Heterologous expression of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> gene of glutamyl endopeptidase in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains defective in regulatory proteins

Expression of the gene of glutamyl endopeptidase from Bacillus intermedius (gseBi) cloned on the plasmid pV has been studied in Bacillus subtilis recombinant strains with mutations of the regulatory proteins involved in sporogenesis and spore germination. It has been established that inactivation of the regulatory protein Spo0A involved in sporulation initiation resulted in a decrease in the expression of the gseBi gene by 65% on average. A mutation in the gene of the sensor histidine kinase kinA had no effect on the biosynthesis of the enzyme. Inactivation of Ger proteins regulating bacterial spore germination resulted in a 1.5–5-fold decrease in glutamyl endopeptidase activity. It has been concluded that expression of the B. intermedius glutamyl endopeptidase gene from plasmid pV in recombinant cells of B. subtilis is under impaired control by the regulatory system of Spo0F/Spo0A phosphorelay, which participates in sporulation initiation. The regulatory Ger proteins responsible for spore germination also affect expression of the gene of this enzyme.     

Heavy-Metal Stress and Developmental Patterns of Arbuscular Mycorrhizal Fungi

The rate of global deposition of Cd, Pb, and Zn has decreased over the past few decades, but heavy metals already in the soil may be mobilized by local and global changes in soil conditions and exert toxic effects on soil microorganisms. We examined in vitro effects of Cd, Pb, and Zn on critical life stages in metal-sensitive ecotypes of arbuscular mycorrhizal (AM) fungi, including spore germination, presymbiotic hyphal extension, presymbiotic sporulation, symbiotic extraradical mycelium expansion, and symbiotic sporulation. Despite long-term culturing under the same low-metal conditions, two species, Glomus etunicatum and Glomus intraradices, had different levels of sensitivity to metal stress. G. etunicatum was more sensitive to all three metals than was G. intraradices. A unique response of increased presymbiotic hyphal extension occurred in G. intraradices exposed to Cd and Pb. Presymbiotic hyphae of G. intraradices formed presymbiotic spores, whose initiation was more affected by heavy metals than was presymbiotic hyphal extension. In G. intraradices grown in compartmentalized habitats with only a portion of the extraradical mycelium exposed to metal stress, inhibitory effects of elevated metal concentrations on symbiotic mycelial expansion and symbiotic sporulation were limited to the metal-enriched compartment. Symbiotic sporulation was more sensitive to metal exposure than symbiotic mycelium expansion. Patterns exhibited by G. intraradices spore germination, presymbiotic hyphal extension, symbiotic extraradical mycelium expansion, and sporulation under elevated metal concentrations suggest that AM fungi may be able to survive in heavy metal-contaminated environments by using a metal avoidance strategy.     

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.     

Heterologous expression of Bacillus intermedius gene of glutamyl endopeptidase in Bacillus subtilis strains defective in regulatory proteins

Expression of the gene of glutamyl endopeptidase from Bacillus intermedius (gseBi) cloned on the plasmid pV has been studied in Bacillus subtilis recombinant strains with mutations of the regulatory proteins involved in sporogenesis and spore germination. It has been established that inactivation of the regulatory protein Spo0A involved in sporulation initiation resulted in a decrease in the expression of the gseBi gene by 65% on average. A mutation in the gene of the sensor histidine kinase kinA had no effect on the biosynthesis of the enzyme. Inactivation of Ger proteins regulating bacterial spore germination resulted in a 1.5-5-fold decrease in glutamyl endopeptidase activity. It has been concluded that expression of the B. intermedius glutamyl endopeptidase gene from plasmid pV in recombinant cells of B. subtilis is under impaired control by the regulatory system of Spo0F/Spo0A phosphorelay, which participates in sporulation initiation. The regulatory Ger proteins responsible for spore germination also affect expression of the gene of this enzyme.     

