Levels and identities of nonrhizobial microorganisms found in commercial legume inoculant made with nonsterile peat carrier.

Sixty samples of commercial North American legume inoculants manufactured for sale in 1994 using nonsterile peat as carrier were tested for Rhizobium (or Bradyrhizobium) content and non-Rhizobium biological contaminant load. Products of three major producers of such inoculants for sale in Canada were examined. Viable Rhizobium content varied from 5.6 x 10(5) to 8.1 x 10(9) cells/g, while the contaminant load varied from 1.8 x 10(8) to 5.5 x 10(10) cfu/g. Most of the inoculants contained more nonrhizobial organisms than they did rhizobia. Identifications were made of the most numerous nonrhizobial bacteria occurring in 100 samples of inoculants collected in 1993 and 1994. The most commonly identified contaminant was Xanthomonas maltophilia. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterobacter cloacae were also found at high levels in some products. Contaminant organisms capable of inhibiting rhizobial growth in plate culture were found in the products of all three manufacturers.

Enhanced N2-fixing ability of a deletion mutant of arctic rhizobia with sainfoin (Onobrychis viciifolia).

Mutagenesis provoked by exposure at elevated temperature of the cold-adapted, arctic Rhizobium strain N31 resulted in the generation of five deletion mutants, which exhibited loss of their smaller plasmid (200 kb), whereas the larger plasmid (> 500 kb) was still present in all mutants. Deletion mutants did not show differences from the wild type in the antibiotic resistance pattern, the carbohydrates and organic acids utilization, and the growth rate at low temperature. However, deletion mutants differed from the wild type and among themselves in the ex planta nitrogenase activity, the nodulation index, and the symbiotic effectiveness. The deletion mutant N31.6rif (r) showed higher nodulation index and exhibited higher nitrogenase activity and symbiotic efficiency than the other deletion mutants and the wild type. The process of deletion mutation resulted in the improvement of an arctic Rhizobium strain having an earlier and higher symbiotic nitrogen fixation efficiency than the wild type.

Purification and partial amino acid sequence of a glutamyl-tRNA synthetase from Rhizobium meliloti.

A glutamyl-tRNA synthetase has been purified to homogeneity from Rhizobium meliloti, using reversed-phase chromatography as the last step. Amino acid sequencing of the amino-terminal region of the enzyme indicates that it contains a single polypeptide, whose molecular weight is about 54,000, as judged by SDS-gel electrophoresis. The primary structures of the amino-terminus region and of an internal peptide obtained by cleavage of the enzyme with CNBr have similarities of 58 and 48% with regions of the glutamyl-tRNA synthase of Escherichia coli; these are thought to be involved in the binding of ATP and tRNA, respectively. The small amount of glutamyl-tRNA synthetase present in R. meliloti is consistent with the metabolic regulation of the biosynthesis of many aminoacyl-tRNA synthetases.

Cloning and sequencing of the gltX gene, encoding the glutamyl-tRNA synthetase of Rhizobium meliloti A2.

The gltX gene, coding for the glutamyl-tRNA synthetase of Rhizobium meliloti A2, was cloned by using as probe a synthetic oligonucleotide corresponding to the amino acid sequence of a segment of the glutamyl-tRNA synthetase. The codons chosen for this 42-mer were those most frequently used in a set of R. meliloti genes. DNA sequence analysis revealed an open reading frame of 484 codons, encoding a polypeptide of Mr 54,166 containing the amino acid sequences of an NH2-terminal and various internal fragments of the enzyme. Compared with the amino acid sequence of the glutamyl-tRNA synthetase of Escherichia coli, the N-terminal third of the R. meliloti enzyme was strongly conserved (52% identity); the second third was moderately conserved (38% identity) and included a few highly conserved segments, whereas no significant similarity was found in the C-terminal third. These results suggest that the C-terminal part of the protein is probably not involved in the recognition of substrates, a feature shared with other aminoacyl-tRNA synthetases.

Large-Scale Production of Rhizobium meliloti on Whey.

