Isolation of Rec mutants of Bacillus subtilis via insertional mutagenesis]

A number of mutants supersensitive to mytomycine C were isolated via insertion of Tn917 into Bacillus subtilis chromosome. Six Rec mutants with lower transformation frequency, though with nondisturbed capacity of DNA binding and uptake, were isolated. Two of these mutations are situated in those chromosomal sites where location of Rec genes had not yet been registered.     

Cloning of the purA16 locus in Rec+ cells of Bacillus subtilis

A portion of purA16 chromosomal locus of Bacillus subtilis was cloned into Rec+ cells of this microorganism with pBD12 plasmid (carrying chloramphenicol and kanamycin resistance determinants) serving as a vector. The hybrid plasmids were stably maintained in cells grown on media supplemented with antibiotics and were lost from cells in the absence of drugs. The cloned fragment could incorporate into the chromosome some with a frequency of 10(-2) per cell per generation. A clone carrying the hybrid plasmid inserted into the chromosome was detected.     

The glucose effect in Bacillus subtilis

An analysis of the glucose downshift mechanism in Bacillus subtilis has shown that the depression of catabolic enzymes characteristic of the 'glucose effect' includes isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. Additionally, phosphofructokinase undergoes what appears to be a reversible modification regulated by glucose transport.     

Peroxidase from Bacillus sp. VUS and its role in the decolorization of textile dyes

Peroxidase was purified by an ion exchange chromatography followed by gel filtration chromatography from dye degrading Bacillus sp. strain VUS. The optimum pH and temperature of the enzyme activity was 3.0 and 65°C, respectively. This enzyme showed more activity with n-propanol than other substrates tested viz. xylidine, 3-(3,4-dihydroxy phenyl) Lalanine (L-DOPA), hydroxyquinone, ethanol, indole, and veratrole. Km value of the enzyme was 0.076 mM towards n-propanol under standard assay conditions. Peroxidase was more active in presence of the metal ions like Li²⁺, Co²⁺, K²⁺, Zn²⁺, and Cu²⁺ where as it showed less activity in the presence of Ca²⁺ and Mn²⁺. Inhibitors like ethylenediamine tetraacetic acid (EDTA), glutamine, and phenylalanine inhibited the enzyme partially, while sodium azide (NaN₃) completely. The crude as well as the purified peroxidase was able to decolourize different industrial dyes. This enzyme decolourized various textile dyes and enhanced percent decolourization in the presence of redox mediators. Aniline was the most effective redox mediator than other mediators tested. Gas chromatography-Mass spectrometry (GC-MS) confirmed the formation of 7-Acetylamino-4-hydroxy-naphthalene-2-sulphonic acid as the final product of Reactive Orange 16 indicating asymmetric cleavage of the dye.     

The effect of chlorpromazine on transformation in Bacillus subtilis

Summary In the presence of the widely used tranquilizer, chlorpromazine, transforming DNA of Bacillus subtilis is photoinactivated by long-wave ultraviolet light. The loss of biological activity is predominantly caused by lack of binding of the DNA to recipient cells and the introduction of single-strand breaks in the treated DNA.     

Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.     

Pleiotropic effect of a rifampin-resistant mutation in Bacillus subtilis.

Rifampin-resistant (Rifr) mutants were isolated spontaneously from Bacillus subtilis strain 168. A fraction of the mutants did not grow on a minimal medium. A high concentration of one of the L-amino acids (glutamic acid, glutamine, arginine, proline, aspartic acid, or asparagine) was required to restore their growth on the medium. Further analysis of one of the mutants (strain RF 161) suggested that the mutant is unable to use ammonia as a nitrogen source and requires amino acids instead. Activity of glutamate synthase was not detected in the crude extract of the mutant. The Rifr mutation was closely located to cysA and the drug resistance was cotransformed with the property of amino acid requirement at 100% frequency. All revertants to prototrophy tested showed the rifampin-sensitive (Rifs) property. The activity of the DNA-dependent RNA polymerase of the mutant was resistant to rifampin. It is concluded that some alteration of RNA polymerase may cause absence of the activity of an enzyme involved in the nitrogen metabolism.     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

The effect of silicon metabolism on genetic transformation in Bacillus subtilis

Germanium dioxide is found to increase the frequencies of the genetical transformation in Bacillus subtilis 30-40 fold. The increased frequency of transformation was registered in Sil- mutant in contrast to Sil+ strain having the decreased one. Bacillus megatherium strain KU-2 and Bacillus oligonitrophilus KU-1 were isolated from soil. These strains possess better ability to utilize the orthoclase and biotite. Germanium dioxide did not induce the transformation frequencies increase in these strains. Sil mutant of Bacillus oligonitrophilus demonstrated no competence to transformation.     

Decoloration of textile dyes by Trametes versicolor and its effect on dye toxicity

Amaranth, Tropaeolin O, Reactive Blue 15, Congo Red, and Reactive Black 5 were completely decolorized with no dye sorption by Trametes versicolor. Cibacron Brilliant Red 3G-P, Cibacron Brilliant Yellow 3B-A, and Remazol Brilliant Blue R were partially decolorized with some dye sorbed to the biomass. The Microtox assay before decoloration showed that Amaranth and Tropaeolin O were not toxic [the percent concentration to decrease 20% of the luminescence of Vibrio fischeri (EC₂₀) was greater than 100%]; Cibacron Brilliant Yellow 3B-A, Reactive Blue 15 and Cibacron Brilliant Red 3G-P were moderately non-toxic (100% > EC₂₀ > 75%); Remazol Brilliant Blue R was toxic (75% > EC₂₀ > 50%); and Congo Red and Reactive Black 5 were moderately toxic (50% > EC₂₀ > 25%). After decoloration the toxicity of the solutions containing Amaranth, Tropaeolin O and Reactive Black 5 was unchanged; Reactive Blue 15, Remazol Brilliant Blue R and Cibacron Brilliant Red 3G-P decreased to non-toxic levels; and Cibacron Brilliant Yellow 3B-A and Congo Red became very toxic (EC₂₀ < 25%).     

Microbial decolourisation and degradation of textile dyes

Dyes and dyestuffs find use in a wide range of industries but are of primary importance to textile manufacturing. Wastewater from the textile industry can contain a variety of polluting substances including dyes. Increasingly, environmental legislation is being imposed to control the release of dyes, in particular azo-based compounds, into the environment. The ability of microorganisms to decolourise and metabolise dyes has long been known, and the use of bioremediation based technologies for treating textile wastewater has attracted interest. Within this review, we investigate the mechanisms by which diverse categories of microorganisms, such as the white-rot fungi and anaerobic bacterial consortia, bring about the degradation of dyestuffs.