Spatiotemporally regulated proteolysis to dissect the role of vegetative proteins during Bacillus subtilis sporulation: cell‐specific requirement of σH and σA

Sporulation in Bacillus subtilis is a paradigm of bacterial development, which involves the interaction between a larger mother cell and a smaller forespore. The mother cell and the forespore activate different genetic programs, leading to the production of sporulation‐specific proteins. A critical gap in our understanding of sporulation is how vegetative proteins, made before sporulation initiation, contribute to spore formation. Here we present a system, spatiotemporally regulated proteolysis (STRP), which enables the rapid, developmentally regulated degradation of target proteins, thereby providing a suitable method to dissect the cell‐ and developmental stage‐specific role of vegetative proteins. STRP has been used to dissect the role of two major vegetative sigma factors, σ^H and σ^A, during sporulation. The results suggest that σ^H is only required in predivisional cells, where it is essential for sporulation initiation, but that it is dispensable during subsequent steps of spore formation. However, evidence has been provided that σ^A plays different roles in the mother cell, where it replenishes housekeeping functions, and in the forespore, where it plays an unexpected role in promoting spore germination and outgrowth. Altogether, the results demonstrate that STRP has the potential to provide a comprehensive molecular dissection of every stage of sporulation, germination and outgrowth.     

Antifungal Activity of Thymol against Aspergillus awamori and Botrytis aclada Isolated from Stored Onion Bulbs

The antifungal activity of thymol against Aspergillus awamori F23 and Botrytis aclada F15 in onions was examined through direct treatment with amended media and gaseous treatment with I-plates (plastic plates containing central partitions). The protective and curative control efficacy of thymol was examined 24 h before and after the inoculation of onion bulbs with the fungal isolates. Mycelial growth, sporulation, and spore germination of the isolates were inhibited on potato dextrose agar amended with various concentrations of thymol or acetic acid (positive control). Overall, thymol produced a stronger inhibitory effect on the mycelial growth and development of the isolates than acetic acid. Following gaseous treatment in I-plates, mycelial growth, sporulation, and spore germination of the isolates were inhibited at higher concentrations of thymol or acetic acid; however, acetic acid showed a little effect on the sporulation and spore germination of the isolates. Following the treatment of onion bulbs with 1000 mg L⁻¹ of thymol 24 h before and after fungal inoculation, lesion diameter was greatly reduced compared with that following treatment with 0.5% ethanol (solvent control). Onion bulbs sprayed with thymol 24 h before fungal inoculation generally showed reduced lesion diameters by isolate F23 but not in isolate F15 compared with those sprayed 24 h after fungal inoculation. Collectively, thymol effectively inhibited the growth and development of A. awamori and B. aclada on amended media and in I-plates. In addition, spraying or fumigation of thymol is more desirable for effectively controlling these postharvest fungal pathogens during long-term storage conditions.     

Genome-Wide Transcriptional Profiling of Clostridium perfringens SM101 during Sporulation Extends the Core of Putative Sporulation Genes and Genes Determining Spore Properties and Germination Characteristics

The formation of bacterial spores is a highly regulated process and the ultimate properties of the spores are determined during sporulation and subsequent maturation. A wide variety of genes that are expressed during sporulation determine spore properties such as resistance to heat and other adverse environmental conditions, dormancy and germination responses. In this study we characterized the sporulation phases of C. perfringens enterotoxic strain SM101 based on morphological characteristics, biomass accumulation (OD₆₀₀), the total viable counts of cells plus spores, the viable count of heat resistant spores alone, the pH of the supernatant, enterotoxin production and dipicolinic acid accumulation. Subsequently, whole-genome expression profiling during key phases of the sporulation process was performed using DNA microarrays, and genes were clustered based on their time-course expression profiles during sporulation. The majority of previously characterized C. perfringens germination genes showed upregulated expression profiles in time during sporulation and belonged to two main clusters of genes. These clusters with up-regulated genes contained a large number of C. perfringens genes which are homologs of Bacillus genes with roles in sporulation and germination; this study therefore suggests that those homologs are functional in C. perfringens. A comprehensive homology search revealed that approximately half of the upregulated genes in the two clusters are conserved within a broad range of sporeforming Firmicutes. Another 30% of upregulated genes in the two clusters were found only in Clostridium species, while the remaining 20% appeared to be specific for C. perfringens. These newly identified genes may add to the repertoire of genes with roles in sporulation and determining spore properties including germination behavior. Their exact roles remain to be elucidated in future studies.     

