Interaction of Apurinic/Apyrimidinic Endonucleases Nfo and ExoA with the DNA Integrity Scanning Protein DisA in the Processing of Oxidative DNA Damage during Bacillus subtilis Spore Outgrowth

Oxidative stress-induced damage, including 8-oxo-guanine and apurinic/apyrimidinic (AP) DNA lesions, were detected in dormant and outgrowing Bacillus subtilis spores lacking the AP endonucleases Nfo and ExoA. Spores of the Δnfo exoA strain exhibited slightly slowed germination and greatly slowed outgrowth that drastically slowed the spores'' return to vegetative growth. A null mutation in the disA gene, encoding a DNA integrity scanning protein (DisA), suppressed this phenotype, as spores lacking Nfo, ExoA, and DisA exhibited germination and outgrowth kinetics very similar to those of wild-type spores. Overexpression of DisA also restored the slow germination and outgrowth phenotype to nfo exoA disA spores. A disA-lacZ fusion was expressed during sporulation but not in the forespore compartment. However, disA-lacZ was expressed during spore germination/outgrowth, as was a DisA-green fluorescent protein (GFP) fusion protein. Fluorescence microscopy revealed that, as previously shown in sporulating cells, DisA-GFP formed discrete globular foci that colocalized with the nucleoid of germinating and outgrowing spores and remained located primarily in a single cell during early vegetative growth. Finally, the slow-outgrowth phenotype of nfo exoA spores was accompanied by a delay in DNA synthesis to repair AP and 8-oxo-guanine lesions, and these effects were suppressed following disA disruption. We postulate that a DisA-dependent checkpoint arrests DNA replication during B. subtilis spore outgrowth until the germinating spore''s genome is free of damage.     

Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant,Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T₁) and reached a maximum after the completion of CaDPA release (T_release) and spore cortex lysis (T_lysis).     

Study of Bacillus subtilis Endospores in Soil by Use of a Modified Endospore Stain

The Schaeffer-Fulton endospore stain was modified so that it would stain Bacillus subtilis endospores in soil smears. The modified stain differentiated among dormant spores, spores undergoing activation, and spores which had germinated but had not yet shown outgrowth. These differentiations were seen for spores in soil and for pure spore preparations in the laboratory. This stain was used to show reversible B. subtilis spore activation promoted by an Ensifer adhaerens-like indigenous bacterium in soil and by pure cultures of E. adhaerens added to spores in the laboratory. Under the specific conditions in the laboratory, spore germination did not proceed beyond the activation stage, and relatively little change occurred in the numbers of both E. adhaerens and B. subtilis. This was also true in soil, although some germination with destruction of spores and vegetative cells did occur if the soil had been nutritionally enriched by preincubation with incorporated ground alfalfa.     

Analysis of the Dynamics of a Bacillus subtilis Spore Germination Protein Complex during Spore Germination and Outgrowth

Germination of Bacillus subtilis spores is normally initiated when nutrients from the environment interact with germinant receptors (GRs) in the spores'' inner membrane (IM), in which most of the lipids are immobile. GRs and another germination protein, GerD, colocalize in the IM of dormant spores in a small focus termed the “germinosome,” and this colocalization or focus formation is dependent upon GerD, which is also essential for rapid GR-dependent spore germination. To determine the fate of the germinosome and germination proteins during spore germination and outgrowth, we employed differential interference microscopy and epifluorescence microscopy to track germinating spores with fluorescent fusions to germination proteins and used Western blot analyses to measure germination protein levels. We found that after initiation of spore germination, the germinosome foci ultimately changed into larger disperse patterns, with ≥75% of spore populations displaying this pattern in spores germinated for 1 h, although >80% of spores germinated for 30 min retained the germinosome foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins essential for dipicolinic acid release changed minimally during this period, although GerD levels decreased ∼50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by ∼2-fold decreases in GerD levels or other factors, such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds.     

Role of the Nfo and ExoA apurinic/apyrimidinic endonucleases in repair of DNA damage during outgrowth of Bacillus subtilis spores

