Chitin Accelerates Activation of a Novel Haloarchaeal Serine Protease That Deproteinizes Chitin-Containing Biomass

The haloarchaeon Natrinema sp. strain J7-2 has the ability to degrade chitin, and its genome harbors a chitin metabolism-related gene cluster that contains a halolysin gene, sptC. The sptC gene encodes a precursor composed of a signal peptide, an N-terminal propeptide consisting of a core domain (N*) and a linker peptide, a subtilisin-like catalytic domain, a polycystic kidney disease domain (PkdD), and a chitin-binding domain (ChBD). Here we report that the autocatalytic maturation of SptC is initiated by cis-processing of N* to yield an autoprocessed complex (N*-I^WT), followed by trans-processing/degradation of the linker peptide, the ChBD, and N*. The resulting mature form (M^WT) containing the catalytic domain and the PkdD showed optimum azocaseinolytic activity at 3 to 3.5 M NaCl, demonstrating salt-dependent stability. Deletion analysis revealed that the PkdD did not confer extra stability on the enzyme but did contribute to enzymatic activity. The ChBD exhibited salt-dependent chitin-binding capacity and mediated the binding of N*-I^WT to chitin. ChBD-mediated chitin binding enhances SptC maturation by promoting activation of the autoprocessed complex. Our results also demonstrate that SptC is capable of removing proteins from shrimp shell powder (SSP) at high salt concentrations. Interestingly, N*-I^WT released soluble peptides from SSP faster than did M^WT. Most likely, ChBD-mediated binding of the autoprocessed complex to chitin in SSP not only accelerates enzyme activation but also facilitates the deproteinization process by increasing the local protease concentration around the substrate. By virtue of these properties, SptC is highly attractive for use in preparation of chitin from chitin-containing biomass.     

Responses to light by a nonphototactic mutant ofDictyostelium discoideum

The responses of the nonphototactic mutant P73 ofDictyostelium discoideum to light stimuli have been investigated and compared with those of the axenin strain Ax2. (1) Pseudoplasmodia of the mutant P73 show no phototactic response to unilateral white light in the range from 2 μW · cm⁻² to 2 × 10⁴ μW · cm⁻². Mixing experiments demonstrated that pseudoplasmodia composed of as little as 10% wild-type amoebas and 90% P73 amoebas showed almost normal phototaxis. (2) Amoebas of the mutant P73 accumulated in light traps of intensities between 10⁴ and 8 × 10⁴ μW · cm⁻² and dispersed from a trap of 10⁵ μW · cm⁻². The sensitivity of both responses was about a factor of 10 lower than that in Ax2. (3) White light inhibited aggregation of P73 amoebas in the range from 10³ to 10⁵ μW · cm⁻², about a factor of 10 higher than that in Ax2.     

Disruption of the chitin synthase gene CHS1 from Fusarium asiaticum results in an altered structure of cell walls and reduced virulence

Natural resistance of wheat against Fusarium head blight (FHB) is inadequate and new strategies for controlling the disease are required. Chitin synthases that catalyze chitin biosynthesis would be an ideal target for antifungal agents. In this study, a class I chitin synthase gene (CHS1) from Fusarium asiaticum, the predominant species of FHB pathogens on wheat in China, was functionally disrupted via Agrobacterium tumefaciens-mediated transformation. Specific disruption of the CHS1 gene resulted in a 58% reduction of chitin synthase activity, accompanied by decreases of 35% in chitin content, 22% in conidiation, and 16% in macroconidium length. The Δchs1 mutant strain had a growth rate comparable to that of the wild-type on PDA medium but had a 35% increase in the number of nuclear cellulae and exhibited a remarkably increased sensitivity to osmosis stresses. Electron microscopy revealed substantial changes occurring in cell wall structures of the macroconidium, ascospore, and mycelium, with the most profound changes in the mycelium. Furthermore, the Δchs1 mutant displayed significantly reduced pathogenicity on wheat spikes and seedlings. Re-introduction of a functional CHS1 gene into the Δchs1 mutant strain restored the wild-type phenotype. These results reveal an important in vivo role played by a CHS1 gene in a FHB pathogen whose mycelial chitin could serve as a target for controlling the disease.     

