Isolation and characterization of Bacillus megaterium mutants containing decreased levels of spore protease.

A proteolytic activity present in spores of Bacillus megaterium has previously been implicated in the initiation of hydrolysis of the A, B, and C proteins which are degraded during spore germination. Four mutants of B. megaterium containing 20 to 30% of the normal level of spore proteolytic activity have been isolated. Partial purification of the protease from wild-type spores by a reviewed procedure resulted in the resolution of spore protease activity on the A, B, and C proteins into two peaks--a major one (protease II) and a minor one (protease I). The protease mutants tested lacked active protease II. All of the mutants exhibited a decreased rate of degradation of the A, B, and C proteins during spore germination at 30 degrees C, but degradation of the proteins did occur. Degradation of the A, B, and C proteins during germination of the mutant spores was decreased neither by blockade of ATP production nor by germination at 44 degrees C. Initiation of spore germination was normal in all four mutants, and all four mutants went through outgrowth, grew, and sporulated normally in rich medium. Similarly, outgrowth of spores of two of the four mutants was normal in minimal medium at 30 degrees C. In the two mutants studied, the kinetics of loss of spore heat resistance and spore UV light resistance during germination were identical to those of wild-type spores. This indicates that the A, B, and C proteins alone are not sufficient to account for the heat or UV light resistance of the dormant spore.     

Genetics analysis of spore germination mutants of Bacillus subtilis 168: the correlation of phenotype with map location.

The isolation and characterization of 29 new germination (Ger) mutants of Bacillus subtilis 168 is described. These were classified, along with previously described mutants, into seven groups according to map location. The mutations in 26 GerA mutants mapped between cysB and thr; detailed mapping of two of these has located them very close to citG. These mutants were deficient in germination in alanine, but responded to the germinative combination of asparagine, glucose, fructose and KCl. One GerB mutant mapped on the origin-proximal side of hisA; it was normal in germination in alanine, but deficient in termination in a mixture of asparagine, glucose, fructose and KCl. Two GerC mutants were linked to lys, but were separable from a temperature-sensitive growth deficiency mapping between lys and trp. The GerC mutants had a similar germination phenotype to the GerA mutants. Three GerD mutants did not germinate in either of the above germinants or in Penassay Broth. They were located on the side of ery distal to cysA. The GerE mutant, which did not germinate in any of the three germinants, was located very close to citF and possessed an altered spore coat. The two GerF mutants were defective in germination in all three germinants and mapped on the origin proximal-side of hisA, but much closer to his than did the GerB mutant. A phosphoglycerate kinase-negative mutant altered in germination mapped between cysB and hisA (GerG). These mutants have established a minimum of seven locations important to germination, and will be useful in the development and appraisal of theories of spore germination.     

PCR detection of potentially pathogenic aeromonads in raw and cold-smoked freshwater fish

AIMS: To determine the mechanism of killing of spores of Bacillus subtilis by ortho-phthalaldehyde (OPA), an aromatic dialdehyde currently in use as an antimicrobial agent. METHODS AND RESULTS: OPA is sporicidal, although spores are much more OPA resistant than are vegetative cells. Bacillus subtilis mutants deficient in DNA repair, spore DNA protection and spore coat assembly have been used to show that (i) the coat appears to be a major component of spore OPA resistance, which is acquired late in sporulation of B. subtilis at the time of spore coat maturation, and (ii) B. subtilis spores are not killed by OPA through DNA damage but by elimination of spore germination. Furthermore, OPA-treated spores that cannot germinate are not recovered by artificial germinants or by treatment with NaOH or lysozyme. CONCLUSIONS: OPA appears to kill spores by blocking the spore germination process. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides information on the mechanism of spore resistance to, and spore killing by, the disinfectant, OPA.     

Isolation of germination mutants of Dictyostelium discoideum

A simple method to separate spores from amoebae of Dictyostelium discoideum has been devized and used to isolate spore germination mutants. A subclass of these mutants is temperature sensitive for germination and growth.     

