The molecular mechanism of protoplasmic incompatibility and its relationship to the formation of protoperithecia in Podospora anserina.

In Podospora anserina, protoplasmic incompatibility due to interactions between non-allelic genes was suppressed by the effect of mutations in two modifier genes, mod-I and mod-2. It is shown that mod-I and mod-2 are involved in the production of three specific proteins, a phenoloxidase and two previously identified proteases (Bégueret & Bernet 1973 a) which are associated with the phenomenon of protoplasmic disintegration. These enzymes, whose messengers are normallly latent during vegetative growth, appear at this stage of the life cycle only as a consequence of incompatible gene interactions. The mode-I and mod-2 genes and each of the five incompatibility loci involved in non-allelic incompatibility systems also participate in the formation of the protoperithecia. This pleiotropic effect suggests that protoplasmic incompatibility is a deviation in the normal physiological processes of protoperithecial formation.     

Incompatibility in the fungus Podospora anserina. Are the mutations abolishing the incompatibility reaction ribosomal mutations?

Summary In the Ascomycete Podospora anserina the incompatibility reaction due to the interaction of non allelic genes exhibits some sensitivity to the antibiotic dihydrostreptomycin as well as to high levels of Magnesium. This incompatibility reaction can be suppressed or made more sensitive to the Magnesium or dihydrostreptomycin effect by mutations in the same genes mod1 and mod2. Properties of mutant strains suggest that mod1 and mod2 are ribosomal genes whose products seem to regulate, in a positive way for the first gene and in a negative way for the second gene, the translation of specific messengers, especially that of some proteolytic enzymes.     

Proteolytic activities during growth and aging in the fungus Podospora anserina: Effect of specific mutations

In Podospora anserina five proteolytic enzymes were characterized by chromatographic procedures. Three of these (proteases A, B and C) were found in the cell extracts of growing cultures and the other two (proteases III and IV) were revealed by studies on protoplasmic incompatibility. During growth, only protease C, an acidic enzyme, was active in crude extracts. From the stationary and the poststationary stages this activity decreased and finally disappeared, whereas a neutral serine protease (activity B) became active in crude extracts. A close relationship was observed between the proteolytic activity of the culture filtrates and the intracellular protease(s) concomitantly active in the crude extracts. None of the proteases associated with protoplasmic incompatibility was detected, both in the extra- and intracellular spaces. Qualitative variations in the proteolytic activities during stationary and post-stationary stages depended on the presence of specific genes and mutations: the mod C mutation suppressing protoplasmic incompatibility, inhibits the progressive decrease of protease C and, furthermore, the presence of non allelic incompatibility genes have for consequence the substitution of serine protease B by serine protease A during the poststationary stage.     

Secretion processing and activation of Erwinia chrysanthemi proteases

E chrysanthemi, a phytopathogenic enterobacterium, secretes several enzymes into the medium such as pectinases cellulases and proteases. It also produces 3 distinct and antigenically related extracellular proteases. The proteases secretion pathway seems to be distinct from that of the other extracellular enzymes since pleiotropic mutants impaired in cellulase and pectinase secretion are unimpaired in protease secretion. E chrysanthemi proteases B and C secretion occurs without an N-terminal signal peptide and is dependent upon specific secretion functions which are encoded by genes adjacent to the protease structural genes. This secretion pathway might be analogous to the alpha-hemolysin secretion pathway in E coli. Protection against intracellular proteolytic activity is achieved by 2 distinct mechanisms: the proteases are synthesized as inactive precursors with an N-terminal extension of 15 aminoacids (protease B) and 17 aminoacids (protease C) absent in the mature active extracellular enzymes; an intracellular specific protease inhibitor is produced by some E chrysanthemi strains.     

