The Oscillopolarographic Behaviour of Deoxyribonucleic Acids Isolated from Bacillus Subtilis and Bacillus brevis

Summary The behaviour of DNA from several strains ofB. subtilis andB. brevis on the dropping mercury electrode in a medium of ammonium formate was studied. Native DNA yields in this medium on the oscillogram dE/dt againstE an anodic indentation for which the residues of deoxyguanylic acid are responsible.B. subtilis DNA produces a substantially smaller indentation thanB. brevis DNA does. It was found that the difference is not conditioned by impurities in the DNA samples, nor by the presence of denatured DNA. The difference in the depth of the indentation produced byB. subtilis andB. brevis DNAs almost disappears after denaturation of these DNAs or in an ammonium formate medium of higher concentration. The assumption was advanced that the different oscillopolarographic behaviour of DNAs obtained fromB. subtilis andB. brevis is connected with the different primary structure of these DNAs.     

Isolation and characterization of a bacteriophage forBacillus licheniformis A5

The isolation of a virulent bacteriophage forBacillus licheniformis A5 is reported. This bacteriophage, designated NLP-1, has an icosahedral head 100 nm in diameter and a contractible tail with a maximum length of 130 nm. Its DNA has a density of 1.741 g/cm³ and aT _m of 78.4°C. Base composition analysis showed that thymine is absent and is replaced by hydroxymethyluracil. NLP-1 appears to belong to theBacillus group of bacteriophages that includes SP01 and SP82. It will infectB. cereus T andB. brevis 8185, but will not infectB. subtilis W23 or 168.     

Effect of salinity on the adaptive capacity of recombinant strains ofEscherichia coli andBacillus subtilis

The effect of different concentrations of salts on natural and recombinant strains ofBacillus subtilis andEscherichia coli was studied. The recombinant strain ofB. subtilis was found to be more osmotolerant than the wild-type strain of this bacterium, whereas the opposite situation was observed for the recombinant and wild-type strains ofE. coli. Some salts exerted a bacteriostatic effect onE. coli andB. subtilis. The adaptive capacity of recombinant strains depended on the number of plasmid copies in the cells. The introduction of recombinant bacteria into model ecosystems resulted in the generation of their variants with increased osmotolerance.     

Relation between single-strand DNA mass and sedimentation distance in alkaline sucrose gradients

Single-strand breaks are introduced into bacteriophage T2 and Bacillus subtilis DNA in dilute solution with gamma rays and the DNA sedimented on alkaline sucrose gradients. Assuming (1) the number of single-strand breaks is linear with dose, and (2) the distance sedimented in alkaline sucrose gradients D is proportional to M^α (M is the single-strand DNA mass), the value of a is determined to be 0.40.     

Determination of poly-β-hydroxybutyrate (PHB) production by some <Emphasis Type="Italic">Bacillus</Emphasis> spp.

In this study, 29 strains of the genus Bacillus were isolated from different soil samples which were taken from grasslands of Ankara, Turkey and were identified as B. brevis, B. sphaericus, B. cereus, B. megaterium, B. circulans, B. subtilis, B. licheniformis and B. coagulans. Two strains, B. sphaericus ATCC 14577 and B. subtilis ATCC 6633 were also included in this study. Poly-β-hydroxybutyrate (PHB) production by these strains was determined by the spectrophotometric method, and it was found that PHB production ranged from 1.06–41.67% (w/v) depending on the dry cell weight. The highest PHB production and productivity percentage was found in B. brevis M6 (41.67% w/v).     

Semi-in vitro Repair of DNA in Germinating Spores of Bacillus subtilis Irradiated with γ-Ray

DNA repair synthesis was studied in germinating spores of Bacillus subtilis made permeable to deoxyribonucleoside triphosphates by treatment with Brij 58. The synthesis is dependent on the presence of all four deoxyribonucleoside triphosphates, but does not require adenosine triphosphate. Repair synthesis in the γ-ray irradiated and Brij 58 treated germinating spores was observed in wild type strain 168Tt, but not in DNA polymerase I-deficient mutant strain D22. Furthermore, the single-strand breaks of DNA in the germinating spores of strain 168Tt induced by γ -ray irradiation were rejoined during postirradiation incubation in the presence of four deoxyribonucleoside triphosphates, nicotinamide adenine dinucleotide and magnesium ion. In the case of a mutant D22, the γ-ray induced DNA single-strand breaks were not rejoined.     

