the T-T-T-T-G-C-A consensus sequence in the -15 region.

Biochem Biophys Res Commun, 1983 Mar 29, 111(3), 798 - 803
A soybean lectin having 4-O-methyl-D-glucuronic acid specificity; Dombrink-Kurtzman MA et al.; Evidence is presented for the presence of a new lectin activity in soybean seeds {Glycine max (L.) Merrill} that has specificity towards the 4-O-methyl-D-glucurono-L-rhamnan exopolysaccharide produced by certain strains of Rhizobium japonicum . Bacterial agglutination and precipitin reactions revealed the lectin activity in phosphate-buffered saline extracts of seeds of all cultivars tested, including the "lectinless" varieties . Reaction of such extracts with carbohydrate haptens demonstrated that the specificity of the binding was towards 4-O-methyl-D-glucuronic acid, D-glucuronic acid and their methyl glycosides.

Eur J Biochem, 1983 Mar 15, 131(2), 325 - 31
Primary structure of the DNA-binding protein HRm from Rhizobium meliloti; Laine B et al.; The amino acid sequence of protein HRm, a DNA-binding HU-type protein of 90 residues (Mr 9303), isolated from Rhizobium meliloti, has been established from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at arginine and aspartic acid residues . The comparison of the primary structure of protein HRm with that of other HU-type proteins shows that two short sequences, of 7 and 6 residues respectively, located in the median part of the molecule, appear highly conserved and may be important in the function of the protein.

J Bacteriol, 1983 Mar, 153(3), 1196 - 201
Methylammonium uptake by Rhizobium sp . strain 32H1; Gober JW et al.; We present evidence that methylammonium is transported into cowpea Rhizobium sp . strain 32H1 cells by a membrane carrier whose natural substrate is ammonium . After growth in low (0.2%) oxygen, which is necessary for nitrogen fixation by these cells, respiring rhizobial cells took up {14C}methylammonium to high intracellular levels . Cells grown in atmospheric (21%) oxygen did not take up methylammonium . Uptake (transport plus metabolism) was maximal in cells harvested in the early stationary phase of batch culture and had a distinct pH optimum of 6.5 to 7.0 . Uptake was inhibited by metabolic poisons that dissipate the proton motive force or inhibit ATP synthesis . Inhibition of uptake by ammonium and the counterflow phenomenon indicated that ammonium and methylammonium share a transport carrier . Of the methylammonium taken up, about 15% was accumulated to intracellular levels 20 times higher than those in the medium; most of the methylammonium was metabolized to gamma-N-methylglutamine.

Ann Microbiol (Paris), 1983 Mar-Apr, 134A(2), 231 - 4
{Comparative evaluation of the use of glucose and fructose by the oxidative pathway in a strain of Rhizobium meliloti}; Hornez JP et al.; The oxidation rate in resting cells of Rhizobium meliloti was investigated with either glucose or fructose as substrate . Fructose uptake was found to be complete, whereas glucose uptake did not fully occur, yielding 2-kitogluconic acid in the medium . At the end of fructose oxidation, significant levels of this substrate and its derivatives had accumulated in the bacteria.

J Bacteriol, 1983 Mar, 153(3), 1155 - 62
Succinate transport by free-living forms of Rhizobium japonicum; McAllister CF et al.; We have demonstrated that the transport of succinate into the cells of Rhizobium japonicum strains USDA 110 and USDA 217 is severely inhibited by cyanide, azide, and 2,4-dinitrophenol, but not by arsenate . These results suggest an active mechanism of transport that is dependent on an energized membrane, but does not directly utilize ATP . The apparent Km for succinate was 3.8 microM for strain USDA 110 and 1.8 microM for strain USDA 217; maximal transport velocities were 1.5 and 3.3 nmol of succinate per min per mg of protein, respectively . The expression of the succinate uptake activity was inducible rather than constitutive, with succinate and structurally related compounds being the most effective inducers . The mechanism showed some specificity for succinate and similar organic acids; fumarate and L-malate were classical competitive inhibitors of the system . In general, the best competing compounds were also the best carbon substrates for induction of succinate uptake activity . EDTA inhibited the transport of succinate, implying a role for divalent cations in the system . When various divalent cations were used to reconstitute EDTA-inhibited activity, Ca2+ was most effective, followed by Mg2+, which restored activity at about half the efficiency of Ca2+ . Growth media that were supplemented with increased Ca2+ concentration supported more rapid growth with succinate as the carbon substrate, and cells from such media showed higher specific activities of succinate transport.

Nature, 1983 Feb 24, 301(5902), 728 - 32
Klebsiella pneumoniae nifA product activates the Rhizobium meliloti nitrogenase promoter; Sundaresan V et al.; Bacteria in the genus Rhizobium normally fix nitrogen only when they interact with leguminous plants to produce on the roots a highly differentiated structure, the nodule, within which the bacteria differentiate into nitrogen-fixing bacteroids . By contrast, the enteric bacterium Klebsiella pneumoniae reduces nitrogen in a free-living state in conditions of low oxygen tension and deficiency of fixed nitrogen . In K . pneumoniae, the overall circuitry by which nitrogen-fixation (nif) genes are regulated has been elucidated . In response to ammonia starvation, the product of the glnG gene activates transcription of the nifLA operon; this activation is dependent on the product of glnF (ref . 4) . The nifA gene product is in turn required for transcription of all the other nif genes, including the nifHDK operon which codes for the subunits of nitrogenase . In contrast, very little is known about the sequence of events involved in the regulated change in rhizobial nif gene expression associated with bacteroid differentiation . In the work described here, we identify the K . pneumoniae and Rhizobium meliloti nifHDK promoters by mapping the in vivo start points of transcription . By defining and comparing the DNA sequences of these two promoters, we find that they share an unexpected degree of homology . Further, by constructing fusions of each of the two promoters to the lacZ gene from Escherichia coli, we show that both promoters are activated by the product of the K . pneumoniae nifA gene.

J Bacteriol, 1983 Feb, 153(2), 1045 - 50
Pleomorphism and acetylene-reducing activity of free-living rhizobia; Kaneshiro T et al.; Cowpea-type Rhizobium sp . strain 32H1 and Rhizobium japonicum USDA 26 and 110 grown on a glutamate-mannitol-gluconate agar medium showed increases in the number of pleomorphic cells coincident with their acetylene-reducing activity . Pleomorphs appeared to be inhibited in growth nonuniformly, because acetylene-reducing cultures were mixtures of rod, branched (V, Y, and T), and other irregularly shaped cells . In contrast, strain USDA 10 consistently failed to reduce acetylene, even though it also could grow and yield pleomorphic cells under various conditions . With minimal inhibitory supplements (5 micrograms per ml of medium) of nalidixic acid and novobiocin as cell division inhibitors, an increase in pleomorphic cells was observed, but the inhibited cultures displayed lower acetylene-reducing activity . A study of pleomorphic cells derived in different ways indicated that not all pleomorphs reduce acetylene.

J Bacteriol, 1983 Feb, 153(2), 635 - 43
Localization of symbiotic mutations in Rhizobium meliloti; Forrai T et al.; A total of 5 Nod- and 57 Fix- symbiotic mutants of Rhizobium meliloti strain 41 have been isolated after either nitrosoguanidine or Tn5 transposition mutagenesis . Chromosomal locations of mutations in 1 Nod- and 11 Fix- derivatives were ascertained by transferring the chromosome (mobilized by plasmid R68.45), in eight fragments, into symbiotically effective recipients and testing the recombinants for symbiotic phenotype . Alternatively, the kanamycin resistance marker of Tn5 was mapped . In five mutants the fix alleles were localized on different chromosomal regions, but six other fix mutations and one nod mutation tested did not map onto the chromosome . It was shown that the chromosome-mobilizing ability (Cma+) of R68.45 was not involved in the mobilization of genes located extrachromosomally . Moreover, Cma- derivatives of R68.45 could mobilize regions of the indigenous plasmid pRme41b but not chromosomal genes . Thus, mobilization of a marker by Cma- R68.45 indicates its extrachromosomal location . With a 32P-labeled DNA fragment carrying Tn5 as a hybridization probe, it was shown that in five extrachromosomally located Tn5-induced fix mutants and one nod mutant Tn5 was localized on plasmid pRme41b . This is in agreement with the genetic mapping data.

Mol Cell Biochem, 1983, 51(1), 61 - 6
Biosynthesis of amino acids from sucrose and Krebs cycle metabolites by Rhizobium lupini bacteroids; Kretovich WL et al.; The possibility of amino acids biosynthesis from sucrose, metabolites of Krebs cycle or glyoxylate and ammonium by intact bacteroids has been studied . The suspension of intact Rhizobium lupini bacteroids in phosphate buffer solution pH 7.8 was shown to catalyse the biosynthesis from sucrose and ammonium of some amino acids, such as alanine, aspartic and glutamic acids, glycine and serine . The yield of alanine and aspartic acid was 2.5-3 times higher than that of other amino acids, which were formed in almost equal quantities . Intact bacteroids were also found to catalyse the biosynthesis of aspartic and glutamic acids, alanine and glycine from ammonium and Krebs cycle metabolites such as fumaric acid (FA), oxaloacetic acid (OAA), pyruvic acid (PA), alpha-ketoglutaric acid (alpha-KGA), malic acid (MA), as well as from glyoxylic acid (GOA) . The biosynthesis of aspartic acid from fumaric acid was dominant . Besides that, the suspension of intact bacteroids catalysed transamination of aspartic and glutamic acids, the transamination of aspartic acid being especially intense with alpha-KGA and GOA . Aspartic acid was synthesized most efficiently through the amination of fumaric acid, while glutamic acid was better synthesized through the transamination of aspartic acid with alpha-KGA than through reductive amination of alpha-KGA . The experimental data proved that intact bacteroids possess Krebs cycle enzymes and primary ammonia assimilation enzymes . This enzyme complex permits bacteroids to detoxify ammonia, which they produce using sucrose and metabolites of Krebs cycle as the sources of carbon . The data obtained are of great interest as they prove the importance of bacteroids in the synthesis of amino acids from ammonium which is formed in the course of N2-fixation, and sucrose available from leaves.

Zentralbl Mikrobiol, 1983, 138(1), 63 - 9
Detection of antibacterial substances in some plant residues and their effect on certain micro-organisms; Abdel-Nasser M et al.; The effect of dry residues from several plants, belonging to different families on certain microorganisms in vitro and in vivo, was studied . Dry residues of paprica leaves, tomato tops, egg plant leaves, guava leaves, onion peels, garlic tops, wheat straw, sugar cane leaves, cotton leaves, Egyptian clover tops, field bean tops or pea tops were examined for the presence of antibacterial substances, using successive extractions with hexane, ethyl ether, ethanol, and water, respectively, for each plant residue . On culture media, the antibacterial effect, expressed as width of inhibition zones, differed according to the type of plant, type of micro-organism, and extraction medium, used for each plant . Water extract from each of the studied plants showed no effect on any of the studied micro-organisms, while the other extracts indicated the presence of antibacterial substances in all the used plants . In most cases, ether extract showed the highest incidence of antimicrobial activities against the majority of test micro-organisms . In general, the antibacterial substances seemed to be more inhibitory to Gram-positive bacteria than to Gram-negative ones . Ethyl-ether extract of the residues of most of these plants markedly affected the growth of more than one of the different Rhizobium species when grown on culture medium, as indicated by the presence of wide zones of inhibition.

J Bacteriol, 1983 Jan, 153(1), 443 - 51
Effects of culture age on symbiotic infectivity of Rhizobium japonicum; Bhuvaneswari TV et al.; The infectivity of the soybean symbiont Rhizobium japonicum changed two- to fivefold with culture age for strains 110 ARS, 138 Str Spc, and 123 Spc, whereas culture age had relatively little effect on the infectivity of strains 83 Str and 61A76 Str . Infectivity was measured by determining the number of nodules which developed on soybean primary roots in the zone which contained developing and preemergent root hairs at the time of inoculation . Root cells in this region of the host root are susceptible to Rhizobium infection, but this susceptibility is lost during acropetal development and maturation of the root cells within a period of 4 to 6 h (T . V . Bhuvaneswari, B . G . Turgeon, and W . D . Bauer, Plant Physiol . 66:1027-1031, 1980) . Profiles of nodulation frequency at different locations on the root were not affected by the age of the R . japonicum cultures, indicating that culture age affected the efficiency of Rhizobium infection rather than how soon infections were initiated after inoculation . Inoculum dose-response experiments also indicated that culture age affected the efficiency of infection . Two strains, 61A76 Str and 83 Str, were relatively inefficient at all culture ages, particularly at low inoculum doses . Changes in infectivity with culture age were reasonably well correlated with changes in the proportion of cells in a culture capable of binding soybean lectin . Suspensions of R . japonicum in water were found to retain their viability and infectivity.

Acta Microbiol Hung, 1983, 30(1), 79 - 82
Purification and electron microscopy of a large plasmid of Rhizobium meliloti 41; Mink M et al.; A large plasmid DNA molecule was purified from Rhizobium meliloti 41 by CsCl-ethidium bromide density gradient centrifugation . Electron microscopic and agarose gel electrophoretic data suggest that addition of alkali effectively removes the chromosomal DNA, the plasmid DNA can be precipitated from the cleared lysate and no gradient centrifugation is needed for plasmid purification.

Arch Microbiol, 1983 Jan, 134(1), 12 - 6
In vitro expression of nitrogenase activity in Parasponia-Rhizobium strain ANU 289; Mohapatra SS et al.; Rhizobium strain ANU 289 derepressed nitrogenase activity under defined in vitro conditions . Acetylene reduction was detected both in agar and liquid stationary culture . The strain is capable of nitrogen-fixing nodulation of legumes {such as siratro (Macroptilium atropurpureum Urb} as well as the non-legumes Parasponia andersonii and P . rugosa . Nitrogenase activity as high as 40-70 nmol C2H4 per mg protein after 7 days of incubation was detected . Strain ANU 289 was similar to Rhizobium strains 32 H1 and CB 756 with regard to oxygen requirement in the gas phase for development of nitrogenase activity between 0 and 10% O2, but showed increased sensitivity to oxygen repression at 20% O2 . Strain ANU 289 also showed pronounced sensitivity to exogenous glutamine compared to strains 32 H1 and CB 756.

Biosystems, 1983-84, 16(3-4), 269 - 89
Parasitic origins of nitrogen-mixing Rhizobium-legume symbioses . A review of the evidence; Sharifi E; This paper is divided into two sections . The first part (I) reviews the literature on the legume-Rhizobium association with emphasis on the processes leading to the establishment of the association . In the second part (II) it is proposed that the legume-Rhizobium association was originally necrotrophic , beginning when the free-living, nitrogen-fixing, saprotrophic Rhizobium developed the ability to infect the plant . The pre-infection events, infection processes and nodulation in the colonization of the legumes by the Rhizobium are similar to those of other parasitic associations . Likewise, the host responses to the Rhizobium entry, infection thread synthesis and bacteroid formation are comparable to those of other plants when they encounter phytopathogens . Evolutionary processes acted in the selection of biotrophy , the fine control and regulation of the extracellular enzymes of the necrotrophic Rhizobium converted the association into biotrophy . The nutritional dependence of the Rhizobium on the legume, the requirement of the plant for combined nitrogen and the Rhizobium potential to meet this requirement drove the biotrophic association into mutualism . This became possible when regulation of the nitrogen-fixing system of the Rhizobium was modified and the oxygen carrying protein leghemoglobin was acquired or evolved by the legume to enhance nitrogen fixation.

J Mol Appl Genet, 1983, 2(3), 249 - 60
Physical and genetic characterization of Rhizobium meliloti symbiotic mutants; Buikema WJ et al.; A set of 19 symbiotic mutants of Rhizobium meliloti obtained by a Tn5 "suicide plasmid" mutagenesis procedure was characterized genetically and physically . As part of this characterization, we showed that R . meliloti strain 1021, like other R . meliloti strains, contains a very large indigenous plasmid (greater than 300 Md) that carries the structural genes for nitrogenase (nifHDK genes) . Among the 19 symbiotic mutations studied, at least six were shown to reside on the megaplasmid . By a "walking procedure" we obtained from a cosmid clone bank a set of overlapping cosmids that contained megaplasmid sequences contiguous to nifHDK . A 90 kb region of contiguous DNA from these cosmids was used to probe the mutant strains for rearrangements within this region . The same six mutations that were located on the megaplasmid mapped within the 90 kb region examined, which included the structural genes for nitrogenase (nifHDK) . A majority of the mutations characterized in this study could not be correlated with a bona fide Tn5 insertion into a symbiotic gene.

J Mol Appl Genet, 1983, 2(3), 225 - 36
Conservation of DNA regions adjacent to nifKDH homologous sequences in diverse slow-growing Rhizobium strains; Hadley RG et al.; Restriction endonuclease-digested deoxyribonucleic acid (DNA) from 17 slow-growing Rhizobium strains was hybridized with 32P-labeled DNA of the Klebsiella pneumoniae nitrogenase structural gene (nifKDH) region . Sixteen of these strains contained two or more fragments that were homologous to K . pneumoniae nifKDH after cleavage with EcoRI or HindIII . Hybridization with nifKDH subclones revealed that most of the Rhizobium fragments were homologous to the HindIII-EcoRI portion of nifKDH (corresponding to the nifDH region), although fragments homologous to the EcoRI-HindIII portion of nifKDH (corresponding to the nifK region) were also present . Comigrating nif-homologous restriction endonuclease fragments were observed for strains isolated from different geographic locations and from nodules of different plant species . Nearly identical hybridization patterns were obtained for five R . japonicum strains which clearly differed in the pattern of restriction endonuclease fragments from their chromosomal DNA . This indicates that there is a high degree of conservation of DNA sequence surrounding the region of nif homology in these strains.

Mol Gen Genet, 1983, 191(3), 430 - 3
The detailed physical map of the temperate phage 16-3 of Rhizobium meliloti 41; Dorgai L et al.; Restriction cleavage maps for enzymes EcoRI, BamHI, PstI, PvuII, XbaI and EcoRV of Rhizobium meliloti temperate phage 16-3 have been established . Together with the earlier maps (HindIII, KpnI, HpaI, BglII) 98 restriction sites, 'evenly' distributed, have been mapped along the phage genome, including the so far unmarked silent region of the chromosome . All the restriction maps have been fitted to each other by computer optimalization . Beyond for conventional techniques a computer program (PMAP) for physical mapping of linear DNA has been employed which made the experimentation, in several cases, extremely efficient.

Mol Gen Genet, 1983, 191(3), 393 - 6
Recombination deficient mutants of Rhizobium meliloti 41; Olasz F et al.; Two mutants deficient in homologous genetic recombination have been isolated from Rhizobium meliloti 41 after Tn5 mutagenesis . Both mutants are defective in the induction of temperate phage 16-3 by UV-light, Mytomycin-C or Bleomycin, their UV sensitivity is more pronounced than that of the wild-type strain, and they lack the 'SOS activity' responsible for induced mutations.

Mol Gen Genet, 1983, 191(2), 288 - 94
Tn5 carries a streptomycin resistance determinant downstream from the kanamycin resistance gene; Putnoky P et al.; In Rhizobium meliloti, Tn5 conferred resistance not only to kanamycin but to streptomycin, as well, in Escherichia coli, however only to kanamycin . Using in vitro recombinant DNA techniques, it was shown that the streptomycin resistance determinant was located downstream from the kanamycin resistance gene in the unique central region of Tn5 . Expression of various cloned fragments of Tn5 suggested that both kanamycin and streptomycin resistance genes were transcribed from the same promoter . E . coli mutants allowing the expression of streptomycin resistance from Tn5 were isolated . The differential expression of the streptomycin resistance gene provides a simple selection/counterselection criterion, using only streptomycin in transfer experiments of Tn5 between E . coli and R . meliloti.

DNA, 1983, 2(2), 149 - 55
Biological nitrogen fixation: primary structure of the Rhizobium trifolii iron protein gene; Scott KF et al.; Biological nitrogen fixation in the Rhizobium-legume symbiosis is dependent on the induction of a bacterially-encoded enzyme complex, nitrogenase . To examine the organization and expression of the genes encoding the components of nitrogenase in this complex system, these genes have been isolated from the legume symbiont Rhizobium trifolii by molecular cloning . DNA sequence analysis of the entire nifH gene (encoding the Fe-protein component of nitrogenase) and of the amino-terminal 141 codons of the nifD gene (encoding the alpha-subunit of the Mo-Fe protein) indicates that these genes are linked on a single operon in this strain . The Fe-protein amino acid sequence shares considerable homology with the sequence from other organisms, in particular the related organism Rhizobium meliloti (90% homology) . The nif structural genes are preceded by a DNA sequence which is repeated at least three times in the Rhizobium trifolii genome.