Characterization of a Dio-9-resistant strain of Saccharomyces cerevisiae.

The ATPase inhibitor Dio-9 effectively suppressed a number of physiological processes in a wild-type strain of Saccharomyces cerevisiae, X2180-1A. Low levels of the antibiotic inhibited cell growth, amino acid transport, hydrogen ion efflux, and ATPase activity. In addition, Dio-9 acted as a permeabilizing agent for the yeast plasma membrane. A mutant yeast strain, XC24, was selected on the basis of its ability to grow on minimal medium containing 200 μg/ml of Dio-9. Strain XC24 had acquired a pH-conditional ability to resist the permeabilizing effects of Dio-9. In addition, amino acid transport and hydrogen ion pumping exhibited a reduced senstivity to Dio-9 at low pH in the mutant strain. Strain XC24 was also resistant to the permeabilizing effects of the basic polymers protamine and deacylated chitin.     

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.     

Mutations of Trp275 and Trp397 altered the binding selectivity of Vibrio carchariae chitinase A

Point mutations of the active-site residues Trp168, Tyr171, Trp275, Trp397, Trp570 and Asp392 were introduced to Vibrio carchariae chitinase A. The modeled 3D structure of the enzyme illustrated that these residues fully occupied the substrate binding cleft and it was found that their mutation greatly reduced the hydrolyzing activity against pNP-[GlcNAc]₂ and colloidal chitin. Mutant W397F was the only exception, as it instead enhanced the hydrolysis of the pNP substrate to 142% and gave no activity loss towards colloidal chitin. The kinetic study with the pNP substrate demonstrated that the mutations caused impaired K_m and k_cat values of the enzyme. A chitin binding assay showed that mutations of the aromatic residues did not change the binding equilibrium. Product analysis by thin layer chromatography showed higher efficiency of W275G and W397F in G4–G6 hydrolysis over the wild type enzyme. Though the time course of colloidal chitin hydrolysis displayed no difference in the cleavage behavior of the chitinase variants, the time course of G6 hydrolysis exhibited distinct hydrolytic patterns between wild-type and mutants W275G and W397F. Wild type initially hydrolyzed G6 to G4 and G2, and finally G2 was formed as the major end product. W275G primarily created G2–G5 intermediates, and later G2 and G3 were formed as stable products. In contrast, W397F initially produced G1–G5, and then the high-M_r intermediates (G3–G5) were broken down to G1 and G2 end products. This modification of the cleavage patterns of chitooligomers suggested that residues Trp275 and Trp397 are involved in defining the binding selectivity of the enzyme to soluble substrates.     

Induction of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> Pyruvate Decarboxylase Genes

A thalium chloride-resistant (TlCl^r) mutant strain and a sodium chloride-resistant (NaCl^r) mutant strain of the diazotrophic cyanobacterium Anabaena variabilis have been isolated by spontaneous and chemical mutagenesis by using TlCl, a potassium (K⁺) analog, and nitrosoguanidine (NTG), respectively. The TlCl^r mutant strain was found to be defective in K⁺ transport and showed resistance against 10 μM TlCl. However, it also showed sensitivity against NaCl (LD₅₀, 50 mM). In contrast, neither wild-type A. variabilis nor its NaCl^r mutant strain could survive in the presence of 10 μM TlCl and died even at 1 μM TlCl. The TlCl^r mutant strain exhibited almost negligible K⁺ uptake, indicating the lack of a K⁺ uptake system. High K⁺ uptake was, however, observed in the NaCl^r mutant strain, reflecting the presence of an active K⁺ uptake system in this strain. DCMU, an inhibitor of PS II, inhibited the K⁺ uptake in wild-type A. variabilis and its TlCl^r and NaCl^r mutant strains, suggesting that K⁺ uptake in these strains is an energy-dependent process and that energy is derived from photophosphorylation. This contention is further supported by the inhibition of K⁺ uptake under dark conditions. Furthermore, the inhibition of K⁺ uptake by KCN, DNP, and NaN₃ also suggests the involvement of oxidative phosphorylation in the regulation of an active K⁺ uptake system. The whole-cell protein profile of wild-type A. variabilis and its TlCl^r and NaCl^r mutant strains growing in the presence of 50 mM KCl was made in the presence and absence of NaCl. Lack of transporter proteins in TlCl^r mutant strain suggests that these proteins are essentially required for the active transport and accumulation of K⁺ and make this strain NaCl sensitive. In contrast, strong expression of the transporter proteins in NaCl^r mutant strain and its weak expression in wild-type A. variabilis is responsible for their resistance and sensitivity to NaCl, respectively. Therefore, it appears that the increased salt tolerance of the NaCl^r mutant strain was owing to increased K⁺ uptake and accumulation, whereas the salt sensitivity of the TlCl^r mutant strain was owing to the lack of K⁺ uptake and accumulation. Received: 7 March 2002 / Accepted: 8 April 2002     