Dynamic light-scattering studies of internal motions in DNA. III. Evidence for titratable joints associated with bound polycations

Dynamic light-scattering techniques are employed to study the internal Brownian motions of a commercial calf thymus DNA, clean and contaminated ?29 DNAs, and a clean ?29 DNA with bound spermidine as a function of pH. The Rouse-Zimm model parameters of both calf thymus and contaminated ?29 DNAs differ substantially from those of clean ?29 DNA in the neutral-pH region. However, this difference is largely removed by adding 0.01M EDTA (which has no effect on clean ?29 DNA) to the calf thymus DNA sample. These findings imply the existence in that preparation of polycation contaminants, presumably basic proteins, that can substantially alter the local mechanical properties of the DNA near their binding sites. The internal motion parameters k_BT/f and b of both calf thymus and contaminated ?29 DNAs are found to exhibit pronounced characteristic variations between pH 8.5 and 10.5, over which range there is essentially no detectable titration to a resolution of about 1% of the base pairs. These variations, which are not observed for clean ?29 DNA, are qualitatively similar to those previously reported for a ?29 DNA with 21 single-strand breaks per chain. This indicates the formation of titratable joints associated with bound polycation contaminants. These basic ligands presumably facilitate local denaturation by stabilizing the titration of one or more protons on base-ring nitrogens near their binding sites. Spermidine binding up to 85–87% of neutralization of the total DNA charge has only a relatively minor effect on the internal motion parameters at neutral pH in 0.01M NaCl. However on raising the pH to 10.2, the internal motion parameter k_BT/f undergoes a marked decrease similar to that observed for both calf thymus and contaminated ?29 DNAs and also ?29 DNA with single-strand breaks. This indicates that spermidine, too, is capable of inducing titratable joints. Evidence is presented that the titratable joints associated with bound polycations on the calf thymus DNA may serve primarily as torsion joints, as was found previously for the titratable joints associated with single-strand breaks.     

The influence of single-strand breaks on the kinetics of denaturation of DNA

The influence of single-strand breaks on the kinetics of the relaxation of DNA in a solution of low ionic strength has been investigated by a temperature jump method. The relaxation of DNA after a jump of 0.7 °C in the melting region has been monitored by measuring the extinction at 260 nm. For essentially monodisperse T4 DNA (M = 130 × 10⁶) two distinct relaxation times have been observed, that depend markedly on the initial extent of denaturation 1 ? θ. The larger relaxation time decreases from 450 sec to about 300 sec, the smaller one from 55 see to 30 when 1 ? θ increases from 0.03 to about 0.8. The dependence of these relaxation times on the average number of single-strand breaks per molecule (p) appears to be very small up to p = 100. However, the relative contribution of the slow process decreases sharply when p increases from 0.6 to 30 and remains nearly constant for larger p. The observations are discussed in the light recent theories of the kinetics of denaturation.     

Toward a bacterial genome technology: integration of theEscherichia coli prophage lambda genome into theBacillus subtilis 168 chromosome

A novel approach to the cloning large DNAs in theBacillus subtilis chromosome was examined. AnEscherichia coli prophage lambda DNA (48.5 kb) was assembled in the chromosome ofB. subtilis. The lambda DNA was first subcloned in four segments, having partially overlapping regions. Assembly of the complete prophage was achieved by successive transformation using three discrete DNA integration modes: overlap-elongation, Campbell-type integration, and gap-filling. In theB. subtilis chromosome, DNA was elongated, using contiguous DNA segments, via overlap-elongation. Jumping from one end of a contiguous DNA stretch to another segment was achieved by Campbell-type integration. The remaining gap was sealed by gap-filling. The incorporated lambda DNA thus assembled was stably replicated as part of the 4188 kbB. subtilis chromosome under non-selective conditions. The present method can be used to accommodate larger DNAs in theB. subtilis chromosome and possible applications of this technique are discussed.     