DNA, 1983, 2(2), 141 - 8
Nitrogenase structural genes are unlinked in the nonlegume symbiont Parasponia rhizobium; Scott KF et al.; Several Rhizobium strains are capable of biological nitrogen fixation in symbiotic association with nonleguminous plants . The gene encoding the iron-protein component of nitrogenase (nifH) from one such strain, Parasponia Rhizobium sp . ANU289, has been isolated and completely sequenced . Unlike previously studied nitrogen-fixing prokaryotes, the Fe-protein subunit is encoded on a separate operon from other components of the nitrogenase enzyme complex . Comparative analysis of Fe-protein amino acid sequences indicates that the symbiotic nitrogen fixation phenotype in Rhizobium may have arisen on at least two separate occasions during its evolution.

Acta Microbiol Pol, 1983, 32(2), 161 - 7
Structure of the rigid-layer of Rhizobium cell wall . III . Electron microscopic evidence for the cellulose microfibrils association with peptidoglycan sacculi; Drozanski W; The morphology of peptidoglycan layer of Rhizobium cell wall was examined by transmission electron microscopy . Peptidoglycans were isolated from intact cells after treatment with sodium dodecyl sulfate, extraction with aqueous 45% phenol and then with a mixture of chloroform-methanol . Finally rigid layers were digested with trypsin and chymotrypsin . The results indicate the presence of lump or bar-like structures on the surface of the cell shaped peptidoglycan sacculi . Evidence is provided suggesting that the cellulose microfibrils arise directly from these excrescences found on the peptidoglycan surface . Digestion with cellulase removed all cellulose microfibrils whereas the lumps and bars remained as an integral part of the Rhizobium peptidoglycan.

Acta Microbiol Pol, 1983, 32(1), 5 - 10
Chromosomal gene controlling symbiotic nitrogen fixation in Rhizobium meliloti L5-30; Malek W et al.; Auxotrophic Rhizobium meliloti strain RM 246 carries two independent mutations: in the biosynthesis of cysteine (cys) and symbiotic nitrogen fixation process (fix) . These two mutations were mapped by transduction between his-240 and ade-4 markers . Cotransduction frequencies show the following order of genes: his-240 fix-1 cys-246 ade-4.

Acta Microbiol Pol, 1983, 32(1), 31 - 5
Isolation and characterization of mutants of cowpea Rhizobium defective in nitrate uptake system; Singh RK et al.; In this communication, two mutants of cowpea Rhizobium CB756 (NR-18 and NR-23), defective in nitrate uptake system, were described . They failed to grow in a medium where nitrate was the sole source of nitrogen and unable to show nitrate reductase activity in cell suspensions . However, they showed nitrate reductase activity (to the level of wild type) in cell-free extract preparations . Thus it appears that in these mutants, the transport genes of nitrate uptake were affected and the synthesis of nitrate induced permease, as its role in nitrate uptake is indicated here, is defective.

Acta Microbiol Pol, 1983, 32(1), 25 - 30
Characterization of the lipopolysaccharide from the nod mutant of Rhizobium trifolii; Russa R et al.; Lipopolysaccharides (LPS) from the non-nodulating Rhizobium trifolii 24SM 15 and from the nodulating R . trifolii 24SM 13 were isolated and examined by means of gas-liquid chromatography and mass spectrometry . Analysis of LPS showed these preparations from both strains examined contained Lipid A, 2-keto-3-deoxyoctonate, neutral sugars, amino sugars, and trace amounts of amino acids . In 24SM 13 LPS prevailed glucose and rhamnose whereas LPS from the non-nodulating strain SM 15 contained mainly mannose, galactose and heptose . Quinovosamine and mannosamine were detected only in the nodulating strain . The ratio of glucosamine phosphate to glucosamine was higher in the LPS of the non-nodulating strain SM 15 than in the corresponding material of the nodulating one . An unknown component producing a peak at the position of glyceryl-S-cysteine on amino acid analysis profiles was detected in SM 15 LPS . The differences in LPS composition were associated with the alterations in the sensitivity to phage 3H, and nodulation ability.

Acta Microbiol Pol, 1983, 32(1), 19 - 24
Symbiotic properties of adenine requiring mutants of Rhizobium meliloti strain L5-30; Malek W et al.; From the effective and prototrophic Rhizobium meliloti strain L5-30 two auxotrophic mutants were isolated: RM4 and RM221 . These two mutants required adenine and adenine with thiamine for their growth, respectively . Both mutants nodulated lucerne plants ineffectively . Electron microscopic observations of the nodule tissue showed that its cells were not occupied by bacteria . Prototrophic revertants and transductants of these mutants showed high symbiotic effectiveness . It is assumed that adenine or adenine and thiamine requirements made impossible release of bacteria from the infection thread.

Acta Microbiol Pol, 1983, 32(1), 11 - 8
Localization of nodulation gene on the chromosome of Rhizobium meliloti; Malek W; Using N-methyl-N'-nitro-N-nitrosoguanidine mutant RM54 of Rhizobium meliloti L5-30 defective in the nodulation process (Nod-) and in the biosynthesis of adenine was obtained . Nod- phenotype of this mutant was not caused by the auxotrophic mutation . The nod gene is located on the chromosome . The wild type strain of R . meliloti and Nod- mutant RM54 harbour two indigenous plasmids having a molecular weight of 90 Mdal and about 300 Mdal.

J Bacteriol, 1982 Nov, 152(2), 928 - 31
Nitrogen fixation (nif) genes and large plasmids of Rhizobium japonicum; Masterson RV et al.; The location of structural nitrogen-fixation genes was determined for the slow- and fast-growing types of Rhizobium japonicum . Slow-growing R . japonicum strains do not harbor structural nif genes, homologous to nifD and nifH, on large plasmids (100 to 200 megadaltons) . In contrast, all fast-growing R . japonicum strains, except PRC194, contain structural nif genes on large plasmids.

J Bacteriol, 1982 Oct, 152(1), 485 - 94
Rhizobium japonicum mutants defective in symbiotic nitrogen fixation; Noel KD et al.; Rhizobium japonicum strains 3I1b110 and 61A76 were mutagenized to obtain 25 independently derived mutants that produced soybean nodules defective in nitrogen fixation, as assayed by acetylene reduction . The proteins of both the bacterial and the plant portions of the nodules were analyzed by two-dimensional polyacrylamide gel electrophoresis . All of the mutants had lower-than-normal levels of the nitrogenase components, and all but four contained a prominent bacteroid protein not observed in wild-type bacteroids . Experiments with bacteria grown ex planta suggested that this protein was derepressed by the absence of ammonia . Nitrogenase component II of one mutant was altered in isoelectric point . The soluble plant fraction of the nodules of seven mutants had very low levels of heme, yet the nodules of five of these seven mutants contained the polypeptide of leghemoglobin . Thus, the synthesis of the globin may not be coupled to the content of available heme in soybean nodules . The nodules of the other two of these seven mutants lacked not only leghemoglobin but most of the other normal plant and bacteroid proteins . Ultrastructural examination of nodules formed by these two mutants indicated normal ramification of infection threads but suggested a problem in subsequent survival of the bacteria and their release from the infection threads.

J Bacteriol, 1982 Oct, 152(1), 422 - 30
Electron transport components involved in hydrogen oxidation in free-living Rhizobium japonicum; O'Brian MR et al.; Membranes from free-living Rhizobium japonicum were isolated to study electron transport components involved in H2 oxidation . The H2/O2 uptake rate ratio in membranes was approximately 2 . The electron transport inhibitors antimycin A, cyanide, azide, hydroxylamine, and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) inhibited H2 uptake and H2-dependent O2 uptake significantly . H2-reduced minus O2-oxidized absorption difference spectra revealed peaks at 551.5, 560, and 603 nm, indicating the involvement of cytochromes c, b, and a-a3, respectively . H2-dependent cytochrome reduction was completely inhibited in the presence of 0.15 mM HQNO . This inhibition was relieved by the addition of 0.1 mM menadione . Evidence is presented for the involvement of two b-type cytochromes in H2 oxidation . One b-type cytochrome was not reduced by ascorbate and had an absorption peak at 560 nm . The reduction of this cytochrome by H2 was not inhibited by cyanide . A second b-type cytochrome, cytochrome b', was not reduced by H2 in the presence of cyanide . This cytochrome had an absorption peak at 558 nm . Carbon monoxide difference spectra with H2 as reductant provided evidence for the involvement of cytochrome o as well as cytochrome a3 in H2 oxidation . H2 uptake activity in cell-free extracts was inhibited by UV light irradiation . Most of the activity of the UV-treated extracts was restored with the addition of ubiquinone . The restored activity was inhibited by cyanide . A branched electron transport pathway from H2 to O2 is proposed.

J Bacteriol, 1982 Sep, 151(3), 1473 - 84
Soluble aldehyde dehydrogenase and metabolism of aldehydes by soybean bacteroids; Peterson JB et al.; A soluble aldehyde dehydrogenase (EC 1.2.1.3) was partially purified from Rhizobium japonicum bacteroids and from free-living R . japonicum 61A76 . The enzyme was activated by NAD+, NADH, and dithiothreitol, and it reduced NAD(P)+ . Acetaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, and succinic semialdehyde were substrates . The Km for straight-chain aldehydes decreased with increasing carbon chain length . The aldehyde dehydrogenase was inhibited by 6-cyanopurine, but not by metronidazole . These compounds inhibited acetylene reduction, but not respiration, by isolated bacteroids.

J Gen Virol, 1982 Sep, 62 (Pt 1), 145 - 52
Identification of structural proteins of Rhizobium meliloti temperate phage 16-3; Erdei S et al.; The structural proteins of Rhizobium meliloti temperate phage 16-3 have been analysed by means of polyacrylamide gel electrophoresis, isoelectric focusing and agarose gel electrophoresis . Five major and five minor proteins were identified and characterized with respect to their size, isoelectric point and their distribution between the head ad tail of the phage particle . The synthesis of structural proteins was studied by one- and two-dimensional gel electrophoresis.

J Bacteriol, 1982 Sep, 151(3), 1069 - 72
Transport and catabolism of D-mannose in Rhizobium meliloti; Arias A et al.; Rhizobium meliloti L5-30 grows on D-mannose as the sole carbon source . The catabolic pathway of D-mannose was characterized . The following activities were present: mannose transport system, mannokinase, and mannosephosphate isomerase . Several mannose-negative mutants were selected; they were classified into three functional groups: group I, mannokinase and mannosephosphate isomerase defective: group II, mannokinase defective; and group III, mannosephosphate isomerase defective . Mannose uptake was an active process, since it was inhibited by azide, dinitrophenol, and cyanide, but not by fluoride or arsenate . Growth on succinate repressed mannose uptake activity . The mannose transport system was present in all the mutants . Uptake studies showed that mannose-negative mutants did not metabolize this sugar.

J Bacteriol, 1982 Sep, 151(3), 1633 - 6
Rhizobium meliloti mutants altered in ammonium utilization; Osburne MS; Derivatives of Rhizobium meliloti 2011 required trace amounts of glutamate to use ammonium as the nitrogen source for growth, although they could use serine as the sole nitrogen source . Specific activities of ammonium assimilatory enzymes were similar to those in strain Rm2011 . The mutants were deficient in nitrogen fixation.

J Bacteriol, 1982 Sep, 151(3), 1621 - 3
Succinate dehydrogenase mutant of Rhizobium meliloti; Gardiol A et al.; A succinate dehydrogenase mutant strain of Rhizobium meliloti was isolated after nitrosoguanidine mutagenesis . It failed to grow on succinate, glutamate, acetate, pyruvate, or arabinose but grew on glucose, sucrose, fructose, and other carbohydrates . The mutant strain showed delayed nodulation of lucerne plants, and the nodules were white and ineffective . A spontaneous revertant strain of normal growth phenotype induced red and effective nodules.

J Biol Chem, 1982 Aug 10, 257(15), 8724 - 30
Heme biosynthesis in Rhizobium . Identification of a cloned gene coding for delta-aminolevulinic acid synthetase from Rhizobium meliloti; Leong SA et al.; A symbiotically important gene system in rhizobial species is the heme biosynthetic pathway . A mutant having reduced levels of delta-aminolevulinic acid synthetase, the first unique enzyme in this pathway, was obtained in Rhizobium meliloti 102F34 by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis . Enzyme activity ranged from 3-13% of the wild type . Alfalfa plants inoculated with the Rhizobium synthetase mutant grew no better than uninoculated controls and formed only small white nodules which had no acetylene-reducing capacity . A cloned segment of Rhizobium genomic DNA capable of complementing this lesion was identified in a previously described gene bank from R . meliloti prepared with the broad host range plasmid cloning vector pRK290 (Ditta . G., Stanfield, S., Corbin, D., and Helinski, D . R . (1980) Proc . Natl . Acad . Sci . U . S . A . 77, 7347-7351) . Symbiotic effectiveness could be restored in the mutant by supplementing plants with exogenous delta-aminolevulinic acid or by introducing into the mutant the wild type delta-aminolevulinic acid synthetase gene cloned into the pRK290 plasmid . The recombinant plasmid carrying the synthetase gene was also able to weakly complement an Escherichia coli hemA mutant . Transposon mutagenesis of this plasmid with Tn5 further localized the delta-aminolevulinic acid synthetase gene to a 1.4-kilobase region contained within a 4-6-kilobase Bam HI fragment . Full complementation of hemA was observed when this fragment was subcloned under E . coli trp and Tet promoter control on a pBR322 replicon . A temperature-sensitive mutant of this latter plasmid, which was unable to complement hemA at high temperature, produced enzyme having temperature-sensitive synthetase activity in vitro . This result confirmed that the cloned complementing DNA contained the structural gene for delta-aminolevulinic acid synthetase and not a biosynthetic regulatory gene.

J Bacteriol, 1982 Aug, 151(2), 560 - 8
Plasmids and stability of symbiotic properties of Rhizobium trifolii; Djordjevic MA et al.; A conjugal plasmid which encodes both peak nodulation genes and nitrogenase genes, and which is labeled with the transposon Tn5, was transferred to a wild-type Rhizobium trifolii strain to examine the stability and expression of the host range and fixation (Fix+) phenotypes . Transconjugates were isolated which were shown to initially form nitrogen-fixing nodules (Nod+ Fix+) on both clovers and peas . These hybrid strains were then repeatedly passaged through either pea or clover nodules or onto a solid agar medium to determine whether these broadened-host-range characteristics were stably maintained . An instability was noted in the capacity of some of these hybrids to form nitrogen-fixing nodules on all of the host plants used . The broadened nodulation ability was, however, more readily maintained . In some cases, the changes in the Nod+ Fix+ phenotype could be attributed to demonstrable changes in the plasmid profile of the hybrid strains, whereas in other cases no demonstrable plasmid alterations could be detected.

J Bacteriol, 1982 Aug, 151(2), 989 - 95
Uptake hydrogenase activity and ATP formation in Rhizobium leguminosarum bacteroids; Nelson LM et al.; The role of uptake hydrogenase was studied in Rhizobium leguminosarum bacteroids from the nodules of Pisum sativum L . cv . Homesteader . Uptake hydrogenase activity, measured by the 3H2 uptake method, was dependent on O-consumption and was similar to H2 uptake measured by gas chromatography . Km for O2 of 0.0007 atm (0.0709 kPa) and a Km for H2 of 0.0074 atm (0.7498, kPa) were determined . H2 increased the rate of endogenous respiration by isolates with uptake hydrogenase (Hup+) but had no effect on an isolate lacking uptake hydrogenase (Hup-) . A survey of 14 Hup+ isolates indicated a wide range of H2 uptake activities . Four of the isolates tested had activities similar to or higher than those found in two Hup+ Rhizobium japonicum strains . H2 uptake was strongly coupled to ATP formation in only 5 of the 14 isolates . H2 increased the optimal O2 level of C2H2 reduction by 0.01 atm and permitted enhanced C2H2 reduction at O2 levels above the optimum in both a coupled and an uncoupled isolate . At suboptimal O2 concentrations a small enhancement of C2H2 reduction by H2 was seen in two out of three isolates in which H2 oxidation was coupled to ATP formation . Thus, the main function of uptake hydrogenase in R . leguminosarum appears to be in the protection of nitrogenase from O2 damage.

Nucleic Acids Res, 1982 Jul 24, 10(14), 4147 - 57
On the operon structure of the nitrogenase genes of Rhizobium leguminosarum and Azotobacter vinelandii; Krol AJ et al.; The transcription of the nitrogenase genes in Rhizobium leguminosarum was studied by analysing total cellular RNA from bacteroids for the presence of nitrogenase messenger RNA . The RNA was separated by agarose gel electrophoresis and blotted onto nitrocellulose filters . Messenger RNA for nitrogenase was detected by hybridization with probes derived from plasmid pSA30, a recombinant plasmid carrying the nitrogenase genes of Klebsiella pneumoniae . In the same way nitrogenase mRNA was detected in RNA isolated from Azotobacter vinelandii and from Klebsiella pneumoniae, but only if both were cultured under conditions of derepression of the nitrogenase genes . The size of the RNA hybridizing with the probes indicates that in all three organisms the genes for the subunits of the two components of nitrogenase constitute a single operon.

J Bacteriol, 1982 Jul, 151(1), 83 - 8
Positive selection of nodulation-deficient Rhizobium phaseoli; Raleigh EA et al.; A high proportion of Rhizobium phaseoli mutants that survived infection with phage F1 were found to be nodulation deficient . Two that were examined in detail had internal defects in addition to the expected surface defects . One internal defect was in the enzyme phosphoglucose isomerase . The use of phages to select appropriate mutants should apply generally in any system in which surface components are involved.

J Bacteriol, 1982 Jul, 151(1), 411 - 9
Structural studies of alfalfa roots infected with nodulation mutants of Rhizobium meliloti; Hirsch AM et al.; Alfalfa roots infected with four nodulation defective (Nod-) mutants of Rhizobium meliloti which were generated by transposon Tn5 mutagenesis were examined by light and electron microscopy . In one class of Nod- mutants, which we can nonreactive, the bacteria did not induce root hair curling or penetrate host cells . In a second class of Nod- mutants, which we call reactive, the bacteria induced some root hair curling and entered root epidermal cells, although no infection threads were formed . In addition, reactive Nod- mutants induced extensive root hair proliferation and hypertrophied roots . This study presents the details of the phenotype of the association between each mutant strain and alfalfa roots.

J Bacteriol, 1982 Jul, 151(1), 36 - 43
Identification and mobilization by cointegrate formation of a nodulation plasmid in Rhizobium trifolii; Scott DB et al.; A nodulation plasmid, pRtr-514a, of molecular size 180 megadaltons (Mdal) was identified in Rhizobium trifolii strain NZP514 . This plasmid was absent in both spontaneous and heat-cured Nod- derivatives of NZP514, and these strains were unable to induce root hair curling . The ability to nodulate clover was transferred from the wild-type strain to a Nod- derivatives, PN104, with the broad-host-range plasmid R68.45 (39 megadaltons) at a cotransfer frequency of about 4 X 10(-3) . Most of the Nod+ transconjugants were resistant to kanamycin, tetracycline, and carbenicillin and had received a plasmid approximately 36 or 70 Mdal larger than pRtr514a but did not contain a plasmid of the size of R68.45, indicating that pRtr-514a was mobilized as a cointegrate plasmid containing either one or possibly two copies of R68.45 . Use of these cointegrate-containing strains as donors in further crosses with the Nod- derivative strain PN118 resulted in high-frequency transfer of Nod+ (10(-3) to 10(-4), with cotransfer frequencies with kanamycin of up to 100% . Introduction of R68.45 into a derivative of NZP514 containing the broad-host-range plasmid pJP4 (52 Mdal) resulted in a high frequency of transconjugants carrying a cointegrate plasmid composed of pRtr-514a and pJP4 . When used as donors to Nod- derivatives, such strains cotransferred Nod+ with kanamycin plus mercury at a frequency of 67% . The identification of stable cointegrates between pRtr-514a and the broad-host-range plasmids R68.45 and pJP4 should enable several genetic manipulations to be carried out with this nodulation plasmid, including the transfer of the plasmid to most gram-negative bacterial genera.

