Gut chitin synthase and sterols from larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae)

Gut chitin synthase was characterized and the sterols and ecdysteroids in the sugarcane rootstalk borer weevil, Diaprepes abbreviatus, were identified. An in vitro cell-free chitin synthase assay was developed using larval gut tissues from D. abbreviatus. Subcellular fractionation experiments showed that the majority of chitin synthase activity was located in 10,000g pellets. The gut chitin synthase requires Mg²⁺ to be fully active: 7–8-fold increases in activity were obtained with 10 mM Mg²⁺ present in reaction mixture. Calcium also stimulated activity (4–5-fold with 10 mM Ca²⁺), while Cu⁺² completely inhibited at 1 mM. Other monovalent and divalent cations had little or no effect on activity. The pH and temperature optima were 7 and 25°C, respectively. Gut chitin synthesis was activated ca. 50% by trypsin treatments. GlcNAc stimulated chitin synthase activity, but Glc, GlcN and glycerin did not. Polyoxin D, UDP, and ADP inhibited the chitin synthase reaction with I₅₀'s of 75 μM, 2.3 mM, and 3.6 mM, respectively. Nikkomycin Z was a potent inhibitor of chitin synthase (91% inhibition at 10 μM). Tunicamycin and diflubenzuron had no effect on the enzyme. The apparent Km and V_max for the gut chitin synthase were, respectively, 122.5 ± 7.4 μM and 426 ± 19.7 pmol/h/mg protein utilizing UDP-GlcNAc as the substrate. Sterol analyses indicated that cholesterol was the major dietary and larval sterol. HPLC/RIA data indicated that 20-hydroxyecdysone was the major molting hormone.     

Characterization of chitin and chitin synthase from the cellulosic cell wall fungusSaprolegnia monoi¨ca

The presence of chitin in hyphal cell walls and regenerating protoplast walls ofSaprolegnia monoi¨ca was demonstrated by biochemical and biophysical analyses. α-Chitin was characterized by X-ray diffraction, electron diffraction, and infrared spectroscopy. In hyphal cell walls, chitin appeared as small globular particles while cellulose, the other crystalline cell wall component, had a microfibrillar structure. Chitin synthesis was demonstrated in regenerating protoplasts by the incorporation of radioactiveN-acetylglucosamine into a KOH-insoluble product. Chitin synthase activity of cell-free extracts was particulate. This activity was stimulated by trypsin and inhibited by the competitive inhibitor polyoxin D (K_i 20 μM). The reaction product was insoluble in 1M KOH or 1M acetic acid and was hydrolyzed by chitinase into diacetylchitobiose. Fungal growth and cell wall chitin content were reduced when mycelia were grown in the presence of polyoxin D. However, hyphal morphology was not altered by the presence of the antibiotic indicating that chitin does not seem to play an important role in the morphogenesis ofSaprolegnia.     

Nikkomycin Z counteracts rylux BSU and Congo red inhibition ofSaccharomyces cerevisiae growth but does not prevent formation of aberrant cell walls

Rylux BSU and Congo red bind to chitin, interfere with proper cell-wall assembly, and stimulate chitin synthesis by increasing, most probably, chitin synthase 3 (ChS3) levels inSaccharomyces cerevisiae. On the other hand, the antibiotic nikkomycin Z inhibits chitin synthesis competitively. As ChS3 is the critical target of nikkomycin Z, its effect was tested in cells inhibited in growth by Rylux BSU or Congo red. Nikkomycin Z counteracted this inhibition but did not counteract aberrant cell-wall formation. These results indicate that chitin synthesis stimulation is the key step in Rylux BSU and Congo red inhibition and support the idea that increase in chitin synthesis represents a compensatory response to damaged cell-wall structure. As Rylux BSU and Congo red bind to newly synthesized chitin, further damage is caused in the wall and the response works in this case contrariwise. Nikkomycin Z breaks this vicious circle by counteracting the chitin synthesis stimulation.     