Effect of violamycin BI on the process of excision repair in Escherichia coli K 12 following UV irradiation]

The influence of violamycin B I on the process of excision repair of DNA-damages after UV-irradiation has been studied by observing the capacity to rejoin single-strand breaks introduced in the DNA at the beginning of the repair process. The number of single-strand breaks remaining unrepaired in the DNA was higher in presence of violamycin B I. Sedimentation analysis of the DNA of unirradiated cells showed in presence of violamycin B I only a small change in the molecular weight. As a possible reason for the lower capacity of the cells to accomplish repair steps following incision in presence of violamycin B I an inhibition of the function of repair enzymes by interaction of the antibiotic with the DNA-template is discussed.     

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.     

Glycogen in Bacillus subtilis: molecular characterization of an operon encoding enzymes involved in glycogen biosynthesis and degradation

Although it has never been reported that Bacillus subtilis is capable of accumulating glycogen, we have isolated a region from the chromosome of B. subtilis containing a glycogen operon. The operon is located directly downstream from trnB, which maps at 275 on the B. subtilis chromosome, it encodes five poly-peptides with extensive similarity to enzymes involved in glycogen and starch metabolism in both prokaryotes and eukaryotes. The operon is presumably expressed by an Eσ^E-controlled promoter, which was previously identified downstream from trnB. We have observed glycogen biosynthesis in B. subtilis exclusively on media containing carbon sources that allow efficient sporulation. Sporulation-independent synthesis of glycogen occurred after integration of an Eσ^A controlled promoter upstream of the operon.     

The inhibition by the tumor necrosis factor of the death and DNA fragmentation of lymphoid cells in irradiated rats]

The influence of a tumor necrosis factor, administered 16 h before irradiation of rats, on the radiation response of thymus and bone marrow cells has been investigated. Three and 6 h after irradiation the following indices were analyzed: the number of apoptotic cells in the thymus; the accumulation of polydeoxyribonucleotides and the appearance of single-strand breaks in DNA of bone marrow and thymus cells; and the electrophoretic properties of thymocyte DNA. The injection of a tumor necrosis factor reduced the number of polydeoxyribonucleotides, inhibited internucleosome DNA fragmentation, and did not influence the formation of single-strand breaks in DNA.     

Sensitized NADH formation of single-stranded breaks in plasmid DNA upon the action of near UV-radiation

It has been shown that NADH photosensitize in vitro single-strand breaks formation in double-strand plasmid DNA pBR 322 upon near-UV (320-400 nm) irradiation. The number of single-strand breaks depends both on UV light dose and sensitizer concentration. Addition of catalase and sodium benzoate strongly decreases the single-strand breaks formation. The results show an important role of hydrogen peroxide (H2O2) and hydroxyl radical (.OH) in inducing single-strand breaks in plasmid DNA irradiated by near-UV radiation in the presence of NADH.     

Characterization of new <Emphasis Type="SmallCaps">l,d</Emphasis>-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis has various cell wall hydrolases, however, the functions and hydrolase activities of some enzymes are still unknown. B. subtilis CwlK (YcdD) exhibits high sequence similarity with the peptidoglycan hydrolytic l,d-endopeptidase (PLY500) of Listeria monocytogenes phage and CwlK has the VanY motif which is a d-alanyl-d-alanine carboxypeptidase (Pfam: http://www.sanger.ac.uk/Software/Pfam/). The β-galactosidase activity observed on cwlK-lacZ fusion indicated that the cwlK gene was expressed during the vegetative growth phase, and Western blotting suggested that CwlK seems to be localized in the membrane. Truncated CwlK fused with a histidine-tag (h-ΔCwlK) was produced in Escherichia coli and purified on a nickel column. The h-ΔCwlK protein hydrolyzed the peptidoglycan of B. subtilis, and the optimal pH, temperature and NaCl concentration for h-ΔCwlK were pH 6.5, 37°C, and 0 M, respectively. Interestingly, h-ΔCwlK could hydrolyze the linkage of l-alanine-d-glutamic acid in the stem of the peptidoglycan, however, this enzyme could not hydrolyze the linkage of d-alanine-d-alanine, suggesting that CwlK is an l,d-endopeptidase not a d,d-carboxypeptidase. CwlK could not hydrolyze polyglutamate from B. natto or peptidoglycan of Staphylococcus aureus. This is the first report describing the characterization of an l,d-endopeptidase in B. subtilis and also the first report in bacteria of the characterization of a PLY500 family protein encoded in chromosomal DNA. Tatsuya Fukushima and Yang Yao contributed equally to this work.     

