Air-liquid interface biofilms of Bacillus cereus: formation, sporulation, and dispersion

Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.     

Air-Liquid Interface Biofilms of Bacillus cereus: Formation, Sporulation, and Dispersion

Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.     

Use of antibiotics in selecting a nystatin producer]

The lethal and mutagenic effect of streptomycin and nystatin on Act. noursei, strain 408 producing nystatin was studied. The survival of the spores of strain 408 on the medium with streptomycin decreased with an increase in the antibiotic concentration. Streptomycin had a selective effect on the nystatin-producing organism decreasing the frequency of morphologically changed and low active variants and revealing highly active and antibiotic stable variants. The survival of the spores of strain 408 on the medium with nystatin (20,000 units/ml) amounted to 35 per cent. Nystatin had an inhibitory effect on the organism producing it which was evident from delayed growth and significant modification variation of the colonies, as well as from a marked increase in the number of the variants characterized by low antibiotic production.     

Biocatalytic decolourisation of molasses by Phanerochaete chrysosporium

Bioremediation potential of Phanerochaete chrysosporium strains NCIM 1073, NCIM 1106 and NCIM 1197 to decolourise molasses in solid and liquid molasses media was studied. Strains varied in the pattern of molasses decolourisation on solid medium by Giant colony method. Under submerged cultivation conditions, strain NCIM 1073 did not decolourise molasses while, strains NCIM 1106 and NCIM 1197 could decolourise molasses up to 82% and 76%, respectively. Under stationary cultivation conditions, none of the strains could decolourise molasses. This was overcome by increasing the surface area of the culture in flat bottom glass bottles under stationary cultivation conditions. Under submerged cultivation conditions, growth was more or less same in all strains. However, the lignin peroxidase and manganese peroxidase activities were significantly less in the strain NCIM 1073. Under stationary cultivation conditions, none of the strains could produce enzymes lignin peroxidase, manganese peroxidase and laccase. However, all of them could produce lignin peroxidase and manganese peroxidase when cultivated in flat bottom glass bottles under stationary cultivation conditions.     

Antimicrobial activity of Laetiporus sulphureus strains grown in submerged culture]

Cultural conditions for growth and fruit body formation were elaborated to four strains of Laetiporus sulphureus isolated from nature. All strains demonstrated antimicrobial activity against a wide spectrum of gram-positive and gram-negative bacteria during agar and submerged cultivation including methicillin-resistant strain of Staphylococcus aureus (MRSA) and glycopeptide-resistant strain of Leuconostoc mesenteroides. Antifungal activity was not found. The level of antimicrobial activity during submerged cultivation reached maximum after seven days of growth on specific medium with soybean meal and corn liquid; the next four weeks its increasing was not so manifested. Antimicrobial activity correlated with orange pigment secretion and cultural liquid acidification to pH 2.0-2.8 that indicates on acid nature of synthesized products.     

Oxidation of Acetate by Various Strains of Bacillus popilliae

A number of strains of Bacillus popilliae were examined for their ability to oxidize acetate. Some of these would not sporulate in vitro, and some were oligosporogenous. The ability to oxidize acetate varied widely among the strains tested. A culture derived from spores of the parent strain produced in vivo and one of the asporogenous strains derived from it failed to produce any significant levels of (14)CO(2) from [(14)C]acetate. Oligosporogenous strains derived from the same parent culture all produced (14)CO(2) from both [1-(14)C] and [2-(14)C]acetate but at relatively low rates. The highest rates of acetate oxidation were observed with three strains which did not produce spores in vitro. When cultured under appropriate conditions, one of these strains displayed a secondary growth response concomitant with a decrease in the titratable acidity and an increase in the pH of the medium. The data indicate that B. popilliae has a complete citric acid cycle but that the activity of the cycle is strongly repressed in wild-type strains under the usual conditions used for in vitro cultivation.     

Biological indicators for low temperature steam and formaldehyde sterilization: the effect of defined media on sporulation, growth index and formaldehyde resistance of spores of Bacillus stearothermophilus strains

Preliminary screening was carried out on spores of 29 strains of Bacillus stearothermophilus to determine their potential as biological indicator organisms for low temperature steam and formaldehyde sterilization. Each strain was sporulated on four chemically defined media. Fourteen strains produced satisfactory sporulation on one or more of the media but there was considerable variation in the extent of sporulation. The growth index of the spores, which was dependent on both the strain of organism and the sporulation medium, ranged from 1% to 90%. The spores were appraised on the basis of their resistance to inactivation by 0.5% w/v formaldehyde in aqueous solution at 70°C. The survivor curves obtained could be characterized into five types on the basis of the shape of the curve. Only five strains of Bacillus stearothermophilus produced spores with the characteristics of high resistance, linear semi-logarithmic survivor curve and high growth index that would be required of a potential biological indicator organism.     