Arch Microbiol, 1982 Jul, 132(1), 51 - 6
Enhancement of specific nitrogenase activity in Azospirillum brasilense and Klebsiella pneumoniae, inhibition in Rhizobium japonicum under air by phenol; Werner D et al.; Specific nitrogenase activity in Azospirillum brasilense ATCC 29145 in surface cultures under air is enhanced from about 50 nmol C2H4 X mg protein -1 X h-1 to 400 nmol C2H4 by the addition of 1 mM phenol . 0.5 and 2 mM phenol added increase the rate 5-fold and 4-fold . This enhancement effect is observed only between 2 and 3 days after inoculation, with only a small reduction of the growth of the cells by the phenol added . In surface cultures under 1% O2, nitrogenase activity is slightly reduced by the addition of 1-0.01 mM phenol . Utilization of succinate is enhanced during the period of maximum enhancement of nitrogenase activity by 60% by addition of 1 mM phenol . The cells did not produce 14CO2 from {U-14C} phenol, neither in surface cultures nor in liquid cultures and less than 0.1% of the phenol was incorporated into the cells . A smaller but significant enhancement of nitrogenase activity by about 100% in surface cultures under air was found with Klebsiella pneumoniae K 11 after addition of 1 mM phenol . However, in Rhizobium japonicum 61-A-101 all phenol concentrations above 0.01 mM reduced nitrogenase activity . With 1 mM phenol added activity was reduced to less than 10% with no effect on the growth in the same cultivation system . With this Rhizobium japonicum strain significant quantities of phenol (25 mumol in 24 h by 2 X 10(12) cells) were metabolized to 14CO2, with phenol as sole carbon source . With Azospirillum brasilense in liquid culture under 1% and 2% O2 in the gas phase, no enhancement of nitrogenase activity by phenol was noticed.

Antibiotiki, 1982 Jun, 27(6), 418 - 21
{Antibiotic resistance of nodular bacteria}; Khmel'nitskii MI et al.; The effect of several antibiotics on 4 rapid-growing Rhizobia, i . e . R . phaseoli, R . leguminosarum, R . trifolii and R . meliloti and 3 slow growing Rhizobia, i . e . R . vigna, R . japonicum and R . lupini was studied . It was shown that 4 of the species, i . e . R . meliloti, R . japonicum, R . vagna and R . lupini had multiple drug resistance . A tetracycline resistant mutant MC of R . japonicum was isolated.

J Bacteriol, 1982 Jun, 150(3), 999 - 1007
Molecular mechanism for loss of nodulation properties of Rhizobium trifolii; Zurkowski W; Of 18 Rhizobium trifolii strains tested, 12 showed a high frequency of loss of nodulation ability after incubation in cultures at elevated temperatures . A correlation between loss of nodulation ability and loss of a large plasmid was demonstrated for R . trifolii . In some nonnodulating (Nod-) mutants, deletions occurred instead of total elimination of the plasmid molecule . The maximum curing effect was observed in bacteria incubated at 35 degrees C . After 4 or more days of incubation at this temperature, the viability of bacteria decreased markedly, and the number of nonnodulating mutants increased significantly . At the elevated temperature DNA synthesis was stopped completely after 2 h, whereas protein synthesis proceeded for a few days . Microscopic observations showed that during the first 3 days of incubation at the elevated temperature, the bacterial cells increased markedly in size . These large irregular cells then divided and produced Nod- clones . Nonnodulating clones did not result from the selection of temperature-resistant mutants . The presence of P-group plasmids in Rhizobium strains strongly inhibited the loss of nodulation ability during incubation at 35 degrees C . The observed phenomenon did not result from integrative suppression . It is possible that a product(s) of the genes of R-plasmids acts as a stabilizing agent on the replication process of the indigenous Rhizobium plasmids.

Gene, 1982 Jun, 18(3), 289 - 96
Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants; Friedman AM et al.; We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK290 (Ditta et al., 1980) by inserting a 1.6-kb Bg/II fragment containing lambda cos into the unique Bg/II site in pRK290 . The new vector, pLAFR1, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts . We constructed a clone bank of Rhizobium meliloti DNA in pLAFR1 using a partial EcoRI digest . The mean insert size was 23.1 kb . When the clone bank was mated (en masse) from Escherichia coli to various R . meliloti auxotrophic mutants, tetracycline-resistant (Tcr) transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007 . pLAFR1 cosmids were mobilized from R . meliloti prototrophic colonies into E . coli and then reintroduced into R . meliloti auxotrophs . In most cases, 100% of these latter Tcr transconjugants were prototrophic.

Cell, 1982 Jun, 29(2), 551 - 9
Directed transposon Tn5 mutagenesis and complementation analysis of Rhizobium meliloti symbiotic nitrogen fixation genes; Ruvkun GB et al.; An 18 kb region adjacent to and surrounding the genes for nitrogenase (nif) was cloned from the genome of the symbiotic nitrogen-fixing species Rhizobium meliloti . A total of 31 Tn5 insertions in the nif region were constructed and assayed for their effect on symbiotic nitrogen fixation (Fix phenotype) . Fix- insertions were found in two clusters, one 6.3 kb region not containing essential symbiotic genes . The locations of at least three transcription units containing Fix genes were deduced from complementation analysis between genomic nif::Tn5 insertions and nif::Tn5 insertions on mobilizable cloning vectors . The locations of R . meliloti genes nifH, nifD and nifK, which code for the single subunit of the nitrogenase Fe protein and for the two subunits of the nitrogenase MoFe protein respectively, were determined by DNA hybridization to cloned Klebsiella pneumoniae nif genes and by comparison of partial R . meliloti DNA sequences with K . pneumoniae nif gene sequences . R . meliloti nifH, D and K are located in the 6.3 kb fix-::Tn5 cluster and are transcribed in the order nifH, nifD, nifK, which is the same order as in K . pneumoniae.

J Biol Chem, 1982 May 10, 257(9), 4677 - 9
Absence of accumulation of ppGpp and RNA during amino acid starvation in Rhizobium meliloti; Belitsky B et al.; Lack of three different amino acids or treatment with the analogue DL-serine hydroxamate does not induce the accumulation of ppGpp and pppGpp, the 3'-pyrophosphates of GDP and GTP, respectively, in Rhizobium meliloti strain 41 . Surprisingly, RNA accumulation is controlled under the above mentioned conditions stringently . Moreover, no significant RNA accumulation was found during chloramphenicol, tetracycline, and streptomycin treatment, suggesting that R . meliloti, unlike any other bacteria in investigated so far, is not able to accumulate RNA without ongoing protein synthesis . On the other hand, lack of carbon source and ammonium starvation result in a significant ppGpp accumulation.

J Bacteriol, 1982 May, 150(2), 465 - 70
Transformation of Rhizobium meliloti 41 with plasmid DNA; Kiss GB et al.; Plasmid pGV1106, a derivative of the wide-host-range plasmid S-a of the W incompatibility group, was introduced into Rhizobium meliloti 41 by plasmid-mediated mobilization to overcome the restriction of foreign DNA . The mobilized plasmid pKK2 differed from the original pGV1106 by an extra piece of DNA of 1.3 kilobase pairs which supposedly originated from pJB3JI used for mobilization . If pKK2 was isolated from R . meliloti 41, it could be successfully reintroduced by transformation . The transformation frequency was low (10 to 54 colonies per micrograms of plasmid DNA) but reproducible, and several lines of evidence showed that it was the consequence of plasmid DNA uptake . The small size (10.3 kilobases) and elevated copy number (10 to 15 copies per cell) of pKK2 make it a potentially useful cloning vector for the study of symbiotic nitrogen fixation genes of R . meliloti 41.

J Bacteriol, 1982 Apr, 150(1), 421 - 4
Genetic transformation of Rhizobium leguminosarum by plasmid DNA; Bullerjahn GS et al.; We demonstrated the genetic transformation of Rhizobium leguminosarum by R68.45 plasmid DNA by freezing and thawing cell suspensions in the presence of R68.45 plasmid DNA and 20 mM MgCl2 . Clones resistant to kanamycin and tetracycline were recovered at a frequency of 10(-8) per recipient cell . No colonies that were doubly drug resistant were recovered in parallel control experiments.

J Bacteriol, 1982 Apr, 150(1), 402 - 6
Megaplasmids in the plant-associated bacteria Rhizobium meliloti and Pseudomonas solanacearum; Rosenberg C et al.; Using analytical and preparative methods, we demonstrated the presence of megaplasmids with molecular weight larger than 450 x 10(6) in the two plant-associated bacteria Rhizobium meliloti and Pseudomonas solanacearum . Such giant plasmids were found in 8 of 9 P . solanacearum strains tested and in all of the 18 R . meliloti strains tested.

J Bacteriol, 1982 Apr, 150(1), 161 - 7
Rhizobium japonicum mutants that are hypersensitive to repression of H2 uptake by oxygen; Maier RJ et al.; The synthesis of an H2 oxidation system in free-living Rhizobium japonicum wild-type strain SR is repressed by oxygen . Maximal H2 uptake rates were obtained in strain SR after derepression in 11 microM or less dissolved oxygen . Oxygen levels above 45 microM completely repressed H2 uptake in strain SR . Five R . japonicum mutant strains that are hypersensitive to repression or H2 oxidation by oxygen were derived from strain SR . The mutants were obtained by screening H2 uptake-negative mutants that retained the ability to oxidize H2 as bacteroids from soybean nodules . As bacteroids, the five mutant strains were capable of H2 oxidation rates comparable to that of the wild type . The mutants did not take up H2 when derepressed in 22 microM dissolved oxygen, whereas strain SR had substantial activity at this oxygen concentration . The O2 repression of H2 uptake in both the wild-type and two mutant strains, SR174 and SR200, was rapid and was similar to the effect of inhibiting synthesis of H2 uptake system components with rifampin . None of the mutant strains was able to oxidize H2 when the artificial electron acceptors methylene blue or phenazine methosulfate were provided . The mutant strains were not sensitive to killing by oxygen, they took up O2 at rates similar to strain SR, and they did not produce an H2 uptake system that was oxygen labile . Cyclic AMP levels were comparable in strain SR and the five mutant strains after subjection of the cultures to the derepression conditions.

J Bacteriol, 1982 Mar, 149(3), 872 - 9
Mechanism of regulation of glucose transport in Rhizobium leguminosarum; de Vries GE et al.; Multiple glucose transport systems were distinguished in Rhizobium leguminosarum . We found nonlinear Lineweaver-Burk plots for the uptake of glucose, 2-deoxy-D-glucose, and alpha-methyl-D-glucoside, and this implied the existence of at least two uptake mechanisms . Different patterns of inhibition of 2-deoxy-D-glucose uptake and alpha-methyl-D-glucoside uptake at 0.1 mM by various carbohydrates revealed differences in the stereospecificities of the transport systems . Osmotic shock treatment abolished transport activities, and two independent glucose-binding activities were detected in the supernatants . Induction of glucose transport was repressed strongly by L-malate, even in the presence of excess D-glucose . Rhizobium bacteroids showed no significant glucose uptake activity at different oxygen concentrations . These results suggested that glucose transport is repressed by dicarboxylic acids during R . leguminosarum symbiosis.

J Bacteriol, 1982 Mar, 149(3), 1135 - 7
Regulation of tryptophan genes in Rhizobium leguminosarum; Holmgren E et al.; Twelve tryptophan auxotrophs of Rhizobium leguminosarum were characterized biochemically . They were grown in complex and minimal media with several carbon sources, in both limiting and excess tryptophan . Missing enzyme activities allowed assignment of all mutant to the trpE, trpD, trpB, or trpA gene, confirming earlier results with the same mutants (Johnston et al., Mol . Gen . Genet . 165:323-330, 1978) . In regulatory experiments, only the first enzyme of the pathway, anthranilate synthase, responded (about 15-fold) to tryptophan excess or limitation.

Can J Biochem, 1982 Mar, 60(3), 272 - 8
Organization and expression of leghaemoglobin genes; Brisson N et al.; Leghaemoglobin genes in soybean (Glycine max) are present as a moderately reiterated family of sequences . Since there are identical restriction site patterns of these sequences in DNA isolated from leaf, root, or nodule tissue, the data suggest that no major changes in the organization or methylation of leghaemoglobin genes occur during their induction . Cloned soybean leghaemoglobin-cDNA cross hybridized with RNA from root nodules of kidney bean (Phaseolus vulgaris), and to a lesser extent, of pea (Pisum sativum) indicating sequence homology in the leghaemoglobin genes of these species . Hybridization to the genomic DNA restriction fragments of two other species, Glycine soja and Vicia faba, also indicated interspecies sequence homologies . Several restriction fragments appear to be common to all species examined . The induction of these genes occurs following infection of the plant by Rhizobium and is independent of the appearance of nitrogenase activity in the nodules . The level of expression is, however, influenced by various mutations in Rhizobium that result in the development of ineffective (nonnitrogen fixing) nodules.

J Bacteriol, 1982 Mar, 149(3), 1021 - 6
Effects of iron deficiency on heme biosynthesis in Rhizobium japonicum; Roessler PG et al.; The effects of iron deficiency on heme biosynthesis in Rhizobium japonicum were examined . Iron-deficient cells had a decreased maximum cell yield and a decreased cytochrome content and excreted protoporphyrin into the growth medium . The activities of the first two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase (EC 2.3.1.37) and delta-aminolevulinic acid dehydrase (EC 4.2.1.24), were diminished in iron-deficient cells, but were returned to normal levels upon addition of iron to the cultures . The addition of iron salts, iron chelators, hemin, or protoporphyrin to cell-free extracts did not affect the activity of these enzymes . The addition of levulinic acid to iron-deficient cultures blocked protoporphyrin excretion and also resulted in high delta-aminolevulinic acid synthase and delta-aminolevulinic acid dehydrase activities . These results suggest the possibility that rhizobial heme biosynthesis in the legume root nodule may be affected by the release of iron from the host plant to the bacteroids.

J Bacteriol, 1982 Mar, 149(3), 1005 - 12
Carriers in electron transport from molecular hydrogen to oxygen in Rhizobium japonicum bacteroids; Eisbrenner G et al.; An investigation has been conducted to identify electron transport carriers that participate in the oxidation of H2 by H2 uptake-positive strains of Rhizobium japonicum bacteroids . We have observed that the reduced form of dibromothymoquinone at a concentration of 0.2 mM strongly inhibited H2 uptake, endogenous respiration, and C2H2 reduction by bacteroid suspensions . Reduced dibromothymoquinone, however, failed to inhibit the transfer of electrons from H2 to methylene blue under anaerobic conditions, indicating that the hydrogenase per se is insensitive to this inhibitor . Metronidazole, at 1 mM, affected rates of H2 uptake and endogenous respiration only slightly, but strongly inhibited C2H2 reduction . Evidence for H2-dependent cytochrome reduction in an H2 uptake-positive strain of R . japonicum bacteroids is presented . In kinetic studies, the rates of reduction of the type b and c cytochromes in the presence of H2 were shown to be severalfold higher than the rates due to endogenous respiration alone . With hydrogenase-deficient mutants of R . japonicum, no measurable effect of H2 on cytochrome reduction was observed . Our results indicate that ubiquinone and cytochromes of types b and c are involved in the oxyhydrogen reaction in R . japonicum.

J Biol Chem, 1982 Feb 25, 257(4), 1870 - 5
Application of two new methods for cleavage of polysaccharides into specific oligosaccharide fragments . Structure of the capsular and extracellular polysaccharides of Rhizobium japonicum that bind soybean lectin; Mort AJ et al.; The extracellular polysaccharide produced by the bacterium Rhizobium japonicum has been implicated in the recognition between symbionts which takes place in the association of R . japonicum with soybean . The complete primary structure of the polysaccharide produced by R . japonicum strain 3I1b 138 has been determined by a combination of conventional and unconventional methods . The polymer contains glucose, mannose, and galacturonic acid in the molar ratio of 2:1:1 and contains a varying proportion of galactose and 4-O-methylgalactose, the sum of these two being equivalent to the amount of mannose (Mort, A . J., and Bauer, W . D . (1980) Plant Physiol, 66, 158-163) . The polymer was specifically degraded and its galacturonic acid residues by treatment with lithium in ethylenediamine to a tetrasaccharide . By sequential specific glycosidase hydrolysis and methylation analysis the tetrasaccharide was shown to have Structure 1: (formula: see text) . Anhydrous liquid hydrogen fluoride at -23 degrees C degraded the polymer to a trisaccharide and monosaccharides . The galactose and 4-O-methylgalactose released by the HF as monosaccharides were found to be partially acetylated . By 1H NMR spectrometry and methylation analysis the trisaccharide was found to have Structure 2: (formula: see text) . From the composition and the structural analyses of the tetra- and trisaccharides produced in high yields by specific degradations of the polysaccharide, the polymer is deduced to have a pentasaccharide repeating unit with Structure 3: (formula: see text).

J Biol Chem, 1982 Feb 25, 257(4), 2092 - 6
Reconstitution of H2 oxidation activity from H2 uptake-negative mutants of Rhizobium japonicum bacteroids; Maier RJ et al.; An in vitro reconstitution of both methylene blue and oxygen-dependent H2 uptake activity from extracts of Hup- (H2 uptake-negative) mutant strains of Rhizobium japonicum bacteroids is described . Cell-free extracts prepared from bacteroids formed from two different Hup- mutants were mixed, and active H2 oxidizing particles formed . Extracts from each mutant alone did not oxidize H2 . The source of the components required for the complementation were soluble . Mixing of membrane particles from the two mutants did not result in reconstituted activity . The development of activity required an incubation period of several hours under anaerobic conditions, and maximal activity was obtained approximately 10 h after the mixing of the two extracts . Along with the development of H2 uptake activity with time, the soluble extract mixture became turbid . The turbidity could be correlated with an increase in the appearance of membrane structures, including closed vesicles . After reconstitution, 65% of the methylene blue-dependent H2 uptake activity was recovered in a particulate fraction.

Folia Microbiol (Praha), 1982, 27(4), 278 - 80
Effect of inoculation on element uptake by plants grown on saline soils; Douka CE et al.; A relationship between inoculation and elemental uptake of Medicago sativa inoculated with Rhizobia meliloti (isolated from a saline area) was found . The plant uptake of the elements with atomic number between 19 and 42 was significantly higher in plants grown on inoculated soils, with the exception of molybdenum . Preliminary evidence shows that the concentration of some elements was affected by inoculation.

J Bacteriol, 1982 Jan, 149(1), 59 - 64
Use of plasmid R68.45 for constructing a circular linkage map of the Rhizobium trifolii chromosome; Megias M et al.; Plasmid R68.45 was used to promote conjugal transfer of chromosomal markers in Rhizobium trifolii RS55 . Analysis of two-factor and three-factor crosses among R . trifolii strains enabled construction of a circular linkage map of the R . trifolii chromosome, containing 17 nutritional and resistance markers.

J Mol Appl Genet, 1982, 1(6), 585 - 96
Symbiotic nitrogen fixation: molecular cloning of Rhizobium genes involved in exopolysaccharide synthesis and effective nodulation; Chakravorty AK et al.; A transposon (Tn5)-induced mutant (strain ANU437) of Rhizobium trifolii was isolated in which no water-soluble exopolysaccharide (EPS) could be detected . This mutant was also incapable of forming nitrogen-fixing root nodules on clover plants . Molecular cloning has demonstrated that the Tn5 transposon was responsible for both of these mutant phenotypes and that there is a direct correlation between EPS synthesis in this bacterial strain and its ability to carry out symbiotic nitrogen fixation . In the mutant ANU437, Tn5 was located in a 9.4-kb EcoRI fragment that was cloned into the amplifiable plasmid pBR322 . The recombinant plasmid was used as a hybridization probe to isolate the corresponding wild-type DNA sequence of R . trifolii from a lambda Charon 28 genomic clone bank . This DNA sequence was subcloned into the broad host range conjugative plasmid RP4 and introduced into the Escherichia coli strain RR1 . It was then transferred to the mutant ANU437 by conjugation . The acquisition of the wild-type DNA sequence by the mutant ANU437 resulted in the restoration of its ability to synthesize normal levels of EPS and to form nitrogen-fixing nodules on white and subterranean clovers.

