Changes in Terminal Respiratory Pathways of Bacillus subtilis During Germination, Outgrowth, and Vegetative Growth

The chemical and enzymatic properties of the cytochrome system in the particulate preparations obtained from dormant spores, germinated spores, young vegetative cells, and vegetative cells of Bacillus subtilis PCI219 were investigated. Difference spectra of particulate fractions from dormant spores of this strain suggested the presence of cytochromes a, a(3), b, c(+c(1)), and o. All of the cytochrome components were present in dormant spores and in germinated spores and vegetative cells at all stages which were investigated. Concentrations of cytochromes a, a(3), b, and c(+c(1)) increased during germination, outgrowth, and vegetative growth, but that of cytochrome o was highest in dormant spores. As the cytochrome components were reducible by reduced nicotinamide adenine dinucleotide (NADH), they were believed to be metabolically active. Difference spectra of whole-cell suspensions of dormant spores and vegetative cells were coincident with those of the particulate fractions. NADH oxidase and cytochrome c oxidase were present in dormant spores, germinated spores, and vegetative cells at all stages after germination, but succinate cytochrome c reductase was not present in dormant spores. Cytochrome c oxidase and succinate cytochrome c reductase activities increased with growth, but NADH oxidase activity was highest in germinated spores and lowest in vegetative cells. There was no striking difference between the effects of respiratory inhibitors on NADH oxidase in dormant spores and those on NADH oxidase in vegetative cells.     

Protein synthesis elongation factor Tu present in spores of<Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> can be phosphorylated<Emphasis Type="Italic">in vitro</Emphasis> by the spore protein kinase

In vitro phosphorylation of EF-Tu was shown in cell-free extract from dormant spores of Streptomyces coelicolor by a protein kinase present in spores. EF-Tu phosphorylation was observed on both intrinsic S. coelicolor factor and externally added purified EF-Tu from S. aureofaciens, on two isoforms. Putative serine and threonine residues as potential phosphorylation targets were determined in primary sequence and demonstrated on 3D structure model of EF-Tu.     

Protease and peptidase activities in growing and sporulating cells and dormant spores of Bacillus megaterium.

Peptidase and protease activities on many different substrates have been determined in several stages of growth of Bacillus megaterium. Extracts of log-phase cells, sporulating cells, and dormant spores of B. megaterium each hydrolyzed 16 different di- and tripeptides. The specific peptidase activity was highest in dormant spores, and the activity in sporulating cells and log-phase cells was about 1.2-fold and 2- to 3-fold lower, respectively. This peptidase acticity was wholly intracellular since extracellular peptidase activity was not detected throughout growth and sporulation. In contrast, intracellular protease activity on a variety of common protein substrates was highest in sporulating cells, and much extracellular activity was also present at this time. The specific activity of intracellular protease in sporulating cells was about 50- and 30-fold higher than that in log-phase cells and dormant spores, respectively. However, the two unique dormant spores proteins known to be the major species degraded during spore germination were degraded most rapidly by extracts of dormant spores, and slightly slower by extracts from log-phase or sporulating cells. The specific activities for degradation of peptides and proteins are compared to values for intracellular protein turnover during various stages of growth.     

Characterization of Ribosomes from Dormant Spores of Bacillus cereus

Characterization of ribosomes from dormant spores and vegetative cells of Bacillus cereus strain T has been carried out. Polyuridylic acid binding activity, ribonuclease activity associated with ribosomes, thermal denaturation profile, and sedimentation coefficients are essentially identical for both ribosomal preparations. However, ribosomal protein content of dormant spore ribosomes is about 70% of that of vegetative ribosomes. Polyacrylamide gel electrophoresis of ribosomal proteins shows that some ribosomal proteins are missing from dormant spore ribosomes. Sucrose density gradient centrifugation of ribosomes shows the existence of defective ribosomal subunits, in addition to 30S and 50S subunits, in dormant spore ribosomes. These results indicate that the ribosomes from dormant spores are distinctively different from those of vegetative cells.     

