Disruption of Botrytis cinerea class I chitin synthase gene Bcchs1 results in cell wall weakening and reduced virulence.

To get a better insight into the relationship between cell wall integrity and pathogenicity of the fungus Botrytis cinerea, we have constructed chitin synthase mutants. A 620 bp class I chitin synthase gene fragment (Bcchs1) obtained by PCR amplification was used to disrupt the corresponding gene in the genome. Disruption of Bcchs1 occurred at a frequency of 8%. Nine independent mutants were obtained and the Bcchs1 mutant phenotype compared to that of transformants in which the gene was not disrupted. These disruption mutants were dramatically reduced in their in vitro Mg2+, Mn2+, and Co2+-dependent chitin synthase activity. Chitin content was reduced by 30%, indicating that Bcchs1p contributes substantially to cell wall composition. Enzymatic degradation by a cocktail of glucanases revealed cell wall weakening in the mutant. Bcchs1 was transcribed at a constant level during vegetative exponential growth, suggesting that it was necessary throughout hyphal development. Bcchs1 mutant growth was identical to undisrupted control transformant growth, however, the mutant exhibited reduced pathogenicity on vine leaves. It can be assumed that disruption of Bcchs1 leads to cell wall weakening which might slow down in planta fungal progression.

Synthesis and evaluation of a C-glycosyl nucleoside as an inhibitor of chitin synthase.

As part of our ongoing program devoted to inhibit chitin synthases, we have prepared a novel C-glycosyl nucleoside as metabolically stable substrate analog of UDP-GlcNAc. The synthetic strategy relies on the consecutive coupling of nucleoside and amino C-glycosyl moieties with L-tartaric acid. However, this compound inhibited only weakly chitin synthase I, with an IC(50) value of 20 mM.

Identification of four chitin synthase genes in the rice blast disease agent Magnaporthe grisea.

Four chitin synthase gene fragments were isolated from Magnaporthe grisea by use of polymerase chain reaction. A pair of degenerate primers based on conserved zymogen-type chitin synthase sequences amplified a approximately 600-bp product containing three chitin synthase gene fragments. A second pair of degenerate primers, based on conserved sequences between the chitin synthases 3 of Saccharomyces cerevisiae and Candida albicans, amplified a single 770-bp fragment. The four corresponding amino acid sequences each fell into one of the chitin synthase classes I-IV, as deduced from sequence analysis. Northern analysis demonstrated that the class II gene was expressed transiently in early phases of growth, whereas the class I and III genes as well as the class IV gene were expressed throughout the entire life cycle. Furthermore, the class III gene was the most expressed.

Structural comparisons lead to the definition of a new superfamily of NAD(P)(H)-accepting oxidoreductases: the single-domain reductases/epimerases/dehydrogenases (the 'RED' family).

Using both primary- and tertiary-structure comparisons, we have established new structural similarities shared by reductases, epimerases and dehydrogenases not previously known to be related. Despite the low sequence identity (down to 10%), short consensus segments are identified. We show that the sequence, the active site and the supersecondary structure are well conserved in these proteins. New homologues (the protochlorophyllide reductases) are detected, and we define a new superfamily composed of single-domain dinucleotide-binding enzymes. Rules for the cofactor-binding specificity are deduced from our sequence alignment. The involvement of some amino acids in catalysis is discussed. Comparison with two-domain dehydrogenases allows us to distinguish two general mechanisms of divergent evolution.

Polyhydroxynaphthalene reductase involved in melanin biosynthesis in Magnaporthe grisea. Purification, cDNA cloning and sequencing.

During the biosynthesis of fungal melanin, tetrahydroxynaphthalene reductase catalyzes the NADPH-dependent reduction of 1,3,6,8-tetrahydroxynaphthalene (T4HN) into (+)-scytalone and 1,3,8-trihydroxynaphthalene into (-)-vermelone. The enzyme from Magnaporthe grisea, the fungus responsible for rice blast disease, has been purified to homogeneity. It is a tetramer of four identical 30-kDa subunits. A full-length cDNA clone of about 1 kb encoding T4HN reductase has been isolated from a cDNA library constructed in the lambda ZAP II vector and characterized. The clone contains a 846-bp open reading frame. Translation of the DNA sequence gave a 282-residue amino acid sequence with a calculated molecular mass of 29.9 kDa. Sequences corresponding to the amino-terminal part and three internal proteolytic peptides were present in the translated sequence. T4HN reductase exhibits characteristics of the short-chain alcohol dehydrogenase family. The reductase shares 56% identity with a putative ketoreductase involved in aflatoxin biosynthesis in Aspergillus parasiticus.