J Mol Appl Genet, 1982, 1(5), 405 - 18
ISRm1: A Rhizobium meliloti insertion sequence that transposes preferentially into nitrogen fixation genes; Ruvkun GB et al.; After transposon Tn5 mutagenesis, a high proportion of Rhizobium meliloti symbiotic mutants do not contain Tn5 insertions in symbiotic genes . Instead, the mutations in these strains are correlated with the presence of an endogenous insertion sequence (ISRm1) in nitrogen fixation (nif) or symbiotic genes which are adjacent to the nif genes . ISRm1 is 1.4 kb and transposes to at least three restriction fragments in the nif region at a frequency between 10(-2) and 10(-3) . A nif region restriction fragment containing ISRm1 was cloned from one of the mutant strains unable to fix nitrogen symbiotically (Fix-) and the resulting plasmid was used as a hybridization probe . ISRm1 is present at least ten times in the R . meliloti genome but is not present in any other R . meliloti strains, E . coli strains, or Rhizobium species tested . We demonstrated that the Fix- phenotype correlated with ISRm1 transposition is indeed caused by ISRm1 insertion by conjugating a cloned fragment containing ISRm1 into a wild type Fix+ R . meliloti host and replacing the normal genomic nif fragment with the nif::ISRm1 fragment . The resulting strain was Fix-.

J Mol Appl Genet, 1982, 1(4), 315 - 26
Molecular cloning of Rhizobium trifolii genes involved in symbiotic nitrogen fixation; Scott KF et al.; DNA sequences responsible for the development and maintenance of symbiotic nitrogen fixation have been identified and isolated from Rhizobium trifolii . Symbiotically-defective strains were generated by random mutagenesis with the transposon Tn5 . The defective genes which give rise to the mutant phenotype have been cloned into bacterial plasmids and used as hybridization probes to isolate the corresponding wild-type genes from a clone bank of R . trifolii DNA . Symbiotic genes cloned in this manner are able to correct the lesion caused by the insertion of the transposon in their respective mutants and so restore the nitrogen fixation phenotype . The correction of the mutation is shown to occur by two distinguishable mechanisms--either by complementation or by homologous recombination . This approach provides a reliable method for isolation and mapping of bacterial DNA sequences involved in symbiotic nitrogen fixation.

J Bacteriol, 1982 Jan, 149(1), 221 - 8
Clustering of nitrogen fixation (nif) genes in Rhizobium meliloti; Corbin D et al.; A cloned 17.3-kilobase region of the Rhizobium meliloti genome with homology to the Klebsiella pneumoniae nitrogenase structural genes was studied . Limits on the extent of homology were determined . Transposon mutagenesis of this region of the genome verified that it contained functional nif genes, Some transposon insertions resulted in a defective symbiotic phenotype, whereas others had no noticeable effect on symbiotic competence . The relative position of insertions yielding these two phenotypic classes suggested that at least three distinct units of gene expression are present in this region . Hybridization of RNA from alfalfa root nodules and from vegetatively grown Rhizobium to this cloned DNA showed that at least 11.1 kilobases of the region was transcribed actively and that transcription was specific for the symbiotic state.

J Bacteriol, 1982 Jan, 149(1), 114 - 22
Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis; Meade HM et al.; We have physically and genetically characterized 20 symbiotic and 20 auxotrophic mutants of Rhizobium meliloti, the nitrogen-fixing symbiont of alfalfa (Medicago sativa), isolated by transposon Tn5 mutagenesis . A "suicide plasmid" mutagenesis procedure was used to generate TN-5-induced mutants, and both auxotrophic and symbiotic mutants were found at a frequency of 0.3% among strains containing random TN5 insertions . Two classes of symbiotic mutants were isolated: 4 of the 20 formed no nodules at all (Nod-), and 16 formed nodules which failed to fix nitrogen (Fix-) . We used a combination of physical and genetic criteria to determine that in most cases the auxotrophic and symbiotic phenotypes could be correlated with the insertion of a single Tn5 elements . Once the Tn5 element was inserted into the R . meliloti genome, the frequency of its transposition to a new site was approximately 10-8 and the frequency of precise excision was less than 10-9 . In approximately 25% of the mutant strains, phage Mu DNA sequences, which originated from the suicide plasmid used to generate the Tn5 transpositions, were also found in the R . meliloti genome contiguous with Tn5 . These later strains exhibited anomalous conjugation properties, and therefore we could not correlate the symbiotic phenotype with a Tn5 insertion . In general, we found that both physical and genetic tests were required to fully characterize transposon-induced mutations.

J Biol Chem, 1981 Dec 25, 256(24), 12905 - 10
The isolation and characterization of a root lectin from soybean (Glycine max (L), cultivar Chippewa); Gade W et al.; A lectin has been isolated from the roots of 5-day soybean (Glycine max (L) cultivar Chippewa) seedlings, and its properties have been compared to those of the soybean seed lectin . The sugar-binding activities of the two lectins, both in terms of specific hemagglutinating activity and sugar specificity, are indistinguishable . Molecular properties of the two lectins, measured as relative molecular weights, isoelectric and electrophoretic patterns, amino acid compositions, immunochemical cross-reactivity, and chromatographic behavior on Sepharose-concanavalin A adsorbents suggest that the seed and the root lectin are very similar but not identical . On the basis of these comparisons, we conclude that models regarding biological functions of soybean lectin derived from studies using the seed lectin can be extended to include the root lectin in this cultivar . Studies on the distribution of the lectin in the root tissue suggest that it is associated with the outer surface of the root and is concentrated in the segments of the root at which hair and early secondary roots are observed . Since this is the region at which Rhizobium binding occurs and at which nodulation probably is initiated, all the reported observations on the root lectin are consistent with its proposed role in the specific interaction of the developing soybean with its symbiont.

J Bacteriol, 1981 Nov, 148(2), 728 - 908 p
Competitive advantage provided by bacterial motility in the formation of nodules by Rhizobium meliloti; Ames P et al.; The effect of motility on the competitive success of Rhizobium meliloti in nodule production was investigated . A motile strain formed more nodules than expected when mixed at various unfavorable ratios with either flagellated or nonflagellated nonmotile derivatives . We conclude that motility confers a selective advantage on rhizobia when competing with nonmotile strains.

J Bacteriol, 1981 Nov, 148(2), 697 - 711
Growth-phase-dependent immunodeterminants of Rhizobium trifolii lipopolysaccharide which bind trifoliin A, a white clover lectin; Hrabak EM et al.; The lipopolysaccharide (LPS) from Rhizobium trifolii 0403 was isolated at different stages of growth and was examined for its (i) ability to bind a white clover lectin (trifoliin A), (ii) immunochemical properties, and (iii) composition . There was significantly more binding of trifoliin A to purified LPS and cells in the early stationary phase than to cells in the exponential phase . Immunofluorescence and enzyme-linked immunosorbent assays indicated that new antigenic determinants of the LPS appeared for brief periods on cells at the end of the lag phase and again at the beginning of the stationary phase . These new antigens were not detected on cells in midexponential or late stationary phase . Monovalent fragments of immunoglobulin G antibodies raised against the unique antigenic determinants in the LPS competitively blocked the binding of trifoliin A to cells in the early stationary phase . Gas chromatographic analysis showed that the relative quantity of several glycosyl components in the LPS increased as the culture advanced from the midexponential to the early stationary phase . In addition, LPS from cells in the early stationary phase had a higher aggregate molecular weight . Quinovosamine (2-amino-2,6-dideoxyglucose) was identified by combined gas chromatography-mass spectrometry as a sugar component of the LPS which had not been previously reported . D-Quinovosamine, N-acetyl-D-quinovosamine, and its n-propyl-beta-glycoside were effective hapten sugars which inhibited the binding of trifoliin A, anti-clover root antibody, and homologous antibody to these new determinants in the LPS . White clover plants had more infected root hairs after incubation with an inoculum of cells in the early stationary phase than after incubation with cells in the midexponential phase . The profound influence of the growth phase on the composition of lectin-binding polysaccharides of Rhizobium may be a major underlying cause of conflicting data among laboratories testing the lectin-recognition hypothesis . In addition, these chemical modifications may reflect mechanisms which regulate Rhizobium-root hair recognition in this nitrogen-fixing symbiosis.

J Bacteriol, 1981 Oct, 148(1), 193 - 202
Succinate transport in Rhizobium leguminosarum; Finan TM et al.; The transport of succinate was studied in an effective streptomycin-resistant strain of Rhizobium leguminosarum . High levels of succinate transport occurred when cells were grown on succinate, fumarate, or malate, whereas low activity was found when cells were grown on glucose, sucrose, arabinose, or pyruvate as the sole carbon source . Because of the rapid metabolism of succinate after transport into the cells, a succinate dehydrogenase-deficient mutant was isolated in which intracellular succinate accumulated to over 400 times the external concentration . Succinate transport was completely abolished in the presence of metabolic uncouplers but was relatively insensitive to sodium arsenate . Succinate transport was a saturable function of the succinate concentration, and the apparent Km and Vmax values for transport were determined in both the parent and the succinate dehydrogenase mutant . Malate and fumarate competitively inhibited succinate transport, whereas citrate and malonate had no effect . Succinate transport mutants were isolated by transposon (Tn5) mutagenesis . These mutants were unable to transport succinate or malate and were unable to grow on succinate, malate, or fumarate as the sole carbon source . The mutants grew normally on pyruvate, oxaloacetate, citrate, or arabinose, and revertants isolated on succinate minimal medium had regained the ability to grow on malate and fumarate . From these data, we conclude that R . leguminosarum possesses a C4-dicarboxylic acid transport system which is inducible and mediates the active transport of succinate, fumarate, and malate into the cell.

Ann Microbiol (Paris), 1981 Sep-Oct, 132B(2), 231 - 9
Effect of fungicides on growth and on DNA, RNA and protein anabolism of a Rhizobium sp; Garg FC et al.; Eleven fungicides were examined for their effect on the growth and anabolism of Rhizobium sp . in pure culture . Of these, only 4 were found to inhibit growth of rhizobia at a concentration as low as 10 micrograms/ml . Growth inhibition by these fungicides appeared to be primarily due to inhibition of respiration, although thiophanate and anilazine also affected DNA and RNA synthesis, respectively . Inhibition is, however, transitory and the bacterium has the ability to overcome this initial inhibition without mutation.

Arch Microbiol, 1981 Jul, 129(5), 391 - 4
The pathways of ammonium assimilation in Rhizobium meliloti; Ali H et al.; Two pathways of ammonium assimilation are known in bacteria, one mediated by glutamate dehydrogenase, the other by glutamine synthetase and glutamate synthase . The activities of these three enzymes were measured in crude extracts from four Rhizobium meliloti wild-type strains, 2011, M15S, 444 and 12 . All the strains had active glutamine synthetase and NADP-linked glutamate synthase . Assimilatory glutamate dehydrogenase activity was present in strains 2011, M15S, 444, but not in strain 12 . Three glutamate synthase deficient mutants were isolated from strain 2011 . They were unable to use 1 mM ammonium as a sole nitrogen source . However, increased ammonium concentration allowed these mutants to assimilate ammonium via glutamate dehydrogenase . It was found that the sole mode of ammonium assimilation in strain 12 is the glutamine synthetase-glutamate synthase route; whereas the two pathways are functional in strain 2011.

J Bacteriol, 1981 May, 146(2), 614 - 20
Revertible hydrogen uptake-deficient mutants of Rhizobium japonicum; Lepo JE et al.; We have developed mutants of Rhizobium japonicum which are deficient in H2 uptake capacity (Hup-) and which spontaneously revert to the parent type at a frequency consistent with that of a single-point mutation (ca . 1.0 x 10(-09)) . The mutagenesis by nitrous acid and the selection of the Hup- phenotype by using penicillin and chemolithotrophy as enrichment for chemolithotrophy-deficient strains are described . Two mutants retain low but reproducible levels of ribulose bisphosphate-dependent CO2 fixation when grown on a low-carbon medium under an atmosphere of 1% O2, 4% H2, 5% CO2, and 90% N2 . Neither O2 nor the artificial electron acceptors phenazine methosulfate or methylene blue supported detectable H2 uptake by the free-living Hup- mutants or by their bacteroids . Plant growth experiments under bacteriologically controlled conditions were conducted to assess the mutants' performance as inocula for soybean plants . Plants inoculated with Hup- strains had lower dry weights and contained less total N than did plants inoculated with the parent Hup+ strain . Use of either the Hup- mutants or the Hup+ parent strain as inocula, however, did not significantly affect the acetylene-reducing activity or the fresh weight of nodules . These results, obtained with apparently isogenic lines of H2 uptake-deficient R . japonicum, provide strong support for a beneficial role of the H2 uptake phenotype in legume symbiosis.

Biochim Biophys Acta, 1981 Mar 26, 653(1), 98 - 107
Regulation of the expression of leghaemoglobin genes in effective and ineffective root nodules of soybean; Verma DP et al.; The expression of leghaemoglobin genes in effective and ineffective (unable to fix nitrogen) root nodules of soybean developed by Rhizobium japonicum strains 61A76, 61A24 and SM5 was measured by using a cDNA probe or a cloned leghaemoglobin sequence and in vitro translation of Lb-mRNA . Hybridization of the poly(A)-containing nodule polysomal RNA from 3-week-old nodules with a kinetically purified Lb-cDNA or with plasmid (pLbl) containing a leghaemoglobin sequence showed that Lb-mRNA is present in ineffective nodules formed by strains SM5 and 61A24 at reduced levels . Of the two major classes of electrophoretically distinguishable leghaemoglobins in soybean, LbS was not synthesized in 3-week-old strain 61A24-induced nodules while both sorts of leghaemoglobin were synthesized and accumulated in ineffective nodules formed by strain SM5 of Rhizobium . Ineffective nodules formed by strain 61A24 are green inside and do not appear to accumulate leghaemoglobin as measured by the haemochromogen assay, although low levels of apoleghaemoglobin were detected using leghaemoglobin antibodies . SM5-induced nodules were found to have about half as much as leghaemoglobin of the of effective (61A76-induced) nodules . This study demonstrates that while the appearance of leghaemoglobin is independent of nitrogenase activity in bacteroids, its synthesis is influenced to different degrees both by a mutation (SM5) and incompatibility (61A24) of Rhizobium . The primary regulation appears to be at the level of transcription or processing of mRNA since ineffective nodules contain Lb-mRNA approximately in proportion to the amount of apoleghaemoglobin present in these nodules.

Biochim Biophys Acta, 1981 Mar 13, 658(1), 1 - 9
Effect of NH4+ on nitrogenase activity in nodule breis and bacteroides from Pisum sativum L; Salminen SO; Nodule breis and bacteroid preparations were made from Pisum sativum L . (cv . Trapper) inoculated with a single strain of Rhizobium leguminosarum . The detached nodules were triturated under helium flow . The resultant breis could support C2H2 reduction in N-Tris{hydroxymethyl}methyl-2-aminoethane-sulfonic acid buffer (Tes) without any additions for over an hour . NH4+ was found to inhibit C2H2 reduction and H2 evolution . The inhibition was not dependent on the counterion and was evident immediately after the addition of NH4+ to the reaction mixture . L-Methionine-D,L-sulfoximine, added to inhibit assimilation of NH4+, had no effect on the inhibition . Addition of pyruvate enhanced the rate of C2H2 reduction in breis and partially overcame the inhibition of NH4+ . Pyruvate was found necessary for measurable activity in bacteroid preparations . When ATP and an ATP-generating system were used in breis the effect of NH4+ was not observed.

J Bacteriol, 1981 Mar, 145(3), 1129 - 36
Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation (nif) genes; Prakash RK et al.; A single large plasmid was isolated from multiplasmid-harboring strains Rhizobium leguminosarum 1001 and R . trifolii 5 . These single plasmids, as well as the largest plasmid detectable in R . phaseoli 3622, hybridized with part of the nif structural genes of Klebsiella pneumoniae . In contrast, the plasmids of R . meliloti strains V7 and L5-30 did not show hybridization with the nif genes of K . pneumoniae, indicating that these genes might be located either on the chromosome or on a much larger plasmid which as yet has not been isolated . Studies of the homology between plasmids of fast-growing Rhizobium species showed that a specific deoxyribonucleic acid sequence, which carries the structural genes for nitrogenase, is highly conserved on a plasmid in R . leguminosarum, R . trifolii, and R . phaseoli . Furthermore, it was found that this type of plasmid in the different species shares extensive deoxyribonucleic acid homology, suggesting that strains in the R . leguminosarum cluster have preserved a nif plasmid.

Biokhimiia, 1981 Mar, 46(3), 520 - 4
{Adenosine-3':5'-monophosphate phosphodiesterase from Rhizobium}; Lisova NE et al.; The phosphodiesterase (PDE) activity of adenosine-3':5'-monophosphate was detected in the cells of tubercular bacteria of Rhizobium lupini and Rhizobium japonicum . The specific activity of three Rhizobium forms, e.g . bacteroids from lupine root tubercles, free-nitrogen-fixing culture and vegetative cells grown on a mannitol--yeast agar, were compared . In the bacteroids PDE is represented both by soluble and membrane-bound forms . The optimal enzyme activity is revealed in an alkaline medium, whereas the curve of PDE activity dependence on pH has a broad maximum . PDE is inhibited by methylxanthines, the inhibiting effect being stronger than that of theophylline.

J Bacteriol, 1981 Feb, 145(2), 1063 - 74
Localization and partial characterization of soybean lectin-binding polysaccharide of Rhizobium japonicum; Tsien HC et al.; Immunoelectron microscopy was combined with partial characterization of isolated exopolysaccharide to study binding of soybean lectin by Rhizobium japonicum strain USDA 138 . Lectin-binding activity resided in two forms of exopolysaccharide produced during growth: an apparently very high-molecular-weight capsular form and a lower-molecular-weight diffusible form . At low-speed centrifugation, the capsular form cosedimented with cells to form a viscous, white, cell-gel complex which was not diffusible in 1% agar, and the diffusible form remained in the cell-free supernatant . Electron microscopic observation of the cell-gel complex after labeling with soybean lectin-ferritin conjugate revealed that capsular polysaccharides, frequently attached to one end of the cells, were receptors for lectin . The outer membrane of the cell bound no lectin . Various preparations of exopolysaccharide isolated from the culture supernatant were tested for lectin binding, interaction with homologous somatic antigen, and the presence of 2-keto-3-deoxyoctonate and were chromatographed in Sepharose 4B and 6B gel beds . Lectin binding was restricted to a polysaccharide component designated as lectin-binding polysaccharide . This polysaccharide, as present in the cell-free culture supernatant, was a diffusible acidic polysaccharide devoid of 2-keto-3-deoxyoctonate, with a molecular weight of 2 X 10(6) to 5 X 10(6) . It was concluded that the soybean lectin-binding component of R . japonicum is an extracellular polysaccharide and not a lipopolysaccharide and that the diffusible lectin-binding polysaccharide probably differs from the very high-molecular-weight lectin-binding polysaccharide of the loose capsule (slime) only in the degree of polymerization.

J Bacteriol, 1981 Feb, 145(2), 1025 - 30
Plasmid transfer within and between serologically distinct strains of Rhizobium japonicum, using antibiotic resistance mutants and auxotrophs; Pilacinski WP et al.; Methionine-requiring and pantothenic acid-requiring auxotrophs of Rhizobium japanicum USDA 31, as well as highly antibiotic-resistant mutants of R . japonicum strains USDA 31, USDA 110, USDA 138, and Webster 48, were isolated . These mutants were used to transfer the P-1 group plasmids R68.45 and RP4 within and between strains USDA 31, USDA 110, and Webster 48 . Attempts to demonstrate transfer of either plasmid to strain USDA 138 were unsuccessful.