Analysis of Airborne Actinomycete Spores with Fluorogenic Substrates

The reactions between seven fluorogenic substrates and different groups of enzymes, esterases, lipases, phosphatases, and dehydrogenases, were studied in a search for a new method for the detection of actinomycete spores. Fluorescence measurement was chosen as a fast and sensitive method for microbial analysis. The focus of the research was on the spores of important air contaminants: Streptomyces albus and Thermoactinomyces vulgaris. For the measurement of the enzymatic activity, the chosen fluorogenic substrate was added to a mixture of spores and nutrient media, and the resulting fluorescence was measured with a spectrofluorometer. Fluorogenic substrates were found to show enzymatic activities even for dormant spores. Comparison of the enzymatic activities of dormant spores with those of vegetative cells showed similarity of the enzymatic profiles but higher activity for vegetative cells. The increase of enzymatic activity from dormant spores to vegetative cells was not linear but fluctuating. The largest fluctuations were found after 4 to 5 h of incubation. The enzymatic activities of S. albus were 10 to 50 times lower than those of T. vulgaris, except for the dehydrogenase activity, which was seven times higher. These results indicate that analysis with fluorogenic substrates has the potential for becoming a fast and sensitive method for the enumeration and identification of airborne actinomycete spores.     

Quantitative Analysis of Polymyxin B Released from Polymyxin B-Treated Dormant Spores of Bacillus subtilis and Relationship between Its Permeability and Inhibitory Effect on Outgrowth

Polymyxin B, one of the cyclic polypeptide antibiotics, binds to the coat of Bacillus subtilis dormant spores and inhibits them from growing after germination. When about 2.8 × 10⁸ cells/ml of polymyxin B-treated dormant spores were incubated in heart infusion broth, 3.6 μg/ml of polymyxin B were released into the liquid medium during germination. Incubation of the same concentration of polymyxin B-treated ones in 100 mM CaCl₂ solution released 4.0 μg/ml of the antibiotic. The effect of various concentrations of polymyxin B on germination, outgrowth and vegetative growth of the dormant spores was investigated; the results showed that concentrations of 4.0 μg/ml and higher of the antibiotic inhibited their outgrowth and vegetative growth after germination. Young vegetative cells were less sensitive to the antibiotic than germinated spores. In addition to these results, immunoelectron microscopy with colloidal gold particles indicated that polymyxin B permeated into the core of the germinated spores and inhibited them from outgrowing.     

Synthesis of EF-Tu and distribution of its mRNA between stroma and thylakoids during the cell cycle of Chlamydomonas reinhardii

In Chlamydomonas reinhardii the elongation factor EF-Tu is encoded in the chloroplast DNA. We identified EF-Tu in the electrophoretic product pattern of chloroplast-made proteins and showed that this protein is only synthesized in the first half of the light period in synchronized cells. The newly synthesized EF-Tu contributed little to the almost invariable content of EF-Tu in chloroplasts during the light period of the cell cycle. However, increasing cell volume and the lack of EF-Tu synthesis in the second half of the light period led to a decrease in the concentration of EF-Tu in chloroplasts. At different times in the vegetative cell cycle, the RNA was extracted from whole chloroplasts and from free and thylakoid-bound chloroplast polysomes. The content of mRNA of EF-Tu in chloroplasts and the distribution between stroma and thylakoids were determined. During the light period, the content of the mRNA for EF-Tu varied in parallel to the rate of EF-Tu synthesis. However, in the dark, some mRNA was present even in the absence of EF-Tu synthesis. Most of the mRNA was bound to thylakoids during the whole cell cycle. This suggests that synthesis of EF-Tu is associated with thylakoid membranes.     

