This study was conducted to investigate changes in in vitro dry matter digestibility (IVDMD), volatile fatty acids (VFA) production and cell-wall constituent degradation in wheat straw treated with six white-rot fungi: Daedalea quercina, Hericium clathroides, Phelinus laevigatus, lnonotus andersonii, lnonotus obliquus, and lnonotus dryophilus. The incubation of wheat straw for 30 days at 28°C improved IVDMD from 41.4 (control) to 59.2% for D. quercina, 56.3% for H. clathroides, 50.2% for P. laevigatus, 51.4% for l. andersonii, 52% for l. obliquus, and 55.9% for l. dryophilus. In contrast, the growth of fungi was accompanied by the dry matter loss of wheat straw: 43% for D. quercina, 12% for H. clathroides, and 22-25% for the other fungi. It is evident that the increase in digestibility by D. quercina was not offset by a loss of dry matter. The total VFA production during the rumen fermentation of fungus-treated straw was slightly increased by H. clathroides and l. dryophilus only. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were reduced in fungus-treated straw. Out of the three fractions (hemicellulose, cellulose, and lignin), hemicellulose and lignin showed the largest proportionate loss after inoculation with the fungi D. quercina, H. clathroides, P. laevigatus, and l. obliquus. The other two fungi showed the largest proportionate loss in cellulose and hemicellulose contents. The results of this study suggest that the digestion enhancement of wheat straw colonized by white-rot fungi is regulated by complex factors including the degradation of structural carbohydrates and lignin.
It has been postulated that the psf gene on a small plasmid, pUH1 (5.8kb), regulates positively the synthesis of capsular poly-γ-glutamic acid (γPGA) in Bacillus subtilis (natto) Asahikawa. We found that this strain harbored a second plasmid, named pNAGL1 (ca. 50kb), in addition to pUH1. The growth conditions that cure pUH1 or pNAGL1 were established. The plasmid-free NAF4 strain derived from B. subtilis (natto) Asahikawa was found to produce γPGA which was the same as the parent strain in terms of quantity and chemical properties having the same molecular mass and content of D-glutamic acid. Furthermore, as in the case of the parent cells, the D-glutamic acid in γPGA, which is known to increase up to ca. 80% of the total glutamic acid as Mn2+ ion concentration increases in growth medium, was found to make up 80% of the total glutamic acid of the γPGA produced by NAF4 cells grown in the presence of 0.1mM MnCl2. Thus, these results led us to conclude that the plasmids do not encode any gene important for γPGA production.
The plant pathogenic fungus Cochliobolus heterostrophus produces melanin, a black pigment, via 1, 8-dihydroxynaphthalene. The deficiency of C. heterostrophus Brn1- mutant was complemented with the cosmid clone pCOS/ML6, screened by heterologous hybridization with the genes involved in the melanin biosynthesis of Alternaria alternata. We determined the DNA sequence of the Brn1 gene and its flanking regions. The Brn1 gene contains one open reading frame consisting of three exons separated by two introns. A comparison of the nucleotide and predicted amino acid sequence of the Brn1 gene with those of other fungal reductase genes involved in melanin biosynthesis indicates significant similarity as well as the pathway of melanin biosynthesis.
Extracellular and cell-bound esterases produced by Acidiphilium sp. AIU 409 were homogeneously purified from culture broth and cells, respectively, and some properties were investigated. Both esterases more rapidly hydrolyzed p-nitrophenyl acyl esters containing long-chain fatty acids from C 8:0 to C 18:0 than those containing short-chain fatty acids from C 2:0 to C 6:0. The Km values for p-nitrophenyl long-chain fatty acid esters from C 8:0 to C 18:0 were approximately 1.3-1.5mM. The enzymes were stable at 50°C for 2 days between pH 3.0 and 6.5, and optimum pH and temperature were 5.0 and 70°C, respectively. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride and SDS. The molecular mass of both enzymes was estimated to be approximately 64kDa by SDS-PAGE. The 23 amino acid sequence from the NH2-terminus was also the same in both enzymes. These results suggest that extracellular esterase might be composed of the same components as cell-bound esterase.
To study the organization and biosynthesis of the yeast cell wall, hypo-osmolarity-sensitive mutants of Saccharomyces cerevisiae were analyzed. Cells of JS4 were irregular in shape and fragile. Calcofluor staining and quantitative analysis indicated that the chitin content was reduced. By DNA cloning and genetic analysis, the mutation hpo1-1 was found to be allelic to GLN1 which encodes glutamine synthetase. The glutamine content was significantly low in JS4, and the mutant was recovered from the cell wall defect by supplying glutamine in the medium. Partial inhibition of glutamine synthetase by phosphinothricin also induced defects in the cell wall. These results indicate that the shortage of glutamine affects cell wall integrity prior to other cellular functions.
The cells of Haloarcula vallismortis, an extreme halophilic archaebacterium, were permeabilized by various chemical, physical, and biological treatments. Biological permeabilization by lysozyme and papain showed effective results as observed by studying the in situ activity of halophilic glyceraldehydes -3-phosphate dehydrogenase (hGAPDH) as the model enzyme. Detergents N-cetyl-N, N, N-trimethyl ammonium bromide (CTAB) and digitonin also showed significant results. Other strains of halobacteria could also be permeabilized by lysozyme. The cell morphology did not show any significant change after permeabilization as observed by phase contrast microscopy. The enzyme characteristics of hGAPDH were studied in situ using permeabilized H. vallismortis cells. The properties, like optimum pH, Km for GAP and NAD+, inhibition by heavy metals, sulphydryl reagents, and other compounds, showed remarkable similarity with those studied in vitro.
Heterothallic fission yeast (Schizosaccharomyces pombe) cells preincubated with sex pheromone, P- or M-factor of the obverse mating-type cells, in mannose synthetic medium (MSM) results in remarkably increased sexual co-flocculation with obverse mating-type cells almost without time lag, i.e., within 10min. By contrast, comparable flocculation requires over 1h if untreated control cells are mixed with obverse mating-type cells. The agglutinin of P cells is more inducible than that of M cells. These pheromonal inductions of sexual co-flocculation are inhibited by the addition of cycloheximide or tunicamycin during preincubation but not by chloramphenicol or hydroxyurea. These results demonstrate that, in addition to (a) the repression of cell division (G1 arrest) and (b) the activation of cell wall autolytic processes (mating-specific elongation of cells: formation of their conjugation tubes), mating pheromones of fission yeast have another important role; (c) to induce sexual co-flocculation (agglutinability). Using our experimental system of preincubation with sexual pheromones, we show that M-agglutinin is heat-stable and its induction is inhibited by tunicamycin, but that P-agglutinin is heat-labile and its induction is only partially inhibited by tunicamycin.