Antonie Van Leeuwenhoek, 1981, 47(6), 481 - 97
Cellular glycogen, beta-1,2,-glucan, poly beta-hydroxybutyric acid and extracellular polysaccharides in fast-growing species of Rhizobium; Zevenhuizen LP; Synthesis of acidic exopolysaccharides, neutral cellular polysaccharides and poly-beta-hydroxybutyric acid (PHB) by Rhizobium is strongly dependent on cultural conditions and the strains used . Exopolysaccharide production by R . leguminosarum, R . Phaseoli and R . trifolii closely parallels growth, whereas R . meliloti mainly excretes (low mol wt) polysaccharides when cell propagation is limited by lack of a necessary growth element (nitrogen) and an excess of carbon source is still present in the medium . In all strains, accumulation of cellular glycogen, beta-1,2-glucan and PHB is initiated only under growth-limiting conditions . When the external carbon source is exhausted, glycogen and PHB are metabolized by the cells, sustaining their longevity and thus act as true reserve materials; on the other hand, beta-1,2-glucan and excreted polysaccharides are not utilized on further incubation of the culture . Differences exist in the nature and relative amounts of the products synthesized by strains of different species of Rhizobium . R . leguminosarum, R . phaseoli and R . trifolii synthesize a uronic containing exopolysaccharide, PHB and/or glycogen, non-metabolizable capsular polysaccharide and low amounts of beta-1,2-glucan . R . meliloti synthesizes a uronic acid-free exopolysaccharide, PHB and /or glycogen and high concentrations of beta-1,2-glucan . Exopolysaccharides, beta-1,2-glucan and glycogen preparations were obtained by isolation and purification from cells of fast-growing species of Rhizobium and chemically characterized.

J Supramol Struct Cell Biochem, 1981, 16(1), 29 - 41
Bacterial attachment as related to cellular recognition in the Rhizobium-legume symbiosis; Dazzo FB; Bacterial attachment is viewed as a cellular recognition event during the infection of legumes by the nitrogen-fixing symbiont, Rhizobium . Studies on the biochemical basis of selective attachment are reviewed, and suggest that this recognition process is accomplished by specific glycoprotein lectin-polysaccharide interactions on the surfaces of the symbionts . An understanding of host specificity may lead to ways to broaden the host range of nitrogen-fixing symbioses.

Mikrobiologiia, 1981 Jan-Feb, 50(1), 128 - 33
{Bacterial population dynamics in a soil--plant system}; Kirillova NP et al.; The dynamics of Rhizobium leguminosarum and Arthrobacter crystallopoietes populations introduced into soil at different levels of density was studied in a zone near barley roots . Microbial life with a high rate of growth was found only at the root surface . For A . crystallopoietes, the ultimate bacterial incidence at the root surface was found to depend on the original level of population density . For Rh . leguminosarum, the ultimate incidence was shown to reach an identical level irrespective of the original population density . Apparently, the identical level of stabilization in Rh . leguminosarum and the dependence of the ultimate incidence of A . crystallopoietes on the original population density reflect the peculiarities of ecological strategies displayed by these microorganisms . The introduction of A . crystallopoietes and Rh . leguminosarum into soil had no effect on the dynamics of soil bacteria assayed by the inoculation method.

Can J Microbiol, 1981 Jan, 27(1), 36 - 43
Light and electron microscopic studies of nodule structure of alfalfa; Patel JJ et al.; Light and electron microscopy was used to establish the structural organisation of the developing nodule of alfalfa . In these nodules three distinct regions were noted: (1) the base region, site of original infection where the nodule is attached to the root and now composed of degenerating nodule tissue, (2) the central region, or active region composed of nodule cells containing tightly packed bacteroids surrounding a central vacuole, and (3) the meristematic region, a site of new growth, behind which newly formed cells are continually invaded . The ongoing infection process accompanying continued nodule development provided the opportunity to study the release of Rhizobium cells from the infection threads . In the nodules of alfalfa it would appear that the Rhizobium cells are released from infection thread into the nodule tissue in two different ways: (i) release with infection thread membrane and (ii) release in thin-walled vesicular structures . Thus it is concluded that Rhizobium cells are surrounded by the infection thread membrane when they are released from the infection thread into nodule tissue.

J Supramol Struct Cell Biochem, 1981, 16(2), 133 - 8
Presence of trifoliin A, a Rhizobium-binding lectin, in clover root exudate; Dazzo FB et al.; Trifoliin A, a Rhizobium-binding glycoprotein from white clover, was detected in sterile clover root exudate by a sensitive immunofluorescence assay employing encapsulated cells of Rhizobium trifolii 0403 heat-fixed to microscope slides . Its presence in root exudate was further examined by immunoaffinity chromatography . The binding of trifoliin A to cells was specifically inhibited by the hapten, 2-deoxyglucose . Significantly higher quantities of trifoliin A were detected in root exudate of seedlings grown hydroponically in nitrogen-free medium than in rooting medium containing 15 mM NO-3, a concentration which completely suppressed root hair infection by the nitrogen-fixing symbiont . The presence of trifoliin A in root exudate may make it possible for recognition processes to occur before the microsymbiont attaches to its plant host.

Z Allg Mikrobiol, 1981, 21(8), 601 - 6
Root exudates in relation to growth and nitrogenase activity of Rhizobium japonicum; Odunfa SA et al.; Root exudates of cowpea and soybean were collected in aerated water cultures . The ability of these exudates to support nitrogenase activity of R . japonicum was studied on defined media . When a complete nitrogenase-inducing medium was supplemented with concentrated root exudate, there was an increased nitrogenase activity . When the cowpea root exudate was substituted for glutamine or sodium succinate in the medium, nitrogenase activity was not detected . Growth was good when the root exudate was substituted for sodium succinate but was poor when the exudate replaced glutamine . There was nitrogenase activity in R . japonicum when the cowpea root exudate was substituted for arabinose in the 20E medium . Gas chromatographic analyses of cowpea root exudates revealed that ribose, arabinose, glucose, and sucrose were the main constituents . The significance of this finding in the asymbiotic nitrogen fixation by Rhizobium spp . is discussed.

Mol Gen Genet, 1981, 184(2), 326 - 33
Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti; Rosenberg C et al.; Large plasmids of molecular weight varying from 90 to around 200 x 10(6) have earlier been detected in most Rhizobium meliloti strains using an alkaline denaturation - phenol extraction procedure . With a less destructive method (Eckardt 1978) it was possible additionally to detect one plasmid of molecular weight clearly greater than 300 x 10(6) (= megaplasmid) in all of twenty-seven R . meliloti strains of various geographical origins and nodulation groupings investigated . Four strains (RCR 2011, A145, S26 and CC2013) were found to carry one megaplasmid and no smaller plasmids . Hybridization experiments with Klebsiella pneumoniae and R . meliloti cloned nitrogenase structural genes D and H showed that these genes are located on the megaplasmid and not on the smaller plasmids . All of the ten independent spontaneous non-nodulating derivatives of three strains of R . meliloti were shown to have suffered a deletion in the nifDH region of the megaplasmid . These results indicate that a gene controlling an early step in nodule formation is located in the nifDH region of the megaplasmid . This indicates that the same replicon carries genes controlling early and late functions in symbiosis.

Mol Gen Genet, 1981, 184(2), 318 - 25
Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R . meliloti; Banfalvi Z et al.; R . meliloti strain 41 (Rm41) was shown to harbour two indigenous plasmids with molecular weights of 140 Mdal (pRme41a) and more than 300 Mdal (pRme41b), respectively . Using a heat-treatment procedure, derivatives of Rm41 defective in nodulation (Nod-) or nitrogen fixation (Fix-) have been readily obtained . In some Nod- mutants the deletion of a segment of plasmid pRme41b was found . Based on the demonstrated homology between the nitrogen fixation (nif) genes of Klebsiella pneumoniae and of R . meliloti the Rhizobium nif region has been cloned into the cosmid vector pHC79, then recloned into pBR322 and the restriction map of the nif region has been determined . 32P-labelled nick-translated probe prepared from the cloned nif DNA fragment hybridized to pRme41b of Rm41 but for most Nod- mutants this hybridization was not detected . Hybridization of a cosmid containing Rm41 DNA to total DNA digest from the wild-type bacterium and from a series of Nod- mutants revealed that at least a 2 kb DNA fragment including the nif structural genes was missing from most of the Nod- mutants . These results, together with the genetic analyses of these symbiotic mutations suggest that some nod and fix genes are located on pRme41b.

J Environ Sci Health B, 1981, 16(3), 293 - 307
Potential effects of Thiram on Medicago - R . meliloti symbiotic association; Sirois JC et al.; The effects of Thiram and 2 commercial Thiram formulations on the growth and respiration of rhizobia were tested to compare the extent of bacteriostasis under controlled conditions . Although bacteriostasis was measurable at all concentrations tested, liquid cultures grew to maximum optical density in Thiram suspensions containing less than 10 micrograms/ml . Percentage germination, root elongation, and subsequent nodulation by R . meliloti of 2 cultivars of alfalfa, were determined in thiram suspensions to determine potential physiological effects of the fungicide on the host plant . Conditions were identified which produced enhancement or inhibition of germination, root elongation and development of nodular nitrogenase activity . At concentrations of the fungicide recommended for seed application, only minor, temporary bacteriostasis was observed as a possible negative effect while germination rates of fungi-contaminated seed were markedly increased.

J Bacteriol, 1981 Jan, 145(1), 533 - 40
Rhizobium japonicum mutant strains unable to grow chemoautotrophically with H2; Maier RJ; Rhizobium japonicum strain SR grows chemoautotrophically on a mineral salts medium when incubated in an H2- and CO2-containing atmosphere . Mutant strains unable to grow or that grow very poorly chemoautotrophically with H2 have been isolated from strain SR . The mutant isolation procedure involved mutagenesis with ethyl methane sulfonate, penicillin selection under chemoautotrophic growth conditions, and plating of the survivors onto medium containing carbon . The resulting colonies were replica plated onto medium that did not contain carbon, and the plates were incubated in an H2- and CO2-containing atmosphere . Mutant strains unable to grow under these conditions were chosen . Over 100 mutant strains with defects in chemoautotrophic metabolism were obtained . The phenotypes of the mutants fall into various classes . These include strains unable to oxidize H2 and strains deficient in CO2 uptake . Some of the mutant strains were capable of oxidizing H2 only when artificial electron acceptors were provided . Two mutant strains specifically lack activity of the key CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase . Other mutant strains lack both H2-oxidizing ability and ribulose 1,5-bisphosphate carboxylase activity.

Cold Spring Harb Symp Quant Biol, 1981, 45 Pt 1, 87 - 91
ISR1: an insertion element isolated from the soil bacterium Rhizobium lupini; Priefer UB et al.; The insertion element ISR1 was isolated from the soil bacterium R . lupini . In this strain, ISR1 shows a very strong affinity to plasmid RP4 . It causes RP4 mutations at the strikingly high frequency of 10(-2) to 10(-1), either by the integration itself or by generating deletions . In E . coli, ISR1 seems to be inactive . No evidence could be obtained for a promoter site on ISR1 or for an ISR1-encoded protein . Our results indicate, however, an ISR1-specific termination signal for either transcription or translation.

Mol Gen Genet, 1981, 182(2), 189 - 95
DNA sequence homology in Rhizobium meliloti plasmids; Jouanin L et al.; Plasmids were recovered by an alkaline procedure from six symbiotically effective strains of Rhizobium meliloti of diverse geographical origin, reported to harbour only one middle-size large plasmid (ranging from 89 to 143 Megadaltons) . Each purified plasmid was digested with eight restriction endonucleases; cleavage patterns were very complex: only KpnI and XbaI gave a limited number of bands . Fingerprints were very different, whatever the restriction enzyme or the geographical origin of the strains . However, Southern DNA-DNA hybridizations revealed that the plasmids showed homologous sequences having a high thermal stability . We gave evidence that some of these sequences are common to all the plasmids of R . meliloti . The biological function of these common sequences is unknown . Hybridization with cloned nitrogen fixation (nif) genes from Klebsiella pneumoniae had demonstrated that nif genes were not located on the middle -- size plasmids of R . meliloti studied in this paper.

Nature, 1981 Jan 1, 289(5793), 85 - 8
A general method for site-directed mutagenesis in prokaryotes; Ruvkun GB et al.; The genetic analysis of genes from prokaryotic species for which experimental genetic systems have not yet been developed is often limited by the difficulty of producing mutations in those genes . We report here a general technique applicable to Gram-negative prokaryotes for site-directed mutagenesis of cloned DNA fragments which we have applied to the study of the symbiotic nitrogen fixation genes of Rhizobium meliloti . In particular, we mutagenized cloned R . meliloti restriction fragments in Escherichia coli with transposon Tn5 and then replaced the wild-type parental DNA sequences with the mutant DNA sequences in the R . meliloti genome . Using this method we show that an R . meliloti DNA restriction fragment, cloned previously on the basis of homology to Klebsiella pneumoniae nif genes, contains gene(s) essential for symbiotic nitrogen fixation . In addition, we use this method to construct a physical genetic map of a subset of the R . meliloti nif genes.

Acta Microbiol Pol, 1981, 30(4), 371 - 85
Structure of the rigid-layer of rhizobium cell wall . II . Evidence for a covalent bond between peptidoglycan and cellodextrins; Drozanski W et al.; Covalent linkages between peptidoglycan and cellodextrins in the cell walls of Rhizobium were defined by the analysis of lysozyme split products . Digestion of peptidoglycan with lysozyme resulted in the liberation, beside disaccharide tetrapeptide fragments composed of glucosamine, muramic acid, alanine, glutamic acid and diaminopimelic acid in a molar ratio 1:1:2:1:1, also significant amounts of glucose and its polymers . The neutral carbohydrates composed of glucose, were further purified and determined as cellobiose, cellotriose and cellotetrose . Peptidoglycans pretreated with cellulase, which librated glucose and cellobiose, still contains glucose linked by lysozyme sensitive but cellulase insensitive bond.

Acta Microbiol Pol, 1981, 30(4), 355 - 70
Structure of rigid-layer of Rhizobium cell wall . I . Purification on the peptidoglycan from the cellulose microfibrils; Drozanski W et al.; The bag shaped peptidoglycan layer of Rhizobium cell wall was isolated from intact cells after treatment with sodium dodecylsulfate and trypsin, chymotrypsin or pepsin digestion . Results of chemical analysis of acid hydrolyzed peptidoglycan revealed beside two amino sugars: glucosamine and muramic acid, three major amino acids; alanine, glutamic acid and 2,6-diaminopimelic acid and also significant amount of glucose . Evidence were provided that the polyglucose found in peptidoglycan preparations of three strains of Rhizobium trifolii, one of Rhizobium leguminosarum and one of Rhizobium meliloti consist of cellulose microfibrils . The content of cellulose present in Rhizobium peptidoglycans ranged from 60 to 80% . Methods of peptidoglycan purification from the cellulose microfibrils are described.

Biochim Biophys Acta, 1980 Dec 11, 610(2), 360 - 70
Turnover of nitrogenase and leghemoglobin in root nodules of Pisum sativum; Bisseling T et al.; Turnover rates of the two nitrogenase components and leghemoglobin in root nodules of pea plants nodulated with Rhizobium leguminosarum were determined with three different methods: 1, Kinetics of 35S incorporation into protein; 2, pulse-chase experiments; 3, chloramphenicol inhibition of bacteroid protein synthesis . Methods 1 and 3 revealed that the turnover rates of the two nitrogenase components and leghemoglobin are identical to the average rate of bacteroid and plant nodule protein turnover . The t1/2 times of component I and II and leghemoglobin were about 2 days . Pulse-chase experiments with 35SO(2-)4 appeared to be rather unsuitable for determination of turnover rates in pea root nodules.

Biokhimiia, 1980 Dec, 45(12), 2139 - 45
{Glucose metabolism in isolated bacteroids of lupine nodules}; Romanov VI et al.; The ability of the bacteroids Rhizobium lupini isolated from the nodules at the stage of active nitrogen fixation to consume {1-14C}- and {2-14C} glucose was studied . 5 min after glucose addition to the cell suspension the label is rapidly incorporated into the organic acids, amino acids and sugars, which is indicative of glucose penetration inside the bacteroids and of a high rate of its catabolism . The rapid incorporation of the label into all Krebs cycle metabolites and the activities of citrate (si)-synthase, isocitrate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, which are positively correlated with the rate of the nitrogen fixation process in the course of plant vegetation provide evidence for the occurrence of the tricarboxylic acid cycle in the bacteroids.

Can J Microbiol, 1980 Dec, 26(12), 1489 - 97
Interaction of lectins from soybean and peanut with rhizobia that nodulate soybean, peanut, or both plants; Pueppke SG et al.; Four of 14 strains of Rhizobium japonicum from soybean nodulated peanut (Arachis hypogaea L . cultivar Jumbo Virginia), and 3 of 8 Rhizobium sp . strains from peanut nodulate soybean (Glycine max (L.) Merr . cultivar Harosoy 63) . Cells of three peanut rhizobia bound fluorescent- and radioisotope-labeled soybean lectin . Two of these strains failed to nodulate soybean, and conversely, two peanut strains that nodulated soybean did not bind to soybean lectin . Both culture medium and age had pronounced effects of the number of peanut rhizobia cells that bound fluorescent-labeled soybean lectin . Harosoy 63 soybean root exudates stimulated the growth of peanut rhizobia, but had no consistent influence on the number of cells that bound soybean lectin . Although extracellular soybean lectin receptors were present in culture fluids from each of the peanut rhizobia whose cells bound the lectin, the titer of receptors was greatest for strains 3G4b5 . The affinity constants for the adherence of soybean lectin to Rhizobium sp . 3G4b5 cells from cultures of various ages ranged from 4.2 X 10(6) to 4.9 X 10(6) M-1, and the number of lectin binding sites per cell decreased as cells aged . Cells of the soybean and peanut rhizobia did not bind fluorescent- or radioisotope-labeled peanut lectin . The results indicate that there is no relationship between the ability of peanut and soybean rhizobia to nodulate the reciprocal host plant and their ability to bind to the lectin of that plant.

Proc Natl Acad Sci U S A, 1980 Dec, 77(12), 7347 - 51
Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti; Ditta G et al.; A broad host range cloning vehicle that can be mobilized at high frequency into Gram-negative bacteria has been constructed from the naturally occurring antibiotic resistance plasmid RK2 . The vehicle is 20 kilobase pairs in size, encodes tetracycline resistance, and contains two single restriction enzyme sites suitable for cloning . Mobilization is effected by a helper plasmid consisting of the RK2 transfer genes linked to a ColE1 replicon . By use of this plasmid vehicle, a gene bank of the DNA from a wild-type strain of Rhizobium meliloti has been constructed and established in Escherichia coli . One of the hybrid plasmids in the bank contains a DNA insert of approximately 26 kilobase pairs which has homology to the nitrogenase structural gene region of Klebsiella pneumoniae.

Nucleic Acids Res, 1980 Oct 10, 8(19), 4337 - 47
Expression of large plasmids in the endosymbiotic form of Rhizobium leguminosarum; Krol AJ et al.; Isolated plasmid DNA from Rhizobium leguminosarum was hybridised with cellular RNA from broth-cultured bacteria and endosymbiotic bacteroids . From these hybridisation, experiments it is concluded that plasmid genes are strongly expressed in bacteroids and only weakly or not at all in bacteria . From the hybridisation of plasmid DNA with the cloned structural nif genes of Klebsiella pneumoniae it is shown that at least part of the nif genes are located on a plasmid.

Eur J Biochem, 1980 Oct, 111(2), 473 - 8
The electron transport chain of Rhizobium trifolii; de Hollander JA et al.; The presence of a branched respiratory chain in Rhizobium trifolii grown in batch culture on a defined medium was demonstrated . The branches of the respiratory chain differ in sensitivity to inhibition by cyanide and in affinity for oxygen . The branching point is localized at cytochrome b-555 . Electron flow from cytochrome b-555 via cytochrome c-549 and cytochrome a-597 towards oxygen has a relatively low affinity for oxygen and is sensitive to inhibition by cyanide . The second terminal oxidase is cytochrome b-561, which is directly linked to cytochrome b-555 . This pathway has a higher affinity for oxygen . The pathway via cytochrome a-597 is more efficient in energy conservation than the pathway via cytochrome b-561, which lacks site III of oxidative phosphorylation.

Genetics, 1980 Oct, 96(2), 321 - 9
Correlation between map position and phenotype of CTI mutants in the c cistron of Rhizobium meliloti phage 16-3; Duda B et al.; Nine temperature-sensitive clear mutations (Cti) in the C cistron (coding for the repressor protein) of Rhizobium meliloti temperate phage 16-3 were characterized according to the inductive temperature, the immunity of cells lysogenic for these mutant phages to superinfection by homoimmune weak virulent mutants, the phenotype of double-ti mutants and interallelic complementation . The results indicate that mutations of similar phenotypic expression are clustered on the genetic map . Furthermore, it seems probable that the C cistron of the original phage 16-3 is identical to that of the independently isolated phage strain 36.

