Bacillus Cultures Research Articles

Influence of extracellular products on the growth of mixed microbial populations in Mg2+-limited chemostat cultures.

SUMMARY: Magnesium-limited mixed cultures, each containing two microbial species, were produced by exchange of small numbers of organisms between magnesium-limited chemostat cultures of Aerobacter aerogenes, Pseudomonas fluorescens, Bacillus subtilis, B. megaterium, Staphylococcus epidermis and Torula utilis. The fate of each species in these mixed cultures was determined following cross-inoculation. Gram-negative organisms invariably outgrew the Gram-positive ones, but the ability of B. subtilis and B. megaterium to outgrow each other, or to outgrow the yeast, depended on the inoculum size. This dependence resulted from the presence of specific extracellular products in the Bacillus cultures which stimulated their growth and uptake of magnesium. The concentration of these extracellular growth-promoting substance(s) in magnesium-limited cultures of B. subtilis varied with population density. Thus, when the magnesium concentration in the medium was decreased from 0.9 to 0.15 μg./ml. (thereby causing the steady-state population density to be decreased to one-eightieth the initial value), B. subtilis could no longer maintain itself against proportionately low concentrations of T. utilis. But addition of extracellular fluids from dense magnesium-limited B. subtilis cultures to magnesium-limited T. utilis cultures enabled small numbers of the Bacillus now to outgrow the yeast. From these two observations it is concluded that magnesium assimilation by B. subtilis is more dependent on extracellular substance(s) than is magnesium assimilation by either T. utilis or the Gram-negative organisms. Modification of the basic theory of microbial growth in a chemostat culture, to take account of product-stimulated substrate-assimilation, is suggested.

Nutritional factors influencing the development of competence in the Bacillus subtilis transformation system.

Cultures of Bacillus subtilis developed competence for the uptake of deoxyribonucleic acid in a chemically defined medium with a predictable, reproducible pattern. The gross effects of individual amino acids were determined. Seven amino acids, most of which are reported to be major components of the cell wall, were shown to impair the development of maximal levels of competence. When the synthetic growth medium was supplemented with a mixture of the nine amino acids which we found to stimulate the development of competence, the level of transfection was increased to 10 to 15% of the population. The actual level of competence in these populations was assayed by transformation of unlinked bacterial markers and by two different transfection assays. The results indicate that calculations from cotransfer of unlinked markers overestimates the degree of competence in highly competent populations of B. subtilis, whereas the number of plaques obtained in transfection is an under-estimate of the actual level of competence. The results are interpreted to indicate that neither method of analysis gives a true estimate of the competent population, but that more than 80% of the cells may be competent.

INTERACTION BETWEEN PARAQUAT AND MICROBES IN SOILS*

Summary. Laboratory experiments were conducted to determine if paraquat had any effect on soil micro‐organisms and consequently on soil fertility.Numbers of bacteria and moulds tended to increase with incubation time as residual paraquat decreased. Lag periods of 1–5 days were observed in the development of soil microflora and concomitant degradation of paraquat. Under controlled culture conditions, Aerobacter aerogenes, Agrobacterium tumefaciens, Pseudomonas fluorescens and Bacillus cereus were able to utilize paraquat as sole carbon and nitrogen sources in synthetic media.Paraquat at 0–25, 0–5, 2–5 and 25 ppm cation was added to four different soils to study changes in microbial activities concomitant with decreases in herbicide concentration. Analytical recoveries were correlated inversely with clay mineral content, cation‐exchange capacity and organic matter.Mineralization of peptone nitrogen by amnnonification and nitrification was stimulated by 0–25 ppm; higher rates up to 25 ppm had little influence. A bimodal effect or growth inversion was observed in several instances, with increased microbial development at intermediate concentrations. Paraquat at recommended field rates appeared to have no appreciable influence on general microbial activities of importance to soil fertility.Interaction entre U paraquat et les microbes dans les sols

Spontaneous transformation in Bacillus subtilis.