Sequence characterization of 5S ribosomal RNA from eight gram positive procaryotes

Summary The available comparative data on procaryotic 5S rRNA was extended through sequencing studies of eight gram positive procaryotes. Complete nucleotide sequences were presented for 5S rRNA fromBacillus subtilis, B. firmus, B.pasteurii, B.brevis, Lactobacillus brevis andStreptococcus faecalis. In addition, 5S rRNA oligonucleotide catalogs and partial sequence data were provided forB.cereus andSporosarcina ureae. These sequences and catalogs were discussed in terms of known features of procaryotic 5S rRNA architecture.     

d-Glucose does not catabolite repress a transketolase-deficientd-ribose-producingBacillus subtilis mutant strain

WhenBacillus subtilis strain ATCC 21951, a transketolase-deficientd-ribose-producing mutant, was grown ond-glucose plus a second substrate which is metabolized via the oxidative pentose phosphate cycle (d-gluconic acid,d-xylose,l-arabinose ord-xylitol),d-glucose did not catabolite repress metabolism of the second carbon source. Thed-ribose yield obtained with the simultaneously converted carbon substrates, significantly exceeded that when onlyd-glucose was used. In addition, the concentration of glycolytic by-products and the fermentation time significantly decreased. Based on these findings, a fermentation process was developed withB. subtilis strain ATCC 21951 in whichd-glucose (100 g L^–1) andd-gluconic acid (50 g L^–1) were converted into 45 g L^–1 ofd-ribose and 7.5 g L^–1 of acetoin. A second process, based ond-glucose andd-xylose (100 g L^–1 each), yielded 60 g L^–1 ofd-ribose and 4 g L^–1 of acetoin plus 2,3-butanediol. Both mixed carbon source fermentations provide excellent alternatives to the less efficientd-glucose-based processes used so far.     

Restriction and modification in B. subtilis. Purification and general properties of a restriction endonuclease from strain R.

Summary All Bacillus subtilis R-type strains showing the phenomena of restriction and modification contain an endonuclease that inactivates in vitro the biological activity of a variety of DNAs lacking R-specific modification, such as transfecting SPP1, SPO2 and 105 DNA, and transforming B. subtilis 168-type DNA. The corresponding DNAs carrying R-specific modification are resistant to the enzyme. The enzyme has been purified approximately 400-fold and is essentially free from contaminating double strand-directed unspecific exo-or endonuclease activity. Only Mg²⁺ is required as cofactor. The substrate DNAs are cleaved at specific sites. The double-stranded fragments produced from SPP1 DNA (molecular weight 2.5×10⁷) have an average molecular weight of about 3×10⁵.     

Single- and double-strand breaks in 5-bromouracil-substituted DNA of B. subtilis and phage PBSH after irradiation with long-wave length UV and their correlation to intramolecular energy transfer

5-Bromouracil-substituted DNA was isolated form B. Subtilis and phage PBSH. The three DNA fractions of Different densities (TT, TB, and BB) were irradiated with u.v. (313nm). The number of single-strand and double-strand breaks was determined. The breakage rates are given. It was found that in hybrid DNA (TB) double-strand breaks occur depending linearly on dose. In BB–DNA the observed double-Strand bresks can be divided into two fractions with a linear and quadratic dose dependence respectively. The results can be explained by assuming intramolecular energy transfer from the BU-containing strand to its complementary strand.     

Antimicrobial activity of 3-methyl-5-furfurylidene-2-thioxo-4-thiazolidones

Substituted 3-methyl-5-furfurylidene-2-thioxo-4-thiazolidones1 –17 were tested for their action againstBacillus pumilus, B. brevis andB. megaterium and againstAspergillus flavus, A. fumigatus andA. niger. Most of the compounds possessed an activity against both bacteria and fungi. These compounds were also tested for their effectiveness as virus inhibitors employingNicotiana glutinosa as assay host against tobacco mosaic virus. Most of them exhibited potential antiviral activity bothin vitro andin vivo.