J Bacteriol, 1980 Oct, 144(1), 12 - 6
Biochemical characterization of a fructokinase mutant of Rhizobium meliloti; Gardiol A et al.; A double mutant strain (UR3) of Rhizobium meliloti L5-30 was isolated from a phosphoglucose isomerase mutant (UR1) on the basis of its resistance to fructose inhibition when grown on fructose-rich medium . UR3 lacked both phosphoglucose isomerase and fructokinase activity . A mutant strain (UR4) lacking only the fructokinase activity was derived from UR3; it grew on the same carbon sources as the parent strain, but not on fructose, mannitol, or sorbitol . A spontaneous revertant (UR5) of normal growth phenotype contained fructokinase activity . A fructose transport system was found in L5-30, UR4, and UR5 grown in arabinose-fructose minimal medium . No fructose uptake activity was detected when L5-30 and UR5 were grown on arabinose minimal medium, but this activity was present in strain UR4 . Free fructose was concentrated intracellularly by UR4 > 200-fold above the external level . A partial transformation of fructose into mannitol and sorbitol was detected by enzymatic analysis of the uptake products . Polyol dehydrogenase activity was detected in UR4 grown in arabinose-fructose minimal medium . The induction pattern of polyol dehydrogenase activities in this strain might be due to slight intracellular fructose accumulation.

J Bacteriol, 1980 Sep, 143(3), 1480 - 90
Developmental fate of Rhizobium meliloti bacteroids in alfalfa nodules; Paau AS et al.; Nitrogen-fixing bacteroids are degraded during nodule senescence . This is in contrast to recent implications that viable bacteroids can be released into soil from legume nodules . Rhizobia originating from persistent infection threads in senescing nodule plant cells seem to be the source of viable cells required for perpetuation of the Rhizobium spp . population in the soil . Our conclusions were derived from electron microscopic examination of stages of development and senescence of alfalfa root nodules.

Can J Microbiol, 1980 Sep, 26(9), 1096 - 103
Changes in the outer cell wall of Rhizobium during development of root nodule symbiosis in soybean; Bal AK et al.; Treatment of soybean root nodule tissue with a nonionic detergent . Nonidet P-40, after aldehyde fixation, results in a selective solubilization of membranes . The cell wall membrane of bacteroids and of free-living Rhizobium is resistant to this treatment . Fragments of "extra" membrane present inside the membrane envelope enclosing the bacteroids are also resistant to the detergent and are morphologically similar to the outer membrane of the cell wall of Rhizobium grown in broth culture . These observations, along with electrophoretic profiles of detergent resistant membranes from nodules, free-living Rhizobium, and isolated nodule bacteroids, suggest that the Rhizobium cell wall membrane undergoes significant changes during establishment of the root nodule symbiosis.

Z Naturforsch {C}, 1980 Sep-Oct, 35(9-10), 729 - 32
Screening for mutants of Rhizobium japonicum with defects in nitrogen fixing ability; Horcher R et al.; Mutants of Rhizobium japonicum with reduced ex planta nitrogenase activity could be isolated with high frequency by direct screening of ultra-violet mutagenized bacteria growing as spots on the surface of an appropriate agar medium permitting derepression of nitrogenase synthesis . Small glass chambers fitted with a serum cap were pushed into the agar around each spot of growth, forming a small enclosed gas space which was made to 10% acetylene, permitting assessment of nitrogenase activity by the acetylene reduction test . Four mutants were isolated from a total of 305 screened spots . Two mutants had almost no ex planta activity, one of which had no symbiotic activity despite normal nodulation (ineffective), the other had only somewhat reduced activity symbiotically . Two other mutants with less than half wild-type activity ex planta were normal in symbiosis.

Can J Microbiol, 1980 Sep, 26(9), 1157 - 61
Homologous serological analysis of Rhizobium meliloti strains by immunodiffusion; Sinha RC et al.; The homologous titers of antisera prepared against 24 Rhizobium meliloti strains ranged from 8 to 64 in immunodiffusion tests when intact cells were used as test antigens . The antisera titers against a number of strains were higher when heated or ultrasonicated cell preparations were used as sources of antigens . The minimum concentration of intact cells required to produce a positive reaction varied between strains and the heat treatment of the cells of some strains increased the detectability of surface antigens . Titers of antigens in sonicated preparations of several strains were higher than those of either intact or heated cells . The minimum concentration of antigens detectable for different strains was independent of the antisera titers . Serological reactions of strains using intact cells were categorized in several groups based on the shape, position, and number of precipitin bands formed at different cell densities . The sonicated cell preparations produced additional bands and most strains contained both heat-stable and heat-labile antigens in such preparations . Some of these antigens were similar but not identical to the surface antigens whereas others appeared to be unrelated.

J Bacteriol, 1980 Sep, 143(3), 1234 - 40
Ammonium assimilation in Rhizobium meliloti; Osburne MS et al.; We have characterized a mutant of Rhizobium meliloti strain 2011 which cannot use ammonium as a nitrogen source . This mutant, RTm2620, was found to have significantly altered glutamate synthase activity . Both the mutant and the wild-type strains had glutamate dehydrogenase activity, which, although stimulated in the presence of glutamate and ammonium, was apparently insufficient to allow ammonium assimmilation . We conclude that the glutamine synthetase-glutamate synthase pathway may be the normal mode of ammonium assimilation by this strain in the free-living state . Independent revertants of Rm2620 were isolated and fell into two classes . Class I revertants regained partial glutamate synthase activity and had the same levels of glutamate dehydrogenase activity as Rm2620 . Class II revertants retained the altered glutamate synthase activity but acquired a very high level of assimilatory glutamate dehydrogenase activity . Both classes were found to be altered in their symbiotic properties, although the original Rm2620 mutant was normal in this regard.

J Biol Chem, 1980 May 10, 255(9), 4213 - 23
Purification and characterization of a ferredoxin from Rhizobium japonicum bacteroids; Carter KR et al.; An eight-iron, eight-sulfur ferredoxin from Rhizobium japonicum bacteroids of soybean root nodules has been purified to apparent homogeneity as judged by disc gel electrophoresis . The purification procedure included chromatography on DEAE-cellulose, Bio-Gel P-60, and hydroxylapatite . Specific activities of several purified preparations of bacteroid ferredoxin ranged from 1700 to 1900 nmol of C2H4 produced . min-1 . mg-1 in the reaction mediating electron transfer between illuminated chloroplasts and bacteroid nitrogenase . A molecular weight of 6740 for the protein was determined by low speed sedimentation equilibrium and a molecular weight of 6500 was estimated from the mobility of bacteroid ferredoxin relative to the mobility of standard proteins during sodium dodecyl sulfate disc gel electrophoresis . All of the common amino acids were present except arginine, methionine, and tryptophan . The absorbance spectrum of the oxidized protein exhibited maxima at 285 nm and 380 nm with a shoulder near 305 nm . The A380/A285 ratio was 0.76 and the extinction coefficient at 380 nm for the oxidized protein was found to be 30,800 M-1 . Equilibration of bacteroid ferredoxin with methyl viologen at various potentials revealed a midpoint oxidation-reduction potential of -484 mV . Spectrophotometric examination of iron-sulfur clusters extruded from bacteroid ferredoxin with benzenethiol and the transfer of its iron-sulfur clusters to other ferredoxins established the presence of two {4Fe-4S} clusters in a molecule of bacteroid ferredoxin . The EPR spectrum of oxidized ferredoxin consisted of a small signal at g = 2.02 integrating to 0.19 spin/molecule . The EPR spectrum of ferredoxin reduced with 5-deazaflavin exhibited a signal with features at g values of 1.88, 1.94, 2.01, and 2.07, and integrated to 1.7 spins/molecule . The EPR properties of bacteroid ferredoxin are characteristic of a ferredoxin operating between the 1+ and 2+ oxidation levels . Bacteroid ferredoxin mediated electron transfer to clostridial hydrogenase, but was not reduced by the clostridial phosphoroclastic system in the presence of pyruvate . Bacteroid ferredoxin reduced by illuminated 5-deazariboflavin also supported a high rate of C2H2 reduction by bacteroid nitrogenase which was free of Na2S2O4 . It was concluded, on this basis, that bacteroid ferredoxin has the capability of functioning as the electron donor for nitrogenase in R . japonicum.

Biochim Biophys Acta, 1980 May 7, 629(2), 292 - 304
Lectins and the soybean-Rhizobium symbiosis . I . Immunological investigations of soybean lines, the seeds of which have been reported to lack the 120 000 dalton soybean lectin; Su LC et al.; Seeds of six soybean lines (Glycine max (L.) Merr . cv . Columbia, D68-127, Norredo, Sooty, T-102, Wilson 5) have been reported to lack the 120 000 dalton soybean lectin . Immunodiffusion and radioimmunoassay using anti-soybean lectin immunoglobulin failed to detect the lectin in seeds of five lines, but D68-127 seeds contained as much soybean lectin as the control line, Harosoy 63 . The D68-127 seed lectin could be purified by affinity chromatography on Sepharose-N-caproylgalactosamine, and was indistinguishable from the conventional soybean lectin by the following criteria: electrophoretic migration in acidic and alkaline buffers, subunit molecular weight and composition, analytical isoelectric focusing, gel filtration chromatography . Phosphate-buffered saline extracts of roots, hypocotyls, stems, and leaves of 3--66-day-old Norredo and Harosoy 63 plants lacked soybean lectin, as determined by hemagglutination and radioimmunoassay (detection limit: 1.4 micrograms soybean lectin/g dry weight tissue) . Cotyledons of Harosoy 63 (but not Norredo) contained large quantities of the lectin, which diminished as the plants aged . 5-day-old roots and hypocotyls of 20 soybean lines did not contain soybean lectin . Roots of Columbia, Norredo, Sooty, T-102, Wilson 5, and Harosoy 63 (control) were nodulated by a variety of strains of Rhizobium japonicum and Rhizobium sp.

Can J Microbiol, 1980 May, 26(5), 572 - 6
The effect of rhizobiophages on populations of Rhizobium trifolii in the root zone of clover plants; Barnet YM; The effect of rhizobiophages CT3 and CT4 on Rhizobium trifolii SU91 and SU36, respectively, was examined . These phage-susceptible strains were used, either singly or paired with a competing phage-resistant type as inoculum for Trifolium subterraneum plants growing in vitro in agar medium . Addition of bacteriophage to single strain experiments produced no significant effects on total bacterial numbers, nodulation, or plant dry weight . However, when phages were added to the paired inocula, significant decreases in counts of the susceptible strain were observed, frequently accompanied by a rise in numbers of the resistnt type . An increase in the proportion of ineffective variants of the susceptible rhizobia was also observed, although plant dry weights and nodule numbers were not altered . These effects were dependent only upon a difference in phage susceptibility of the bacteria and were obtained even when the competing strain was only partially resistant and a relativly poor competitor.

Cell, 1980 May, 20(1), 153 - 63
Identification of "nodule-specific" host proteins (nodoulins) involved in the development of rhizobium-legume symbiosis; Legocki RP et al.; Infection of legume roots with Rhizobium species results in the development of a root nodule structure in which the bacteria form an intracellular symbiosis with the plant . We report here that the infection of soybean (Glycine max L.) roots with Rhizobium japonicum results in the synthesis by the plant of at least 18-20 polypeptides other than leghemoglobin during the development of root nodules . Identification of these "nodule-specific" host polypeptides (referred to as nodulins) was accomplished by two-dimensional gel analysis of the immunoprecipitates formed by a "nodule-specific" antiserum with in vitro translation products of root-nodule polysomes that are free of bacteroidal contaminations . Nodulins account for 7-11% of the total 35S-methionine-labeled protein synthesized in the host cell cytoplasm, and the majority of them are of 12,000-20,000 molecular weight . These proteins are absent from the uninfected roots, bacteroids and free-living Rhizobium, and appear to be coded for the plant genes that may be obligatory for the development of symbiosis in the legume root nodules . Analysis of nodulins in ineffective (unable to fix nitrogen) nodules developed due to Rhizobium strains SM5 and 61A24 showed that their synthesis is reduced and their expression differentially influenced by mutations in rhizobia . Two polypeptides of bacterial origin were also found to be cross-reactive with the "nodule-specific" antiserum, suggesting that they are secreted by Rhizobium into the host cell cytoplasm during symbiotic nitrogen fixation.

Can J Microbiol, 1980 May, 26(5), 606 - 12
Differentiation of Rhizobium japonicum strain derivatives by antibiotic sensitivity patterns, lectin binding, and utilization of biochemicals; Meyer MC et al.; Several strains of Rhizobium japonicum have been reported to consist of mixtures of stable derivativess having distinct colony morphologies and physiological characteristics . We isolated derivatives from strains of R . japonicum and systematically compared them with previously isolated derivatives with respect to the utilization of biochemicals, antibiotic sensitivity, and soyben lectin binding . With the exception of a pair of derivatives from 3Ilb110, one of which utilized pyruvate and one of which did not, sibling derivatives had essentially identical biochemical utilization patterns . The sibling derivatives of parental strains 3Ilb110 and 3Ilb140 exhibited marked variation in their sensitivities to several antibiotics, including gentamicin, sul famethoxazole, and tetracycline . Compared with the derivative with small colony morphology, derivatives with large colony morphology were in general more sensitive to these antibiotics . With one exception, the binding of soybean lectin to the derivatives was quantitatively the same as that to the parental strain . The anomaly was 110-Y which, in contrast to its parental strain and sibling derivatives, failed to bind detectable amounts of the lectin . 110-Y, as well as all the other derivatives and parental strains, nodulated Disoy soybean.

Can J Microbiol, 1980 Apr, 26(4), 562 - 5
Dynamics of Rhizobium competition for nodulation of Pisum sativum cv . Afghanistan; Broughton WJ et al.; Nodulation of Pisum sativum cv . Afghanistan by Rhizobium leguminosarum strain Tom can be blocked by the nonnodulating R . leguminosarum strain PF2 . This system was used to study the dynamics of rhizobial binding to seedling root surfaces . Appreciable numbers of 35S-labeled rhizobia bound to the root surface within 2h after inoculation . PF2 colonized the root surfaces earlier and in greater numbers that did Tom . Radioactive extracellular polysaccharides and lipopolysaccharides from either organism failed to bind to the seedling roots in measurable quantities.

Can J Microbiol, 1980 Apr, 26(4), 542 - 5
Sensitivity of Rhizobium to selected isoflavonoids; Pankhurst CE et al.; Eleven isoflavonoids were tested in both soft agar and liquid media for inhibitory activity against eight Rhizobium strains . Results of the two bioassays showed that pisatin, coumestriol, biochanin A, formononetin, genistein, rotenon, and vestitol lacked significant inhibitory activity . Phaseolin and maackiain were moderately inhibitory (ED50 (mean effective dose) = 30-100 microgram/mL), and medicarpin and kievitone were strongly inhibitory (ED50 = 10-60 microgram/mL) towards slow-growing R . japonicum and R . lupini and also towards two fast-growing Lotus rhizobia . In contrast, R . trifolii, R . leguminosarum, and R . phaseoli were not affected by these compounds ED50 greater than 100 microgram/mL), and R . meliloti was inhibited only by kievitone (ED50 = 30-60 microgram/mL) . At a concentration of 100 microgram/mL, medicarpin and kievitone were found to be bactericidal to R . japonicum, R . lupini, and the fast-growing Lotus rhizobia.

J Bacteriol, 1980 Mar, 141(3), 1454 - 6
Rhizobium leguminosarum mutants incapable of normal extracellular polysaccharide production; Napoli C et al.; Mutants of Rhizobium leguminosarum which are deficient in normal polysaccharide production have been isolated and characterized . A correlation between diminished production of extracellular polysaccharide and reduced infection and nodulation efficiency has been observed.

Can J Microbiol, 1980 Mar, 26(3), 338 - 42
Free-living and symbiotic characteristics of chlorate resistant mutants of Rhizobium; de Vasconcelos L et al.; This work investigated the usefulness of chlorate resistance as a method for the selection of nitrate reductase negative (NR-) strains from Rhizobium japonicum (61A76) and evaluated the symbiotic, characteristics of these strains . Chlorate resistent strains were selected from populations seeded on CS 7 agar containing 10 or 20 mM KC10, and incubated in 2% air- 98% N2-CO2 (95:5) . Over 200 resistant strains were isolated, 58% of which lacked the dissimilatory nitrate reductase . In 12 selected isolates, some strains had also lost the assimilatory nitrate reductase, but all retained hydrogenase activity . Chlorate resistant strains inoculated to soybean seedlings were equal to or better than the parent strain in terms of nodule mass and acetylene reduction . Those strains lacking both assimilatory and dissimilatory nitrate reductase showed the best symbiotic characteristics, suggesting that chlorate resistance in R . japonicum could be a useful method for the selection of strains with superiod nitrogen-fixing characteristics.

J Bacteriol, 1980 Mar, 141(3), 1109 - 14
Polyol metabolism by Rhizobium trifolii; Primrose SB et al.; In Rhizobium trifolii 7000, the polyols myo-inositol, xylitol, ribitol, D-arabitol, D-mannitol, D-sorbital, and dulcitol are metabolized by inducible nicotinamide adenine dinucleotide-dependent polyol dehydrogenases . Five different polyol dehydrogenases were recognized: inositol dehydrogenase, specific for inositil; ribitol dehydrogenase, specific for ribitol; D-arabitol dehydrogenase, which oxidized D-arabitol, D-mannitol, and D-sorbitol; xylitol dehydrogenase, which oxidized xylitol and D-sorbitol; and dulcitol dehydrogenase, which oxidized dulcitol, ribitol, xylitol, and sorbitol . Apart from inositil and xylitol, all of the polyols induced more than one polyol dehydrogenase and polyol transport system, but the heterologous polyol dehydrogenases and polyol transport systems were not coordinately induced by a particular polyol . With the exception of xylitol, all of the polyols tested served as growth substrates . A mutant of trifolii 7000, which was constitutive for dulcitol dehydrogenase, could also grow on xylitol.

J Bacteriol, 1980 Mar, 141(3), 1209 - 16
Physiological roles of glutamine synthetases I and II in ammonium assimilation in Rhizobium sp . 32H1; Ludwig RA; The two glutamine synthetases of Rhizobium sp . 32H1 appear to be structurally and functionally distinct . Glutamine synthetase I was reversibly adenylylated, and its synthesis was repressed only twofold by ammonium . When in the unadenylylated configuration, it was the enzyme which allowed the organism to grow, albeit marginally, on ammonium as a nitrogen source . There is no evidence to suggest that the second enzyme, glutamine synthetase II, is regulated by adenylylation . However, this enzyme was repressed at least 50-fold by even low amounts of ammonium . Glutamine synthetase II does not seem to function in ammonium assimilation but rather in purine biosynthesis.

Can J Microbiol, 1980 Feb, 26(2), 107 - 14
Fractionation and characterization of two morphologically distinct types of cells in Rhizobium japonicum broth culture; Shantharam S et al.; Differential centrifugation of stationary phase broth culture of Rhizobium japonicum yielded two distinct morphological types of bacterial cells, rods, and small coccoid forms with capsulated and non-capsulated cells in each group . The rods usually had polar capsules which resulted in "star" formation . The coccoid bacteria were either free with thick capsular material surrounding the cells or held together in a common capsular sheath forming clusters and chains . 125I soybean . lectin bound to the two types of cells . The binding sites were localized in the capsular material as revealed by colloidal gold- and ferritin-labelled lectin . Both fractions were capable of nodule formation in the soybean.

J Bacteriol, 1980 Feb, 141(2), 979 - 80
Infection and nodulation of clover by nonmotile Rhizobium trifolii; Napoli C et al.; Nonmotile mutants of Rhizobium trifolii were isolated to determine whether bacterial motility is required for the infection and nodulation of clover . The nonmotile mutants were screened for their ability to infect and nodulate clover seedlings in Fahraeus glass slide assemblies, plastic growth pouches, and vermiculite-sand-filled clay pots . In each system, the nonmotile mutants were able to infect and nodulate clover.

J Bacteriol, 1980 Feb, 141(2), 722 - 7
Behavioral mutants of Rhizobium meliloti; Ames P et al.; Rhizobium meliloti exhibited behavior resembling that of other chemotactic bacteria . Nonflagellated, nonmotile but flagellated, and nonchemotactic mutants were isolated, and genetic mapping placed all three types of lesions within a small region of the R . Meliloti chromosome . Results from nodulation experiments performed with these mutants indicate that members of the three classes (Fla-, Mot-, and Che-) infect and produce as many nodules on alfalfa plants as does the wild-type strain.