Cultures ofBacillus subtilisrelease transforming DNA during the early exponential and stationary phases of growth. The pattern of release of transforming DNA was followed by measuring transformation in a system consisting of a non-transformable DNA donor and a differently marked transformable recipient. Transformation in this system seems to be at least as efficient as that induced by purified DNA. Fluorescence microscopy revealed that released DNA remained bound extracellular to intact cells. The release of DNA during early exponential growth seemed to be correlated with the cells' proneness to lysis; both DNA release and cell lysis were inhibited by chloramphenicol. In stationary cells, the release of DNA was neither correlated with a similar proneness to lysis nor inhibited by chloramphenicol.

EVALUATION OF TESTS AND GROUPING OF CULTURES BY A TWO-STAGE PRINCIPAL COMPONENT METHOD

A two-stage multivariate procedure for analysis and condensation of large sets of +/− data has been tested on data derived from application of 75 tests to 59 named cultures of Aerobacter, Aeromonas, Bacillus, Escherichia, and Pseudomonas. Seventeen clusters of associated attributes (attribute-complexes) were found by Adansonian R-analysis. As a result of successive separate principal component analyses, 47 principal component vectors were found which cumulatively accounted for 88% of aggregate variance and collectively defined an attribute-complex subspace. Four leading principal component vectors were extracted from attribute-complex space and accounted for 43% of the original variance; cultures were allotted 4-part ordination scores in respect of these vectors and these scores were treated as coordinates of item-points for purposes of cluster analysis. Cultures were separated into appropriate major categories but minor groupings were not entirely satisfactory; deficiences in the data and geometrical distortion during condensation were implicated. Major discriminants were defined in terms of important individual attributes, of combinations of attributes, and of attribute complexes.

Fine structure of Bacillus megaterium during microcycle sporogenesis.

Ultrathin sections were prepared from cultures of Bacillus megaterium QM B1551 undergoing microcycle sporogenesis (initial spore to primary cell to second-stage spore without intervening cell division) on a chemically defined medium. The cytoplasmic core of the dormant spore was surrounded by plasma membrane, cell-wall primordium, cortex, outer cortical layer, and spore coats. Early in the cycle, the coat opened at the germinal groove, the cortex swelled, ribosomes and a chromatinic area associated with large mesosomes (which may later be incorporated into the expanding plasma membrane) appeared in the core, and the cell wall became defined at the site of the cell wall primordium. Poly-beta-hydroxybutyrate granules began to appear in the primary cell at about 3 hr. By 7 hr, the forespore of the second-stage spore was delineated by typical double membranes. Between 7 and 12 hr, second-stage cell-wall primordium and cortex developed between the separating forespore membranes. The inner membrane became the plasma membrane of the second-stage spore, and the outer membrane eventually disintegrated within the second-stage spore cortex. A densely staining double layer (spore-coat primordium) developed external to the outer forespore membrane. The inner spore coat and the outer cortical layer of the second-stage spore developed from this primordium. The outer part of the spore coat, probably of sporangial origin, was laid down on the external surface of the inner spore coat. By 12 hr, second-stage spores were almost mature. By 20 hr, the mature endospores, with a thickened outer coat, were often still enclosed by degenerate primary cell wall and by the outer cortical layer and spore coat of the initial spore.

Synchronous growth and sporulation of Bacillus megaterium.

Filtration of late log-phase cultures of Bacillus megaterium ATCC 19213 grown on defined sucrose salts medium (SS) or SS plus glutamate medium (SSG) through nine layers of Whatman no. 40 filter paper in a fritted-glass disc Büchner funnel resulted in filtrates containing cells which showed synchronous growth and proceeded to sporulation. SS cells completed one synchronous division after filtration; sporulation ensued after the cessation of growth. SSG cells completed two synchronous divisions and sporulation occurred during the second division. A high degree of synchrony of vegetative growth of SSG cells was evident by the stepwise pattern of growth, by the doubling of cell numbers at each division, the high division index, and by the rapid formation of sporulation cell types and homogeneity of cell types in the filtered cultures when compared with asynchronous cultures. Because the described system gives both good growth and sporulation synchrony, the method should be useful in delineating early events in sporulation and their regulation.