Peptide Synthesis by Extracts from Bacillus subtilis Spores

Cell-free peptide synthesis by extracts from vegetative cells and spores of Bacillus subtilis was analyzed and compared. The initial rate of phenylalanine incorporation in a polyuridylate-directed system was found to be in a similar range for the two extracts. However, spore extracts frequently incorporated less total phenylalanine as did the vegetative cell system. Optimal conditions for amino acid incorporation by spore extracts were found to be similar to those of vegetative cell extracts. Polyphenylalanine synthesis was stimulated by preincubation of both extracts prior to the addition of polyuridylic acid (poly U) and labeled phenylalanine. Both systems showed a dependence on an energy-generating system and were inhibited by chloramphenicol and puromycin. Ribonuclease, but not deoxyribonuclease, inhibited the reaction significantly. The presence of methionine transfer ribonucleic acid (tRNA(F)) and methionyl-tRNA(F) transformylase was demonstrated in spore extracts. An analysis of several aminoacyl-tRNAs in spores revealed that the relative amounts of these tRNAs were similar to those found in vegetative cells. Only lysine tRNA was found to be present in relatively greater amounts in spores. These results indicate that dormant spores of B. subtilis contain the machinery for the translation of genetic information.     

Dielectric Study of the Physical State of Electrolytes and Water Within Bacillus cereus Spores

Dielectric measurements revealed that dormant spores of Bacillus cereus have extremely low conductivities at high frequencies (50 MHz) and so must contain remarkably low concentrations of mobile ions both within the core and in the surrounding integuments. Activation, germination, and outgrowth were all accompanied by increases in conductivity of the cells and their suspending medium, and this result indicated that intracellular electrolytes had become ionized and leaked from the spores. High-frequency dielectric constants of spores were consistent with normal states for cell water. These values increased during successive stages of development from dormant spore to vegetative bacillus, and they could be directly related to increases in cell water content. In all, the results refuted a model of the dormant spore involving freely mobile, ionized electrolytes and supported a model involving electrostatically bound electrolytes.     

Germination-initiated spores of Bacillus brevis Nagano retain their resistance properties.

Initiated spores and vegetative cells of the gramicidin S-producing Bacillus brevis Nagano were compared with respect to their resistance to various forms of stress (osmotic shock-starvation, exposure to ethanol, sonic oscillation, and heat). The resistance of initiated spores to all of these stress situations was considerably greater than that of vegetative cells and approached that of dormant spores. The period during which the initiated spores remained resistant to heat was extended by addition of gramicidin S. The antibiotic may therefore be of survival value to the species in nature by slowing down the development of initiated spores in the outgrowth phase of germination, thereby extending the period during which the cells are resistant to environmental stress.     

Percent Charging of Transfer Ribonucleic Acid and Levels of ppGpp and pppGpp in Dormant and Germinated Spores of Bacillus megaterium

The levels of transfer ribonucleic acids (tRNAs) specific for 14 amino acids were almost identical in dormant spores and in spores germinated from 6 to 75 min. Germinated spore tRNAs specific for all amino acids tested were between 63 and 93% charged, and there was no significant change in this value from 6 to 75 min of germination. In contrast, tRNAs isolated from dormant spores specific for nine different amino acids were almost completely(>93%) uncharged. However, some dormant spore tRNAs, i.e., those for arginine, histidine, isoleucine, and valine, showed significant (21 to 72%) levels of aminoacylation. Dormant spores contained no detectable guanosine penta- (pppGpp), tetra- (ppGpp), or triphosphate (GTP). However, these nucleotides appeared in the first minutes of germination, and thereafter all increased in parallel with a ratio of pppGpp plus ppGpp to GTP of 0.07 to 0.11, which is characteristic of unstarved vegetative cells.     

Heat shock affects permeability and resistance of Bacillus stearothermophilus spores

Heat shock of dormant spores of Bacillus stearothermophilus ATCC 7953 at 100 or 80 degrees C for short times, the so-called activation or breaking of dormancy, was investigated by separating the resulting spores by buoyant density centrifugation into a band at 1.240 g/ml that was distinct from another band at 1.340 g/ml, the same density as the original spores. The proportion of spores at 1.240 g/ml became larger when the original dormant spores were heated for a longer period of time, but integument-stripped dormant spores were quickly and completely converted to spores with a band at 1.240 g/ml. The spores with bands at both 1.240 and 1.340 g/ml were germinable faster than the original dormant spores and thus were considered to be activated. The spores with a band at 1.240 g/ml, which were considered to be fully activated, were apparently permeabilized, with a resulting complete depletion of dipicolinic acid, partial depletion of minerals, susceptibility to lysozyme action, permeation of the gradient medium, changed structural appearance in electron micrographs of thin-sectioned spores, and partly decreased heat resistance (D100 = 453 min) compared with the original dormant spores (D100 = 760 min). However, the fully activated spores with a band at 1.240 g/ml, although devoid of dipicolinic acid, still were much more resistant than germinated spores or vegetative cells (D100 = 0.1 min). The spores with a band at 1.340 g/ml, which were considered to be partly activated, showed no evidence of permeabilization and were much more heat resistant (D100 = 1,960 min) than the original dormant spores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat shock affects permeability and resistance of Bacillus stearothermophilus spores.

