Asporogenous Mutants Research Articles

Characterization of Bacillus pumilus Strains with Targeted Gene Editing for Antimicrobial Peptides and Sporulation Factor.

Due to their capacity to produce antimicrobial peptides that can prevent the growth of diseases, many Bacillus spp. are beneficial to plants. In this study, we looked into the antagonistic activity of the B. pumilus 3-19 strain and its derivatives following targeted genome editing. Two peptide genes with antibacterial action, bacilysin (bac) and bacteriocin (bact), and the sigF gene, which encodes the sigma factor of sporulation, were specifically inactivated using the CRISPR-Cas9 system in the genome of B. pumilus 3-19. Antibacterial activity against B. cereus and Pantoea brenneri decreased as a result of the inactivation of target genes in the B. pumilus 3-19 genome, with a noticeable effect against bacilysin. The growth dynamics of the culture changed when the bac, bact, and sigF genes were inactivated, and the altered strains had less proteolytic activity. An asporogenic mutant of B. pumilus 3-19 was obtained by inactivating the sigF gene. It has been proven that bacilysin plays a unique part in the development of B. pumilus 3-19's antagonistic action against soil microorganisms.

Production of asporogenous mutants of Bacillus sphaericus 2362 in continuous culture.

To report the production of asporogenous mutants (Spo-) of Bacillus sphaericus 2362 in continuous culture. Microbial culture samples were taken at 0.05 h-1 dilution rate and plated out on nutrient agar plates. Translucent colonies were obtained with vegetative morphology under phase contrast microscope. Heat resistance evaluations at different temperature settings showed that the Spo- mutants had lower heat resistance than the Spo+ wild type. Western blots analyses carried out on both wild type and the mutants indicated the presence of binary protein toxins of 42 and 51 kDa in both. Bioassays carried out on the wild type and the Spo- mutants against mosquitoes showed the mutants to be 100-fold less toxic in comparison to the wild type. Existence and production of asporogenous mutants of Bacillus sphaericus 2362 in continuous culture at low dilution rates is demonstrated by this study. The organism's ability to produce toxins appears to be significantly reduced by the mutational process. The production of asporogenous mutants had not been reported previously among strains of Bacillus sphaericus. The present report on the toxigenic capability of asporogenous mutants also raises the possibility of using continuous culture to significantly improve the productivity of toxin production in future.

In vivo and In vitro function of the intracellular proteolytic apparatus in nongrowing bacillus megaterium under heat stress

In Bacillus megaterium sporulating at 35 degreesC, up to 90% of 10-min pulse-labeled proteins were degraded. Degradation proceeded in two waves. Short-lived proteins, i.e., intrinsically labile proteins and proteins made short-lived because of starvation, were mostly degraded during the reversible sporulation phase. Their amount corresponded to 20% or slightly more during 2 h. The second wave of protein degradation, which followed during the irreversible sporulation phase at 35 degreesC, increased the amount of total degradable pulse-labeled proteins to about 90%. This wave was absent in the isogenic asporogenic mutant 27-36 or in the wild strain, whose sporulation was inhibited by increased temperature. The proportion of degradable proteins was thus reduced to less than 40% in the asporogenic mutant incubated at 35 degreesC and to 46% in the wild strain whose sporulation was suppressed by the temperature of 47 degreesC. Unlike sporulating cells, these cells were thus capable of degrading short-lived and denatured proteins, but were not able to degrade most of other proteins. The in vitro protein degradation was substantially enhanced by increasing the Ca2+ concentration, suggesting a role of Ca2+-dependent proteinase(s) in the process.

Isolation of an asporogenic (spoOA) protective antigen-producing strain of Bacillus anthracis.

We found that Congo red agar allows identification of sporulation-deficient Bacillus anthracis. Using Congo red agar, we isolated an asporogenic derivative of the protective antigen-producing strain B. anthracis delta Sterne-1(pPA102). Polymerase chain reaction and Southern hybridization analyses of DNA from the asporogenic mutant revealed that a deletion was present in spoOA, an essential gene for the initiation of sporulation. The deletion also encompassed the spoIVB homologue and a portion of the recN homologue. The avirulent spoOA strain delta Sterne-1(pPA102)CR4 is suitable for the safe production of protective antigen without endospore contamination of the vaccine production facility.