Germination and outgrowth are critical steps for returning Bacillus subtilis spores to life. However, oxidative stress due to full hydration of the spore core during germination and activation of metabolism in spore outgrowth may generate oxidative DNA damage that in many species is processed by apurinic/apyrimidinic (AP) endonucleases. B. subtilis spores possess two AP endonucleases, Nfo and ExoA; the outgrowth of spores lacking both of these enzymes was slowed, and the spores had an elevated mutation frequency, suggesting that these enzymes repair DNA lesions induced by oxidative stress during spore germination and outgrowth. Addition of H₂O₂ also slowed the outgrowth of nfo exoA spores and increased the mutation frequency, and nfo and exoA mutations slowed the outgrowth of spores deficient in either RecA, nucleotide excision repair (NER), or the DNA-protective α/β-type small acid-soluble spore proteins (SASP). These results suggest that α/β-type SASP protect DNA of germinating spores against damage that can be repaired by Nfo and ExoA, which is generated either spontaneously or promoted by addition of H₂O₂. The contribution of RecA and Nfo/ExoA was similar to but greater than that of NER in repair of DNA damage generated during spore germination and outgrowth. However, nfo and exoA mutations increased the spontaneous mutation frequencies of outgrown spores lacking uvrA or recA to about the same extent, suggesting that DNA lesions generated during spore germination and outgrowth are processed by Nfo/ExoA in combination with NER and/or RecA. These results suggest that Nfo/ExoA, RecA, the NER system, and α/β-type SASP all contribute to the repair of and/or protection against oxidative damage of DNA in germinating and outgrowing spores.     

Bacillus subtilis Cells Lacking Penicillin-Binding Protein 1 Require Increased Levels of Divalent Cations for Growth

Bacillus subtilis strains lacking penicillin-binding protein 1 (PBP1), encoded by ponA, required greater amounts of Mg²⁺ or Ca²⁺ for vegetative growth or spore outgrowth than the wild-type strain and strains lacking other high-molecular-weight (HMW) PBPs. Growth of ponA cells in a medium low in Mg²⁺ also resulted in greatly increased cell bending compared to wild-type cells or cells lacking other HMW PBPs. The addition of high levels of Mg²⁺ to growth media eliminated these phenotypes of a ponA mutant. In contrast to the effects of divalent cations, NaCl did not restore ponA cell growth in a divalent-cation-deficient medium. Surprisingly, wild-type cells swelled and then lysed during both vegetative growth and spore outgrowth when 500 mM NaCl was included in a divalent-cation-deficient medium. Again, Mg²⁺ addition was sufficient to allow normal vegetative growth and spore outgrowth of both wild-type and ponA cells in a medium with 500 mM NaCl. These studies demonstrate that (i) while HMW PBPs possess largely redundant functions in rich medium, when divalent cations are limiting, PBP1 is required for cell growth and spore outgrowth; and (ii) high levels of NaCl induce cell lysis in media deficient in divalent cations during both vegetative growth and spore outgrowth.     

Mechanism of the Heat Sensitization of Bacillus subtilis Spores by Ethidium Bromide

Pretreatment with ethidium bromide (5 μg/ml) followed by a water wash had no effect on unheated Bacillus subtilis spores, but the viability of these spores after heating was much lower than that of similarly heated spores exposed to water alone. The fate of water- or ethidium bromide-treated spores, unheated or heated, was followed by allowing them to germinate and outgrow in a minimal or a complex liquid medium. Spores exposed to ethidium bromide and then heated (85°C, 10 min) exhibited a developmental block during germination and outgrowth. Many of them were blocked at the stage when the bacterium emerged from the germinated spore. When 0.35 μg of ethidium bromide per ml was added to heated spores in the germination-growth medium, the outgrowth of heated spores was inhibited to the same extent as were pretreated spores. Ethidium bromide acted in the first hour of germination of heated spores since addition after this time was ineffective in inhibiting recovery events. Repair of heat-damaged spore DNA was detected during the first 2 h of germination. The addition of ethidium bromide (final concentration, 0.35 μg/ml) inhibited DNA repair during early outgrowth. Increased sensitivity of spores to heat after pretreatment with sublethal concentrations of ethidium bromide was due to the inhibition of the repair of heat-damaged DNA.     

Genetically controlled removal of "spore photoproduct" from deoxyribonucleic acid of ultraviolet-irradiated Bacillus subtilis spores

Previous genetic analysis indicated that at least two genes determine the ultraviolet (UV) sensitivity of Bacillus subtilis spores. The present study shows that these genes independently control two distinguishable processes for removing UV-induced spore photoproduct (5-thyminyl-5,6-dihydrothymine, or TDHT) from spore deoxyribonucleic acid. The first, is a spore repair mechanism by which TDHT is removed rapidly without appearing in acid-soluble form. This mechanism, which is demonstrated in both UV-resistant and excision-deficient strains, operates to a certain extent during germination without requiring vegetative growth. The second, demonstrated in a mutant which lacks the first mechanism, removes TDHT relatively slowly and only if germinated spores are allowed to develop toward vegetative cells. The latter mechanism appears identical to excision-resynthesis repair, since the mutation abolishing it renders the irradiated vegetative cells incapable of removing cyclobutane-type pyrimidine dimers. Blocking either one of these mechanisms only slightly affects the UV sensitivity of spores, but blocking both prevents TDHT removal and gives high UV sensitivity.     

