Mannitol-1-phosphate dehydrogenase (EC 22.214.171.124) was purified to homogeneity from anaerobically grown cells of Brochothrix thermosphacta on mannitol. The enzyme was a monomer of apparent molecular weight 50, 000. Reduction of fructose-6-phosphate was optimal at pH 6.5 (Km Fru-6-p, 0.25mM; Vmax, 65 units/mg protein) as compared to a pH maximum of 9.5 for the oxidation of mannitol-1-phosphate (Km Mno-1-p, 50μM; Vmax, 18 units/mg protein). The oxidation reaction was inhibited by ATP and ADP, although these nucleotides had no effect on the reverse reaction. ADP was a stronger inhibitor than ATP (Ki: 0.7 and 1.6mM, respectively). Possible role of this enzyme in maintaining redox balance in this organism is discussed.
The distribution of rhizobia, bacteria which nodulate roots of leguminous plants, was surveyed for 91 species of the family Leguminoceae. One hundred and twenty-one strains of rhizobia were isolated, and 29 strains were obtained from culture collections. The total of 150 strains were classified based on their small subunit (ssu) ribosomal RNA (rRNA) sequences. The analyses of the partial sequences (157 bases from positions 1220 to 1377 in Escherichia coli numbering system) revealed the presence of three major groups which corresponded with the genera Rhizobium, Bradyrhizobium, and Azorhizobium and the presence of 17 varieties (eight varieties for the genus Rhizobium, eight for the genus Bradyrhizobium, and one for the genus Azorhizobium). Several phenotypic characteristics and DNA base compositions were determined for 27 representative strains from 17 varieties. Also, genomic relatedness among the 27 strains was estimated by DNA-DNA hybridization. The strains which were different in their ssu rRNA partial sequences never showed close relationships in DNA-DNA hybridization. Further, sequences covering most of ssu rRNA were compared among 17 representative strains of the 17 varieties including type strains of seven Rhizobium species, Bradyrhizobiumjaponicum and Azorhizobium caulinodans, and phylogenetic relationships among rhizobia were discussed. From phylogenetic analyses it is inferred that the nodulation genes transferred among rhizobia.
Growth and arginine and proline regulation of heterocyst and nitrogenase formation have been studied in the diazotrophic cyanobacterium Anabaenacycadeae and its glutamine auxotrophic mutant strain lacking glutamine synthetase (GS) activity. The wild-type strain having normal GS activity utilized arginine and proline as sole nitrogen source without producing heterocyst and nitrogenase activity. In contrast, the glutamine auxotrophic mutant lacking GS activity could not utilize these amino acids as sole nitrogen source. However, similar to the wild-type strain it showed complete repression of heterocyst and nitrogenase activity in the presence of both amino acids. Both wild-type and mutant strain showed high level of arginase and proline oxidase activities but arginine- and proline-dependent NH4+ generation was confined to only the mutant strain lacking GS activity. These results suggest that (1) GS does not control the formation of N2-fixing heterocysts; and (2) GS activity is necessarily required for the assimilation of arginine and proline as sole nitrogen source.
Attempts were made to separate and purify cellulose-binding proteins (CBPs) from the lysate of cells of Fibrobacter succinogenes S85, utilizing their affinity to cellulose. After Avicel cellulose was incubated in the culture treated with Tween 20 at 37°C, cellulose was washed with buffer containing Softes 12 as a detergent to remove the cell debris and proteins incapable of adhering to cellulose. Proteins strongly adhering to cellulose after washing were eluted with buffer containing 10% cellobiose. The eluent contained mainly two major proteins and one minor protein. When the eluent, concentrated by ultrafiltration, was dialyzed against distilled water, small particles of aggregate appeared in it. The dialyzed eluent was separated into soluble and insoluble fractions by high speed centrifugation. Proteins present in both the soluble fraction and the solution of the insoluble fraction dissolved in buffer containing Softes 12 were purified by treatment with a saturated solution of ammonium sulfate. Protein purified from the soluble fraction was designated as CBP1 and protein from the insoluble fraction as CBP2. The molecular weight of CBP1 and CBP2 was approximately 120 and 225 kilodaltons (kDa), respectively. The culture containing a cell lysate of the bacterium exhibited such enzyme activities as β-glucosidase, cellobiosidase and CMCase, whereas both CBP1 and CBP2 did not show these enzyme activities. Both CBP1 and CBP2 displayed adhering ability to cellulose powder, but not to starch granules.
Several properties of chemotaxis by a psychrophilic marine bacterium, Vibrio sp. strain ABE-1, were examined by swarm formation on a soft agar. This bacterium exhibited a large chemotactic response to succinate, acetate and fumarate. Small but significant response to amino acids such as aspartate, glutamate and serine was also observed. The maximum chemotactic response to succinate was obtained in the presence of 400mM NaCl, 10mM KCl and 25mM NH4Cl. Chemotaxis of this bacterium was observed in the range of pH from 6.0 to 9.0, and the optimum pH for chemotaxis was between 7.5 and 8.0. Chemotactic response by this bacterium was dependent on temperature, the optimum temperature was 15°C, and very high chemotactic response (about 60% of the maximum response) was retained even at low temperature such as 5°C. On the other hand, no response was detectable at 25°C.