Isolation of an asporogenic (spoOA) protective antigen-producing strain of Bacillus anthracis

We found that Congo red agar allows identification of sporulation-deficient Bacillus anthracis. Using Congo red agar, we isolated an asporogenic derivative of the protective antigen-producing strain B. anthracis deltaSterne-1(pPA102). Polymerase chain reaction and Southern hybridization analyses of DNA from the asporogenic mutant revealed that a deletion was present in spoOA, an essential gene for the initiation of sporulation. The deletion also encompassed the spoIVB homologue and a portion of the recN homologue. The avirulent spoOA strain deltaSterne-1(pPA102)CR4 is suitable for the safe production of protective antigen without endospore contamination of the vaccine production facility.Key words: Bacillus anthracis, protective antigen, spoOA, vaccine, Congo red.

Regulation of maltose metabolism in stationary phase cultures of an asporogenous mutant of Bacillus licheniformis

An asporogenous strain of Bacillus licheniformis accumulated maltose by an energy dependent transport mechanism during an extended stationary phase. Maltose transport was sensitive to the effects of the uncoupler tetrachlorosalicylanide (TCS), and was also inhibited by glucose. Maltose stimulated synthesis of a p-nitrophenyl-a-D-glucoside-hydrolysing enzyme (pNPGase) in log phase and in stationary phase cells. In the presence of glucose this induction was inhibited. Glucose was used preferentially to maltose in stationary phase cells. The uptake of maltose from the medium, and the synthesis of pNPGase, were immediately and completely inhibited in the presence of glucose. These results are consistent with a mechanism of inducer exclusion mediating the repressive effect of glucose upon pNPGase synthesis in stationary phase cells. Catabolite repression of α-amylase synthesis by glucose was also demonstrated in late stationary phase mutant cells.

Mosquito larvicidal binary toxin localization in recombinant bacteria: identification using an immunogold labelling technique

An immunogold labelling technique has been developed and used to characterize the localization of mosquito larvicidal binary toxin in Bacillus sphaericus 2362, asporogenous mutants of Bacillus sphaericus 2362, Bacillus megaterium pWH1520 TK and E. coli DH5α pAR5. Different patterns of binary toxin concentration occur between the wild type and recombinant strains which may be related to their effective toxicities. © Rapid Science Ltd. 1998

Analysis of the relationship between the decrease in pH and accumulation of 3-phosphoglyceric acid in developing forespores of Bacillus species.

Analysis of the pH decrease and 3-phosphoglyceric acid (3PGA) accumulation in the forespore compartment of sporulating cells of Bacillus subtilis showed that the pH decrease of 1 to 1.2 units at approximately 4 h of sporulation preceded 3PGA accumulation, as observed previously in B. megaterium. These data, as well as analysis of the forespore pH decrease in asporogenous mutants of B. subtilis, indicated that sigma G-dependent forespore transcription, but not sigma K-dependent mother cell transcription, is required for the forespore pH decrease. Further analysis of these asporogenous mutants showed an excellent correlation between the forespore pH decrease and the forespore's accumulation of 3PGA. These latter results are consistent with our previous suggestion that the decrease in forespore pH results in greatly decreased activity of phosphoglycerate mutase in the forespore, which in turn leads to 3PGA accumulation. In further support of this suggestion, we found that (i) elevating the pH of developing forespores of B. megaterium resulted in rapid utilization of the forespore's 3PGA depot and (ii) increasing forespore levels of PGM approximately 10-fold in B. subtilis resulted in a large decrease in the spore's depot of 3PGA. The B. subtilis strain with a high phosphoglycerate mutase level sporulated, and the spores germinated and went through outgrowth normally, indicating that forespore accumulation of a large 3PGA depot is not essential for these processes.

Expression of parasporal crystal protein (δ—endotoxin) gene(s) ofBacillus thuringiensis var.israelensis in sporogenic and asporogenic mutant strains ofBacillus cereus

The expression of parasporal crystal protein (δ-endotoxin) coding gene(s) ofBacillus thuringlensis var.israelensis and its association, if any, with sporulation was studied in sporogenicBacillus cereus and its asporogenic mutant strains. Five asporogenous mutants ofBacillus cereus blocked at different stages of sporulation, were isolated from a streptomycin-resistant strain, The transconjugants isolated from the plasmid transfer experiments betweenBacillus thuringiensis var.israelensis and streptomycin resistantBacillus cereus and its asporogenous mutants, showed larvicidal activity. The crystal protein gene(s) are, therefore, expressed both in sporulating and in non-sporulating mutant strains ofBacillus cereus suggesting that the expression of crystal protein gene(s), is independent of sporulation specific functions inBacillus cereus.

