Transformation ofBacillus subtilis with the treatment by alkali cations

Summary ExposingBacillus subtilis cultures to high concentrations of alkali cations, especially K⁺, allows efficient transformation by plasmids. The method allows transformation with unfractionated plasmid DNA, monomeric plasmid DNA as well as linear plasmid DNA.B. subtilis strains, not amenable to natural transformation, were also transformed by the present method.     

Stability ofBacillus subtilis DNA in the environment

The stability of genetic material was measured by determining the transforming activity remaining in samples ofBacillus subtilis DNA subjected to different simulated environmental conditions. Destruction of transforming activity is rapid when the DNA is stored outdoors. The DNA is somewhat more stable in the laboratory when sterile and/or without possible enzyme sources. The DNA is still labile at 4°C if dilute.     

Characterization of temperate phages ofBacillus subtilis

Nine known temperature phages ofBacillus subtilis, including four that are newly isolated (ϱ6, ϱ10, ϱ14, and ϱ18), have been compared. Analysis by serology, immunity, host range, and adsorption site similarity place the phages into four groups: Group I, ϕ105, ϱ6, ϱ10, and ϱ14, which are 80–90% related; Group II, SPO2; Group III, ϕ3T and ϱ11, 100% related; and Group IV, SP16. The phage ϱ18 is largely uncharacterized, but is heteroimmune to other groups.     

Reversion of anomalous forms ofBacillus subtilis

Anomalous forms ofBacillus subtilis A 32 produced by prolonged cultivation in a chemostat under nitrogen limitation are described. A change in the cultivation conditions brings about a transformation of these forms to bacillar rods. The transformation is gradual and lasts for several generations.     

Restricted turnover of the cell wall ofBacillus subtilis

Cultures ofBacillus subtilis in balanced growth exhibited a constant rate of turnover of peptidoglycan for 2.5–3.5 generations. Turnover was measured by determining the retention of a labeled precursor of peptidoglycan. When fluorescein-conjugated concanavalin A was used to monitor the fate of cell surface teichoic acid, label disappeared from the cylinders more rapidly than from caps and septa. The results suggest that cell wall poles are partially resistant to turnover.     

Autolysis ofBacillus subtilis by glucose depletion

In cultures in minimal medium, rapid lysis of cells ofBacillus subtilis was observed as soon as the carbon source, e.g. glucose, had been completely consumed. The cells died and ultraviolet-absorbing material was excreted in the medium. The results suggest that the cells lyse because of the presence of autolytic enzymes. In the presence of glucose the damage to the cell wall caused by these enzymes is repaired immediately.     

Bacteriophages ofBacillus subtilis: Comparison of different isolation techniques and possible use for classification ofBacillus subtilis strains

When different techniques are used for the isolation of bacteriophages ofBacillus subtilis a number of different phages may be obtained. Furthermore defective phages are found in old cultures of all strains ofB. subtilis tested so far. The possible use of the phages and the defective phages for classifyingB. subtilis strains into a number of groups according to their susceptibility to different phages and according to the presence of certain defective phages in the cells is discussed.     

Use of yeast two-hybrid system for detection ofBacillus subtilis FtsZ protein partners

Yeast two-hybrid system was modified to allow easy detection of prokaryotic protein-protein interactions. Three plasmids (pGBR1, pGBR2, pGBR3) with theClaI restriction site shifted in the three possible reading frames in fusion withGAL4 activating domain were constructed. The modified plasmids were used for identification of protein partners of FtsZ fromBacillus subtilis. Among partners of FtsZ the FtsA protein and a globular part of the SpoIIE protein were identified. The protein interactions were quantified by measurements of β-galactosidase activity in yeast cells using 4-methylumbelliferyl β-d-glactopyranoside as fluorogenic substrate.     

Stringent control of ribonucleic acid synthesis in Bacillus subtilis treated with granaticin.