Heat shock of dormant spores of Bacillus stearothermophilus ATCC 7953 at 100 or 80 degrees C for short times, the so-called activation or breaking of dormancy, was investigated by separating the resulting spores by buoyant density centrifugation into a band at 1.240 g/ml that was distinct from another band at 1.340 g/ml, the same density as the original spores. The proportion of spores at 1.240 g/ml became larger when the original dormant spores were heated for a longer period of time, but integument-stripped dormant spores were quickly and completely converted to spores with a band at 1.240 g/ml. The spores with bands at both 1.240 and 1.340 g/ml were germinable faster than the original dormant spores and thus were considered to be activated. The spores with a band at 1.240 g/ml, which were considered to be fully activated, were apparently permeabilized, with a resulting complete depletion of dipicolinic acid, partial depletion of minerals, susceptibility to lysozyme action, permeation of the gradient medium, changed structural appearance in electron micrographs of thin-sectioned spores, and partly decreased heat resistance (D100 = 453 min) compared with the original dormant spores (D100 = 760 min). However, the fully activated spores with a band at 1.240 g/ml, although devoid of dipicolinic acid, still were much more resistant than germinated spores or vegetative cells (D100 = 0.1 min). The spores with a band at 1.340 g/ml, which were considered to be partly activated, showed no evidence of permeabilization and were much more heat resistant (D100 = 1,960 min) than the original dormant spores.(ABSTRACT TRUNCATED AT 250 WORDS)     

The spectrum of spontaneous rifampin resistance mutations in the rpoB gene of Bacillus subtilis 168 spores differs from that of vegetative cells and resembles that of Mycobacterium tuberculosis

Mutations causing rifampin resistance in vegetative cells of Bacillus subtilis 168 have thus far been mapped to a rather restricted set of alterations at either Q469 or H482 within cluster I of the rpoB gene encoding the beta subunit of RNA polymerase. In this study, we demonstrated that spores of B. subtilis 168 exhibit a spectrum of spontaneous rifampin resistance mutations distinct from that of vegetative cells. In addition to the rpoB mutations Q469K, Q469R, and H482Y previously characterized in vegetative cells, we isolated a new mutation of rpoB, H482R, from vegetative cells. Additional new rifampin resistance mutations arising from spores were detected at A478N and most frequently at S487L. The S487L change is the predominant change found in rpoB mutations sequenced from rifampin-resistant clinical isolates of Mycobacterium tuberculosis. The observations are discussed in terms of the underlying differences of the DNA environment within dormant cells and vegetatively growing cells.     

Permeability of dormant spores of Bacillus subtilis to gramicidin S

Abstract Gramicidin S, dissolved in ethanol, penetrated into the inside of the dormant spores of Bacillus subtilis , had a partial inhibitory effect on l-alanine-initiated germination and completely inhibited their outgrowth and vegetative growth. The activity of particulate NADH oxidase of the antibiotic-treated dormant spores was also influenced significantly. Abnormal morphological changes were observed in germinated spores from gramicidin S-treated dormant spores. An immunoelectron microscopy method with colloidal gold-IgG complex showed that the penetration site of gramicidin S inside dormant spores was mainly the core region. These facts suggest that gramicidin S induces the damage of not only the outer membrane-spore coat complex but also the inner membrane surrounding the spore protoplast, and is able to penetrate into the core region of B. subtilis dormant spores.     