The inhibitory effect of albomycin (grisein) on growth of Bacillus cereus and Escherichia coli.

It was confirmed that albomycin inhibits the growth ofBacillus cereus andEscherichaa coli depending on a concentration of the antibiotic and physiological state of the culture. Salts of trivalen iron in a test medium reduce the time of the effect of albomycin, however, they also increase the growth of the control culture ofBacillus cereus and increase a value of maximal out-growth. An exponential growth of the culture is restored following a short lag when albomcycin is removed from a test medium. The antibiotic effect of albomycin A1 onBacillus cereus andEscherichia coli is removed by adding a 10-fold concentration of albomycin A3. The effect of Neilands ferrichrome is similar. On comparison withEscherichia coli, albomycin does no inhibit the growth ofBacillus cereus when cultivated at 28°C. The addition of CrCl3 solution partially restored the antibiotic activity of albomycin deprived of iron. It was found by microbiological tests that a retored growth of the whole culture was a result of albomycin inactivation. However, we did not find what change in the albomycin molecule is responsible for this inactivation.

Biochemical changes in lysogenic Bacillus stearothermophilus after bacteriophage induction.

Welker, N. E. (University of Illinois, Urbana), and L. Leon Campbell. Biochemical changes in lysogenic Bacillus stearothermophilus after bacteriophage induction. J. Bacteriol. 90:1129-1137. 1965.-Cultures of Bacillus stearothermophilus 1503-4R (TP-1) continued to grow at an unaltered rate after induction with mitomycin C (MC). MC-induced cultures exhibited a 2.5-fold increase in cell number before lysis occurred. Prior to lysis, cells were observed to elongate and to contain areas of lesser density. Protein synthesis was slightly inhibited in MC- or ultraviolet light (UV)-induced cultures for a period of 5 to 10 min, and then proceeded at a rate identical to that in the noninduced culture. Ribonucleic acid (RNA) synthesis was not affected by MC induction. UV induction caused RNA synthesis to occur in two stages: in the first stage, the rate of RNA synthesis was one-third that observed in the noninduced culture and lasted for a period of 15 min; the second stage of RNA synthesis then proceeded at a rate identical to that in the noninduced culture. The synthesis of deoxyribonucleic acid (DNA) in an MC- or UV-induced culture occurred in two stages. In the first stage, DNA synthesis in induced cultures occurred at a rate of one-half (MC) and one-third (UV) of that observed in the noninduced culture. The first stage of DNA synthesis in MC- or UV-induced cultures lasted for 25 to 30 min and 15 to 20 min, respectively. In the second stage, the rate of DNA synthesis in MC- or UV-induced cultures occurred at a rate three times that of the noninduced culture. UV induction appeared to have a greater inhibitory effect than MC induction on protein, RNA, and DNA synthesis as well as phage yield. The differential rate (K) of inducible and constitutive alpha-amylase synthesis was inhibited by 75 and 100%, respectively, for a period of 20 min after MC induction. After 20 min, the K values for alpha-amylase synthesis were identical to those obtained in the absence of MC induction. The synthesis of TP-1 phage DNA occurred rapidly and was complete 25 min after MC induction, whereas bacterial DNA was degraded or its rate of synthesis was decreased. During the second stage of DNA synthesis, only bacterial DNA was synthesized, but at a rate greater than that found in the noninduced culture.

INDUCED BIOSYNTHESIS OF ALPHA-AMYLASE BY GROWING CULTURES OF BACILLUS STEAROTHERMOPHILUS.