Temperature-sensitive flocculation during growth ofSaccharomyces cerevisiae

Summary Cell wall surface proteins were extracted from a temperature-sensitive flocculent strain ofSaccharomyces cerevisiae. Electrophoretic analysis identified two protein bands (28 and 43 kDa) present when grown at 21°C. These proteins were initially absent when the strain was grown at 37°C, but intensified after 6 days of growth concomitant with the onset of flocculation.     

chs-4, a class IV chitin synthase gene fromNeurospora crassa

InSaccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by theCSD2/CAL1/DIT101/KT12 gene. We have identified, isolated and structurally characterized aCSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomyceteNeurospora crassa and have used a “reverse genetics” approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of aN. crassa gene (designatedchs-4) encoding a polypeptide belonging to a class of chitin synthases which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those ofS. cerevisiae andCandida albicans. N. crassa strains in whichchs-4 had been inactivated by the Repeat-Induced Point mutation (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in thechs-4 ^RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme inN. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase.     

Mating reaction inSaccharomyces cerevisiae

The effect of proteolytic enzymes on sexual agglutinability of haploid cells of the yeastSaccharomyces cerevisiae was examined. Sexual agglutinability of cells of botha and α types was lost on treatment with alkaline protease and two kinds of neutral proteases ofBacillus subtilis, pronase and α-chymotrypsin. Agglutinability of α type cells was lost after treatment with acid protease ofRhizopus chinensis and trypsin, but that ofa type cells was not. These results indicate that the sex-specific substance responsible for the sexual agglutination (agglutination factor) ina type cells differs from that in α type cells. Agglutination factors were solubilized from cell-wall fractions of both mating types by Glusulase treatment. These crude factors specifically inhibited the agglutinability of cells of the opposite mating type with little effect on the agglutinability of cells of the same mating type.     

FLO11 expression in clinical and non-clinical Saccharomyces cerevisiae strains and its association with virulence

In Saccharomyces cerevisiae, FLO11 encodes a protein associated with phenotypic traits considered important for virulence. Here, we report the analysis of FLO11 gene expression using RT-LightCycler PCR in several S. cerevisiae strains of different origin (clinical and non-clinical) and with different degrees of in vivo virulence. An association between in vivo virulence and FLO11 expression was observed for the majority of strains when cells were grown at 37 °C in brain heart infusion (BHI) broth to mimic conditions encountered during brain colonization. However, there was a lack of correlation for two of the strains and this was probably due to the loss of a repression sequence in the FLO11 promoter and/or to changes in repetitive sequences in the ORF. The results indicate that the method proposed here, in conjunction with determination of other virulence factors, could usefully predict which S. cerevisiae strains are better suited to colonize in vivo systems.     

Properties of chitin synthase in homogenates from Chironomus cells

Homogenates of Chironomus cells synthesize chitin as effectively as intact cells. Chitin is produced in a dose-dependent manner, when GlcN, GlcNAc, or UDP-GlcNAc is used as precursor. Due to the lability of UDP-GlcNAc incorporation of this substrate is underestimated. No allosteric effect is observed when GlcN or GlcNAc is used as a substrate. Chitin synthesis is stimulated by Mg²⁺ and inhibited by uridine monophosphate (UMP), uridine diphosphate (UDP), and uridine triphosphate (UTP). The apparent temperature optimum is 30°C, the apparent pH optimum is 5.5–6. Addition of the chitinase inhibitor allosamidin does not enhance chitin synthesis significantly. The time course of chitin formation reveals a lag period of about 12 h, which can be overcome by trypsin treatment. Addition of protease inhibitors prevents chitin synthesis.     