Influence of cellular differentiation on ultraviolet induced DNA damage and its repair mechanisms in B. cereus

Susceptibility to UV irradiation of B. cereus BIS-59 spores undergoing germination at various stages-dormant spores to vegetative cell stage and their ability to recover from radiation damage were studied. For a given dose of radiation, the number of spore photoproducts (SPP) formed in the DNA of dormant spores was about 5-times greater than that of thymine dimers (TT) formed in the DNA of vegetative cells. At intermediate stages of the germination cycle, there was a rapid decline in the UV radiation-induced SPP formed in DNA with a concomitant increase in the UV radiation-induced TT formed in DNA. Bacterial spores undergoing germination (up to 3 hr) in the low nutrient medium (0.3% yeast extract) displayed much higher resistance to UV radiation than those germinating in the rich nutrient medium, even though there was no discernible difference under the two incubation conditions in respect of the extent of germination and the time at which the outgrowth stage appeared (3 hr). This was due to the formation TT in the DNA of spores germinating in the low nutrient as compared to that of spores germinating in the rich-nutrient medium. In UV-irradiated dormant spores, SPP formed in the spore DNA did not disappear even after prolonged incubation in the non-germinating medium. However, when the UV-irradiated dormant spores were germinated in low or rich nutrient medium, a significant proportion of SPP in DNA was eliminated. The dormant spores incubated in either of the germinating media for 15 min and then UV-irradiated were capable of eliminating SPP (presumably by monomerization) even by incubation in a non-germinating medium and in the complete absence of protein synthesis (buffer holding recovery), thereby implying that spore-repair enzymes were activated in response to initial's germination. The acquisition of photo-reactivation ability appeared in spores subjected to germination only in the rich-nutrient medium at the outgrowth stage and required de novo synthesis of the required enzymes.     

Proteolytic activity of subcellular fractions from Streptomyces griseus no. 45-H.

Subcellular fractions were prepared from Streptomyces griseus No. 45-H at different stages of life cycle, and their proteolytic activity was examined. The highest proteolytic activity was found in the 24- and 72- h-old vegetative hyphae, the lowest in the resting spores. Spores contained about 9--30% of the proteolytic activity of vegetative cells. At the age of 16 h about 80%, at 26 h 70%, at 72 h 40%, and in spores about 60% of the proteolytic activity was particulate. The greatest part of the proteolytic activity could be inhibited by EDTA, lower levels of serine and sulfhydryl protease activities were detected in the cell-free extracts of vegetative cells.     

Structure of DNA in Spores of Bacillus cereus

The structure of DNA extracted from dormant and germinating spores of B. cereus T was investigated using circular dichroism and other methods. No significant differences between DNAs extracted from vegetative cells and from spores of various stages could be found by analyses of CD spectra, CsCl density gradient centrifugation, melting profiles and template activity. All the DNA preparations were in B conformation and had the same density (1.695), Tm (83°C) and template activity in the reaction of DNA-dependent RNA polymerase. An abnormal DNA fraction of high density which was formerly found in B. cereus spores or a stable DNA complex with protein and/or RNA was not detected in the present extracts of spores. Preliminary X-ray analyses of intact spores indicate that the structure of DNA in spores is not so different from B form.     

Response of Micromonospora echinospora (NCIMB 12744) spores to heat treatment with evidence of a heat activation phenomenon

The effects of heat treatment on spores of the actinomycete Micromonospora echinospora were investigated. The percentage of culturable spores in untreated spore stocks was found to be approximately 20%. A 60 degrees C treatment of spores in phosphate buffer for 10 min led to an approximately five-fold increase in the number of culturable units. This indicated that a large proportion of the spores were constitutively dormant. Within 10 min and in the absence of an external energy-yielding substrate, the heat treatment was found to stimulate spore respiration suggesting that endogenous storage compounds were being utilized. Heating spores at 70 degrees C shortened the time period required for activation; holding times greater than 10 min, however, resulted in a reduction of culturable cells. Classic thermal death characteristics were seen at temperatures of 80 degrees C and above with D-values of 21.43, 2.67, 0.45 and 0.09 min being recorded at 70, 80, 90 and 100 degrees C, respectively. Spores of this organism, while being weakly heat resistant in comparison with bacterial endospores, are significantly more resistant than vegetative cells.