Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase).

D-hydantoinases catalyze an enantioselective opening of 5- and 6-membered cyclic structures and therefore can be used for the production of optically pure precursors for biomedical applications. The thermostable D-hydantoinase from Geobacillus stearothermophilus ATCC 31783 is a manganese-dependent enzyme and exhibits low activity towards bulky hydantoin derivatives. Homology modeling with a known 3D structure (PDB code: 1K1D) allowed us to identify the amino acids to be mutated at the substrate binding site and in its immediate vicinity to modulate the substrate specificity. Both single and double substituted mutants were generated by site-directed mutagenesis at appropriate sites located inside and outside of the stereochemistry gate loops (SGL) involved in the substrate binding. Substrate specificity and kinetic constant data demonstrate that the replacement of Phe159 and Trp287 with alanine leads to an increase in the enzyme activity towards D,L-5-benzyl and D,L-5-indolylmethyl hydantoins. The length of the side chain and the hydrophobicity of substrates are essential parameters to consider when designing the substrate binding pocket for bulky hydantoins. Our data highlight that D-hydantoinase is the authentic dihydropyrimidinase involved in the pyrimidine reductive catabolic pathway in moderate thermophiles.

Characterization of a non-pigment producing Monascus purpureus mutant strain.

A characterization of a non-pigment producing mutant Monascus purpureus M12 compared with its parental strain Monascus purpureus Went CBS 109.07 has been performed aiming to investigate the relation between pigment biosynthesis and other characteristics of these fungi. A comparison has been made of morphological features, some physiological properties and biochemical activities of both strains. The albino mutant exhibits an anamorph life cycle, high conidia forming capability, slower radial growth rate and temperature sensitivity. The assimilation capacity of both strains for mono-, disaccharides and some alcohols is in the same range (Yx/c 0.2 - 0.35), while the red strain has a higher fermentation capacity. In a selected albino mutant, the growth rate, metabolic activity and capacity for production of typical for Monascus fungi secondary metabolites were reduced considerably. Hydrolytic activity towards natural substrates expressed through glucoamylase and protease was approximately 10 fold lower in the non pigment producing strain (0.05 - 0.08 U/mg protein and 0.01 - 0.07 U/mg protein respectively) compared with the red one. Important qualitative differences between both strains was found in fatty acid composition and in the production of citrinin and monacolin. The mutant strain possessed C17, C20 and C22 fatty acids and did not produce citrinin.

Genotyping study of Scedosporium apiospermum isolates from patients with cystic fibrosis.

Usually a saprophyte, Scedosporium apiospermum often colonizes the respiratory tracts of patients with cystic fibrosis (CF). In order to improve our understanding of the molecular epidemiology of the airway colonization, 129 sequential and multiple isolates collected from January 1998 to March 1999 from nine CF patients monitored in three hospitals in France were typed by random amplification of polymorphic DNA with primers GC70, UBC-701, and UBC-703. Among these primers, UBC-703 was the most discriminating, allowing the differentiation of 14 genotypes. Combining the results obtained with this three-primer set resulted in the differentiation of 16 genotypes. No common genotype was found among the different patients, and no clustering according to geographic origin of the isolates was seen. In addition, five of the patients were colonized by a single genotype. The others usually exhibited a predominant genotype accompanied by one or two others, which were found occasionally and were genetically close to the predominant genotype. Thus, our study demonstrates the persistence of the fungus despite antifungal treatments and therefore reinforces the need for the development of new antifungals that are more efficient against this species.

Typing of Scedosporium apiospermum by multilocus enzyme electrophoresis and random amplification of polymorphic DNA.

The genetic diversity among epidemiologically unrelated strains of the human pathogenic fungus Scedosporium apiospermum or its teleomorph, Pseudallescheria boydii, from different areas in Europe, was investigated by multilocus enzyme electrophoresis (MLEE) and random amplification of polymorphic DNA (RAPD). Fourteen enzyme activities were analysed by starch gel electrophoresis, corresponding to 27 polymorphic loci and 43 iso-enzymes. Among the enzymes studied, propionate esterase, carboxyl esterase, superoxide dismutase, carbonate dehydratase and malate dehydrogenase were the most polymorphic, allowing the classification of the strains into 6-11 groups each. Combination of the data obtained for the different enzyme activities studied allowed differentiation of the strains. Similarly, a high polymorphism was also revealed by each of the 20 RAPD primers tested, but no single primer was able to differentiate all the strains. The most efficient primers were GC70, UBC-701 and UBC-703, which revealed 17 distinct genotypes each, and combination of the results obtained with this three-primer set allowed complete discrimination of the strains. The dendrograms obtained from MLEE or RAPD by the unweighted pair-group method using arithmetic average cluster analysis did not reveal any clustering according to the geographic origin of the strains or their pathogenicity.