Can J Microbiol, 1980 Feb, 26(2), 179 - 82
Nodulation of soybeans carrying the nodulation-restrictive gene, rj1, by an incompatible Rhizobium japonicum strain upon mixed inoculation with a compatible strain; Devine TE et al.; The rj1 gene in soybeans prevents nodulation by most strains of Rhizobium japonicum . Several strains, however, are known to nodulate rj1 plants in vermiculite or sand culture . Pure broth cultures of one of these strains (61 NalR) and a strain producing the typical non-nodulating response with rj1 (I-110 ARS) were mixed and used as inoculum on Clark rj1 soybeans in a growth chamber experiment . Both strains carried drug resistance markers and were identified using selective media . Analysis of the nodules formed indicated that 32% of the nodules contained both strains, 36% contained only the usually non-nodulating strain I-110 ARS, and 32% contained the usually infective strain (61 NalR) . These results indicate that under conditions of high inoculum density the roots of Clark rj1 plants did not distinguish between Rhizobium strains 61 NalR and I-110 ARS . Subsequent tests with Rhizobium isolates from the nodules containing only strain I-110 ARS indicated that these rhizobia had not undergone a permanent genetic change in nodulation potential but were infective only because of temporary association with strain 61 NalR.

J Bacteriol, 1980 Feb, 141(2), 664 - 70
Chemoautotrophic growth of hydrogen-uptake-positive strains of Rhizobium japonicum; Lepo JE et al.; Recently reported research from this laboratory has demonstrated the autotrophic growth of certain hydrogen-uptake-positive strains of Rhizobium japonicum and defined minimal conditions for such growth . Ribulose 1,5-bisphosphate carboxylase has been detected in autotrophically growing cells, but at low specific activity . Moreover, growth rates were low, and growth ceased at low cell densities . We report here improved autotrophic growth rates of R . japonicum SR through the use of a modified mineral salts/vitamins medium and a programmed increase in oxygen tension as autotrophic growth proceeds . Under these conditions, ribulose, 1,5-biphosphate carboxylase activity increased greater than 10-fold and crude-extract-uptake-hydrogenase activities were from 20 to 47 times those heretofore reported for free-living R . japonicum . It is likely that previous assays for these enzymes were done on preparations of cells in which their synthesis had been partially repressed . The contribution of CO2 fixation to organic carbon accumulation in autotrophic cells was assessed as sufficient to support observed growth . Enzymological determination of the product of carbon fixation has established a stoichiometric ratio of 1.9 mol of 3-phosphoglycerate per mol of CO2 fixed and unequivocally assigns the role of carbon fixation catalysis to ribulose 1,5-bisphosphate carboxylase . Ammonium served best as a nitrogen source, nitrate was less effective, and gaseous nitrogen would not support autotrophic growth . Ecological, evolutionary, and practical considerations of autotrophy in the rhizobia are briefly discussed in the light of our findings.

Zentralbl Bakteriol Naturwiss, 1980, 135(4), 296 - 301
Growth of rhizobia in protein hydrolysates; Gulati SL; Sixteen nitrogen-rich agricultural and industrial wastes and by-products, such as vegetable waste, lasora (Cordia myxa L.), meat waste, water-hyacinth (Eichhornia crassipes), and pea (Pisum sativum L.) husks were partially hydrolysed to amino acids with the help of acetic acid of 1, 5, 10, 15, and 20% concentration . In general, 10-20% acetic acid gave the highest amounts of amino acids . Each protein hydrolysate, separately or/and in proper combination, was substituted with the nutrient yeast extract in equal concentration (mg/ml) in yeast extract mannitol (YEM) medium . The growth of Rhizobium spp . was observed over a period of 5 or 7 days with determination of turbidity at 700 nm . High optical densities, but somewhat inferior to the control, were obtained in the case of medium consisting of hydrolysate of water-hyacinth, vegetable waste, pea husk, mixed leaves, and corn steep liquor substrates separately at different concentration of acid, but the medium consisting of hydrolysates of pea husk and water-hyacinth (5% acetic acid) in equal proportions showed similar growth to that with the YEM medium in the case of both Rhizobium spp.

Zentralbl Bakteriol Naturwiss, 1980, 135(3), 201 - 4
Nodulation studies in species of genus Cassia; Gaur YD; An examination of eight species of Cassia for nodulation under natural unsterilized soils of different types from varied agroclimatic regions, as well as under aseptic conditions, inoculated with rhizobia, representing all the 8 cross inoculation groups, showed lack of nodulation in all the tested species . The absence of specific strains of rhizobia and the morpho-physiological features of the non-nodulating species of Cassia were considered the possible non-nodulating causes.

Microbios, 1980, 27(107), 27 - 32
Specific adsorption of bacteria to clover root hairs, related to the presence of the plasmid pWZ2 in cells of Rhizobium trifolii; Zurkowski W; Rhizobium trifolii strains containing plasmid pWZ2, the genetic element indispensable for nodule induction, showed a strong specific adsorption of cells to clover roots . Elimination of the plasmid pWZ2 from bacteria caused loss of adsorption capability, and this property could be recoverd by introduction of the plasmid into bacterial cells . The results obtained indicate that the plasmid pWZ2 contains gene(s) controlling the first stage of Rhizobium-legume symbiosis which is a specific adsorption of nodule bacteria to a suitable plant host.

Arch Virol, 1980, 64(1), 17 - 24
Morphology and general characteristics of viruses active against cowpea Rhizobium CB756 and 32H1; Singh RB et al.; Two newly isolated viruses, RS1 and RS2, infective on two strains of cowpea Rhizobium capable of N2-fixation in vitro, were characterized . RS1 parasitizes CB756 but RS2 infects both 32H1 and CB756 . RS1 has an isometric, polyhedral head and a long contractile tail, while RS2 has an oblate, polyhedral head and a long flexible non-contractile tail; RS1 is considerably larger than RS2 . The phages were relatively stable between pH 5 and 9 (1 hour incubation) . RS1 appeared to be more thermal sensitive and exhibited one component inactivation, while RS2 showed two component inactivation at 58, 60 and 62 degrees C . RS1 had a slower adsorption rate (3.3 X 10(-10) ml minutes-1) than RS2 (1.2 X 10(-9) ml minutes-1, on 32H1) . The latent period of RS1 and RS2 was 180 and 225 minutes, and the burst size was 15 and 9 particles/cell, respectively.

Zentralbl Bakteriol Naturwiss, 1980, 135(4), 302 - 7
A simple device of an improvised fermentor for production of rhizobial culture; Gulati SL; A simple new batch fermentor, suitable for both laboratory and industrial small scale cultivation of micro-organisms, particularly rhizobia, was designed and constructed . It was found to operate as a conventional stirring tank reactor . The device reliably ensures the sterility of cultivation, irrespective of its duration . Its installation costs are relatively low, it does not show a high consumption of energy for stirring, the operation is very simple and does not require maintenance of complicated equipment . On this basis, a procedure of design for this fermentor is outlined over the more complex scale up problems of a tank fermentor.

Zentralbl Bakteriol Naturwiss, 1980, 135(4), 290 - 5
A strain marker in Rhizobium trifolii based on the absorption of congo-red; Bromfield ES et al.; It is widely recognized that most rhizobia absorb congo-red weakly when grown on agar media containing this dye . In contrast to this generalized behaviour, an ineffective strain of Rhizobium trifolii (Coryn) has been found which strongly absorbs the dye, producing growth of a very deep-red colour on yeast-extract mannitol agar, supplemented with congo-red (0.025 g/l) . Assays for dye absorption characteristics on this medium with 21 strains of R . trifolii showed that Coryn could be readily differentiated from all the other strains examined . Supplementary fluorescent antibody tests with Coryn confirmed the reliability of differential absorption of congo-red as a strain-marker system . Data are presented which demonstrate the potential of this characteristic as a strain identification method in nodulation and competition studies under aseptic growth conditions, using mixed inocula.

Folia Microbiol (Praha), 1980, 25(2), 148 - 54
Indirect methods of determination of activity of root-nodule bacteria; Mishustin YN et al.; Significant correlations exist between the ability of root-nodule bacteria to fix molecular nitrogen and some of their properties (accumulation of riboflavin, cobalamine, ATP, cell pyrolysis, effect on plant vegetation characteristics) which can be used for quickly assessing the activity of the process in individual strains of rhizobia.

Mol Gen Genet, 1980 Jan, 177(2), 329 - 31
Autotransmissible resident plasmid of Rhizobium meliloti; Bedmar EJ et al.; A resident plasmid of wild-type strains of Rhizobium meliloti of 59.6 megadaltons has been shown to be transferred at a high frequency to "cured" strains of this bacterial species . This plasmid, named pEZ1, that confers phage-sensitivity to cells carrying it is also transmissible to Escherichia coli and from it to "cured" R . meliloti strains.

Proc Natl Acad Sci U S A, 1980 Jan, 77(1), 191 - 5
Interspecies homology of nitrogenase genes; Ruvkun GB et al.; Cloned nitrogen fixation (nif) genes from Klebsiella pneumoniae hybridize to DNA from 19 out of 19 widely divergent nitrogen-fixing bacterial strains but do not hybridize to DNA from 10 different non-nitrogen-fixing species . K . pneumoniae nif DNA fragments that hybridize to DNA from other species contain part of the three structural genes that code for nitrogenase polypeptides . We have utilized this homology to clone an EcoRI restriction endonuclease fragment from Rhizobium meliloti that hybridizes to the K . pneumoniae nif structural genes . Some of the species whose DNA hybridizes with K . pneumoniae nif DNA have been postulated to have diverged from K . pneumoniae 3 x 10(9) years ago . Nitrogenase genes are the only known example of such highly conserved prokaryotic translated genes . Nitrogenase genes are either extraordinarily conserved in evolution or have been exchanged between different nitrogen-fixing species relatively recently in evolutionary time.

J Bacteriol, 1980 Jan, 141(1), 121 - 8
Isolation and characterization of an R-prime plasmid from Rhizobium meliloti; Kiss GB et al.; Using a simple enrichment procedure, we isolated an R-prime derivative of plasmid R68.45 carrying a 17.8-megadalton segment of the Rhizobium meliloti 41 chromosome . The chromosomal segment carried on this plasmid (pGY1) includes the markers cys-24+, cys-46+, and att16-3 . Plasmid pGY1 mobilized the chromosome in a polarized way starting from the region of homology, but cannot promote chromosome transfer from other sites . The att16-3 site on pGY1 allowed the integration of phage 16-3 into pGY1, and a composite plasmid of 91.8 megadaltons was formed . This vector (pGY2) is suitable for the introduction of Rhizobium bacteriophage 16-3 into other gram-negative bacteria.

Mol Gen Genet, 1980, 178(3), 511 - 6
Generalized transduction in Rhizobium meliloti; Sik T et al.; The phage 11 of R . meliloti performs generalized transduction . This was confirmed by the variety of single markers transferred and by separating transducing particles containing BUdR-labelled bacterial DNA . The transduction frequencies depended on the marker . Linked alleles were mapped by cotransduction on fragments of bacterial DNA equal in size to the phage DNA . With crosses between antibiotic resistancy and auxotrophic markers a partial map was constructed with str, cml, pur-19, and leu-44 sites . With a few multi-auxotrophic mutants linkage data of conjugation were compared with the linkage by cotransduction.

Mol Gen Genet, 1980, 179(1), 163 - 7
Heterozygosis of phage 16-3 of Rhizobium meliloti: moderate level of mismatch repair or gene conversion; Orosz L et al.; Analysis of clear/turbid mottled (heterozygotic plaques) of Rhizobium meliloti temperate phage 16-3 indicates that the efficiency of repair at three sites (ti3, ti4, and ti5) in the C cistron is 2 to 20-fold less than that observed in E . coli phage lambda . In agreement with this conclusion, heterozygotic plaques were observed at similar frequency in crosses where point and small deletion mutants were combined, suggesting that in Rhizobium, DNA molecules with short single-stranded loops can escape from repair as efficiently as the simple mismatches.

Mol Gen Genet, 1980, 180(2), 405 - 10
Transposition of Tn 1 to the Rhizobium meliloti genome; Casadesus J et al.; A derivative of the IncP1 plasmid RP4, carrying the thermoinducible prophage Mucts62, was obtained in Escherichia coli K12 J53 (RP4) . It was impossible to maintain the hybrid plasmid RP4::Mucts62 in Rhizobium meliloti GR4 . Thus, it was used as a vehicle for introducing the ampicillin-resistant transposon Tn1 into the R . meliloti genome . Transposition of Tn1 did not generate auxotrophic strains, suggesting that the insertion of Tn1 into the R . meliloti genome was relatively specific . Two chromosomal hot spots for Tn1 insertion were identified by cotransductional analysis, after general transduction by phage DF2 . Plasmid-curing experiments, carried out by heat treatment, revealed that symbiotic plasmid(s) also contain at least one site for Tn1 insertion.

Zentralbl Bakteriol Naturwiss, 1980, 135(2), 99 - 118
{Effectiveness of symbiotic n2-fixation in leguminous plants, as affected by inoculation with rhizobia, by substrate, n-fertilizing, and 14c-sucrose application (author's transl)}; Merbach W et al.; Cultivation experiments (Mitscherlich-vessels, quartz sand, 15N-labelled soil, 15N-fertilizer) showed, that various strains of Rhizobium lupini (white and yellow lupines) and of Rhizobium leguminosarum (field beans and peas) induced a different N2-fixation of the inoculated plants, the most effective Rhizobium strains being 367a, Cz, T3, 271 (Rh . lupini), and Azotogen (Rh, leguminosarum) . Yellow lupines and field bean plants were supplied with N2 from the air considerably better than white lupines and peas after inoculation with the most effective Rhizobium strains . Application of mineral N to the white lupines and peas not only substituted the inhibited N2-fixation, but increased N amounts in the plants . White lupines fixed more N2 under soil conditions than in quartz sand . An experiment with steam-sterilized and 15-labelled soil as a comparative substrate showed, that this finding was mainly caused by an additional Rhizobium infection from the soil . Contrary to field beans and yellow lupines which fix N2 up to ripeness, white lupines and peas finished N2-fixation in the time of flowering . Mineral-N applied at that time was an additional source of N for last-named plants and they utilized it for production of higher protein yields . Continual spraying of white lupine plants with 14C-labelled sucrose solution after the time of flowering caused continuance of N2-fixation up to the stage of ripeness . It is assumed that the cause of this effect was the competition of growing seeds and nodules for the photosynthates . The supply of nodules was inadequate without external sucrose application . Mineral N inhibited the sucrose-induced N2-fixation of white lupine nodules and their consumption of photosynthates . Consequently, the applied 14C was transported into seeds to a larger extent . The investigations allow the following conclusion: Effective N2-fixation requires nodules being a powerful sink for assimilates on the basis of a highly efficient photosynthetic system of the host plant.

Acta Microbiol Pol, 1980, 29(4), 331 - 41
Typing of Rhizobium with two different phage dilutions; Staniewski R; The sensitivity of 223 Rhizobium strains to 28 phages in concentration 4xRTD and 0.4xRTD was compared . Depending on their sensitivity to the different phages the strains were divided into three groups: 1 . Rhizobium trifolii strains, Rhizobium leguminosarum for pea, vetch and horse bean and Rhizobium phaseoli, 2 . R . trifolii strains and R . leguminosarum for pea and vetch, 3 . R . meliloti strains, R . lupini and R . meliloti mutants . The use of 4xRTD distinguished 50 different phage types among the 223 strains tested . To the lower phage concentration, i.e . 0.4xRTD responded 155 Rhizobium strains and this concentration gave 48 new sensitivity patterns to phages besides 19 determined with 4xRTD . The use of lower phage concentration resulted in further differentiation within some phage types of the first group though most the strains of this group were not lysed by phages in this concentration . Much greater differentiation was observed among the R . trifolii mutants of the second group . Wild-type and mutant R . meliloti strains of the third group showed similar number of phage sensitivity patterns in the case of both 0.4xRTD and 4xRTD.

Biochim Biophys Acta, 1979 Dec 17, 565(2), 365 - 78
The biosynthesis of nitrogenase MoFe protein polypeptides in free-living cultures of Rhizobium japonicum; Scott DB et al.; The biosynthesis of the constituent polypeptides of nitrogenase component I (Rj 1) in free-living cultures of Rhizobium japonicum (strain 110) was investigated under different growth conditions . Cells were pulse-labelled and the proteins analysed by one and two-dimensional gel electrophoresis . The positions of the constituent Rj 1 polypeptides were identified by co-electrophoresis with purified Rj 1 isolated from bacteroids of soybean nodules, and by comparison with an immunoprecipitate from a culture induced for nitrogenase . The synthesis of the proteins preceded any detectable enzyme activity and increased with time, reaching a maximum after 3 days . At this time, between 6 and 8% of the total sodium dodecyl sulfate-soluble protein synthesized was Rj 1 . Exposure to air led to a dramatic decrease in the rate of Rj 1 synthesis, with almost complete regression after 20 min . In the presence of KNO3, there was no nitrogenase activity, but the proteins were present in similar amounts (7%) as the control culture . When mannitol and glycerol were used as the sole carbon sources, the amount of Rj 1 synthesized was extremely low.

Gene, 1979 Nov, 7(3-4), 349 - 53
Host dependence of RP1-specified resistance to ampicillin: differential expression in Escherichia coli and Rhizobium leguminosarum; Ram J et al.; Rhizobium leguminosarum L4 is able to serve as a host for the plasmid RP1 . Properties of R . leguminosarum {RP1} plasmid carrier suggest that the expression of RP1-coded Apr gene(s) is inhibited in this host, although the determinants of transfer and resistance to kanamycin and tetracycline are expressed . This system exemplifies a differential expression of plasmid genes in a new host.

Mol Gen Genet, 1979 Nov, 176(3), 439 - 48
Restriction mapping of DNA of temperate Rhizobium meliloti phage 16-3: comparison of genetic and physical maps indicates a long, genetically silent chromosomal arm; Dallmann G et al.; The complete restriction map of DNA (61.57 Kb) of temperate Rhizobium meliloti phage 16-3 has been constructed for enzymes BglII, HindIII, HpaI, KpnI, and a partial map for EcoRI . The strategy employed for mapping included the analysis of double, triple and partial digests; comparison of wild type and deletion mutants; and detailed analysis of subfragments, exploiting the presence of cohesive ends of the phage . Comparison of the genetic and physical maps indicates that one arm of the chromosome is genetically silent and/or contains nonessential genes.

Biochim Biophys Acta, 1979 Oct 11, 570(2), 221 - 30
Purification and properties of the particulate hydrogenase from the bacteroids of soybean root nodules; Arp DJ et al.; The uptake hydrogenase (hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1) from the bacteroids of soybean root nodules infected with Rhizobium japonicum 110 has been purified and characterized . Bacteroids were prepared, then broken by sonication . The particulate enzyme was solubilized by treatment with Triton X-100 and further purified by polyethylene glycol fractionation, DEAE-cellulose and Sephadex G-100 chromatography . The specific activity has been increased 196-fold to 19.6 units/mg protein . The molecular weight is 63 300 as determined by gel filtration and 65 300 as determined by SDS-polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer . The enzyme is O2 sensitive, with a half-life of 70 min when exposed to air . The pH optimum of the solubilized enzyme is near 5.5; the Km for H2 is 1.4 microM . Suitable electron acceptors are methylene blue, ferricyanide, 2,6-dichlorophenolindophenol, and cytochrome c . Benzyl viologen is reduced slowly; methyl viologen, NAD(P)+, FAD, FMN, and O2 are not reduced . The optimum temperature for activity is 65-70 degrees C with an activation energy of 9.2 kcal . H2 evolution by the enzyme has been demonstrated . The hydrogenase is well-suited to function in an environment where all the available H2 is generated in situ.