Regulation of conidiation and adenylyl cyclase levels by the Galpha protein GNA-3 in Neurospora crassa

We have identified a new gene encoding the G protein alpha subunit, gna-3, from the filamentous fungus Neurospora crassa. The predicted amino acid sequence of GNA-3 is most similar to the Galpha proteins MOD-D, MAGA, and CPG-2 from the saprophytic fungus Podospora anserina and the pathogenic fungi Magnaporthe grisea and Cryphonectria parasitica, respectively. Deletion of gna-3 leads to shorter aerial hyphae and premature, dense conidiation during growth on solid medium or in standing liquid cultures and to inappropriate conidiation in submerged culture. The conidiation and aerial hypha defects of the Deltagna-3 strain are similar to those of a previously characterized adenylyl cyclase mutant, cr-1. Supplementation with cyclic AMP (cAMP) restores wild-type morphology to Deltagna-3 strains in standing liquid cultures. Solid medium augmented with exogenous cAMP suppresses the premature conidiation defect, but aerial hypha formation is still reduced. Submerged-culture conidiation is refractory to cAMP but is suppressed by peptone. In addition, Deltagna-3 submerged cultures express the glucose-repressible gene, qa-2, to levels greatly exceeding those observed in the wild type under carbon-starved conditions. Deltagna-3 strains exhibit reduced fertility in homozygous crosses during the sexual cycle; exogenous cAMP has no effect on this phenotype. Intracellular steady-state cAMP levels of Deltagna-3 strains are decreased 90% relative to the wild type under a variety of growth conditions. Reduced intracellular cAMP levels in the Deltagna-3 strain correlate with lower adenylyl cyclase activity and protein levels. These results demonstrate that GNA-3 modulates conidiation and adenylyl cyclase levels in N. crassa.     

Role of gramicidin S in the sporulation of the R+ and R- variants of Bacillus brevis var. G.-B.]

The effect of gramicidin S added to the cultivation medium on sporulation of the gramicidin S-producing P+ variant and gramicidin S-nonproducing P- variant of Bacillus brevis var. G.-B. was studied. Gramicidin S added to the synthetic medium with glucose in an amount of 30 and 100 microgram/ml 4 and 7 hours after inoculation with the vegetative cells of R- variant had no effect on the growth of the culture but retarded its sporulation. When gramicidin S was added in an amount of 100 microgram/ml 4 hours after inoculation, the sporulation rate of R- variant strongly decreased, rohile sporulation was not suppressed as it was noted before with respect to R+ variant. Active stimulation of Bacillus brevis var. G.-B. sporulation was observed after addition of gramicidin S 13 hours after development of R+ and R- variants without the antibiotic biosynthesis. Synthesis of gramicidin S by R+ strain was suppressed by the specific inhibitor beta-phenyl-beta-alanine. The amount of gramicidin S added to the medium during the sporulation process of R+ and R- variants decreased. On addition of 30 microgram/ml of the antibiotic it was practically not detectable when the culture showed the greatest number of the spores. Therefore, gramicidin S added to the medium is probably adsorbed by the cells of Bac. brevis var. G.-B. and affects sporulation of R- and R+ variants thus accelerating or retarding this process depending on the cultivation conditions.     

Overexpression of the diguanylate cyclase CdgD blocks developmental transitions and antibiotic biosynthesis in Streptomyces coelicolor

Cyclic dimeric GMP(c-di-GMP) has emerged as the nucleotide second messenger regulating both development and antibiotic production in high-GC, Gram-positive streptomycetes. Here, a diguanylate cyclase(DGC), CdgD, encoded by SCO5345 from the model strain Streptomyces coelicolor, was functionally identified and characterized to be involved in c-di-GMP synthesis through genetic and biochemical analysis. cdgD overexpression resulted in significantly reduced production of actinorhodin and undecylprodigiosin, as well as completely blocked sporulation or aerial mycelium formation on two different solid media. In the cdgD-overexpression strain, intracellular c-di-GMP levels were 13-27-fold higher than those in the wild-type strain. In vitro enzymatic assay demonstrated that CdgD acts as a DGC, which could efficiently catalyze the synthesis of c-di-GMP from two GTP molecules. Heterologous overproduction of cdgD in two industrial Streptomyces strains could similarly impair developmental transitions as well as antibiotic biosynthesis. Collectively, our results combined with previously reported data clearly demonstrated that c-di-GMP-mediated signalling pathway plays a central and universal role in the life cycle as well as secondary metabolism in streptomycetes.     