Two chitin synthases in Saccharomyces cerevisiae

Disruption of the yeast CHS1 gene, which encodes trypsin-activable chitin synthase I, yielded strains that apparently lacked chitin synthase activity in vitro, yet contained normal levels of chitin (Bulawa, C. E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W. L., and Robbins, P. W. (1986) Cell 46, 213-225). It is shown here that disrupted (chs1 :: URA3) strains have a particulate chitin synthetic activity, chitin synthase II, and that wild type strains, in addition to chitin synthase I, have this second activity. Chitin synthase II is measured in wild type strains without preincubation with trypsin, the condition under which highest chitin synthase II activities are obtained in extracts from the chs1 :: URA3 strain. Chitin synthase II, like chitin synthase I, uses UDP-GlcNAc as substrate and synthesizes alkali-insoluble chitin (with a chain length of about 170 residues). The enzymes are equally sensitive to the competitive inhibitor Polyoxin D. The two chitin synthases are distinct in their pH and temperature optima, and in their responses to trypsin, digitonin, N-acetyl-D-glucosamine, and Co2+. In contrast to the report by Sburlati and Cabib (Sburlati, A., and Cabib, E. (1986) Fed. Proc. 45, 1909), chitin synthase II activity in vitro is usually lowered on treatment with trypsin, indicating that chitin synthase II is not activated by proteolysis. Chitin synthase II shows highest specific activities in extracts from logarithmically growing cultures, whereas chitin synthase I, whether from growing or stationary phase cultures, is only measurable after trypsin treatment, and levels of the zymogen do not change. Chitin synthase I is not required for alpha-mating pheromone-induced chitin synthesis in MATa cells, yet levels of chitin synthase I zymogen double in alpha factor-treated cultures. Specific chitin synthase II activities do not change in pheromone-treated cultures. It is proposed that of yeast's two chitin synthases, chitin synthase II is responsible for chitin synthesis in vivo, whereas nonessential chitin synthase I, detectable in vitro only after trypsin treatment, may not normally be active in vivo.     

Stability and zymogenic nature of chitin synthase fromCandida albicans

Chitin synthase preparations from both yeast and mycelialCandida albicans were chiefly in a zymogenic form, activatable by trypsin treatment. This was especially marked for preparations that had been solubilized with digitonin treatment. Endogenous activation of chitin synthase zymogen was observed over many days in preparations stored in glycerol (33%, wt/v) at?12°C and over many hours in preparations stored at 30°C. Gel chromatography of enzyme preparations suggested that zymogen was preferentially retarded on the columm matrices in comparison with active enzyme.     

Opposite regulation by temperature of the intracellular Ca2+-dependent serine proteinase in growing and nongrowingBacillus megaterium

Specific activity of the cytoplasmic Ca²⁺-dependent serine proteinase (ISP1) in exponentially growing cultures decreased with increasing growth temperature. On the other hand, a temperature shift-up applied to a non-growing population incubated in a sporulation medium induced a rise of its activity. The ISP1 activity was assayed in the presence of 30 mmol/L CaCl₂ to release the enzyme inhibition and/or stimulate its processing. Immunoblotting applied to the 1-D SDS-PAGE electrophoretogram detected the ISP1 in growing cells mainly in bands withM of 41 and 38 kDa. The intensity of the latter decreased with increasing growth temperature. In nongrowing cells another intensively reacting band ofM 40 kDa appeared. In contrast to the commonly accepted opinion that starvation brings about a rise of the ISP1 synthesis or activation, its increase during incubation in sporulation medium was found only in cells pregrown at 35 and 42°C, where the enzyme activity in growing culture was low. No increase of the ISP1 specific activity in sporulation medium was detected in cells pregrown at 24 or 31°C, where the activity in growing cells was high.     

Correlation of the fluorescent banding pattern and ultrastructure of a human chromosome

After incubation with Con A, cultured melanoma cells B16-C2W stuck firmly to the dish wall and could be detached neither with trypsin and pronase treatment nor with EDTA treatment, whereas the control cells were easily released from the dish wall by the same treatments. This effect became evident within 5 min of incubation at 37 °C with Con A in a greater concentration than 5 μg/ml. Resistance to trypsinization arose more rapidly as the temperature increased up to 22 °C and changed substantially around 15 °C. By addition of α-methyl-d-mannoside which combines specifically with Con A, the resistant cells became de novo susceptible to trypsinization within 5 min of incubation at 37 °C. The reversing effect of the inhibitor was also temperature dependent. The appearance of resistance to trypsinization was observed without divalent cations (Ca²⁺ and Mg²⁺), but was inhibited by pretreatment with 10^?4 M 2,4-dinitrophenol. The temperature-dependence and the concentration of Con A required for agglutination of freed cells was the same as for induction of resistance to trypsinization.     