A detailed study of gerJ mutants of Bacillus subtilis

A total of nine gerJ mutants have now been isolated in Bacillus subtilis. All are defective in their spore germination properties, being blocked at an intermediate (phase grey) stage. The dormant spores are sensitive to heating at 90 degrees C and two of the mutants (generated by transposon insertion) produce spores sensitive at 80 degrees C. The spores of these two more extreme mutants had a visibly defective cortex when studied by electron microscopy, as did some of the other mutants. During sporulation, the acquisition of spore resistance properties and the appearance of the sporulation-specific penicillin-binding protein PBP5* were delayed. A strain probably carrying a lacZ fusion to the gerJ promoter demonstrated increased expression between t2 and t4. We propose that the gerJ locus is involved in the control of one or more sporulation-specific genes.     

Proteolytic processing of the protease which initiates degradation of small, acid-soluble proteins during germination of Bacillus subtilis spores.

Degradation of small, acid-soluble spore proteins during germination of Bacillus subtilis spores is initiated by a sequence-specific protease called GPR. Western blot (immunoblot) analysis of either Bacillus megaterium or B. subtilis GPR expressed in B. subtilis showed that GPR is synthesized at about the third hour of sporulation in a precursor form and is processed to an approximately 2- to 5-kDa-smaller species 2 to 3 h later, at or slightly before the time of accumulation of dipicolinic acid by the forespore. This was found with both normal levels of expression of B. subtilis and B. megaterium GPR in B. subtilis, as well as when either protein was overexpressed up to 100-fold. The sporulation-specific processing of GPR was blocked in all spoIII, -IV, and -V mutants tested (none of which accumulated dipicolinic acid), but not in a spoVI mutant which accumulated dipicolinic acid. The amino-terminal sequences of the B. megaterium and B. subtilis GPR initially synthesized in sporulation were identical to those predicted from the coding genes' sequences. However, the processed form generated in sporulation lacked 15 (B. megaterium) or 16 (B. subtilis) amino-terminal residues. The amino acid sequence surrounding this proteolytic cleavage site was very homologous to the consensus sequence recognized and cleaved by GPR in its small, acid-soluble spore protein substrates. This observation, plus the efficient processing of overproduced GPR during sporulation, suggests that the GPR precursor may autoproteolyze itself during sporulation. During spore germination, the GPR from either species expressed in B. subtilis was further processed by removal of one additional amino-terminal amino acid (leucine), generating the mature protease which acts during spore germination.     

Role of the gerP Operon in Germination and Outgrowth of Bacillus anthracis Spores

Germination of Bacillus anthracis spores occurs when nutrients such as amino acids or purine nucleosides stimulate specific germinant receptors located in the spore inner membrane. The gerP_ABCDEF operon has been suggested to play a role in facilitating the interaction between germinants and their receptors in spores of Bacillus subtilis and Bacillus cereus. B. anthracis mutants containing deletions in each of the six genes belonging to the orthologue of the gerP_ABCDEF operon, or deletion of the entire operon, were tested for their ability to germinate. Deletion of the entire gerP operon resulted in a significant delay in germination in response to nutrient germinants. These spores eventually germinated to levels equivalent to wild-type, suggesting that an additional entry point for nutrient germinants may exist. Deletions of each individual gene resulted in a similar phenotype, with the exception of ΔgerPF, which showed no obvious defect. The removal of two additional gerPF-like orthologues was necessary to achieve the germination defect observed for the other mutants. Upon physical removal of the spore coat, the mutant lacking the full gerP operon no longer exhibited a germination defect, suggesting that the GerP proteins play a role in spore coat permeability. Additionally, each of the gerP mutants exhibited a severe defect in calcium-dipicolinic acid (Ca-DPA)–dependent germination, suggesting a role for the GerP proteins in this process. Collectively, these data implicate all GerP proteins in the early stages of spore germination.     