Whey, a by-product of the cheese industry, can sustain the growth of fast-growing rhizobia. To avoid any latency of growth, rhizobial inoculum must be prepared under inducing conditions. In unsupplemented whey, the number of cells of Rhizobium meliloti Balsac reached 5 x 10 CFU/ml in 48 h of incubation. This is comparable to the yield obtained with yeast-mannitol broth, the standard medium for the growth of rhizobia. In raw whey supplemented with yeast extract (1.0 g/liter) and phosphate (0.5 g/liter), the number of cells reached 10 CFU/ml in 48 h of incubation. This is a twofold increase compared with the population normally obtained in industrial production. Whey represents a relatively inexpensive and efficient substrate medium for the large-scale production of fast-growing rhizobia.

Effect of oxygen on batch and continuous cultures of a nitrogen-fixing Arthrobacter sp.

Growth, acetylene reduction, and respiration rate were studied in batch and continuous cultures of Arthrobacter fluorescents at different oxygen partial pressures. The optimum pO2 values for growth and acetylene reduction were 0.05 and 0.025 atm, respectively, but microorganisms can tolerate higher pO2 values. The growth of cultures provided with combined nitrogen was dependent on oxygen availability, and strict anaerobic conditions did not support growth. Acetylene reduction of a population grown in continuous culture and adapted to low pO2 (0.02 atm) was much more sensitive to oxygenation than that of a population adapted to high pO2 (0.4 atm). Their maximum nitrogenase activity, at their optimal pO2 values, were quite different. The respiratory activity of nitrogen-fixing cultures increased with increasing oxygen tensions until a pO2 of 0.2 atm. At higher pO2 values, the respiration rate began to decrease.

[Identification of actinomycetes with antifungal activity which do not affect Rhizobium meliloti]. / Identification des actinomycètes antagonistes de champignons et n'affectant pas le Rhizobium meliloti.

Thirteen isolates of actinomycetes that have broad antifungal activity and do not affect two efficient strains of Rhizobium meliloti were identified as: Nocardia autotrophica, Streptomyces antimycoticus, S. anulatus, S. capoamus, S. lydicus, S. murinus, S. roseo-luteus, and S. thermotolerans.

[Actinomycetes antagonistic to fungi and not affecting Rhizobium meliloti]. / Actinomycètes antagnoistes de champignons et n'affectant pas le Rhizobium meliloti.

The effects of 481 actinomycetes isolated from agricultural soils supporting good growth of alfalfa or clover on two efficient strains of Rhizobium meliloti A2 and S14 were studied. Strain A2 was inhibited by 28% of the isolates and strain S14 was inhibited by 31% of them. No significant difference was found between the resistance of both actinomycete strains. The effects of the 288 isolates not affecting R. meliloti on six fungi were also studied. The most sensitive fungus was Stemphylium sarcinaeforme inhibited by 20% of the isolates, while Fusarium culmorum was the most resistant fungus and was inhibited by only 6% of the isolates. Thirteen isolates inhibited four to six fungi. In an autoclaved greenhouse soil, isolate 181 which inhibited the six fungi tested significantly reduced the population of the phytopathogenic fungus F. oxysporum f. sp. medicaginis and eliminated the inhibitory effect showed by this fungus on strain A2 of R. meliloti.

[Physical and chemical properties of tryptophenases of five species of Enterobacteriaceae]. / Propriétés physiques et chimiques des tryptophanases de cinq espèces d'Entérobactériaceae

The molecular weight, sedimentation coefficient, and amino acids composition were determined on five tryptophanases (TPases) from Escherichia coli B and E. aurescens, Shigella alkalescens, and Proteus vulgaris and P. morganii. These TPases have identical sedimentation profile and coefficient (9.6 S), and the same molecular weight (220 000). Each enzyme is constituted of four identical subunits having a molecular weight of 55 000. The amino acids composition of these TPases is very similar, with the exception of P. morganii and P. vulgaris TPases which present significative variations in basic amino acids and tryptophan content. The species differentiation of the coli group cannot be made on their TPase characteristics only, contrary to P. morganii and P. vulgaris which can be differentiated between them and from the coli group.