Sporulation temperature affects initiation of germination and inactivation by high hydrostatic pressure of Bacillus cereus

The influence of sporulation temperature (20, 30 and 37 °C) on the heat resistance and initiation of germination and inactivation by high pressure on Bacillus cereus ATCC 14579 spores was investigated. Spores sporulated at 37 °C were the most heat-resistant. However, spores sporulated at 20 °C were more resistant to the initiation of germination and inactivation by high pressure. Spores were more sensitive to pressure at higher treatment temperatures. At 25 °C, there was an optimum pressure (250 MPa) for the initiation of germination for the three suspensions; at higher temperatures an increase of pressure up to 690 MPa caused progressively more germination. Resistance to the germinability and inactivation by high pressure of the spore population was distributed heterogeneously. Semilogarithmic curves of the ungerminated and survival fraction of B. cereus spores were concave. The resistant fraction of the spore population was lower at higher treatment temperatures. At 60 °C after 30 s of treatment at 690 MPa almost 5 log cycles of the population of B. cereus sporulated at 20 °C was germinated, and more than 7 log cycles of the population of B. cereus sporulated at 30 and 37 °C. The same treatment inactivated 4, 6 and 7 log cycles of the population of B. cereus sporulated at 20, 30 and 37 °C, respectively.     

苏云金芽孢杆菌gerA操纵子在芽孢萌发中的作用

芽孢萌发的营养诱导剂通过与特异的萌发受体结合激活下游的萌发过程,从而使芽孢经过一系列的遗传变化及生化反应恢复营养生长.从苏云金芽孢杆菌(Bacillus thuringiensis)中克隆到一个与枯草芽孢杆菌(Bacillus subtilis)gerA操纵子和蜡状芽孢杆菌(Bacillus cereus)gerR操纵子同源的gerA操纵子.苏云金芽孢杆菌gerA操纵子含有3个开放读码框:gerAA、gerAC和gerAB,该操纵子在产孢起始3个小时后开始转录.gerA的破坏阻断了L-丙氨酸诱导的芽孢萌发并且延迟了肌苷诱导的萌发.在L-丙氨酸诱导芽孢萌发的过程中D-环丝氨酸能够提高芽孢的萌发率.     

Patterning of development in Dictyostelium discoideum: factors regulating growth, differentiation, spore dormancy, and germination.

Cellular communication dictates all stages of growth and development in the cellular slime molds. Dictyostelium discoideum utilizes a number of signal molecules for cell-to-cell communication, including growth and density factors, cAMP, ammonia, differentiation-inducing factor, discadenine, and spore autoactivator. A source and sink model is presented in which the assimilation of ammonia plays a major role in determining cell fate and pattern formation. This model emphasizes a recycling of ammonia by prespore cells, the accumulation of free hydrophilic and neutral amino acids, and their incorporation into proteins associated with sporulation and (or) germination. If spore cAMP signalling is regulated by the relative concentrations of discadenine and autoactivator, and its disruption triggers the initiation of the spore germination cascade, then the accumulation of intracellular cAMP may be necessary for both sporulation and dormancy maintenance.     

Changes in growth patterns and intracellular calcium concentrations in Aspergillus awamori treated with amphotericin B

Growth patterns and intracellular Ca²⁺ concentrations in the mutant strain Aspergillus awamori 66A containing a recombinant aequorin gene were studied in the presence of a permeabilizing fungicidal agent amphotericin B. The cell response, i.e., changes in the growth and development of the fungus (initiation of spore germination, mycelial growth, and intensity of sporulation) was dose-dependent. Low concentrations of amphotericin B (2.5 μM) stimulated spore germination: the number of germinating spores was 2–3 times higher than in the control (without the fungicide). At higher amphotericin concentrations (20 μM) spore germination was inhibited. Amphotericin B had a dose-dependent effect on mycelial growth and sporulation intensity on solid Vogel medium. Intracellular Ca²⁺ concentrations in the presence of amphotericin B were investigated using the luminescence of the photoprotein aequorin. High concentrations of amphotericin B (10 and 20 μM) were shown to cause an instantaneous increase in Ca²⁺ concentrations compared to the control and lower amphotericin concentration (2.5 μM). Ca²⁺ concentrations remained elevated throughout the experiment and correlated with the inhibition of mycelial growth and development.     