Chitin synthetase activity in a developmental mutant of Phycomyces blakesleeanus

Chitin synthetase activity was analyzed in vitro and in vivo in two morphogenetic stages, namely, dormant spore cells and germlings of the wild type strain and the developmental mutant S356 of Phycomyces blakesleeanus. In vitro experiments showed a much higher specific activity in dormant spores of the mutant strain than in those of the wild-type. This difference was restricted to the dormant spore phase since germlings exhibited comparable levels of activity to those detected in the wild-type strain. Although no correlation was observed between chitin synthesis in vitro and in vivo in mutant spores, germination of these cells was accompanied by an earlier expression of chitin synthetase in vivo. Germination of mutant spores in liquid medium produced morphologically aberrant germlings. Contrary to the extended mycelial growth of the wild-type strain in solid medium, the mutant grew with a typical colonial morphology. Results are discussed in relation to the possible basis of the mutant phenotype.     

Evidence for a specific fructose carrier inRhodotorula glutinis based on kinetic studies with a mutant defective in glucose transport

The mutant R₃₃ of the obligatory aerobic yeastRhodotorula glutinis exhibited a defect ind-glucose uptake. Detailed kinetic studies ofd-glucose andd-fructose transport in wild-type and mutant strains provided evidence for the existence in the plasma membrane of a carrier specific for fructose. The transport ofd-fructose in the mutant exhibited saturation kinetics up to 1 mmol/Ld-fructose; at higher concentrations the rate ofd-fructose uptake decreased. In the wild-type strain biphasicd-fructose uptake kinetics were observed; the low-affinity component was not found in the mutant, but the high-affinity transport system persisted. During the exponential phase of growth (ond-glucose) the high-affinityd-fructose system was repressed in the wild-type strain. Mutual competition betweend-fructose andd-glucose as well as the pH dependence of transport of the two hexoses further supported the following conclusion: In the wild-type strain,d-fructose is taken up both by the specific fructose carrier (K _T=0.22 mmol/L) and the glucose carrier (K _T=9.13 mmol/L). The former does not translocated-glucose, the latter is damaged by the mutation. Finally H⁺ co-transport and plasma membrane depolarization induced by the onset ofd-fructose transport indicated that the fructose carrier is an H⁺ symporter.     

Concomitant loss of respiratory chain NADH: ubiquinone reductase (complex I) and citric acid accumulation in Aspergillus niger

Summary Growth, citric acid production and enzymatic activity of the mitochondrial respiratory enzymes of a wild-type and a citric-acid-producing mutant of Aspergillus niger have been compared during fermentation under citric-acid-accumulating and non-accumulating conditions. Under non-accumulating conditions, both strains showed standard growth and no citric acid production. The mutant strain was characterized by delayed onset of growth and lowered cell yield. Under citric-acid-accumulating conditions the wild-type strain exhibited decelerated growth and a maximal citric acid concentration of 12 g l^–1. Reduced, but continuing growth and citric acid production of 32 g l^–1 was observed for the mutant strain. In general, the mutant strain exhibited reduced activity for the proton-pumping respiratory complexes and enhanced activity for the alternative respiratory enzymes. In contrast to the stable activity of complex I in the wild-type strain, this complex was selectively lost in the mutant strain at the onset of citric acid production, while the alternative NADH dehydrogenases were kept at enhanced and constant activity. A possible causal connection between the loss of complex I and citric acid accumulation is discussed. Offsprint requests to: J. Wallrath     