Can J Microbiol, 1979 Oct, 25(10), 1169 - 74
Nitrate reductase activities of rhizobia and the correlation between nitrate reduction and nitrogen fixation; Manhart JR et al.; All species of Rhizobium except R . lupini had nitrate reductase activity . Only R . lupini was incapable of growth with nitrate as the sole source of nitrogen . However, the conditions necessary for the induction of nitrate reductase varied among species of Rhizobium . Rhizobium japonicum and some Rhizobium species of the cowpea strains expressed nitrate reductase activities both in the root nodules of appropriate leguminous hosts and when grown in the presence of nitrate . Rhizobium trifolii, R . phaseoli, and R . leguminosarum did not express nitrate reductase activities in the root nodules, but they did express them when grown in the presence of nitrate . In bacteroids of R . japonicum and some strains of cowpea Rhizobium, high N2 fixation activities were accompanied by high nitrate reductase activities . In bacteroids of R . trifolii, R . leguminosarum, and R . phaseoli, high N2 fixation activities were not accompanied by high nitrate reductase activities.

Can J Microbiol, 1979 Oct, 25(10), 1191 - 6
{Effect of 2-ketogluconic acid synthesis on the exopolysaccharide production in a Rhizobium meliloti strain}; Courtois B et al.; Two categories of carbon substrates are defined for Rhizobium meliloti: the first favours the synthesis of exopolysaccharides (fructose belongs to this category) while the other is not suitable (glucose belongs to this category) . With fructose, resting cells synthesize polysaccharides during more than 100 h and this synthesis is at its best in aerobic conditions at 30 degrees C . With glucose, 2-ketogluconic acid accumulates and rapidly stops the synthesis . The method used to stop this acidification allows with glucose a synthesis which can be compared to the one obtained with fructose.

Can J Microbiol, 1979 Sep, 25(9), 979 - 86
Properties of Rhizobium trifolii isolates surviving exposure to specific bacteriophage; Barnet YM; Six strains of Rhizobium trifolii were exposed to specific bacteriophages and the properties of 10 surviving clones of each studied . Two temperate bacteriophages produced clones which were lysogenic but showed no changes in colony form, symbiotic properties, or somatic antigens . Of forty clones selected by exposure to four other bacteriophages, none were lysogenic although there was some indication of unusually long association of phage with bacteria in infected cultures . Seventeen of these clones were changed in symbiotic properties, 15 in colony morphology, and 13 in somatic cross-reaction with the parent . Unexpectedly, despite stringent selection conditions, 28 were still either partially or completely susceptible to the selecting phage.

Can J Microbiol, 1979 Sep, 25(9), 974 - 8
Effect of a bacteriophage on colonisation and nodulation of clover roots by paired strains of Rhizobium trifolii; Evans J et al.; The effect of a virulent bacteriophage on the competitive behaviour of paired strains of Rhizobium trifolii in the root zone of clover plants was examined . The presence of bacteriophage reduced the population density of susceptible strains on the root surface and, in nodulation, favoured resistant or even partially resistant strains which were otherwise less able to form nodules . These effects occurred with different growth temperatures, host species, and ratios of strains in the inoculum.

Can J Microbiol, 1979 Sep, 25(9), 968 - 73
Effect of a bacteriophage on the colonisation and nodulation of clover roots by a strain of Rhizobium trifolii; Evans J et al.; The presence of a virulent bacteriophage in the root zone of clover growing in seedling agar under controlled environments (14--17 and 19--23 degrees C) produced changes in the persistence and symbiotic effectiveness of a susceptible strain of Rhizobium trifolii . The phage reduced the rhizoplane population of rhizobia and led to the appearance of variant substrains which were less susceptible to the bacteriophage and mostly ineffective in symbiotic nitrogen fixation . Some were also changed in colonial morphology and nutritional requirements . At the higher temperature, the frequency of bacterial variants increased and the number of nodules due to the parent strain decreased . A large initial population of bacteriophage was able to reduce, but generally did not completely suppress, nodulation.

Mikrobiologiia, 1979 Sep-Oct, 48(5), 901 - 5
{Effect of indolylacetic acid on formation of bacteroid forms of Rhizobium leguminosarum}; Lobanok EV et al.; The purpose of this work was to study the effect of indolylacetic acid (IAA) on the strains of Rhizobium leguminosarum, effective and noneffective with respect to symbiotic nitrogen fixation (L4 and 245a, and 14--73, respectively) . IAA at a concentration of 50 mcg/ml and higher inhibited the growth of the bacterium, temporarily delayed celular division, and induced intensive formation of elongated bacteroid-like cells, predominantly Y-shaped or having a clavate shape . Many bacteroid-like cells were capable of division after a certain delay.

J Bacteriol, 1979 Sep, 139(3), 1075 - 8
Effect of glucose on polyol metabolism by Rhizobium trifolii; Ronson CW et al.; The effect of glucose on polyol metabolism by Rhizobium trifolii was studied . Phenomena similar to catabolite repression and catabolite inhibition were observed . The catabolism of glucose to at least glucose 6-phosphate was required for the effects to be exerted.

Rev Argent Microbiol, 1979 Sep-Dec, 11(3), 83 - 8
{Production of soy bean inoculants . Behavior of supports based on peat from Tierra del Fuego sterilized by vapor and ethylene oxide}; Balatti AP et al.; The survival of Rhizobium japonicum was studied in neutralized and sterilized peats from Ushuaia and Rio Grande . The carriers were sterilized by ethylene oxide and by autoclaving . Similar counts for Rhizobium (5 x 10(8) cel/g) were obtained in peat-cultures sterilized by both methods, after eight months . A good nodulation and nitrogen fixation capacity was observed with inoculated soybean plants . Using the strain Rhizobium japonicum E-45, no appreciable difference in symbiotic effectiveness was found between the inoculants prepared with the two peats.

Biochim Biophys Acta, 1979 Jul 26, 563(2), 496 - 507
Sequence complexities of the poly(A)-containing mRNA in uninfected soybean root and the nodule tissue developed due to the infection by Rhizobium; Auger S et al.; In order to determine the number of structural genes expressed specifically in root nodules, the total complexities of poly(A) + polysomal RNA populations from uninfected roots and mature nodules were compared . Hybridization kinetics of nodule poly(A) + RNA (NRNA) to its cDNA (NcDNA) revealed a very abundant component comprising 18--20% of the NRNA . This component was shown to be leghaemoglobin (Lb) by the similarity of its kinetics of hybridization to that of purified Lb-cDNA . The hybridization kinetics of uninfected root cDNA (RcDNA) to root poly(A) + RNA (RRNA) indicate that this abundant RNA fraction is absent in RRNA . The extent of sequence homologies between root and nodule tissue was determined by heterologous hybridizations of RcDNA to NRNA and vice versa . The data suggest that the mRNA populations of the two tissues are substantially homologous, though shifts in abundance of certain sequences are quite marked . The hybridization of purified Lb-cDNA to total polysomal RNA from developing nodules shows that the increase in concentration of Lb-mRNA sequences parallels the leghaemoglobin synthesis in this tissue . Lb sequences appear to increase between 8 and 13 days after infection with Rhizobium.

Mol Gen Genet, 1979 Jul 13, 174(2), 203 - 9
Rough and fine linkage mapping of the Rhizobium meliloti chromosome; Casadesus J et al.; A circular linkage map of the Rhizobium meliloti chromosome, obtained from R68.45-mediated crosses, has been revised by cotransductional analysis, after general transduction by DF2 phage . Three short chromosomal regions have been mapped by cotransduction . Comparison between conjugal and cotransductional data suggests that R68.45-mediated linkage measures are indeed rough . Cotransduction seems to be a useful tool for improving the linkage map of R . meliloti.

Can J Microbiol, 1979 Jul, 25(7), 803 - 7
Fingerprinting bacterial chromosomal DNA with restriction endonuclease EcoRI: comparison of Rhizobium spp . and identification of mutants; Mielenz JR et al.; Total cellular DNA from Rhizobium trifolii, R . melitoti, and R . japonicum strains 110 and 117 were prepared . DNA fragments generated with restriction endonuclease EcoRI from these DNA samples were compared in agarose gels after electrophoresis . DNA cleavage patterns generated from R . japonicum strain 110, R . trifolii, and R . meliloti were clearly distinguishable from each other . Restriction endonuclease cleavage patterns of DNA from R . japonicum strain 110 and presumptive R . trifolii mutant strains that nodulate soybean were found to be similar . Rhizobium trifolii mutant strains were also lysed by a phage specific for R . japonicum strain 110 . These results show that "R . trifolii mutant strains" are indeed derivatives of R . japonicum strain 110 and not R . trifolii.

J Bacteriol, 1979 Jul, 139(1), 316 - 7
General transduction in Rhizobium meliloti by a thermosensitive mutant of bacteriophage DF2; Casadesus J et al.; A simiplified version of bacteriophage DF2 transduction, using the thermosensitive mutant DF2tsC, is described . The transduction frequencies of Rhizobium meliloti markers by DF1tsC are of about 10(-6) or higher . The development of transductants at 34 degrees C does not require the addition of antiserum for phage neutralization.

Biokhimiia, 1979 Jul, 44(7), 1240 - 5
{Metlegoglobin reductase from lupine nodules . Purification and properties}; Bashirova NF et al.; The purification procedure and properties of metlegoglobin reductase from the soluble fraction of lupine (Lupinus luteus L.) nodules and from the proteins secreted by bacteroids Rhizobium lupini in vitro are described . The properties of both forms of enzyme were found to be similar . A metlegoglobin reductase preparation purified 125-fold with a yield of 21% was obtained . The enzyme is strictly specific to the cofactor (NADH) . No substrate specificity was revealed . The enzyme reduces oxidized cytochrome c, Mb+, Lb+, Hb+ and exygen . The pH optimum for the enzyme is 7,4 . The enzyme is inhibited by p-chloromercurybenzoate . In some properties the enzyme from lupine nodules is close to methemoglobin reductase from the erythrocytes . It was shown that apart from metlegoglobin reductase, bacteroids secrete some other proteins, which is indicative of a close interrelationship between the bacteroids and the plant in a symbiotic nitrogen-fixing system.

J Bacteriol, 1979 Jul, 139(1), 256 - 63
Effect of cyclic guanosine 3',5'-monophosphate on nitrogen fixation in Rhizobium japonicum; Lim ST et al.; The addition of exogenous cyclic guanosine 3',5'-monophosphate (cGMP) at a concentration of 0.1 mM to a free-living culture of Rhizobium japonicum 3I1b110 was found to completely inhibit the expression of nitrogenase activity and markedly inhibit the expression of hydrogenase and nitrate reductase activities . The effect was specific for cGMP . Experiments on the in vivo incorporation of radioactive methionine and subsequent analysis of the labeled proteins on polyacrylamide gels showed that the biosynthesis of nitrogenase polypeptides was inhibited . It appears that the time of addition of cGMP is important since the effect was only seen during the early stages of nif gene expression . The intracellular level of cGMP was found to respond to physiological changes in the cell, and there was a fall in cGMP concentrations when nitrogenase was induced . Microaerophilic-aerobic shift experiments showed that intracellular levels increased from 0.25 pmol/mg of cell protein under microaerophilic conditions to 2.6 pmol/mg of cell protein under aerobic conditions, suggesting that the cellular pool size of cGMP may be under redox control.

Biochim Biophys Acta, 1979 Jun 20, 563(1), 216 - 26
Polypeptide synthesis by Rhizobium bacteroids and bacteria; Shaw BD et al.; When Rhizobium bacteroids (strain NZP 2257) from lupin nodules were isolated and incubated aerobically at high osmolarity, they incorporated {35S}-methionine into a characteristic set of polypeptides; many of these polypeptides coelectrophoresed on SDS-polyacrylamide gels with the bacteroid polypeptide bands stained by Coomassie blue . The labelled polypeptides were stable for several hours in pulse-chase experiments . Changes in the concentration of H+, K+ and Mg2+ in the incubation mixture affected overall incorporation of label, but not the relative incorporation into different polypeptides . A similar set of bacteroid polypeptides was labelled in situ when detached nodules were fed {35S}methionine . Distinctive labelling patterns were observed with bacteroid suspensions from mature and immature nodules, with a transitional pattern at the time when nitrogenase activity appeared . Two of the major labelled components in mature bacteroids had estimated molecular weights of 60- and 34-kilodaltons similar to values reported by others for the constituent polypeptides of nitrogenase . Bacteroids of the same Rhizobium strain grown in different plant hosts gave similar polypeptide labelling patterns in purified suspensions, but bacteroids of different Rhizobium strains gave different patterns . The polypeptide labelling patterns obtained using broth-cultured Rhizobium bacteria from various growth stages and growth media differed from those obtained using bacteroids of the same strain.

Mol Gen Genet, 1979 Jun 20, 173(3), 289 - 98
Use of RP4-prime plasmids constructed in vitro to promote a polarized transfer of the chromosome in Escherichia coli and Rhizobium meliloti; Julliot JS et al.; RP4-prime plasmids containing chromosomal fragments of either Escherichia coli or Rhizobium meliloti were constructed in vitro . When introduced into E . coli or R . meliloti respectively, they promoted a polarized transfer of the chromosome as demonstrated either by the gradient of transfer of various markers or by the study of the genetic constitution of recombinants . In E . coli, mobilization was shown to be dependent upon the presence of a functional rec A system . Inheritance of markers was due to their integration into the chromosome of the recipient as shown by the need for a functional rec A system in the recipient E . coli or by mobilization of recessive markers in R . meliloti . The system described could be applied to genetic mapping in any Gram negative bacteria.

Can J Microbiol, 1979 Jun, 25(6), 675 - 9
Thymidine incorporation into Rhizobium meliloti; Behki RM et al.; Thymidine is rapidly catabolized to thymine, beta-aminoisobutyric acid, and carbon dioxide by Rhizobium meliloti cells . The incorporation of labelled thymidine into the DNA of R . meliloti cells can be enhanced by the addition of low concentrations (10-20 micrograms/mL) of deoxyadenosine or other nucleosides (adenosine, uridine, guanosine) . However, at high concentrations ( greater than 50 micrograms/mL) these compounds inhibit thymidine incorporation . Conditions to obtain highly radioactive DNA of Rhizobium are described.

Biochim Biophys Acta, 1979 May 24, 562(3), 515 - 26
Development of the nitrogen-fixing and protein-synthesizing apparatus of bacteroids in pea root nodules; Bisseling T et al.; Some aspects of root nodule development of Pisum sativum inoculated with Rhizobium leguminosarum were examined . 1 . Nitrogenase activity (measured as acetylene reduction) appears to be preceded by leghemoglobin synthesis (measured immunologically) . 2 . Syntheses of component I and component II of nitrogenase are not strictly coordinated . Synthesis of component I starts before component II . 3 . Plant and bacteroid protein synthesis (measured by {35S}sulfate labeling) in root nodules declines rapidly during nodule development . Corresponding with this decline is a decrease in quantity and quality of rRNA.

Biochim Biophys Acta, 1979 May 16, 584(3), 479 - 92
Regulation of hydrogen utilisation in Rhizobium japonicum by cyclic AMP; Lim ST et al.; Utilisation (uptake) of hydrogen gas by whole cells of Rhizobium japonicum was found to be influenced by the carbon source(s) present in the growth medium, with activity being highest in a medium containing sugars . Tricarboxylic acid cycle intermediates, such as malate, significantly reduced H2 utilisation . No reduction in the hydrogenase activity is observed when the enzyme is assayed directly by the tritium exchange method, indicating that the decrease in hydrogen uptake activity is not due to repression of hydrogenase biosynthesis . Cyclic AMP was found to alleviate the inhibition of H2 uptake by malate, and this requires new protein synthesis . Addition of chloramphenicol or rifampicin simultaneously with cyclic AMP eliminated the stimulation of H2 uptake in the malate medium . These results show that in R . japonicum cyclic AMP plays a major role in the regulation of H2 metabolism.

Appl Environ Microbiol, 1979 May, 37(5), 867 - 70
Multiple antibiotic resistance in Rhizobium japonicum; Cole MA et al.; A total of 48 strains of the soil bacterium Rhizobium japonicum were screened for their response to several widely used antibiotics . Over 60% of the strains were resistant to chloramphenicol, polymyxin B, and erythromycin, and 47% or more of the strains were resistant to neomycin and penicillin G, when tested by disk assay procedures . The most common grouping of resistances in strains was simultaneous resistance to tetracycline, penicillin G, neomycin, chloramphenicol, and streptomycin (25% of all strains tested) . The occurrence of multiple drug resistance in a soil bacterium that is not a vertebrate pathogen suggests that chemotherapeutic use of antibiotics is not required for the development of multiple drug resistance.

Can J Microbiol, 1979 May, 25(5), 565 - 78
Structure of nitrogen-fixing nodules formed by Rhizobium on roots of Parasponia andersonii Planch; Trinick MJ; The structure of nitrogen-fixing nodules produced by Rhizobium infection of the non-legume Parasponia andersonii was examined by light and electron (both SEM and TEM) microscopy . Comparisons were made with the nodules previously described on P . rugosa . Like the nodules on different non-legumes formed by other types of endophytes, the Rhizobium nodules on Parasponia resembled modified roots by having a central vascular bundle surrounded by an endophyte-infected zone . The intimate association between the Rhizobium and the host nodule cell was compared with the Rhizobium association found in legumes . The rhizobia were not released from the infection thread as happens in the legume . The infection thread, which propagates the Rhizobium infection to new cells, was transformed within a nodule cell from a darkly stained (light microscopy) or very electron-dense (TEM) structure to a number of thread types . The walls of the threads varied greatly in thickness and often the thread structures were without rigid walls and were only enclosed by a plasma membrane . If the rhizobia are transformed into bacteroids, as in the legumes, it would have to occur when the threads had reached their mature size, when bacterial division had ceased . Nitrogen fixation was considered to occur in all thread types.

Appl Environ Microbiol, 1979 May, 37(5), 862 - 6
Transfer of R factors to and between genetically marked sublines of Rhizobium japonicum; Kuykendall LD; Plasmids R1822 and pRD1 of the P-1 incompatibility group, for which Rhizobium japonicum had not previously been shown to serve as host, were introduced into a strain of R . japonicum . Acquisition of R68 and R68.45 plasmids by this Rhizobium was equivocal . Transfer of R1822 from Pseudomonas aeruginosa and of pRD1 from Escherichia coli to R . japonicum was unambiguous, because the exconjugants subsequently cotransferred the three R-factor resistance determinants (kanamycin, tetracycline, and penicillin) between genetically marked sublines of strain I-110 . Under optimal conditions the transfer of R1822 and pRD1 occurred at frequencies of approximately 10(-3) in plate matings of strains bearing as many as five dissimilar genetic markers . In matings with R1822 on membrane filters, recombinants were formed at incidences as high as 4%.

Proc R Soc Lond B Biol Sci, 1979 Apr 11, 204(1155), 219 - 33
The Rhizobium--legume symbiosis; Beringer JE et al.; The rhizobia are soil microorganisms that can interact with leguminous plants to form root nodules within which conditions are favourable for bacterial nitrogen fixation . Legumes allow the development of very large rhizobial populations in the vicinity of their roots . Infections and nodule formation require the specific recognition of host and Rhizobium, probably mediated by plant lectins . Penetration of the host by a compatible Rhizobium species usually provokes host root cell division to form the nodule, and a process of differentiation by both partners then ensues . In most cases the rhizobia alter morphologically to form bacteroids, which are usually larger than the free-living bacteria and have altered cell walls . At all stages during infection, the bacteria are bounded by host cell plasmalemma . The enzyme nitrogenase is synthesized by the bacteria and, if leghaemoglobin is present, nitrogen fixation will occur . Leghaemoglobin is a product of the symbiotic interaction, since the globin is produced by the plant while the haem is synthesized by the bacteria . In the intracellular habitat the bacteria are dependent upon the plant for supplies of energy and the bacteroids, in particular, appear to differentiate so that they are no longer able to utilize the nitrogen that they fix . Regulation of the supply of carbohydrate and the use of the fixed nitrogen thus appear to be largely governed by the host.

Can J Microbiol, 1979 Mar, 25(3), 352 - 61
Nitrogen fixation by Rhizobium sp . 32H1 . A morphological and ultrastructural comparison of asymbiotic and symbiotic nitrogen-fixing forms; van Brussel AA et al.; The induction of nitrogenase (C2H2) activity in asymbiotically cultured Rhizobium sp . 32H1 was found to be associated with morphological changes in the cells which were more pronounced than those seen in bacteroids . Polyphosphate granules were found in both bacteroids and cultured cells, but poly-beta-hydroxybutyrate vesicles were almost absent in bacteroids but were present in cultured cells . Freeze-etching techniques revealed no differences between the asymbiotically cultured nitrogen-fixing forms and bacteroids in that both the cell wall and cytoplasmic membrane cleavage planes were normal for gram-negative bacteria.

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