Welker, N. E. (Western Reserve University, Cleveland, Ohio), and L. Leon Campbell. Induced biosynthesis of alpha-amylase by growing cultures of Bacillus stearothermophilus. J. Bacteriol. 86:1196-1201. 1963.-The maximal differential rate (K) of alpha-amylase synthesis was usually two to three times that of the sucrose control culture, over an inducer concentration range of 5 x 10(-4) to 1 x 10(-3)m. With maltotetraose, higher concentrations decreased the K value, whereas higher concentrations of maltose were needed to obtain maximal K values. Glucose, in concentrations from 10(-5) to 10(-2)m, had no effect on the differential rate of enzyme synthesis. Cultures growing on maltotriose, maltotetraose, maltopentaose, and maltohexaose exhibited the same growth rate (k) and differential rate of alpha-amylase synthesis over a concentration range of 2.92 x 10(-4) to 1.46 x 10(-2)m. Growth of cultures in various concentrations of pure maltose revealed that with concentrations of maltose ranging from 2.92 x 10(-3) to 1.46 x 10(-2)m the K value for alpha-amylase production increased 18-fold. The amount of maltose utilized, during the growth period, at each concentration of maltose, was constant. Diauxic type growth was observed when maltose was used in addition to another carbon source (i.e., glucose, glycerol, fructose, or sucrose). Maltose was not utilized until the other carbon source had been metabolized. Phenyl-, methyl-, and ethyl-alpha-d-glucoside and methyl-beta-d-maltoside were good inducers of alpha-amylase and would not serve as a carbon source in a chemically defined medium supplemented with 0.1% casein hydrolysate. These compounds were therefore gratuitous inducers of alpha-amylase. Isomaltose, panose, butyl-alpha-d-glucoside, and methyl-alpha-d-maltotetraoside were not effective as inducers of alpha-amylase. Fructose had an inhibitory effect on constitutive (41%) and inducible (55%) alpha-amylase formation; glucose had no effect.

EFFECTS OF COPPER ON BACILLUS SUBTILIS.

Weed, Lawrence L. (Western Reserve University, Cleveland, Ohio). Effects of copper on Bacillus subtilis. J. Bacteriol. 85:1003-1010. 1963.-Variants have been isolated from liquid cultures of Bacillus subtilis 168 after exposure to copper. The variations manifested are in terms of loss of capacity to be transformed from tryptophan auxotrophy to prototrophy as shown by variant NTCu, and in terms of colony size and altered base composition of the deoxyribonucleic acid as shown by variant SC-22. In addition to the data on altered morphology and chemical composition of the variants, detailed studies on the reversion of one of the variants to B. subtilis are presented.

GROWTH OF CELLULAR FORMS IN CULTURES OF CHROMATIN BODIES ISOLATED FROM BACILLUS MEGATERIUM.

Chatterjee, B. R. (Baylor University College of Medicine, Houston, Texas) and Robert P. Williams. Growth of cellular forms in cultures of chromatin bodies isolated from Bacillus megaterium. J. Bacteriol. 85:623-627. 1963.-Chromatin bodies isolated from old cultures of Bacillus megaterium were capable of growing into protoplastlike cells when cultured in broth enriched with horse serum, yeast extract, adenosine triphosphate, and penicillin. A tendency toward formation of rod forms of bacteria was observed in such cultures. Omission of penicillin from the medium resulted in development of short bacterial forms. In 3 of 29 experiments, actual bacillary forms indistinguishable from the parent B. megaterium organism were recovered. Culture of the chromatin bodies in plain nutrient broth did not produce any growth. Inoculation on serum-enriched agar medium of a culture of chromatin bodies, after they had begun multiplication in serum-enriched broth, resulted in development of large bodies characteristic of L forms. Ability of chromatin bodies to grow was not affected by heating for 2 hr at 80 C or by sonic treatment for up to 25 min. The possible role of such resistant chromatin bodies in the latency and persistence of infectious diseases was discussed.

Aerobic sporulating bacteria. I. Glucose dehydrogenase of Bacillus cereus.

Bach, John A. (Michigan State University, East Lansing) and H. L. Sadoff. Aerobic sporulating bacteria. I. Glucose dehydrogenase of Bacillus cereus. J. Bacteriol. 83:699-707. 1962.-A heat-resistant glucose dehydrogenase occurs in cultures of Bacillus cereus which are in the initial stages of sporulation. This enzyme is, kinetically, identical to the glucose dehydrogenase which can be extracted from mature spores, but is considerably more heat resistant than the spore-free enzyme. The two enzymes produce identity lines in two-dimensional immunodiffusion experiments, and their behavior in chromatographic and electrophoretic studies is also identical.A labile glucose dehydrogenase can be extracted from germinated spores of B. cereus. It differs from its stable counterparts in possessing a higher pH optimum for enzymatic activity.