Effects of Ekalux Ec-25 on akinete germination and sporulation ofPithophora kewensis

The effect of the organophosphorus insecticide Ekalux Ec-25 (quinalphos) was studied on akinete germination, and sporulation of the green algaPithophora kewensis (Cladophorales). Initiation of akinete germination was delayed by 1–3 d with 0.025–0.1% concentrations of the pesticide used. The percentage of akinete germination was found to be markedly affected by the treatment with insecticide, except with the concentration of 0.25% Ekalux Ec-25, where percent germination was observed to be higher than in the control. Generally, the time taken for the initiation of akinete formation increased and percent sporulation decreased with the increase of concentrations from 0.025 to 0.1% of Ekalux Ec-25.     

Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.     

Effects of overexpression of nutrient receptors on germination of spores of Bacillus subtilis

The rates of germination of Bacillus subtilis spores with L-alanine were increased markedly, in particular at low L-alanine concentrations, by overexpression of the tricistronic gerA operon that encodes the spore's germinant receptor for L-alanine but not by overexpression of gerA operon homologs encoding receptors for other germinants. However, spores with elevated levels of the GerA proteins did not germinate more rapidly in a mixture of asparagine, glucose, fructose, and K(+) (AGFK), a germinant combination that requires the participation of at least the germinant receptors encoded by the tricistronic gerB and gerK operons. Overexpression of the gerB or gerK operon or both the gerB and gerK operons also did not stimulate spore germination in AGFK. Overexpression of a mutant gerB operon, termed gerB*, that encodes a receptor allowing spore germination in response to either D-alanine or L-asparagine also caused faster spore germination with these germinants, again with the largest enhancement of spore germination rates at lower germinant concentrations. However, the magnitudes of the increases in the germination rates with D-alanine or L-asparagine in spores overexpressing gerB* were well below the increases in the spore's levels of the GerBA protein. Germination of gerB* spores with D-alanine or L-asparagine did not require participation of the products of the gerK operon, but germination with these agents was decreased markedly in spores also overexpressing gerA. These findings suggest that (i) increases in the levels of germinant receptors that respond to single germinants can increase spore germination rates significantly; (ii) there is some maximum rate of spore germination above which stimulation of GerA operon receptors alone will not further increase the rate of spore germination, as action of some protein other than the germinant receptors can become rate limiting; (iii) while previous work has shown that the wild-type GerB and GerK receptors interact in some fashion to cause spore germination in AGFK, there also appears to be an additional component required for AGFK-triggered spore germination; (iv) activation of the GerB receptor with D-alanine or L-asparagine can trigger spore germination independently of the GerK receptor; and (v) it is likely that the different germinant receptors interact directly and/or compete with each other for some additional component needed for initiation of spore germination. We also found that very high levels of overexpression of the gerA or gerK operon (but not the gerB or gerB* operon) in the forespore blocked sporulation shortly after the engulfment stage, although sporulation appeared normal with the lower levels of gerA or gerK overexpression that were used to generate spores for analysis of rates of germination.     

Influence of certain environmental factors on spore germination and spore differentiation inPithophora oedogonia

Of the light- and dark-stored spores ofPithophora oedogonia for over a period of one and half years, the latter exhibited complete failure of germination under controlled culture conditions. pH 8, blue light and 1 klx intensity of white light proved to be ideal conditions as they resulted in optimum germination of akinetes. pH 5, green light and 0.25 klx light intensity delayed the initiation of sporulation and also reduced percentage sporulation. All filaments died before sporulation in pH 4.     

Levels of oxidized and reduced pyridine nucleotides in dormant spores and during growth, sporulation, and spore germination of Bacillus megaterium.