The Bacillus subtilis germinant receptor GerA triggers premature germination in response to morphological defects during sporulation

During sporulation in Bacillus subtilis, germinant receptors assemble in the inner membrane of the developing spore. In response to specific nutrients, these receptors trigger germination and outgrowth. In a transposon‐sequencing screen, we serendipitously discovered that loss of function mutations in the gerA receptor partially suppress the phenotypes of > 25 sporulation mutants. Most of these mutants have modest defects in the assembly of the spore protective layers that are exacerbated in the presence of a functional GerA receptor. Several lines of evidence indicate that these mutants inappropriately trigger the activation of GerA during sporulation resulting in premature germination. These findings led us to discover that up to 8% of wild‐type sporulating cells trigger premature germination during differentiation in a GerA‐dependent manner. This phenomenon was observed in domesticated and undomesticated wild‐type strains sporulating in liquid and on solid media. Our data indicate that the GerA receptor is poised on a knife's edge during spore development. We propose that this sensitized state ensures a rapid response to nutrient availability and also elicits premature germination of spores with improperly assembled protective layers resulting in the elimination of even mildly defective individuals from the population.     

Role of SpoVA proteins in release of dipicolinic acid during germination of Bacillus subtilis spores triggered by dodecylamine or lysozyme

The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of approximately 9 and a temperature optimum of 60 degrees C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl(2). Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca(2+) almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca(2+) and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca(2+)-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca(2+)-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.     

Expression of proteolytic enzymes during Dictyostelium discoideum spore germination

The specific activity of cathepsin B-like, cathepsin D-like, and leucine aminopeptidase enzymes was measured in dormant, aging, and germinating spores of wild-type and mutant Dictyostelium discoideum.The activity of leucine aminopeptidase was relatively constant during spore aging and spore germination. The level of cathepsin D-like activity was highest in young dormant spores but decreased during germination or aging.The level of cathepsin B-like activity remained constant in wild-type spores which were aged for 13 days. The dormant spores of spontaneous germination mutants initially contained low levels of cathepsin B-like activity which increased during aging. Thus, there was no correlation between the level of endogenous cathepsin B activity and the ability to be autoactivated or heat-activated. The level of cathepsin B-like activity does not have a role in the generation of energy for the swelling stage of germination. Finally, the combined level of endogenous and exogenous cathepsin B activity increased more than 20-fold during the emergence of myxamoebae suggesting that the enzyme(s) may play a role at this development stage of germination.     

Spore Dormancy Mutants of Phycomyces

Rivero, F., and Cerdá-Olmedo, E. 1994. Spore dormancy mutants of Phycomyces. Experimental Mycology 18, 221-229. The spores of the Zygomycete Phycomyces blakesleeanus are called dormant because few of them germinate when placed in a medium that sustains mycelial growth and development. Nearly all the spores germinate after activation, that is, exposure to heat or certain chemicals. We have looked for mutants whose spores would not need activation. Nine mutants formed authentic, but transient spores, which germinated spontaneously in the sporangium. Mutant mycelia had lower alcohol and aldehyde dehydrogenase activities and less glycogen than wild-type mycelia. The spontaneous germination and the metabolic alterations are attributed to the same recessive mutations. No differences were found between mutants and wild type in the cyclic AMP and fructose 2,6-bisphosphate concentrations in immature sporangia and the trehalase activity in the mycelia. In another mutant the spore primordia did not form spores, but remained viable for some time in the sporangium. The mutants were difficult to keep in the laboratory (except as lyophils); this stresses the importance of preventing spore germination in the sporangium.     

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.     