Crystals of two mutants ofBacillus thuringiensis show increased toxicity against larvae ofSpodoptera littoralis

Crystals of two asporogenous mutants ofBacillus thuringiensis var.kurstaki strain HD-1 obtained following treatment with ozone and N-methyl-N′-nitro-N-nitrosoguanidine showed increased toxicity against larvae ofSpodoptera littoralis when compared to the wild-type crystal.

A rapid and convenient screening technique for developmental pathway mutants ofSaccharomyces cerevisiae

A brief exposure to iodine vapour was used to screen for mutants of the yeast Saccharomyces cerevisiae affected in development. Besides obtaining a large number of asporogenous mutants, two novel mutations were identified that permitted germination of spores to occur in conditions (sporulation medium) in which the wild-type would not germinate. These two mutations were named gdr1 and gdr2 for germination derepressed. Both alter nutritional control of germination, but not the kinetics of germination in glucose-containing medium.

A rapid and convenient screening technique for developmental pathway mutants of Saccharomyces cerevisiae

A brief exposure to iodine vapour was used to screen for mutants of the yeast Saccharomyces cerevisiae affected in development. Besides obtaining a large number of asporogenous mutants, two novel mutations were identified that permitted germination of spores to occur in conditions (sporulation medium) in which the wild-type would not germinate. These two mutations were named gdr1 and gdr2 for germination derepressed. Both alter nutritional control of germination, but not the kinetics of germination in glucose-containing medium.

A model for asymmetric septum formation during sporulation in Bacillus subtilis

Many differentiation processes in both prokaryotes and eukaryotes begin with an asymmetric division, producing 'daughter' cells that differ in size and developmental fate. This is particularly obvious in the well-studied prokaryotic life cycles of Caulobacter and Bacillus. In no system, however, is the mechanism of asymmetric division understood. Here I propose a model for the mechanism of asymmetric division during sporulation in Bacillus subtilis. The model explains both the timing and asymmetric localization of spore-septum formation. It also explains the morphological phenotypes of various asporogenous (spo) mutants.

Characterization of an asporogenous mutant ofStreptomyces aureofaciens with increased resistance to several aminoglycoside antibiotics

An asporogenous spontaneous mutant of Streptomyces aureofaciens named ASR1 was selected on streptomycin gradient plates. The mutant is very stable and differs in ultrastructure and morphology, it is prototrophic but it lost the ability to grow well on soybean extract medium and produces one-tenth tetracyclines of the parent. The ASR1 mutant has a 3-4-fold increased resistance to streptomycin and is cross-resistant to other aminoglycosides. Comparison of the protein profiles from both strains on SDS gels revealed a very low expression of a 29.5 kDa protein in the ASR1 mutant which is overexpressed in both vegetative cells and spores of the parental strain.

Stability of solvent production by Clostridium acetobutylicum in continuous culture: strain differences

Woolley, R.C. & Morris, J.G. 1990. Stability of solvent production by Clostridium acetobutylicum in continuous culture: strain differences. Journal of Applied Bacteriology69, 718–728.Several strains of Clostridium acetobutylicum, including strains ATCC 824 and DSM 1731, continue to produce solvents during prolonged periods of chemostat culture. In such cultures, dominance is established by asporogenous mutant(s) that retain the ability to produce solvents. Strain NCIB 8052 (which is not identical with ATCC 824) behaved differently in that its chemostat cultures invariably became acidogenic due to ultimate selection of asporogenous mutant(s) unable to produce solvents, incapable of synthesizing granulose, and demonstrating enhanced sensitivity to environmental stresses of various types. These mutants spontaneously reverted, at a low but measurable frequency, to the parental phenotype, indicating thai their multiple loss of capacities was the pleiotropic consequence of a lesion in some global regulatory gene. Their resemblance to previously described cls mutants of strain P262 and the possible nature of the affected regulatory gene are discussed. A simple tetrazolium blue plate assay procedure is described which allows visual discrimination between solvent‐producing and non‐solventogenic colonies of Cl. ocetobutylicum.