Studies on a new marine streptomycete BT-408 producing polyketide antibiotic SBR-22 effective against methicillin resistant Staphylococcus aureus

A new actinomycete strain designated as BT-408 producing polyketide antibiotic SBR-22 and showing antibacterial activity against methicillin resistant Staphylococcus aureus has been characterized and found to be a novel strain of Streptomyces psammoticus. Nutritional and cultural conditions for the production of antibiotic by this organism under shake-flask conditions have been optimized. Glucose and ammonium nitrate were found to be best carbon and nitrogen sources respectively for growth and antibiotic production. Similarly initial medium pH of 7.2, incubation temperature of 30 degrees C and incubation time of 96 h were found to be optimal. Optimization of medium and cultural conditions resulted in 1.82-fold increase in antibiotic yield.     

Production and characteristics of the exocellular polysaccharide of a mutantMycobacterium strain

Mutant strains ofMycobacterium sp. V-649 producing highly mucous colonies on a solid cultivation medium were prepared after treatment with N-methyl-N′-nitro-N-nitrosoguanidine and production of the exocellular polysaccharide was tested. The strains were cultivated in media with suitable sugar sources under submerged conditions. It was found thatMycobacterium sp. V649/15 produces a maximum of 15–19% polymer after a 5–6-d cultivation. Gas chromatography indicated that the exocellular polysaccharide produced by this strain is of glucan type.     

The use of the protoplast method for the selection of oleandomycin producers

The use of protoplasting with subsequent reversion to the cellular form in improvement of the oleandomycin-producing organism provided a 110% increase in the range of culture variation with respect to the antibiotic production property. A regenerant with a potency of 12 to 20 per cent higher than that of the initial strain which produced 30 per cent lower amounts of dark pigments of melanin nature was isolated. Repeated protoplasting and regeneration of the regenerant provided a very low regeneration frequency i.e. 0.0002%. The potency of all the secondary regenerants was low.     

Production,long term preservation and synchronous germination of aerial spores of Streptomyces

Vigorous vegetative growth of various Streptomyces species (S. auroefaciens, S. collinus and S. granaticolor) was achieved in a new semisynthetic liquid medium. Unlike the media commonly used for the cultivation of the submerged mycelia of different streptomycetes, this one does not contain insoluble material which enables direct and reliable measurement of net production of biomass. The medium was formulated to meet the nutritional requirements of all the three species. Is also supported production of antibiotic in each of the strains. A method for bulk preparation of Streptomyces aerial spores, involving cultivation on agar plates covered with cellophane, was developed. Advantage of this method lies in higher yields of spores, their higher purity and easier harvesting. The spores were activated by amild treatment with an Ultra-Turrax homogenizer resulting in the breakage of fibrous sheath, suspended in 20% glycerol, and stored at ?60°C. Thus, treated spores germinated synchronously even after several months of the storage. Hence, such spore material may be used for precise inoculation in a large series of experiments implying synchronous germination, and the inoculations can be carried out from the same batch over a long period.     

Sporulation of Streptomyces griseus in submerged culture.

A wild-type strain of Streptomyces griseus forms spores both on solid media (aerial spores) and in liquid culture (submerged spores). Both spore types are highly resistant to sonication, but only aerial spores are resistant to lysozyme digestion. Electron micrographs suggest that lysozyme sensitivity may result from the thinner walls of the submerged spores. Studies of the life cycle indicate that neither streptomycin excretion nor extracellular protease activity is required for sporulation: the analysis of mutants, however, suggests that antibiotic production may be correlated with the ability to sporulate. A method was devised to induce the rapid sporulation of S. griseus in a submerged culture. This method, which depends on nutrient deprivation, was used to determine that either ammonia or phosphate starvation can trigger sporulation and that the enzyme glutamine synthetase may be useful as a sporulation marker after phosphate deprivation.     