Cytological changes in aging bacterial cultures.

Chatterjee, B. R. (Baylor University College of Medicine, Houston, Texas) and Robert P. Williams. Cytological changes in aging bacterial cultures. J. Bacteriol. 84:340-344. 1962.-Morphological changes occurring in aging broth and agar cultures of Bacillus anthracis were observed with the phase-contrast microscope. In 4 to 5 days, in liquid culture, cells lost their normal shape and become fusiform or spherical. Chromatin bodies were clearly visible inside the cells at this time. Spontaneous conversion into protoplast-like, spherical cells was observed on the 6th or 7th day. These cells were sensitive to osmotic changes, but could be preserved by the addition of sucrose to the medium. Budding and atypical multicellular forms also were observed at this stage. In agar cultures, the conversion into spherical forms was observed with more regularity; the presence of sucrose in the medium favored the process. In very late stages of growth, the spherical cells disintegrated into conglomerated masses of cellular debris with the discrete chromatin bodies enmeshed in them. At this stage, secondary colonies appeared, superimposed on the primary ones, and on transfer to suitable medium gave rise to unstable L colonies.

The influence of the toxins separated by adsorption on wood charcoal from the culture of Bacillus polymyxa on the fermentation of sucrose.

Fractions from toxins adsorbed at charcoal have been separated by extraction. The acetone fraction causes decrease in the formation of reducing sugars and increase in sugar consumption, decrease in yield of 2,3 butanediol. The absolute alcohol fraction causes decrease in the formation of reducing sugar without affecting the sugar consumption and considerable decrease in the yield of 2,3 butanediol. The petroleum ether fraction increases the formation of reducing sugar without increasing the sugar consumption. It greatly decreases the production of 2,3 butanediol. All fractions inhibit formation of acetoin.

Chemical and morphological studies of bacterial spore formation. III. The effect of 8-azaguanine on spore and parasporal protein formation in Bacillus cereus var. alesti.

The purine analogue, 8-azaguanine, was added to cultures of the parasporal crystal-forming organism Bacillus cereus var. alesti at different times during growth and synchronous sporulation. The effect of its incorporation has been studied with particular reference to cell growth, nucleic acid composition, cytology, and the synthesis of the spore and crystal protein. Additions of the analogue during any stage of growth prevented further cell proliferation and all spore and crystal formation. Since both nucleic acids continued to be formed, cells of an increased size developed, containing large masses of chromatin in the form of condensed balls or axial cords. Lipid-containing inclusions also appeared following these additions and were usually aggregated at the centre or poles of the cells. The analogue could be isolated as the ribonucleotide from both the acid soluble and RNA fractions of these inhibited cells. Additions of the analogue following commencement of sporulation did not prevent either spore or crystal formation or affect the nucleic acid content of the sporulating cells. However, as before, the 8-azaguanine was incorporated into both the acid soluble and RNA of the cells, but not into these fractions of the spores ultimately formed. The implications of these findings are discussed in relation to crystal protein synthesis.

Changes in the peripheral nervous system due to experimental fever

Studies of the nervous apparatus of the large veins in animals with experimental fever caused by the injection of a culture ofBacillus mesentericus demonstrated reactive changes of the sensory apparatus in the walls of the superior vena cava and pulmonary veins with intact afferent innervation. Structural changes mainly in preterminal and terminal sections of the medullated fibers were as a rule detected in 3 hours after the injection of the stimulus, reached the maximum in 6–8 hours and disappeared in 24 hours. Neurons of the afferent innervation in walls of the g-nodosa and the spinal ganglions were not changed. The data obtained show that reactive changes caused by experimental fever first appear in the peripheral section of the neuron and are reversible.

