Bacillus Subtilis Marburg Research Articles

D-amino acids in the cell pool of bacteria.

About 30 different bacterial species were tested for the possible presence of freed-amino acids in their cell pool. Gram-positive bacteria particularly the species of the genusBacillus have a fairly large pool of freely extractabled-amino acids. Varied quantities of freed-amino acids were detected inBacillus subtilis B3,Bacillus subtilis Marburg,Bacillus licheniformis, Bacillus brevis, Bacillus stearothermophilus, Lactobacillus fermenti, Lactobacillus delbrueckii, Staphylococcus aureus andClostridium acetobutylicum. The individual components ofd-amino acids were identified in 5Bacillus species referred to above,d-alanine is the major component; the otherd-amino acids identified are aspartic acid, glutamic acid, histidine, leucines, proline, serine and tyrosine. Thed-amino acid pool size inBacillus subtilis B3 varies with different culture conditions. The pool size is maximum when growth temperature is 30°C and it fluctuates with change in pH of the medium. The maximum quantity ofd-amino acids could be recovered when the culture was at mid log phase. O2 supply to the medium has little effect ond-amino acid pool size. The starvation of cells leads to depletion of thed-amino acid pool which is exhausted almost completely within 4 hours by incubation in nutrient-free medium.

Purification et propriétés de la lévane-sucrase exocellulaire de Bacillus subtilis marburg

Two inducible enzymes, namely a levansucrase and a sucrase, are responsible for the saccharolytic activity in Bacillus subtilis Marburg. In the strain QB 13 producing a high level of saccharolytic activity, up to 90% of this activity is excreted. The exocellular activity can be accounted for by levansucrase. This enzyme has been obtained in pure form from the supernatant of an induced culture by fractional precipitations with ethanol and ammonium sulfate, and chromatography on hydroxyapatite; mol. wt. is ca. 40,000; ▪ = 4.16 S. The enzyme consists of a single polypeptide chain with lysin as the N-terminal amino acid. This enzyme catalyzes transfructosylation reactions; sucrose and low-molecular-weight levans may act as D-fructosyl donors; water, D-glucose, sucrose, and levans act as acceptors. The apparent Michaelis constants of levansucrase for sucrose and low-molecular-weight levans (mol. wt. 10,000) are respectively 27mM and 3mM (30 g/l); the yield in levan of the reaction catalyzed by levansucrase reaches 90% in the presence of low-molecular-weight levans acting as initiators. The exocellular levansucrase of Bacillus subtilis Marburg QB 13 appears to be identical with the previously studied enzyme of Bacillus subtilis var. Nigra BS 5.

Characterization of two sucrase activites in Bacillus subtilis marburg

Two proteins responsible for sucrase activity in Bacillus subtilis Marburg have been separated by chromatography on hydroxyapatite. These enzymes have been characterized as transfructosidases, namely a levansucrase and a sucrase. Their molecular weights are very similar (40,000). These enzymes differ by their catalytic properties, their sensitivity to trypsin and SH reagents and by their immunological properties. They are both induced by sucrose ; only sucrase synthesis is decreased in the presence of glucose. Sucrase is endocellular while levansucrase is excreted in the culture medium. L'activité saccharasique produite par Bacillus subtilis Marburg est portée par deux protéines qui ont été séparées par chromatographie sur hydroxyapatite. Ces enzymes ont été caractérisés comme une lévane-sucrase et une saccharase ; ce sont des transfructosidases. Leurs poids moléculaires sont voisins (40.000). Ces enzymes diffèrent par leurs propriétés catalytiques, leurs sensibilité à la trypsine et aux réactifs des groupes SH ainsi que par leur caractéristiques immunologiques. Elles sont toutes deux induites par le saccharose ; seule la synthèse de la saccharase apparaît être sensible à un effet glucose. La saccharase est endocellulaire alors que la lévane-sucrase est excrétée dans le milieu de culture. Die Saccharase-Aktivität, welche von Bacillus subtilis Marburg erzeugt wird, wird von zwei Proteinen, welche durch Chromatographie an Hydroxylapatit getrennt wurden, getragen. Diese Enzyme sind als eine Levan-Sucrase und eine Saccharase gekennzeichnet worden ; es sind Transfruktosidasen. Ihre Molekulargewichte sind ähnlich (40.000). Die Enzyme unterscheiden sich durch ihre katalytischen Eigenschaften, ihre Empfindlichkeit dem Trypsin und den Reagentien der SH-Gruppen gegenüber, sowie durch ihre immunologischen Eigenschaften. Sie werden beide durch die Saccharose induziert ; nur die Synthese der Saccharase scheint einem Glukose-Effekt empfindlich zu sein. Die Saccharase ist endozellulär, während die Levan-Sucrase in das Kulturmedium abgesondert wird.