Heteromorphism for Heterokaryon Incompatibility Genes in Natural Populations of NEUROSPORA CRASSA

Five Neurospora crassa isolates from each of three sites in Louisiana were compared for genotype at five heterokaryon incompatibility (het) loci. The comparisons were made using duplications (partial diploids), based on the fact that duplications heterozygous for het loci have strikingly abnormal phenotypes which greatly facilitate the study of such genes. Duplications were synthesized in crosses between the wild strains (normal chromosome sequence) and testers of defined het genotype and having duplication-producing chromosome rearrangements. Crosses segregating for phenotypes characteristic of duplications heterozygous for het loci indicated allelic differences between testers and wild strains for specific het genes. Whenever a wild strain differed from a tester for a specific het locus, but another wild strain did not, the two wild strains could be inferred to differ from each other.—No two isolates from any site were heterokaryon compatible (of identical het genotype), despite the fact that all isolates from each of two sites occurred within several meters of each other. Heteromorphism was found for all five genes studied at one site, four genes at another site, and three at another. Intra- and interpopulation differences between strains were approximately the same.—Confirmation is also provided that two het genes originally detected in duplications are in fact heterokaryon incompatibility loci.     

In Silico Prediction of Mutant HIV-1 Proteases Cleaving a Target Sequence

HIV-1 protease represents an appealing system for directed enzyme re-design, since it has various different endogenous targets, a relatively simple structure and it is well studied. Recently Chaudhury and Gray (Structure (2009) 17: 1636–1648) published a computational algorithm to discern the specificity determining residues of HIV-1 protease. In this paper we present two computational tools aimed at re-designing HIV-1 protease, derived from the algorithm of Chaudhuri and Gray. First, we present an energy-only based methodology to discriminate cleavable and non cleavable peptides for HIV-1 proteases, both wild type and mutant. Secondly, we show an algorithm we developed to predict mutant HIV-1 proteases capable of cleaving a new target substrate peptide, different from the natural targets of HIV-1 protease. The obtained in silico mutant enzymes were analyzed in terms of cleavability and specificity towards the target peptide using the energy-only methodology. We found two mutant proteases as best candidates for specificity and cleavability towards the target sequence.     

Isolation and characterization of protease overproducing mutants of Trichoderma harzianum

Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes is considered as the main mechanism involved in the antagonistic process. Strain Trichoderma harzianum T334 is a potential biocontrol agent against plant pathogenic fungi with the ability to produce low levels of proteases constitutively. To improve its fungal antagonistic capacity, mutagenetic program was undertaken for the construction of protease overproducing derivates. The mutant strains were obtained by means of UV-irradiation and were selected for p-fluorophenyl-alanine resistance or altered colony morphology. It was revealed by means of specific chromogenic protease substrates that both trypsin-like and chymotrypsin-like protease secretion was elevated in most of the mutant strains. The profiles of isoenzymes were different between the mutants and the wild-type strain, when examined by gel filtration chromatography. Certain mutants proved to be better antagonists against plant pathogens in in vitro antagonism experiments. This study suggests the possibility of using mutants with improved constitutive extracellular protease secretion against plant pathogenic fungi.     

Protoplasmic Incompatibility: Possible Involvement of Proteolytic Enzymes

THE failure to form heterocaryons between non-isogenic strains in fungi, which has been reported for many species, results from protoplasmic disintegration that quickly follows hyphal fusion. Genetic control of this heterogenic incompatibility has been investigated especially in the Ascomycetes Neurospora crassa¹ and Podospora anserina². Cellular (or protoplasmic) incompatibility always arises from a very specific interaction between two genes. Allelic mechanisms, when antagonistic genes are allelic, are found in the two species. In Podospora anserina, however, three incompatibility mechanisms, c/d, c/e and r/v, involve genes of unlike loci^3,4.     

Thermosensitive, extracellular neutral proteases in Bacillus subtilis: isolation, characterization, and genetics.