Isolation and experimental study of 2 new strains of acid-alcohol-fast bacteria from the nasal mucus of lepers

The A. described on December 14, 1951, his first culture of acid-fast bacillus isolated from nasal mucus of a leprous girl. in this paper the A. describes two new strains of acid-fast bacilli gotten also from nasal mucus of other two leprous girls, L3 cases. The first patient (layse S) had her mucus treated by petroff's method on December 16, 1952 and sown onto three tubes of loewenstein medium and in glycerin broth. After two weeks incubation at 37°C all three tubes of Loewenstein showed many punctiforme and pin-head yellowish colonies, whose microscopic examination proved to be of a pure acid-fast bacillus culture. This sample inoculated in rats and mice produced, after 55 days incubation, small tumors from which the culture was easily recovered. On April, 9, 1953 a new sample of nasal mucus of the same patient was sown in three tubes of Loewenstein. After two weeks incubation at 37°C all 3 tubes showed germination of small yellowish colonies of acid-fast bacilli. Within four months being gotten two samples of identical cultures in all smeared tubes of Loewenstein medium sown, proved that such cultures were not an ordinary ambient contamination. Second patient: - After various sowing of different kinds of material from her, february 26, 1953 her nasal mucus treated by soda and sown onto Loewenstein medium, after 25 days incubation showed in only one tube, one small round colony, at first white, becoming creamy after three months. Transplants in various media grew at first slowly and after 2 or 3 generations grew faster. The Layse strain produced pellicle in glycerin broth and Dubos medium; the Maria N. strain did not produce as yet. Both strains (Layse I and II, and N.) gave weak positive Dubos test in half-an-hour and negative after 24 hours reading. Both were strongly positive when stained by Gram, Ziehl-Neelsen and Fontes methods. Both strains gave also positive fluoroscopy. These cultures are being studied. The A. concludes that, according to his experience, the slower growing cultures of acid-fast bacilli isolated from leprosy material, are the more suitable for experimental work. Aknowledgement. The A. thanks to Miss MARIA DE LOURDES SANTANA for her valuable collaboration in the studies of the described cultures.

Characteristics of Antibiotic-Producing Strains of the Ocular Bacterial Flora

Summary The antibiotics produced by a series of 22 ocular Micrococci have proven to be heterogeneous. Nine different antibiotic agents were produced by the 22 strains. These were apparently active by at least 3 distinct and independent metabolic mechanisms. Fourteen of the 22 antibiotics were rather clearly of polypeptide nature, as evidenced by enzyme destruction and other properties. The remaining 8 agents were resistant to enzyme activity, but a few observations suggest that these too, could be peptide structures. The antibacterial spectra of samples of these strains have been examined in some detail. Only gram positive species were affected significantly. Micrococcus strains were inhibited most frequently, while Corynebacterium, Sarcina, and Bacillus cultures were also commonly and highly susceptible. Streptococci, Clostridia and Mycobacteria were moderately susceptible. In general, the spectra were grossly similar for the three groups of antibiotics which acted by differing metabolic mechanisms. A high degree of variability was often shown in the susceptibility of strains of a given species to a given antibiotic producer.

Comparative Immunizing Capacity of BCG and the Proteins and Polysaccharides in Tubercle Bacillus Culture Filtrates

Summary In the presence of large inocula of tubercle bacilli previous sensitization with tuberculin protein provided no protection, and appeared possibly to be detrimental. When small inocula were used a definite increase in survival time could be demonstrated in animals previously vaccinated with BCG. In experiments in which such resistance was demonstrated, parallel series of rabbits sensitized with tuberculin protein A (soluble at its iso-electric point) or with tuberculin protein C (insoluble at its iso-electric point) as well as with the small haptenic tuberculin polysaccharide I or with the larger antigenic tuberculin polysaccharide II, did not show increased resistance to tuberculous infection. Electrophoretic changes were demonstrated in antisera to some of these fractions. Even though, by indirect methods, some of these serum components had been shown to have antibodies which possessed anti-infective properties, nevertheless, the rabbits possessing these antisera were not immune.