Autolysis of isolated cell walls of Bacillus licheniformis N.C.T.C. 6346 and Bacillus subtilis Marburg Strain 168. Separation of the products and characterization of the mucopeptide fragments.

1. Cell walls were isolated from Bacillus licheniformis N.C.T.C. 6346 and Bacillus subtilis Marburg strain 168 trp grown on casein hydrolysate into exponential phase. Autolysis was carried out and the soluble products, separated by chromatography on DEAE-cellulose, from the two wall preparations are broadly similar in composition and are in agreement with autolysis proceeding with hydrolysis of amide bonds between l-alanine and N-acetylmuramic acid residues in the mucopeptide components. 2. Peptides originating from the mucopeptide components were isolated and shown to be a monomer peptide, l-alanyl-d-glutamyl-meso-diaminopimelic acid and a dimer peptide containing two monomer peptides linked through a residue of d-alanine. Approximately one amide group is present for each equivalent tripeptide unit and is probably substituted on diaminopimelic acid residues. 3. Oligosaccharides originating from the mucopeptide components were isolated and after hydrolysis contained almost equimolar amounts of glucosamine and muramic acid and only very small amounts of amino acids. The number-average chain length, estimated by the release of non-reducing end groups of N-acetylglucosamine with exo-beta-N-acetylglucosaminidase, is approximately ten hexosamine residues for oligosaccharides isolated from either organism. The oligosaccharides are polydisperse. 4. N-Acetylglucosamine residues are the only reducing terminals detectable in the oligosaccharides isolated from B. subtilis or B. licheniformis cell-wall autolysates. The number-average chain lengths of the oligosaccharides were determined by estimation of the content of these residues and are higher than those found by enzymic assay. Possible reasons for the discrepancy are discussed.

Characterization of proteinases excreted by Bacillus subtilis Marburg strain during sporulation.

Summary. This study has characterized 3 proteolytic enzymes during sporulation by Bacillus subtilis Marburg strain when grown in nutrient broth. A method of purification is described which permits the separation of 2 different proteinases: one belonging to the metal enzyme group and the other to the serine enzyme group. The third enzyme, probably an esterase, showed a high esterolytic activity, but only low proteolytic activity. Determination of the 3 enzymes in a mixture was accomplished by using specific substrates and inhibitors. They were excreted simultaneously between the end of the growth phase until the appearance of the prespores. During this entire period, 20% of the total proteolytic activity was due to the metal proteinase; 80% of the proteolytic activity and 15% of the esterolytic activity was due to the serine proteinase; 85% of the esterolytic activity was the result of the esterase. These findings will contribute to a more complete phenotypic characterization of those mutants of sporulation that appear to be involved in the production of extracellular hydrolytic enzymes.

PARTICIPATION OF A REGULATOR GENE IN THE α-AMYLASE PRODUCTION OF BACILLUS SUBTILIS

Bacillus natto IAM 1212 produces α-amylase by about 5 times as much as Bacillus subtilis Marburg strain, α-amylase of which is more thermostable and moves more rapidly to anode at pH 8.3 than that of B. natto. When this high producing activity of α-amylase (Amysup character) was transferred to a Marburg strain by the DNA from B. natto, about one-tenth of the transformants with Amysup character produced recipient type, i.e. Marburg type α-amylase and the other natto type α-amylase. In other words, the former acquired only high producing activity of α-amylase. This result suggests that a gene which regulates α-amylase synthesis participates in the α-amylase-producing system of B. subtilis and this regulator gene (amyR) is closely linked to the α-amylase structure gene. Reciprocal crossing also confirmed this assumption. The amyR gene is linked to the aro116 gene which has been reported to be the linked marker of α-amylase structure gene by S. Yuki.