The cell surface tension of 250 strains of bacteria isolated from grassland soil was examined by measuring the contact angle of a liquid droplet on the cell layer. The values of the contact angle of aqueous 0.1M NaCl solution and α-bromonaphthalene on the cell layer were used to calculate the polar and non-polar components of surface tension for the isolates. The polar component of the "slow-growing" isolates was smaller than that of the "fast-growing" isolates. On the other hand, no difference could be observed in the non-polar component between the fast- and slow-growing isolates. These results indicate that slow-growing isolates have more hydrophobic cell surface than fast-growing isolates.
A sucrose-adapted inoculum of Saccharomyces cerevisiae growing in a batch fermenter in a sucrose medium was dosed with a high concentration of the enzyme invertase (β-D-fructosidase) during the mid-exponential phase of growth. The culture then underwent a short lag of about 20min before dramatically decreasing its growth rate. The sugar analysis profiles showed that, during the lag phase, all the sucrose was hydrolyzed to glucose and fructose. The specific growth rate decreased from 0.50h-1 when growing on sucrose (before addition of invertase) to 0.44h-1 on the resulting mixture of glucose and fructose. These results help in explaining the mechanism of utilization of sucrose by actively growing yeast cells in a batch culture and suggest a direct uptake of sucrose molecules into yeast cells.
Sinki, a non-salted fermented radish tap root product, is traditionally consumed as a base for soup and as a pickle in some north-eastern states of India, in Nepal and a few places of Bhutan. A total of 453 strains of lactic acid bacteria were isolated from 40 samples of sinki. They belonged to either Lactobacillus plantarum or Lactobacillus brevis. In the substrate, in addition to these two, Lactobacillus fermentum was present. Sinki was dominated by L. plantarum followed by L. brevis, with their prevalence in 100% of the samples. In order to study the changes in proximate and microbial composition accompanying fermentation, the process conditions for the production of sinki were optimized. Glass jar as a fermentation container and 12 days' fermentation at 30°C were found optimum. During fermentation under optimized conditions, the pH of the fermenting mass dropped from 6.7 to 3.3. This was due to an increase in titratable acidity from 0.04 to 1.28%. The fermentation was initiated by hetero-fermentative L. fermentum, followed by another heterofermentative L. brevis, and finally succeeded by homofermentative L. plantarum.
Two good polygalacturonase (PG) producing strains were selected from 21 Rhizopus spp. isolates from ‘ragi’ and fermented foods and from 4 authentic Rhizopus strains. Strain LKN produced 3.2U/mg protein of crude enzyme extract (0.68U/g substrate) in 3 days solid culture of pectin and cassava starch supplemented wheat bran. Strain A11-enzyme showed a wide pH stability range of pH 2-11 and tolerance at 50°C for 20min.
Myxobacteria are known to lyse a wide variety of microorganisms. Lysis was analyzed to be caused by various enzymes which myxobacteria produce extracellularly. Selecting Rhodotorula glutinis, which is known to be rather resistant to lytic enzymes, we analyzed morphologically lytic phenomena of this microorganism by myxobacteria. During co-cultivation of myxobacteria and Rhodotorula glutinis, myxobacteria crowded around the coagulated Rhodotorula cells. On electronmicroscopic investigation of the yeast cells, the majority of cytoplasmic material disappeared and only membranous material and the cell wall remained. Preliminary biochemical studies have shown that extracellular protease was not found to be a sole factor but the fatty acids extracted from the myxobacterial cells were additionally required.
Bacteriophages specific for Pediococcus halophilus, a group of halophilic lactic cocci used as starters for soy sauce fermentation, were isolated from fermenting soy sauce moromi-mash. Typical two of these phages, φ7116 and φD-86, which propagate on P. halophilus NISL 7116 and D-86 respectively, were purified and studied for their morphology and some infecting properties. Morphotype of φ7116 was Ackermann's A1 having an isometric head of 87-96nm diameter and a contractile tail of 200nm length. φD-86 was found to be morphotype B1, having a 67nm isometric head and a 300nm noncontractile tail. Both the phages could propagate on their each host under any pH or salt conditions in which their hosts could grow. Latent periods were both 5h and burst sizes were 27-28. Their host ranges were substantially strain-specific. They both thermally inactivated at 60°C. Stable pH ranges for φ7116 and φD-86 were 5.0-8.8 and 4.4-10.7 respectively. In diluted NaCl solutions, φD-86 was fairly stable over 0.03-2.6M but φ7116 was unstable specifically between 0.04-0.1M.