Production and characterization of a thermostable protease produced by an asporogenous mutant ofBacillus stearothermophilus

An asporogenous mutant ofBacillus stearothermophilus (TPM-8) which produces 4-fold higher levels of a thermostable neutral protease than does wild-type strain 308-1 was obtained by mutagenesis with ethyl methanesulfonate. The protease produced by both the mutant and wild-type strain is a metalloprotease requiring Zn2+ and Ca2+ for activity and thermostability, respectively. It has a temperature optimum of 80°C at pH 7.0 and is highly thermostable, retaining 60% of its activity after 60 min at 85°C. The properties of the enzyme are similar to those of thermolysin.

Bacillus subtilis expressing a haemolysin gene from Listeria monocytogenes can grow in mammalian cells

Intracellular parasites can be classified into those that reside within a host vacuole and those which grow directly in the host cytoplasm. Members of the latter group, which includes Rickettsia, Shigellae, Trypanosoma cruzi, and Listeria monocytogenes, possess haemolytic activity associated with the ability to enter the host cytoplasm. Therefore mutants of L. monocytogenes lacking a pore-forming haemolysin, listeriolysin O, do not escape from the endosomal compartment and consequently fail to become established in the cytoplasm. To examine the role of listeriolysin O, we cloned the structural gene for the L. monocytogenes haemolysin, hlyA, into an asporogenic mutant of Bacillus subtilis under the control of an IPTG-inducible promoter. After being internalized by the macrophage-like cell line J774, haemolytic B. subtilis disrupted the phagosomal membrane and grew rapidly within the macrophage cytoplasm. These results show that a single gene product is sufficient to convert a common soil bacterium into a parasite that can grow in the cytoplasm of a mammalian cell.

A new chemically-defined medium for RAC-certified and other strains of Bacillus subtilis

Conventional chemically-defined media were found to be inadequate for extensive batch culture growth of an asporogenous mutant of Bacillus subtilis certified as an HV-1 host for recombinant DNA. We developed a chemically-defined medium for rapid and extensive growth of one such strain, BGSC-1S53. The medium contains no components requiring separate sterilization. It is also useful for the prototrophic 168 strain (from which 1S53 was derived), a triple auxotroph of 168, and a highly debilitated (HV-2) 168 strain (BGSC 1S76) upon addition of the specific growth factors required by these strains. Excellent growth was also obtained with W-23, a Bacillus subtilis strain unrelated to the 168 series.

Identification of the promoter for a spore coat protein gene in Bacillus subtilis and studies on the regulation of its induction at a late stage of sporulation.

The cotA (pig) gene of Bacillus subtilis encodes a 65,000 Mr protein that is a component of the spore coat and is responsible for the brown pigment characteristic of colonies in which cells are undergoing sporulation. To study developmental regulation of the cotA gene, we identified its promoter and studied its transcription in a large number of mutants blocked at various stages of sporulation and germination. Deletion analysis showed that induction and efficient transcription of cotA required DNA sequences extending no more than 55 base pairs (bp) upstream (and no more than 130 bp downstream) from the 5' terminus of cotA mRNA. Transcription from the cotA promoter was found to be switched on at approximately the time (4 to 5 h after the onset of sporulation) of spore coat synthesis and deposition. Strikingly, this transcription was substantially inhibited in almost all asporogenous mutants blocked prior to the developmental stage (V) of spore coat formation. cotA transcription was also impaired in several stage V mutants but not in other stage V mutants or in mutants blocked in germination. The germination mutant gerE caused a several-fold overexpression of cotA. The dependence of cotA expression on so many genes required at early to intermediate stages of sporulation suggests that transcription of this spore coat gene is somehow coupled (directly or through several intervening steps) to a morphological or physiological feature(s) of the developing sporangium.

Bacillus sphaericus asporogenous mutants: morphology, protein pattern and larvicidal activity

Asporogenous mutants from Bacillus sphaericus strains 2297 and 1593-4, blocked at different stages of the sporulation process, were isolated. Two mutants (2297 Aspo30A and 2297 Aspo34) which were blocked early in sporulation did not possess any crystalline inclusions and were poorly toxic to Culex pipiens mosquito larvae. Other mutants (2297 Aspo115, 2297 Aspo24 and 1593-4 Aspo12) which were blocked at later stages synthesized crystal-like inclusions next to the forespores, and were highly toxic to mosquito larvae. Electrophoretic protein analysis of alkali extracts from mutants and wild type strains confirmed the absence of toxic crystal-related proteins in early-blocked mutants and their presence in later ones. Western blots with antisera directed against the crystal proteins confirmed those observations.