The GerW Protein Is Not Involved in the Germination of Spores of Bacillus Species

Germination of dormant spores of Bacillus species is initiated when nutrient germinants bind to germinant receptors in spores’ inner membrane and this interaction triggers the release of dipicolinic acid and cations from the spore core and their replacement by water. Bacillus subtilis spores contain three functional germinant receptors encoded by the gerA, gerB, and gerK operons. The GerA germinant receptor alone triggers germination with L-valine or L-alanine, and the GerB and GerK germinant receptors together trigger germination with a mixture of L-asparagine, D-glucose, D-fructose and KCl (AGFK). Recently, it was reported that the B. subtilis gerW gene is expressed only during sporulation in developing spores, and that GerW is essential for L-alanine germination of B. subtilis spores but not for germination with AGFK. However, we now find that loss of the B. subtilis gerW gene had no significant effects on: i) rates of spore germination with L-alanine; ii) spores’ levels of germination proteins including GerA germinant receptor subunits; iii) AGFK germination; iv) spore germination by germinant receptor-independent pathways; and v) outgrowth of germinated spores. Studies in Bacillus megaterium did find that gerW was expressed in the developing spore during sporulation, and in a temperature-dependent manner. However, disruption of gerW again had no effect on the germination of B. megaterium spores, whether germination was triggered via germinant receptor-dependent or germinant receptor-independent pathways.     

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.     

High Salinity Alters the Germination Behavior of Bacillus subtilis Spores with Nutrient and Nonnutrient Germinants

The effect of high NaCl concentrations on nutrient and nonnutrient germination of Bacillus subtilis spores was systematically investigated. Under all conditions, increasing NaCl concentrations caused increasing, albeit reversible, inhibition of germination. High salinity delayed and increased the heterogeneity of germination initiation, slowed the germination kinetics of individual spores and the whole spore population, and decreased the overall germination efficiency, as observed by a variety of different analytical techniques. Germination triggered by nutrients which interact with different germinant receptors (GRs) was affected differently by NaCl, suggesting that GRs are targets of NaCl inhibition. However, NaCl also inhibited GR-independent germination, suggesting that there is at least one additional target for NaCl inhibition. Strikingly, a portion of the spore population could initiate germination with l-alanine even at NaCl concentrations near saturation (∼5.4 M), suggesting that spores lack a salt-sensing system preventing them from germinating in a hostile high-salinity environment. Spores that initiated germination at very high NaCl concentrations excreted their large depot of Ca²⁺-pyridine-2,6-dicarboxylic acid and lost their heat resistance, but they remained in a phase-gray state in the phase-contrast microscope, suggesting that there was incomplete germination. However, some metabolic activity could be detected at up to 4.8 M NaCl. Overall, high salinity seems to exert complex effects on spore germination and outgrowth whose detailed elucidation in future investigations could give valuable insights on these processes in general.     

Bacillus thermoamylovorans Spores with Very-High-Level Heat Resistance Germinate Poorly in Rich Medium despite the Presence of ger Clusters but Efficiently upon Exposure to Calcium-Dipicolinic Acid

High-level heat resistance of spores of Bacillus thermoamylovorans poses challenges to the food industry, as industrial sterilization processes may not inactivate such spores, resulting in food spoilage upon germination and outgrowth. In this study, the germination and heat resistance properties of spores of four food-spoiling isolates were determined. Flow cytometry counts of spores were much higher than their counts on rich medium (maximum, 5%). Microscopic analysis revealed inefficient nutrient-induced germination of spores of all four isolates despite the presence of most known germination-related genes, including two operons encoding nutrient germinant receptors (GRs), in their genomes. In contrast, exposure to nonnutrient germinant calcium-dipicolinic acid (Ca-DPA) resulted in efficient (50 to 98%) spore germination. All four strains harbored cwlJ and gerQ genes, which are known to be essential for Ca-DPA-induced germination in Bacillus subtilis. When determining spore survival upon heating, low viable counts can be due to spore inactivation and an inability to germinate. To dissect these two phenomena, the recoveries of spores upon heat treatment were determined on plates with and without preexposure to Ca-DPA. The high-level heat resistance of spores as observed in this study (D_120°C, 1.9 ± 0.2 and 1.3 ± 0.1 min; z value, 12.2 ± 1.8°C) is in line with survival of sterilization processes in the food industry. The recovery of B. thermoamylovorans spores can be improved via nonnutrient germination, thereby avoiding gross underestimation of their levels in food ingredients.     