[Differentiation of tryptophanases of five species of Enterobacteriaceae by sulfhydryl groups]. / Distinction des tryptophanases de cinq espèces d'Entérobactériaceae par les groupements sulfhydryles

We have studied the sulfhydryl groups (-SH) on the tryptophanases (TPases) from Escherichia coli B. and E. aurescens, Shigella alkalescens, and Proteus vulgaris and P. morganii. The coli group and the P. morganii apo TPases have 20 -SH groups per mole of enzyme, where as P. vulgaris apoTPase has 16. In coli group TPases, there are 16 -SH groups on the mole surface and they are all implicated in the activity and the enzyme-substrate bond. Proteus morganii TPase has 8 surface -SH groups, 4 of which are implicated in the activity; the remaining 12 -SH groups are located inside the mole and take part in the activity and the enzyme-substrate bond. Proteus vulgaris TPase has 4 surface -SH groups which are constructive of the enzyme structure, whereas the 12 remaining -SH groups are located inside the mole and take part in the activity and the enzyme-substrate bond. It is concluded that Proteus TPases are molecules which have inverted quaternary structure in comparison to those of the coli group. The studied TPases have four subunits, each of them is constituted of one polypeptidic chain having a molecular weight of 55,000.

[The effect of pyridoxal phosphate on the tryptophanases of five species of Enterobacteriaceae]. / Effet du phosphate de pyridoxal sur les triptophanases de cinq espèces d'Eenérobactriaceae

Escherichia coli B and E. aurescens, Shigella alkalescens, and Proteus vulgaris et P. morganii tryptophanases (TPases) were studied for the spectral forms of the enzyme. The pH effect on the absorption spectrum and on the enzyme specific activity revealed that the coli group TPases are identical with but differ from Proteus TPases which differ themselves. The coli group TPases attach 4 mol of pyridoxal phosphate (PLP)/mol of enzyme, independently of the pH in the presence of K(plus) ions, and 9 mol of PLP/mol of enzyme must be reduced to achieve complete inactivation. The Proteus TPases attach 4 mol of PLP/mol of enzyme at PH 6.8, and 3 mol of PLP/mol of enzyme at pH 7.8 in K(plus) buffer. In P. morganii, 7 mol of PLP/mol of enzyme must be reduced to inactivate the enzyme, whereas P. vulgaris TPase cannot be completely inactivated by this method. These five TPases attach only 3 mol of PLP/mol of enzyme in a Na(plus) buffer, independently of the pH.

Azobenzene residues from aniline-based herbicides; Evidence for labile intermediates.

Formation of asymmetric azobenzenes from variously substituted aniline pairs in soil and by peroxidase pointed to the existence of labile intermediates. These were tentatively identified as the respective phenylhydroxylamines.

Biochemical decomposition of the herbicide N-(3,4-dichlorophenyl)-2-methylpentanamide and related compounds.

Organisms capable of decomposing N-(3,4-dichlorophenyl)-2-methylpentanamide (Karsil) were isolated, identified, and tested for their ability to hydrolyze this herbicide. Primary products of Karsil decomposition by cells and cell-free extracts of a Penicillium sp. were identified as 2-methyl-valeric acid and 3,4-dichloroaniline. The Karsil acylamidase (EC 3.5.1.a aryl acylamine amidohydrolase) was an induced enzyme. It was partially purified and tested for its ability to hydrolyze 25 related compounds. Some relations between the structures of these compounds and their susceptibility to enzymatic hydrolysis were discerned.

Rapid technique for enumeration and isolation of peroxidase-producing microorganisms.

Peroxidase-positive microorganisms were enumerated on agar plates by use of a p-anisidine-H(2)O(2) spray. Combined with replica-plating, the same technique allowed the selective isolation of peroxidase-producing microbial cultures.