Isolation of cell wall mutants in Aspergillus nidulans by screening for hypersensitivity to Calcofluor White

As a first step toward identifying novel genes of wall metabolism in filamentous fungi, we have screened a collection of Aspergillus nidulans mutants for strains exhibiting hypersensitivity toward the chitin binding agent Calcofluor White (CFW). This strategy has been used previously to identify cell wall mutants in Saccharomyces cerevisiae. We have identified 10 mutants representing eight loci, designated calA through calH, for Calcofluor hypersensitivity. All cal mutants are impaired for sporulation at 30 C or 42 C or both, and in eight of the 10 mutations this sporulation defect shows at least partial osmotic remediability. All cal mutants show elevated sensitivity to one or more of the following agents: Caspofungin, Nikkomycin, Tunicamycin, Congo red and SDS, which are recognized wall-compromising agents or have been shown to be inhibitory to wall integrity mutants in yeast. Seven of the 10 cal mutants show swelling at elevated temperature, which in most cases is osmotically remediable. Spore swelling also can be induced at 30 C in all but one of the cal mutants by germination in the presence of one or more of the following: Caspofungin, Nikkomycin or Tunicamycin. Analysis of wall sugars showed no major changes in mutant strains. We also report that the chitin synthase inhibitor Nikkomycin induces excessive spore swelling during germination in all tested strains that have wild type cell wall metabolism (GR5, A4, A28 and AH12) at 42 C but not at 30 C. This effect mimics that of certain temperature-sensitive swollen cell (swo) mutations.     

Polysaccharide and protein degradation,germination, and virulence against mosquitoes in the entomopathogenic fungus Metarhizium anisopliae

From a genetically uniform wild-type strain of Metarhizium anisopliae pathogenic to mosquitoes, mutants were selected which were altered in the ability to degrade starch, gelatin, or milk. The mutants with enhanced starch degradation (dep), when grown on starch-containing media, proved hypervirulent toward the mosquito Culex pipiens pipiens in standard laboratory tests. Alterations in protein (gelatin or milk) degradation did not correlate with changes in virulence. The dep mutants appear to belong to the same class as mutants selected previously as hypervirulent and characterized by early spore germination. The relationship among polysaccharide degradation, early germination, and virulence is discussed.     

Characterization of Bacillus subtilis mutants with a temperature-sensitive intracellular protease.

A colony screening procedure was devised to detect Bacillus subtilis mutants containing temperature-sensitive trypsin-like intracellular protease activity. The enzyme was characterized as a non-sulfhydryl serine protease on the basis of inhibitor studies. It was also inhibited by D- or L-histidine but not by any other amino acid tested. The long-term survival at 45 degrees C of these mutants in a minimal salts medium was decreased, with rapid lysis occurring within 24 h. A D-histidine function in long-term survival and inhibition accounted for the presence of additional protease mutants among survivors of histidine auxotrophs selected for their ability to utilize D-histidine. In addition to being lysed when incubated at 45 degrees C under nongrowth conditions, all of the protease mutants had a decreased rate of protein turnover and produced spores deficient in a major low-molecular-weight spore coat polypeptide. The morphology of the undercoat layers was altered, but there was no effect on spore heat resistance or on germination. The missing spore coat polypeptide appeared to be processed from a larger precursor by cleavage to produce N-terminal histidine. A defect in this protease could account for the lack of processing and thus the absence of this polypeptide in spore coats.     

Moments of weaknesses – exploiting vulnerabilities between germination and encystment in the Phytomyxea

Attempts at management of diseases caused by protozoan plant parasitic Phytomyxea have often been ineffective. The dormant life stage is characterised by long-lived highly robust resting spores that are largely impervious to chemical treatment and environmental stress. This review explores some life stage weaknesses and highlights possible control measures associated with resting spore germination and zoospore taxis. With phytomyxid pathogens of agricultural importance, zoospore release from resting spores is stimulated by plant root exudates. On germination, the zoospores are attracted to host roots by chemoattractant components of root exudates. Both the relatively metabolically inactive resting spore and motile zoospore need to sense the chemical environment to determine the suitability of these germination stimulants or attractants respectively, before they can initiate an appropriate response. Blocking such sensing could inhibit resting spore germination or zoospore taxis. Conversely, the short life span and the vulnerability of zoospores to the environment require them to infect their host within a few hours after release. Identifying a mechanism or conditions that could synchronise resting spore germination in the absence of host plants could lead to diminished pathogen populations in the field.     

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.