DNA supercoiling and thermal regulation of unsaturated fatty acid synthesis in Bacillus subtilis

Bacillus subtilis growing at 37° C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37°C is transferred to 20°C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20° C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20°C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37°C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.     

TheAspergillus nidulans geneschsA andchsD encode chitin synthases which have redundant functions in conidia formation

We previously isolated three chitin synthase genes (chsA, chsB, andchsC) fromAspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, namedchsD, fromA. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 ofSaccharomyces cerevisiae and Chs3 ofCandida albicans. Disruption ofchsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption ofchsA andchsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption ofchsC andchsD caused no defect. Thus it appears thatchsA andchsD serve redundant functions in conidia formation.     

A multicopy suppressor ofnin1-1 of the yeastSaccharomyces cerevisiae is a counterpart of theDrosophila melanogaster diphenol oxidase A2 gene,Dox-A2

NIN1 is an essential gene for growth of the yeastSaccharomyces cerevisiae and was recently found to encode a component of the regulatory subunit of the 26S proteasome. Thenin1-1 mutant is temperature sensitive and its main defect is in G₁/S progression and G₂/M progression at non-permissive temperatures. One of the two multicopy suppressors ofnin1-1, SUN2 (SUppressor of Nin1-1), was found to encode a protein of 523 amino acids whose sequence is similar to those ofDrosophila melanogaster diphenol oxidase A2 and the mouse mast-cell Tum^? transplantation antigen, P91A. The C-terminal half of Sun2p was found to be functional as Sun2p at 25° C, 30° C, and 34° C but not at 37° C. The open reading frame (ORF) of theDrosophila diphenol oxidase A2 gene (Dox-A2) was obtained from a lambda phage cDNA library using the polymerase chain reaction technique. TheDox-A2 ORF driven by theTDH3 promoter complemented the phenotype of a strain deleted forsun2. ThisDox-A2-dependent strain was temperature sensitive and accumulated dumb-bell-shaped cells, with an undivided nucleus at the isthmus, after temperature upshift. This morphology is similar to that ofnin1-1 cells kept at a restrictive temperature. These results suggest thatSUN2 is a functional counterpart ofDox-A2 and that these genes play a pivotal role in the cell cycle in each organism.     

Production of β-carotene and acetate in recombinant Escherichia coli with or without mevalonate pathway at different culture temperature or pH

Natural β-carotene has received much attention as consumers have become more health conscious. Its production by various microorganisms including metabolically engineered Escherichia coli or Saccharomyces cerevisiae has been attempted. We successfully created a recombinant E. coli with an engineered whole mevalonate pathway in addition to β-carotene biosynthetic genes and evaluated the engineered cells from the aspects of metabolic balance between central metabolism and β-carotene production by comparison with conventional β-carotene producing recombinant E. coli (control) utilizing a native methylerythritol phosphate (MEP) pathway using bioreactor cultures generated at different temperatures or pHs. Better production of β-carotene was obtained in E. coli cultured at 37°C than at 25°C. A two-fold higher titer and 2.9-fold higher volumetric productivity were obtained in engineered cells compared with control cells. Notably, a marginal amount of acetate was produced in actively growing engineered cells, whereas more than 8 g/L of acetate was produced in control cells with reduced cell growth at 37°C. The data indicated that the artificial operon of the whole mevalonate pathway operated efficiently in redirecting acetyl-CoA into isopentenyl pyrophosphate (IPP), thereby improving production of β-carotene, whereas the native MEP pathway did not convert a sufficient amount of pyruvate into IPP due to endogenous feedback regulation. Engineered cells also produced lycopene with a reduced amount of β-carotene in weak alkaline cultures, consistent with the inhibition of lycopene cyclase.