Contrasting Effects of Heat Treatment and Incubation Temperature on Germination and Outgrowth of Individual Spores of Nonproteolytic Clostridium botulinum Bacteria

In this study, we determined the effects of incubation temperature and prior heat treatment on the lag-phase kinetics of individual spores of nonproteolytic Clostridium botulinum Eklund 17B. The times to germination (t_germ), one mature cell (t_C1), and two mature cells (t_C2) were measured for individual unheated spores incubated at 8, 10, 15, or 22°C and used to calculate the t_germ, the outgrowth time (t_C1 − t_germ), and the first doubling time (t_C2 − t_C1). Measurements were also made at 22°C of spores that had previously been heated at 80°C for 20 s. For unheated spores, outgrowth made a greater contribution to the duration and variability of the lag phase than germination. Decreasing incubation temperature affected germination less than outgrowth; thus, the proportion of lag associated with germination was less at lower incubation temperatures. Heat treatment at 80°C for 20 s increased the median germination time of surviving spores 16-fold and greatly increased the variability of spore germination times. The shape of the lag-time (t_C1) and outgrowth (t_C1 − t_germ) distributions were the same for unheated spores, but heat treatment altered the shape of the lag-time distribution, so it was no longer homogeneous with the outgrowth distribution. Although heat treatment mainly extended germination, there is also evidence of damage to systems required for outgrowth. However, this damage was quickly repaired and was not evident by the time the cells started to double. The results presented here combined with previous findings show that the stage of lag most affected, and the extent of any effect in terms of duration or variability, differs with both historical treatment and the growth conditions.Clostridium botulinum is a group of four physiologically and phylogenetically distinct anaerobic spore-forming bacteria (known as groups I, II, III, and IV) that produce the highly toxic botulinum neurotoxin (12). The severity of the intoxication, botulism, ensures considerable effort is directed at preventing the growth of this pathogen in food. Nonproteolytic (group II) C. botulinum is one of the two groups most frequently associated with food-borne botulism. It forms heat-resistant spores and can germinate, grow, and produce toxin at 3°C (8); thus, nonproteolytic C. botulinum is a particular concern in mild heat-treated chilled foods (16, 17).Spores formed by pathogens such as C. botulinum are a significant food safety issue since they are able to resist many of the processes, such as cooking, used to kill vegetative cells. Understanding the transformation from a dormant spore to active vegetative cells is an important part of quantifying the risk associated with such organisms. Considerable effort has been targeted at measuring and relating the kinetic responses of populations of C. botulinum to environmental conditions and such data have been used to create predictive models, for example, ComBase (www.combase.cc). Such approaches have made a considerable contribution to ensuring food safety but problems with using population based predictions may arise when an initial inoculum is very small or additional information beyond point values is required. Spores typically contaminate foods at low concentrations so that growth of C. botulinum, when it occurs, is likely to initiate from just a few spores. In these circumstances the distribution of times to growth in packs will reflect the heterogeneity of times to growth from the contaminating individual spores. There is an intrinsic variability between individual spores within a population, and the relationship between population lag and individual lag is complex. Consequently, individual lag times cannot be predicted from population measurements (3). Knowledge of the underlying distribution would allow greater refinement of risk assessments.The lag period between a spore being exposed to conditions suitable for growth and the start of exponential growth will reflect the combined times of germination, emergence, elongation, and first cell division. Currently, very little is known about the variability and duration of these stages and any relationships between them. Measuring the kinetics of spore germination is usually achieved by measuring a population to identify time to percent completion. Such germination curves represent the summation of responses by individual spores. Some authors have measured the biovariability associated with individual spores, but most studies have examined only germination (4-7, 11, 22) and not subsequent outgrowth. More recently, we have used phase-contrast microscopy and image analysis to follow individual spores of nonproteolytic C. botulinum from dormancy, through germination and emergence, to cell division (21, 23). These experiments showed there is very little, or no, relationship between the time spent in each stage by individual spores. We have now extended this work to determine distributions of times for different stages in lag phase as affected by heat treatment and incubation temperature.     

Survival of Microorganisms in a Simulated Martian Environment: I. Bacillus subtilis var. globigii

Survival of Bacillus subtilis var. globigii in a simulated Martian environment was demonstrated. Previous contact with the simulated Martian soil or atmosphere reduced germination or outgrowth of unheated spores, or both. Inoculation into simulated Martian soil and then flushing with a simulated Martian atmosphere were lethal to both vegetative cells and spores. After one diurnal temperature cycle (26 to -60 C), the majority of of cells present were spores. No further effect of the diurnal cycle on survival was noted in any of the experimental samples.     