Linear and circular dichroism of membranes from Rhodopseudomonas capsulata

Absorption, linear dichroism and circular dichroism spectra of Rhodopseudomonas capsulata (wild-type-St. Louis strain, mutant Y5 and mutant Ala⁺) are particularly sensitive to the nature of the light-harvesting bacteriochlorophyll-carotenoid-protein complexes. Evidence for exciton-type interactions is seen near 855 nm in the membranes from the wild-type and from mutant Y5, as well as in an isolated B-800 + 850 light-harvesting complex from mutant Y5. The strong circular dichroism that reflects these interactions is attenuated more than 10-fold in membranes from the Ala⁺ mutant, which lacks both B-800 + 850 and colored carotenoids and contains only the B-875 light-harvesting complex. These results lead to the conclusion that these two light-harvesting complexes have significantly different chromophore arrangements or local environments.     

Structural Insights into Cellulolytic and Chitinolytic Enzymes Revealing Crucial Residues of Insect β-N-acetyl-D-hexosaminidase

The chemical similarity of cellulose and chitin supports the idea that their corresponding hydrolytic enzymes would bind β-1,4-linked glucose residues in a similar manner. A structural and mutational analysis was performed for the plant cellulolytic enzyme BGlu1 from Oryza sativa and the insect chitinolytic enzyme OfHex1 from Ostrinia furnacalis. Although BGlu1 shows little amino-acid sequence or topological similarity with OfHex1, three residues (Trp⁴⁹⁰, Glu³²⁸, Val³²⁷ in OfHex1, and Trp³⁵⁸, Tyr¹³¹ and Ile¹⁷⁹ in BGlu1) were identified as being conserved in the +1 sugar binding site. OfHex1 Glu³²⁸ together with Trp⁴⁹⁰ was confirmed to be necessary for substrate binding. The mutant E328A exhibited a 8-fold increment in K _m for (GlcNAc)₂ and a 42-fold increment in K _i for TMG-chitotriomycin. A crystal structure of E328A in complex with TMG-chitotriomycin was resolved at 2.5 Å, revealing the obvious conformational changes of the catalytic residues (Glu³⁶⁸ and Asp³⁶⁷) and the absence of the hydrogen bond between E328A and the C3-OH of the +1 sugar. V327G exhibited the same activity as the wild-type, but acquired the ability to efficiently hydrolyse β-1,2-linked GlcNAc in contrast to the wild-type. Thus, Glu³²⁸ and Val³²⁷ were identified as important for substrate-binding and as glycosidic-bond determinants. A structure-based sequence alignment confirmed the spatial conservation of these three residues in most plant cellulolytic, insect and bacterial chitinolytic enzymes.     

Isolation of Copper Biochelates from Methylosinus trichosporium OB3b and Soluble Methane Monooxygenase Mutants

Methylosinus trichosporium OB3b produces an extracellular copper-binding ligand (CBL) with high affinity for copper. Wild-type cells and mutants that express soluble methane monooxygenase (sMMO) in the presence and absence of copper (sMMO^c) were used to obtain cell exudates that were separated and analyzed by size exclusion high-performance liquid chromatography. A single chromatographic peak, when present, contained most of the aqueous-phase Cu(II) present in the culture medium. In mutant cultures that were unable to acquire copper, extracellular CBL accumulated to high levels both in the presence and in the absence of copper. Conversely, in wild-type cultures containing 5 μM Cu(II), extracellular CBL was maintained at a low, steady level during exponential growth, after which the external ligand was rapidly consumed. When Cu(II) was omitted from the growth medium, the wild-type organism produced the CBL at a rate that was proportional to cell density. After copper was added to this previously Cu-deprived culture, the CBL and copper concentrations in the medium decreased at approximately the same rate. Apparently, the extracellular CBL was produced throughout the period of cell growth, in the presence and absence of Cu(II), by both the mutant and wild-type cultures and was reinternalized or otherwise utilized by the wild-type cultures when it was bound to copper. CBL produced by the mutant strain facilitated copper uptake by wild-type cells, indicating that the extracellular CBLs produced by the mutant and wild-type organisms are functionally indistinguishable. CBL from the wild-type strain did not promote copper uptake by the mutant. The molecular weight of the CBL was estimated to be 500, and its association constant with copper was 1.4 × 10¹⁶ M⁻¹. CBL exhibited a preference for copper, even in the presence of 20-fold higher concentrations of nickel. External complexation may play a role in normal copper acquisition by M. trichosporium OB3b. The sMMO^c phenotype is probably related to the mutant’s inability to take up CBL-complexed copper, not to a defective CBL structure.     