Dormant spores of Bacillus megaterium contained no detectable reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) despite significant levels of the oxidized forms of these nucleotides (NAD and NADP). During the first minutes of spore germination there was rapid accumulation of NADH and NADPH. However, this accumulation followed the fall in optical density that is characteristic of the initiation of spore germination. Accumulation of NADH and NADPH early in germination was not blocked by fluoride or cyanide, and it occurred even when germination was carried out in the absence of an exogenous source of reducing power. In addition to pyridine nucleotide reduction, de novo synthesis also began early in germination as the pyridine nucleotide levels increased to those found in growing cells. Midlog-phase cells grown in several different media had 20 to 35 times as much total pyridine nucleotide as did dormant spores. However, as growth and sporulation proceeded, the NADH plus NAD level fell four- to fivefold whereas the NADPH plus NADP level fell by a lesser amount. From min 10 of spore germination until midway through sporulation the value for the ratio of NADH/NAD is about 0.1 (0.03 to 0.18) while the ratio of NADPH/ANDP is about 1.4 (0.3 to 2.4). Comparison of these ratios in log-phase versus stationary phase (sporulation) growth in all three growth media tested did not reveal any common pattern of changes.     

酚类物质对两种潜伏炭疽菌生长和繁殖的影响

通过测定丹宁、间苯二酚、邻苯二酚、绿原酸和咖啡因,作者对潜伏侵染在香蕉果实中的芭蕉炭疽菌Colletotrichum musae(Berk & Curt)Arx和芒果果实中的胶孢炭疽菌C.gloeosporioides Penz.的分生孢子萌发,附着胞和分生孢子的形成,以及菌丝体生长的影响进行了研究。结果表明,这几种酚类物质,在一定浓度下,可抑制两种炭疽菌的生长和发育,其中邻苯二酚对芭蕉炭疽菌的作用浓度最低,而间苯二酚对胶孢炭疽菌的作用浓度最低,丹宁则对两种菌的作用浓度最高,在一定浓度下影响着附着胞的形成。     

Production and characterization of pure Clostridium spore suspensions

Aims:  A general protocol was derived for optimizing the production of pure, high concentration Clostridium endospore suspensions.
Methods and Results:  Two sporulation methods were developed that yielded high concentrations of notably pure Clostridium sporogenes , C. hungatei and C. GSA-1 (Greenland ice core isolate) spore suspensions (10 ml of 10⁹ spores ml⁻¹ with >99% purity each). Each method was derived by evaluating combinations of three sporulation conditions, including freeze drying of inocula, heat shock treatment of cultures, and subsequent incubation at suboptimal temperatures that yielded the highest percentage of sporulation. Pure spore suspensions were characterized in terms of dipicolinic acid content, culturability, decimal reduction time ( D ) value for heat inactivation (100°C) and hydrophobicity.
Conclusions:  While some Clostridium species produce a high percentage of spores with heat shock treatment and suboptimal temperature incubation, other species require the additional step of freeze drying the inocula to achieve a high percentage of sporulation.
Significance and Impact of the Study:  Pure Clostridium spore suspensions are required for investigating species of medical and environmental importance. Defining the conditions for optimal spore production also provides insight into the underlying mechanisms of Clostridium sporulation.     

Role of YpeB in Cortex Hydrolysis during Germination of Bacillus anthracis Spores

The infectious agent of the disease anthrax is the spore of Bacillus anthracis. Bacterial spores are extremely resistant to environmental stresses, which greatly hinders spore decontamination efforts. The spore cortex, a thick layer of modified peptidoglycan, contributes to spore dormancy and resistance by maintaining the low water content of the spore core. The cortex is degraded by germination-specific lytic enzymes (GSLEs) during spore germination, rendering the cells vulnerable to common disinfection techniques. This study investigates the relationship between SleB, a GSLE in B. anthracis, and YpeB, a protein necessary for SleB stability and function. The results indicate that ΔsleB and ΔypeB spores exhibit similar germination phenotypes and that the two proteins have a strict codependency for their incorporation into the dormant spore. In the absence of its partner protein, SleB or YpeB is proteolytically degraded soon after expression during sporulation, rather than escaping the developing spore. The three PepSY domains of YpeB were examined for their roles in the interaction with SleB. YpeB truncation mutants illustrate the necessity of a region beyond the first PepSY domain for SleB stability. Furthermore, site-directed mutagenesis of highly conserved residues within the PepSY domains resulted in germination defects corresponding to reduced levels of both SleB and YpeB in the mutant spores. These results identify residues involved in the stability of both proteins and reiterate their codependent relationship. It is hoped that the study of GSLEs and interacting proteins will lead to the use of GSLEs as targets for efficient activation of spore germination and facilitation of spore cleanup.