The influence of gramicidin S on hydrophobicity of germinating Bacillus brevis spores

Gramicidin S is known to prolong the outgrowth stage of spore germination in the producing culture. Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to cell-surface hydrophobicity and germination of their spores. Parental spores were hydrophobic as determined by adhesion to hexadecane, whereas mutant spores showed no affinity to hexadecane. Addition of gramicidin S to mutant spores resulted in a high cell surface hydrophobicity and a delay in germination outgrowth. The hydrophobicity of parental spores was retained throughout most of the germination period. Hydrophobicity was lost as outgrowing spores entered into the stage of vegetative growth. The data indicate that gramicidin S is responsible for the hydrophobicity of B. brevis spores. It is suggested that in making spores hydrophobic, the antibiotic plays a role in concentrating the spores at interfaces where there is a higher probability of finding nutrients for germination and growth.Abbreviation GS Gramicidin S     

Slow Leakage of Ca-Dipicolinic Acid from Individual Bacillus Spores during Initiation of Spore Germination

When exposed to nutrient or nonnutrient germinants, individual Bacillus spores can return to life through germination followed by outgrowth. Laser tweezers, Raman spectroscopy, and either differential interference contrast or phase-contrast microscopy were used to analyze the slow dipicolinic acid (DPA) leakage (normally ∼20% of spore DPA) from individual spores that takes place prior to the lag time, T_lag, when spores begin rapid release of remaining DPA. Major conclusions from this work with Bacillus subtilis spores were as follows: (i) slow DPA leakage from wild-type spores germinating with nutrients did not begin immediately after nutrient exposure but only at a later heterogeneous time T₁; (ii) the period of slow DPA leakage (ΔT_leakage = T_lag − T₁) was heterogeneous among individual spores, although the amount of DPA released in this period was relatively constant; (iii) increases in germination temperature significantly decreased T₁ times but increased values of ΔT_leakage; (iv) upon germination with l-valine for 10 min followed by addition of d-alanine to block further germination, all germinated spores had T₁ times of less than 10 min, suggesting that T₁ is the time when spores become committed to germinate; (v) elevated levels of SpoVA proteins involved in DPA movement in spore germination decreased T₁ and T_lag times but not the amount of DPA released in ΔT_leakage; (vi) lack of the cortex-lytic enzyme CwlJ increased DPA leakage during germination due to longer ΔT_leakage times in which more DPA was released; and (vii) there was slow DPA leakage early in germination of B. subtilis spores by the nonnutrients CaDPA and dodecylamine and in nutrient germination of Bacillus cereus and Bacillus megaterium spores. Overall, these findings have identified and characterized a new early event in Bacillus spore germination.     

Impact of Sorbic Acid on Germination and Outgrowth Heterogeneity of Bacillus cereus ATCC 14579 Spores

Population heterogeneity complicates the predictability of the outgrowth kinetics of individual spores. Flow cytometry sorting and monitoring of the germination and outgrowth of single dormant spores allowed the quantification of acid-induced spore population heterogeneity at pH 5.5 and in the presence of sorbic acid. This showed that germination efficiency was not a good predictor for heterogeneity in final outgrowth.     

Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination.

During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination of gpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major alpha/beta-type SASP. These data suggest that (i) alpha/beta-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the alpha/beta-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of alpha/beta-type SASP on the chromosome is detrimental to normal spore outgrowth.     

Ordered Synthesis of Proteins During Outgrowth of Spores of Bacillus cereus

The onset of macromolecular synthesis in activated spores of Bacillus cereus occurs under conditions in which the amino acids and nucleotides to be used for building proteins and nucleic acids must be derived only from stored pools and turnover of macromolecules of the spore. Upon addition of the factors required to initiate germination, (14)C-uracil is incorporated with a lag of 30 to 60 sec; (14)C-amino acids, with a lag of 3 to 4 min. The progression of protein synthesis during germination has been studied, and the results suggest three phases of development of the protein synthetic pattern of these germinating spores. The initial synthesis which occurs during the early part of germination is limited to only a few proteins. When the initiated spores are put in a medium containing a complete set of growth requirements and outgrowth ensues, the cells synthesize a large number of different proteins so that the distribution of radioactivity into different fractions appears to be a continuous function. At a later time during outgrowth, the distribution of synthetic rates among the different proteins becomes more representative of that found during vegetative growth.