Two mutants (NT02 and NT17), each producing a thermosensitive neutral protease, were isolated from Bacillus subtilis NP58, a transformant which acquired the property of hyperproduction of neutral protease from Bacillus natto IAM 1212. The neutral proteases produced by these two mutants were partially purified and enzymologically characterized. The two mutant neutral proteases displayed increased thermosensitivity as well as altered pH optima compared with those of the NP58 enzyme. In addition, the hydrolytic activity of the thermosensitive neutral proteases on synthetic peptide substrates was found to be extremely different. These results strongly suggest that the site of mutation in each of the temperature-sensitive strains is located within the structural gene for neutral protease (nprE). Previous studies indicated the existence of a specific regulator gene (nprR) in addition to the structural gene for neutral protease. Phage PBS1-mediated transduction and deoxyribonucleic acid-mediated transformation studies with the parental and mutant strains suggest that the chromosomal order of these genes is recA-pyrA-nprR-nprE-fruB-metC. Moreover, the results of these genetic analyses imply that the mutations to thermosensitivity are located proximate to each other within the nprE gene.     

Suppressor Mutants of Neurospora Crassa That Tolerate Allelic Differences at Single or at Multiple Heterokaryon Incompatibility Loci

Allelic differences at any one of at least 11 heterokaryon incompatibility (het) loci in Neurospora crassa trigger an incompatibility response: localized cell death at sites of hyphal anastomosis. We have isolated spontaneous and insertional suppressor mutants that are heterokaryon-compatible in spite of allelic differences at one or at several het loci. Some intra- and extragenic mutants tolerated allelic differences only at single het loci. Multi-tolerant spontaneous mutants were isolated by selecting simultaneously for tolerance of differences at het-c, -d and -e, or at each of these plus mating-type. Some suppressor mutants were specific for only one allele at the affected het locus; others suppressed both alleles. Insertional mutations were isolated from banks of transformants, each having a plasmid integrated into a random position in the chromosome. One mutant tolerated allelic differences at het-d. A homologous cosmid from a Neurospora genomic bank complemented the mutant phenotype. A second insertional inactivation mutant was tolerant of het-c differences. Inactivation of the wild-type locus corresponding to the integration site was accomplished by repeat-induced point mutation (RIP). The RIP progeny, like the original mutant, were tolerant of differences at het-c. It may be possible to use such suppressor mutants as universal donors of hypovirulence in pathogenic fungi.     

The location and analysis of two heterokaryon incompatibility (het) loci in strains of Aspergillus nidulans

The heterokaryon incompatibility system in Aspergillus nidulans has been investigated by parasexual methods. The use of complementary auxotrophs with a repeated serial transfer method or with a protoplast fusion technique has enabled heterokaryons and diploid strains to be recovered from heterokaryon incompatible combinations of strains. The effects of allelic interaction at heterokaryon incompatibility (het) loci on the morphologies of the heterokaryon and diploid colonies isolated are described. Parasexual analyses conducted among strains belonging to the heterokaryon compatibility groups, h-cGl and h-cB, and the two recombinant compatibility classes, have located the hetA and hetB genes to linkage groups V and VI respectively.     

Two allelic genes responsible for vegetative incompatibility in the fungus Podospora anserina are not essential for cell viability

Summary Vegetative incompatibility is a lethal reaction that destroys the heterokaryotic cells formed by the fusion of hyphae of non-isogenic strains in many fungi. That incompatibility is genetically determined is well known but the function of the genes triggering this rapid cell death is not. The two allelic incompatibility genes, s and S, of the fungus Podospora anserina were characterized. Both encode 30 kDa polypeptides, which differ by 14 amino acids between the two genes. These two proteins are responsible for the incompatibility reaction that results when cells containing s and S genes fuse. Inactivation of the s or S gene by disruption suppresses incompatibility but does not affect the growth or the sexual cycle of the mutant strains. This suggests that these incompatibility genes have no essential function in the life cycle of the fungus.     