Temperature-sensitive mutants ofStaphylococcus aureus: Isolation and preliminary characterization

Temperature-sensitive (ts) mutants ofStaphylococcus aureus were isolated after mutagenesis with nitrosoguanidine and two cycles of enrichment with Penicillin G and D-Cycloserine. The mutants expressed tight, coasting, and leaky phenotypes on solid media. In broth, however, most exhibited coasting for a limited number of generations. The reversion frequency of selected ts mutants was less than 10^–6. Intraperitoneal (i.p.) immunization with ts mutant G/1/2 conferred significant protection (0 dead/6 total vs. 7/7, immunized vs. control; p=0.0006) from lethal i.p. challenge with the parental wild-type (wt)S. aureus suspended in 5% porcine mucin, performed 28 days after i.p. administration of 10⁸ colony-forming units. Protection induced by mutants of coasting phenotype was higher and lasted longer than that induced by mutants of the tight phenotype. The results of this study demonstrate that ts mutants ofS. aureus can be obtained and that ts mutants are able to induce protective immunity from subsequent challenge with the parental wt strain.     

Pea saponins in the pea-Fusarium solani interaction

Saponin-like compounds isolated fromPisum sativum were tested for antifungal activity, effect on pea tissue, and effect on chitin and chitosan synthesis inFusarium solani. Growth ofFusarium solani f. sp.phaseoli and f. sp.pisi macroconidia was inhibited by saponins at concentrations of 150 and 300 μg/ml, respectively. Pod endocarp tissue treated with saponins showed temporary reduction in cell viability (esterase activity); however, there was no significant reduction in resistance toF. solani f. sp.phaseoli, normally incompatible on peas. Macroconidia germinated in the presence of saponin showed decreased incorporation ofN-[³H]acetylglucosamine into chitin and chitosan at concentrations as low as 32 μg/ml. Thus, a reduction in chitin and chitosan synthesis may be associated with inhibition of fungal growth. Saponins may contribute to the disease resistance of peas     

Chitin synthetase mutants of Phycomyces blakesleeanus

Mutants resistant to nikkomycin, an inhibitor of chitin biosynthesis, were isolated after exposure of wild-type spores of the fungus Phycomyces blakesleeanus to N-methyl-N′-nitro-N-nitrosoguanidine. Genetic analysis revealed that nikkomycin resistance was due to mutations in a single gene, chsA. Mutants and wild type grew equally well in the absence of nikkomycin. In contrast to the wild type, whose spore germination and mycelial growth were inhibited by 5 μM nikkomycin, chsA mutants grew reasonably well in the presence of 50 μM nikkomycin. Chitin synthesis in vivo was much less affected by the drug in the mutants than in the wild type. Resistance was not due to impaired uptake or detoxification of the drug. Analysis of the kinetics of chitin synthesis in vitro showed that the mutants had a decreased K_a for the allosteric activator, N-acetylglucosamine, and gross alterations in nikkomycin inhibition kinetics. These results indicate that chsA is the structural gene for chitin synthetase, or at least for the polypeptide that bears the catalytic and allosteric sites.     

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability I. Dihydrofolate reductase,thymidylate synthethase and formyltetrahydrofolate synthetase in amethopterin-resistant and -sensitive strains of Pediococcus cerevisiae

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent K_m values for dihydrofolate in enzymes from the three strains were in the range of 4.8–7.2 μM and for NADPH 6.5–8.0 μM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10–20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthethase and 10-formyltetra-hydrofolate synthetase (formate: tetrahydrofolate ligase (ADP)-forming), EC 6.3.4.3) were similar in the three strains studied.     

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA^- strain were G:C → T:A transversions, occurring within the sequence which in recA⁺ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.