Role of residue Glu152 in the discrimination between transfer RNAs by tyrosyl-tRNA synthetase from Bacillus stearothermophilus.

Residue Glu152 of tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus is close to phosphate groups 73 and 74 of tRNATyr in the structural model of their complex. TyrTS(E152A), a mutant synthetase carrying the change of Glu152 to Ala, was toxic when overproduced in Escherichia coli. The toxicity strongly increased with the growth temperature. It was measured by the ratios of the efficiencies with which the producing cells plated in induced or repressed conditions and at 30 degrees C or 37 degrees C. TyrTS(E152Q), TyrTS(E152D) and the wild-type synthetase were not toxic in conditions where TyrTS(E152A) was toxic. The toxicity of TyrTS(E152A) was abolished by additional mutations of the synthetase that prevent the binding of tRNATyr but not by a mutation that prevents the formation of Tyr-AMP. Because TyrTS(E152A) was active for the aminoacylation of tRNATyr, its toxicity could only be due to faulty interactions with non-cognate tRNAs, either their non-productive binding or their mischarging with tyrosine. TyrTS(E152A) and TyrTS(E152Q) mischarged tRNAPhe and tRNAVal in vitro with tyrosine unlike TyrTS(E152D) or the wild-type enzyme. Thus, several features of the side-chain in position 152 of TyrTS, including its negative charge, are important for the rejection of non-cognate tRNAs. TyrTS(E152A), TyrTS(E152D) and TyrTS(E152Q) had similar steady-state kinetics parameters for the charging of tRNATyr with tyrosine in vitro, with kcat/KM ratios improved 2.5 times relative to the wild-type synthetase. We conclude that the side-chain of residue Glu152 weakens the binding of TyrTS to tRNATyr and prevents its interaction with non-cognate tRNAs.

Overproduction of tyrosyl-tRNA synthetase is toxic to Escherichia coli: a genetic analysis.

The tyrS genes from Escherichia coli and Bacillus stearothermophilus were toxic to E. coli when they were carried by plasmids with very high copy numbers (pEMBL8 and pEMBL9). We quantified this effect by comparing the efficiencies of plating of E. coli derivatives harboring recombinant plasmids in various experimental conditions. The toxicity was apparent at both 30 and 37 degrees C. It increased with the growth temperature, the strength of the tyrS promoter, and the copy number of the plasmidic vector. Two- to threefold enhancement of tyrS expression raised the toxicity 300-fold. Point mutations in tyrS that prevent interaction between its product, tyrosyl-tRNA synthetase, and tRNA(Tyr) but do not alter the rate of formation of tyrosyl-adenylate abolished the toxicity. Thus, the toxic effect was due to high cellular levels of synthetase activity. At 30 degrees C, the cellular concentration of tyrosyl-tRNA synthetase reached 55% of that of soluble proteins and led to decreased beta-galactosidase stability. We discuss possible causes of this toxic effect and describe its applications to the study of the recognition and interaction between the synthetase and tRNA(Tyr).

Vitamin K-dependent carboxylation. Mechanistic studies with 3-fluoroglutamate-containing substrates.

The tripeptides t-butyloxycarbonyl-Xaa-Glu-[3H]Val, where Xaa is either (2R,3S)- or (2R,3R)-3-fluoroglutamate (respectively the erythro and the threo isomer), were synthesized and their behaviour during vitamin K-dependent carboxylation was studied. Neither peptide was carboxylated. The erythro compound gave rise to an HF-elimination product representing 1% of the starting material. This HF elimination did not occur during incubation of the threo compound. The formation of the dehydropeptide, probably by elimination of an F- anion from an intermediate carbanion, favours the ionic pathway for vitamin K-dependent carboxylation.

Interaction of L-threo and L-erythro isomers of 3-fluoroglutamate with glutamate decarboxylase from Escherichia coli.

L-threo-3-Fluoroglutamate and L-erythro-3-fluoroglutamate were tested with glutamate decarboxylase from Escherichia coli. Both isomers were substrates: the threo isomer was decarboxylated into optically active 4-amino-3-fluorobutyrate, whereas the erythro isomer lost the fluorine atom during the reaction, yielding succinic semialdehyde after hydrolysis of the unstable intermediate enamine. The difference between the two isomers demonstrates that the glutamic acid-pyridoxal phosphate Schiff base is present at the active site under a rigid conformation. Furthermore, although the erythro isomer lost the fluorine atom, yielding a reactive aminoacrylic acid in the active site, no irreversible inactivation of E. coli glutamate decarboxylase was observed.