A chitinase indispensable for formation of protoplast of Schizophyllum commune in basidiomycete-lytic enzyme preparation produced by Bacillus circulans KA-304

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia. alpha-1,3-Glucanase, which was isolated from an ammonium sulfate fraction of 0-30% saturation of KA-prep, gave the protoplast-forming activity to an ammonium sulfate fraction of 30-50% saturation of KA-prep, which contained chitinase(s) and beta-glucanase(s) but was inactive in the protoplast formation. Chitinase(s) and beta-glucanase(s) in the ammonium sulfate fraction of 30-50% saturation were separated by DEAE-cellulofine A-500 column chromatography, and the protoplast-forming activity appeared when the chitinase preparation was mixed with the alpha-1,3-glucanase. The beta-glucanase preparation was not effective for the protoplast formation whereas its addition enhanced the protoplast-forming activity of the mixture of alpha-1,3-glucanase and the chitinase preparation. The chitinase preparation contained two chitinases (chitinase I and II). Chitinase I showed the protoplast-forming activity with alpha-1,3-glucanase, but chitinase II did not. Chitinase I, a monomeric protein with a molecular weight of 41,000, was active toward colloidal chitin and ethylene glycol chitin. Chitinase I produced predominantly N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose from colloidal chitin, and the enzyme was inactive to p-NP-beta-D-N-acetylglucosaminide, suggesting that it was an endo-type enzyme. The N-terminal amino acid sequence of chitinase I (A L A T P T L N V S A S S G M) had no sequential identity to those of known chitinases.     

Cloning and expression of an alpha-1,3-glucanase gene from Bacillus circulans KA-304: the enzyme participates in protoplast formation of Schizophyllum commune

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity. The gene of alpha-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of alpha-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of alpha-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (alpha-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases.The recombinant alpha-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant alpha-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-beta-D-thiogalactopyranoside. The recombinant alpha-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.     

Cloning and expression of a Bacillus circulans KA-304 gene encoding chitinase I, which participates in protoplast formation of Schizophyllum commune

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, isolated from KA-prep, brings about the protoplast-forming activity. The gene of chitinase I was cloned from B. circulans KA-304 into pGEM-T Easy vector. The gene consists of 1,239 nucleotides, which encodes 413 amino acids including a putative signal peptide (24 amino acid residues). The molecular weight of 40,510, calculated depending on the open reading frame without the putative signal peptide, coincided with the apparent molecular weight of 41,000 of purified chitinase I estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal domain of the deduced amino acid sequence showed high similarity to that of family 19 chitinases of actinomycetes and other organisms, indicating that chitinase I is the first example of family 19 chitinase in Bacillus species. Recombinant chitinase I without the putative signal peptide was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the purified recombinant enzyme were almost the same as those of chitinase I purified from KA-prep, and showed the protoplast-forming activity when it was combined with alpha-1,3-glucanase from KA-prep. Recombinant chitinase I as well as the native enzyme inhibited hyphal extension of Trichoderma reesei.     

Role of a cell-wall glucan-degrading enzyme in mating of Schizophyllum commune

Mycelial enzyme extracts of Schizophyllum commune were prepared during vegetative growth matings leading to common-A and common-B heterokaryons and the dikaryon, and were examined for hydrolytic activity against an alkaliinsoluble cell-wall glucan (R-glucan) isolated from this mushroom. In extracts from several individual homokaryotic mycelia the R-glucanase activity was low and did not increase when the cultures exhausted glucose in the medium. In common-A matings, a 30-fold increase in specific activity of intracellular R-glucanase was found even in the presence of glucose in the broth. An increase of this magnitude was not observed in the common-B mating nor in the fully compatible cross leading to the dikaryon. Extracts of the dikaryon did show elevated R-glucanase activity after exogenous glucose disappearance and subsequent fruiting. In none of these situations was an enzyme activity detected towards an alkali-soluble cell-wall glucan (S-glucan) prepared from S. commune. Changes in R-glucanase were not parallelled by identical changes in laminarinase, pustulanase, cellobiase, and p-nitrophenyl-beta-d-glucosidase, but comparable increases in specific activities were found for hydrolysis of glycogen and maltose. After interaction of the various mycelia in mating combinations, the S-glucan/R-glucan ratio of the cell wall of the dikaryon was found to be similar to that of the homokaryons, but increased in the common-B interaction and was elevated almost threefold in the common-A heterokaryon.     

N-terminal region of chitinase I of Bacillus circulans KA-304 contained new chitin-biding domain

Chitinase I (CHI1) of Bacillus circulans KA-304 forms protoplasts from Schizophyllum commune mycelia when the enzyme is combined with α-1,3-glucanase of B. circulans KA-304. CHI1 consists of an N-terminal unknown region and a C-terminal catalytic region classified into the glycoside hydrolase family-19 type. An N-terminal region-truncated mutant of CHI 1 (CatCHI1), which was expressed in Escherichia coli Rosetta-gami B (DE3), lost colloidal chitin- and powder chitin-binding activities. The colloidal chitin- and the powder chitin-hydrolyzing activities of CatCHI1 were lower than those of CHI1, and CatCHI1 was not effective in forming the protoplast. A fusion protein of the N-terminal region of CHI1 and green fluorescent protein (Nterm-GFP) was expressed in E. coli, and the fusion protein was adsorbed to colloidal chitin, powder chitin, and chitosan. Fluorescence microscopy analysis showed that Nterm-GFP bound to the S. commune cell-wall.     