A COMPARATIVE STUDY ON DNA MEDIATED PHAGE INFECTION AND BACTERIAL TRANSFORMATION

The infection of Bacillus subtilis Marburg strains with DNA of bacteriophage M2 was studied in comparison with the bacterial DNA transformation. The results of dose response experiment and dilution experiment suggested that the phage-DNA infection could be accomplished only by the simultaneous incorporation of at least four DNA fragments which determine the size of phage-DNA. These results were taken as evidence that there might be no intrinsic difference between the two phenomena.

INCORPORATION OF RADIOACTIVE AMINO ACIDS AND BASES INTO NUCLEIC ACID AND PROTEIN FRACTIONS OF GERMINATING SPORES OF BACILLUS SUBTILIS

Using germinating spores of Bacillus subtilis Marburg, synthesis of macromolecules was studied by assay of radioactive amino acids and bases incorporated into protein and nucleic acid fractions. In spores heated at 70° for 10min, incorporation into protein and RNA fractions occurred simultaneously, but incorporation into DNA fraction about 40-50min later. But, when the spores were heated at 90° for 5min, delay was observed. In the spores, incorporation into RNA fraction occurred earlier than incorporation into protein. Early incorporation into protein was inhibited by actinomycin S, and later incorporation into RNA fraction was inhibited by chloramphenicol, puromycin and p-fluorophenylalanine. These agents also inhibited the incorporation into DNA fraction. On these grounds, the possibility was discussed that synthesis during germination occurred in a sequence of RNA, protein, and DNA, and the RNA synthesized in the early period of germination was essential for the following synthesis of protein, which might, in turn, play an important role in the subsequent synthesis of RNA and DNA during outgrowth.

食品防腐剤の細菌胞子の発育におよぼす影響

Effect of some food preservatives and antibiotics upon the germination of spores of Bacillus subtilis Marburg strain has been studied. Results obtained may be summarized as follows: 1. No appreciable difference in the minimal inhibitory concentrations (MIC) of the drugs was observed between those valuse obtained with vegetative cells and those with spores of the test organism (Table 2). Nitrofurazone (NFS), nitrofurylacryl amide (NFA), and chlortetracycline (CTC) were found to inhibit the growth of test organism, while the remaining drugs so far tested did not exhibit the growth inhibition within respective permissible concentration. 2. Concerning effects of drugs on the germination of spores determined turbidimetrically, none of the test drug showed apparent inhibitory action on the germination of spores, on the other hand, inhibition appeared in the growth after germination, so-called outgrowth stage or post-germinative development. Seemingly the rate of inhibition appeared in the outgrowth stage run parallel with the increment of concentration of a drug (Fig. 1 and 2). 3. No marked difference in the inhibitory action of NFS in the outgrowth stage of spores was observed between pH 6.0 and 8.0 (Fig. 3), while sodium dehydroacetate and sodium sorbate were more effective in acidic medium than in neutral or alkaline (Figs. 4 and 5).

Transformation of genetic traits by the DNA prepared from a lysogenic phage S-1

An experimental system involving both transformation and transduction was recently established in Bacillus subtilis Marburg (1,2,3), but there appears to be no published account of the transformation by the DNA prepared from the transducing phage (SP-10). The bacteriophage S-1 described in this paper is a temperate phage different in some respects from the SP-10. Details of the experimental methods and the specificities of the phage are to be reported elsewhere.

Studies on Production of Nucleic Acid and its Related Compounds by Micro-organisms

Mutants of Bacillus subtilis Marburg capable of synthesizing adenosine from adenine in high yield were described. The mutants of B. subtilis Marburg 160 (tryptophan-dependent, streptomycin-resistant) were isolated via either ultraviolet light irradiation or 2, 6-diaminopurine treatment. In experiments with 2, 6-diaminopurine, mutants were obtained in very high frequency. It was found that mutants with purine dependency besides tryptophan requirement and streptomycin resistance, accumulated large amounts of adenosine when grown in a glucose mineral medium containing large amounts of adenine. The parent strain and mutants with amino acids, thiamine or adenine dependency, on the other hand, accumulated only small amounts of adenosine and large amounts of hypoxanthine from adenine.