The antibiotic granaticin interferes in Bacillus subtilis with the charging process of tRNALeu causing both the arrest of protein synthesis and bacteriostasis [A. Ogilvie, K. Wiebauer & W. Kersten (1975) Biochem. J. 152, 511-515]. A concomitant inhibition of RNA synthesis is observed. This inhibition was studied with mutant strains of B. subtilis. 2. Granaticin inhibits protein and RNA synthesis in stringently controlled B. subtilis (rel+) to about the same extent. In a relaxed mutant strain (rel-) of B. subtilis, protein synthesis is also inhibited, but the accumulation of RNA continues after the addition of the drug. 3. Chloramphenicol, which is known to abolish the stringent control mechanism, added simultaneously with granaticin, allows the synthesis of RNA to proceed in the stringent strain. 4. Guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) accumulate in granaticin-treated stringently controlled B. subtilis but not in the rel- mutant. 5. It is concluded that the inhibition of RNA synthesis granaticin can adequately be explained as a stringent response caused by the interference by the drug with leucyl-tRNA synthetase.     

Sensitivity ofBacillus subtilis sporulation to methylene blue inhibition

The dye methylene blue was found to inhibit sporulation inBacillus subtilis 168. The compound blocked spore formation at concentrations subinhibitory to vegetative growth while allowing synthesis of serine protease, antibiotic, and certain catabolite-repressed enzymes. The sporulation process was sensitive to the inhibitor through T₆, but germination and outgrowth were not affected by the presence of the compound. The inhibition of sporulation may be related to the ability of the compound to inhibit oxidative phosphorylation.     

Colony switching in an alpha-amylase-producing strain ofBacillus subtilis

Four colony variants (two stable and two unstable phenotypes) were observed inBacillus subtilis -10, an -amylase-hyperproducing strain. The stable variants lost the ability to produce -amylase, while the unstable ones reverted to the typical morphology after restreaking. Unstable expanding sectors appeared in typical colonies, and their appearance was influenced by the culture origin and age.     

Formation of some extracellular enzymes during the exponential growth ofBacillus subtilis

The formation of the exoenzymes, neutral and alkaline proteinase as well as alpha-amylase of Bacillus subtilis, is characterized by the same time course. The exoenzyme formation starts in the exponential phase of growth by an excess of C and N sources. We assume that carbon metabolism of pyruvate is responsible for the exoenzyme formation during this growth phase. The proteinase formation at the transient and/or stationary phase of growth is related to amino acid limitation.     

Effect of boseimycin on some enzyme systems ofBacillus subtilis

The effect of boseimycin on the in vitro activity and in vivo synthesis of alkaline phosphatase, aconitase and lactate, isocitrate, glutamate and alanine dehydrogenases was studied in Bacillus subtilis. At a subinhibitory concentration, synthesis of glutamate dehydrogenase was stimulated but alkaline phosphatase, lactate dehydrogenase and aconitase synthesis was inhibited. On the contrary, boseimycin inhibited slightly the activity of lactate dehydrogenase in cell-free extracts. Glutamate dehydrogenase and aconitase activities were not affected.     

Chemical stabilization ofBacillus subtilis α-amylase by modification with D-glucono-δ-lactone

Summary Thermostability ofBacillus subtilis α-amylase at 70°C and higher temperatures is increased by its modification with D-glucono-δ-lactone, regardless whether the modification reaction proceeds in the presence or absence of the substrate. The pH-profile of the spontaneous hydrolysis of the modifier was determined with the aim of finding the optimal modification conditions. The Arrhenius plot of the inactivation rate constants exhibits the isokinetic effect, with the temperature of compensation being approximately 63°C.     

Characteization of bacterial spores from high-temperature growth transformants ofBacillus subtilis

Spores from high-temperature growth transformants ofBacillus subtilis were examined for a number of sporal characteristics. Analyses showed a dramatic increase in both the calcium (Ca) and dipicolinic acid (DPA) contents, a slight increase in the Ca/DPA mole ratio, but a reduction in magnesium (Mg) content and the Mg/Ca mole ratio. The spore wet density increased, whereas the core/core+cortex volume ratio and protoplast water content decreased. Spore heat resistance increased but not to levels normally observed for thermophilicBacillus species. It is concluded that the biophysical and biochemical changes within the spores of the transformants are influenced by both the inherent mesophilic genotype and the transformant's inherited ability to grow at elevated temperatures.Florida Agricultural Experiment Station, Journal Series No. 8189