Proteases from Bacillus: a new insight into the mechanism of action for rhizobacterial suppression of nematode populations

AIMS: The aim of this study was to investigate the role of proteases in Bacillus spp. of rhizobacteria in suppressing nematode populations and to understand their mechanism of action. METHODS AND RESULTS: Rhizobacteria with nematicidal activity were isolated from soil samples of five root knot nematode-infested farms. Among these strains, nematotoxicities of Bacillus strains were intensively analysed. Further assays of nematicidal toxins from Bacillus sp. strain RH219 indicated an extracellular cuticle-degrading protease Apr219 was an important pathogenic factor. The Apr219 shared high similarity with previously reported cuticle-degrading proteases from Brevibacillus laterosporus strain G4 and Bacillus sp. B16 (Bacillus nematocida). The cuticle-degrading protease genes were also amplified from four other nematicidal Bacillus strains isolated from the rhizosphere. In addition to Apr219, a neutral protease Npr219 from Bacillus sp. RH219 was also investigated for activity against nematodes. CONCLUSIONS: The wide distribution of cuticle-degrading proteases in Bacillus strains with nematicidal activity suggested that these enzymes likely play an important role in bacteria-nematode-plant-environment interactions and that they may serve as important nematicidal factors in balancing nematode populations in the soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Increased understanding of the mechanism of action of Bacillus spp. against nematodes could potentially enhance the value of these species as effective nematicidal agents and develop new biological control strategies.     

Regulation of Neutral Protease Productivity in Bacillus subtilis: Transformation of High Protease Productivity

A transformable strain of Bacillus subtilis 6160, a derivative of B. subtilis 168, produces three kinds of casein hydrolytic enzymes (alkaline protease, neutral protease, and esterase) in a culture medium. B. natto IAM 1212 produces 15 to 20 times as much total proteolytic activity as does B. subtilis. Extracellular proteases produced by the two strains were separated into each enzyme fraction by diethylaminoethyl-Sephadex A-50 column chromatography. The difference in the total protease activities of extracellular proteases between the two strains was due to the amount of neutral protease. The ratios of neutral protease activity to alkaline protease activity (N/A) were 1.1 in B. subtilis 6160 and 13.0 in B. natto IAM 1212. Enzymological and immunological properties of alkaline protease and neutral protease obtained from the two strains were quite similar or identical, respectively. Specific activities measured by an immunological analysis of the two neutral proteases against casein were also equal. A genetic character of high protease productivity in B. natto IAM 1212 was transferred to B. subtilis 6160 by the deoxyribonucleic acid-mediated transformation. Among 73 transformants that acquired high protease productivity, 69 produced a higher amount of neutral protease and the ratios of N/A were changed to 15 to 60. Three other strains were transformed in the productivity of neutral protease and alpha-amylase simultaneously, and one showed considerable change in the production of alkaline protease and neutral protease. The specific activities (casein hydrolytic activities/enzyme molecules) of neutral proteases from the representative four transformants were equal to those of the two parental strains. These results suggested the presence of a specific gene(s) that participated in the productivity of neutral protease in B. subtilis.     

Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome

Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases.     

A nonself recognition gene complex in Neurospora crassa

Nonself recognition is exemplified in the fungal kingdom by the regulation of cell fusion events between genetically different individuals (heterokaryosis). The het-6 locus is one of approximately 10 loci that control heterokaryon incompatibility during vegetative growth of N. crassa. Previously, it was found that het-6-associated incompatibility in Oak Ridge (OR) strains involves two contiguous genes, het-6 and un-24. The OR allele of either gene causes "strong" incompatibility (cell death) when transformed into Panama (PA)-background strains. Several remarkable features of the locus include the nature of these incompatibility genes (het-6 is a member of a repetitive gene family and un-24 also encodes the large subunit of ribonucleotide reductase) and the observation that un-24 and het-6 are in severe linkage disequilibrium. Here, we identify "weak" (slow, aberrant growth) incompatibility activities by un-24PA and het-6PA when transformed separately into OR strains, whereas together they exhibit an additive, strong effect. We synthesized strains with the new allelic combinations un-24PA het-6OR and un-24OR het-6PA, which are not found in nature. These strains grow normally and have distinct nonself recognition capabilities but may have reduced fitness. Comparing the Oak Ridge and Panama het-6 regions revealed a paracentric inversion, the architecture of which provides insights into the evolution of the un-24-het-6 gene complex.     