Occurrence of microtubules in the hyphae of Schizophyllum commune during intercellular nuclear migration

Summary During the intercellular nuclear migration of the basidiomycete Schizophyllum commune cytoplasmic microtubules were frequently observed scattered in the hyphae around interphase nuclei and connected with a semiglobular structure at the poles of mitotic and postmitotic nuclei. Thus it seems possible that microtubules, which have been demonstrated to participate in the intracellular nuclear movements in the dikaryotic hyphae of the basidiomycetes, are also involved in the intercellular nuclear movements of these fungi. During hyphal fusion microtubules close to an interphase nucleus were connected with electron-dense structures. It is suggested that these structures are centers for the assembly of microtubules necessary for nuclear movements not associated with nuclear divisions.Abbreviations KCE kinetochore equivalent - ch chromatin - cw cross wall of septum - ge semiglobular end of KCE - gm grey material - m mitochondrion - mp middle plate of KCE - mt microtubules - n nucleus - ne nuclear envelope - nu nucleolus - s electron-dense structure connected with microtubules     

Regulation of the pattern of protein synthesis in Schizophyllum commune by the incompatibility genes

The pattern of protein synthesis in various coisogenic mycelial types of Schizophyllum commune, viz. monokaryon, dikaryon, and homokaryons carrying primary mutations in the A and the B factors, was studied by two-dimensional gel electrophoresis. After pulse-labeling with ³⁵S-methionine, approximately 650 of 710 proteins analyzed were common to all mycelial types. Coisogenic monokaryons differed by only 2%, whereas the largest difference was found between these monokaryons and the dikaryon derived from them (6.6 and 7.7%). The majority of these differences fell into two about equally sized categories, i.e., proteins which were either specifically absent (“switched-off” proteins) or present (“switched-on” proteins) in the dikaryon. “Switched-on” proteins were on the average larger and slightly more acidic than “switched-off” proteins. The double factor mutant which best mimicked the dikaryon in morphology also best resembled the dikaryon in types of proteins synthesized. Unexpected, however, was the large overlap in proteins apparently controlled by each of the two incompatibility factors individually, despite the distinct morphological sequences directed by each of them.     

Ultrastructural Aspects of a Mutant of Schizophyllum commune with Continuous Nuclear Migration

Study of a mutant of the basidomycete Schizophyllum commune with continuous migration of nuclei revealed that the mutant is characterized by vacuolization, bundles of fibrillar-like material, and microtubules. The bundles of fibrillar-like material and microtubules extend through degraded septa to adjacent cells and are found in proximity to nuclei.     

Identification and overexpression of genes encoding cAMP-dependent protein kinase catalytic subunits in homobasidiomycete Schizophyllum commune

This report describes the first cloning and overexpression experiments on genes encoding cAMP-dependent protein kinase catalytic subunits in homobasidiomycete Schizophyllum commune. We used a degenerate PCR approach to identify two novel genes (ScPKAC1 and ScPKAC2) that are very similar to the catalytic subunits in many eukaryotes. The morphological phenotypes of ScPKAC1 and ScPKAC2 overexpressing clones were compared with those of constitutively active ScGP-A overexpressing clones to determine whether ScPKAC1 and ScPKAC2 are located downstream of heterotrimeric G-protein alpha subunit ScGP-A. Overexpression of constitutively active ScGP-A increased intracellular cAMP levels and suppressed aerial mycelium formation. In contrast, overexpressing ScPKAC1 and ScPKAC2 did not affect the intracellular cAMP levels, though aerial mycelium formation was strongly suppressed. These observations suggest that ScPKAC1 and ScPKAC2 proteins are located downstream of the G-protein alpha subunit ScGP-A in the cAMP signaling pathway.     

Specificity-determining regions of a lepidopteran-specific insecticidal protein produced by Bacillus thuringiensis

The lepidopteran-specific, insecticidal crystal proteins of Bacillus thuringiensis vary in toxicity to different species of lepidopteran larvae. We report studies of CryIA(a) and CryIA(c), two related proteins that have different degrees of toxicity to Heliothis virescens yet very similar degrees of toxicity to Manduca sexta. The amino acid differences between these proteins are located primarily between residues 280 and 722. We have constructed a series of chimeric proteins and determined their toxicities to both insects. The most significant findings arise from the replacement of three segments of the cryIA(c) gene with homologous portions of the cryIA(a) gene: codons 332-428, 429-447, and 448-722. Each of these segments contributed substantially and largely additively toward efficacy for H. virescens. However, replacement of the 429-447 segment of cryIA(c) gene with the cryIA(a) sequence resulted in a 27-50-fold reduction in toxicity toward M. sexta whereas the reduction in toxicity to H. virescens was only 3-4-fold. Subdivision of the 429-447 segment and replacements involving residues within this segment reduced toxicity to M. sexta by 5- to more than 2000-fold whereas toxicity to H. virescens was only reduced 3-10-fold. These observations indicate that: 1) different but overlapping regions of the cryIA(c) gene determine specificity to each of the two test insects; 2) some of the examined gene segments interact in determining specificity; and 3) different sequences in the cryIA(a) and cryIA(c) genes are required for maximal toxicity to M. sexta.