Spore germination of Frankia strains isolated from Colletia hystrix and Retanilla ephedra (Rhamnaceae)

Two Frankia strains, ChI1 from Colletia hystrix and ReI6 from Retanilla ephedra, were assayed to determine the germinability of their spores and the stages of the life cycle in different media. In BAP medium, both strains showed approximately 18% spore germination. However, in BAP medium lacking micronutrients and FeEDTA, strain ChI1 spores exhibited approximately 53% germination and strain ReI6 spores about 42%. The survey also showed that the spores were not heat-resistant, the ReI6 spores being more sensitive than ChI1 spores. Both strains exhibited a small increase in their percentage of germination with a 15-pulse ultrasonic treatment.     

Study of the fructosediphosphate aldolase and transketolase activity in the nystatin producer, Actinomyces noursei]

Activity of transketolase, an enzyme of the pentose cycle and fructosodiphosphataldolase, an enzyme of glycolisis was studied in the dynamics of development of the nystatin-producing organism and its inactive mutant under various conditions of their cultivation with a purpose of finding relation between the antibiotic production and general metabolism of Act. noursei. The transketolase activity of the organism was 2-4 times higher than that of the inactive mutant. Addition of 8000 Units/ml of nystatin to the medium markedly suppressed (50-100 per cent) the aldolase activity, however it had no effect on the transkelotase activity. Possibly the antibiotic accumulated in the mycelium played the role of a regulator of the activity of the enzymes, directing the metabolites along the hexosomonophosphate pathway of carbohydrate dissimilation.     

The effect of palitantin,a metabolite ofPenicillium frequentans onLeishmania brasiliensis

A compound with an antiprotozoal effect againstLeishmania brasiliensis was isolated from the submerged culture ofPenicillium frequentans 60A/7 which was collected in this country. The active compound was characterised as palitantin, a metabolite of several strains ofPenicillium the antibiotic activity of which has not yet been reported. The investigated strain produced 200–500mg of palitantin per liter of medium during submerged cultivation and also a small quantity of frequentin: 10–20mg per liter. Frequentin has no activity againstLeishmania brasiliensis and several other investigated protozoa.     

Lethal and mutagenic action of N-nitroso-N-methylbiuret on the producers of levorin, amphotericin and mycoheptin]

The lethal and mutagenic effect of N-nitrozo-N-methylbiuret (NMB) on the organisms producing levorin, amphotericin B and mycoheptin was studied. The mutagen effect depended on the dose, culture and physiological state of the spores. NMB had a low mutagenic effect on the levorin-producing organism characterized by high activity and genetic homogenicity with respect to the colony morphology and antibiotic production. As for the organisms producing amphotericin B and mycoheptin characterized by high genetic heterogenicity, significant variation of all the features studied was observed on their exposure to the mutagen. Inspite of diverse reaction of the organisms producing levorin, amphotericin B and mycoheptin to the effect of NMB mutants with increased antibiotic production were obtained from the three cultures. The lethal and mutagenic effect of NMB on the mycoheptin-producing organism depended on the process of the spore DNA replication. The spores during the DNA replication period were least sensitive to the lethal effect of the mutagen and most mutable with the respect to the colony morphology. For selection of highly active and stable strains exposure to NMB of the spores of the mycoheptin-producing organism during replication of DNA proved to be more effective than that of the spores during the lag-phase.     

Properties of the germination inhibitor of Streptomyces viridochromogenes spores

Germinating spores of Streptomyces viridochromogenes excreted a substance into the surrounding medium which inhibited germination of another sample of the spores. The germination inhibitor (GI) was produced during submerged culture after exponential growth had ceased. The GI was purified 51-fold following extraction from growth liquor with chloroform. It was soluble in alcohol and water and had a molecular weight of less than 1000. The GI blocked growth and respiration of some Gram-positive bacteria and was an inhibitor of the membrane bound, but not solubilized, calcium-dependent ATPase of germinated spores and mycelia of the producing organism. Several sodium-potassium activated ATPases were also inhibited. All four activities (respiration, growth, germination inhibition, ATPase) co-purified during column and thin-layer chromatography. The GI activities released during germination and produced during growth were identical. A role for the GI antibiotic in regulation of dormancy of spores of the producing organism is discussed.