A comparative genetic study of serologically distinct Haemophilus influenzae type 1 immunoglobulin A1 proteases.

The bacterial immunoglobulin A1 (IgA1) proteases are putative virulence factors secreted by a number of human pathogens capable of penetrating the mucosal barrier. Among Haemophilus influenzae strains, the IgA1 protease is found in several allelic forms with different serological neutralizing properties. A comparison of the primary structures of four serologically distinct H. influenzae IgA1 proteases suggests that this variation is caused by epitopes of the discontinuous conformational type. Analysis of the homologies among the four iga genes indicates that the variation results from transformation and subsequent homologous recombination in the iga gene region among H. influenzae strains. We find evidence for gene rearrangements, including transpositions in the iga gene region encoding the secretory part of the IgA1 preprotease. The amino acid sequence of the C terminus of the preprotease (the beta-core), which is assumed to be involved in secretion of the protease by forming a pore in the outer membrane, is highly conserved. In contrast to conserved areas in the protease domain, the nucleotide sequence encoding the beta-core showed a striking paucity of synonymous site variation.     

Molecular genetics of heterokaryon incompatibility in filamentous ascomycetes.

Filamentous fungi spontaneously undergo vegetative cell fusion events within but also between individuals. These cell fusions (anastomoses) lead to cytoplasmic mixing and to the formation of vegetative heterokaryons (i.e., cells containing different nuclear types). The viability of these heterokaryons is genetically controlled by specific loci termed het loci (for heterokaryon incompatibility). Heterokaryotic cells formed between individuals of unlike het genotypes undergo a characteristic cell death reaction or else are severely inhibited in their growth. The biological significance of this phenomenon remains a puzzle. Heterokaryon incompatibility genes have been proposed to represent a vegetative self/nonself recognition system preventing heterokaryon formation between unlike individuals to limit horizontal transfer of cytoplasmic infectious elements. Molecular characterization of het genes and of genes participating in the incompatibility reaction has been achieved for two ascomycetes, Neurospora crassa and Podospora anserina. These analyses have shown that het genes are diverse in sequence and do not belong to a gene family and that at least some of them perform cellular functions in addition to their role in incompatibility. Divergence between the different allelic forms of a het gene is generally extensive, but single-amino-acid differences can be sufficient to trigger incompatibility. In some instances het gene evolution appears to be driven by positive selection, which suggests that the het genes indeed represent recognition systems. However, work on nonallelic incompatibility systems in P. anserina suggests that incompatibility might represent an accidental activation of a cellular system controlling adaptation to starvation.     

Molecular and functional analyses of incompatibility genes at het-6 in a population of Neurospora crassa

Two closely linked genes, un-24 and het-6, associated with the het-6 heterokaryon incompatibility functional haplotype were examined in 40 Neurospora crassa strains from a Louisiana sugarcane field. Partial diploid analyses were used to determine that half of the strains were functionally Oak Ridge (OR) and half were non-OR and indistinguishable from the standard Panama (PA) form. PCR-based markers were developed to identify polymorphisms within both un-24 and het-6. Two common forms of each gene occur based on these molecular markers. Rare forms of both un-24 and het-6 were identified as variants of the non-OR form by a DNA transformation assay. The heterokaryon incompatibility function of haplotypes, based on partial diploid analyses, was perfectly correlated with the PCR-based markers at both loci. This correlation indicates that the two loci are in severe linkage disequilibrium in this population sample and may act as an incompatibility gene complex. Southern hybridizations using OR- and PA-derived cloned probes from the region that spans un-24 and het-6 showed that the apparent absence of recombination in this approximately 25-kbp region is associated with low levels of overall sequence identity between the PA and OR forms.