In-vivo selection of an azole-resistant petite mutant of Candida glabrata.

Two isolates of Candida glabrata from the same stool sample from a bone marrow transplant recipient treated with fluconazole, and designated 1084-L for large colonies on yeast extract-peptone-dextrose-agar and 1084-S for small colonies, were analysed. In-vitro susceptibility tests with a commercially available disk diffusion procedure showed that isolate 1084-L had a susceptibility pattern typical of wild-type strains of C. glabrata with sensitivity to polyenes and the presence of resistant colonies randomly distributed within the inhibition zones for all azole compounds except tioconazole. In contrast, isolate 1084-S, which was found by pulsed-field gel electrophoresis and random amplification of polymorphic DNA to be genetically closely related to isolate 1084-L, exhibited cross-resistance to the azole compounds except tioconazole. Determination of MICs by the E-test method confirmed these results, showing that isolate 1084-S had greater sensitivity to amphotericin B and complete resistance to ketoconazole and fluconazole. Growth on agar plates containing glucose or glycerol as the sole carbon source suggested that the resistant isolate had a respiratory deficiency, which was further demonstrated by flow cytometric analysis of the fluorescence of rhodamine 123-stained blastoconidia. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) established the mitochondrial origin of the respiratory deficiency. However, PCR amplification of the mtDNA with primers ML1 and ML6, as well as transmission electron microscopy, suggested a partial deletion of the mtDNA analogous to that described for rho- petite mutants of Saccharomyces cerevisiae. Together, these results provided evidence that the selection of azole-resistant petite mutants of C. glabrata may occur in vivo after fluconazole administration, which might explain, therefore, clinical failure of antifungal therapy.

In-vitro resistance to azoles associated with mitochondrial DNA deficiency in Candida glabrata.

A commercially available disk diffusion procedure was used in a large-scale study to evaluate the susceptibility of a wide range of Candida isolates to polyenes and azoles. With almost all isolates of C. glabrata resistant colonies were present within the inhibition zones for the azole compounds fluconazole, ketoconazole and miconazole, and less frequently for isoconazole, econazole and clotrimazole. Ten randomly selected isolates were cloned by limiting dilution and the susceptibility of the resulting strains to polyenes and azoles was determined. All strains presented a similar susceptibility pattern with sensitivity to polyenes and the presence of resistant colonies for all azole compounds except tioconazole. For each strain and each antifungal agent, one of these resistant colonies was subcultured and studied for antifungal susceptibility. All these colonies showed similar properties regardless of which antifungal agent allowed their selection, with increased sensitivity to polyenes and cross-resistance to the azole compounds except tioconazole. Similar results were obtained on Shadomy's modified medium and on synthetic medium. Likewise, determination of MICs by the Etest method confirmed the resistance to fluconazole. Comparative growth studies revealed a respiratory deficiency in the mutants caused by mitochondrial DNA (mtDNA) deletions. In addition, 'petite' mutants were obtained from a wild-type strain by exposure to ethidium bromide, and these respiratory mutants were shown to be resistant to azoles. These results demonstrate the relationship between mtDNA deficiency and resistance to azoles, and provide an interesting model to study the mechanisms of action of these antifungal agents.

Genes and enzymes of the acetyl cycle of arginine biosynthesis in Corynebacterium glutamicum: enzyme evolution in the early steps of the arginine pathway.

A cluster of arginine biosynthetic genes of Corynebacterium glutamicum ATCC 13032, comprising argJ, argB and argD as well as part of argC and argF, has been cloned by heterologous complementation of an Escherichia coli argE mutant. The gene order has been established as argCJBDF by sequencing the entire 4.4 kb cloned DNA fragment. The C. glutamicum argB gene can be transcribed in E. coli cells from an internal promoter located in the coding part of the preceding argJ gene, whereas transcription of the argJ gene appears vector-dependent. Expression of the corynebacterial argB gene is repressed by arginine in the native host but not in recombinant E. coli cells. Feedback inhibition of the corresponding N-acetylglutamate kinase activity was observed both in cell extracts of C. glutamicum and in recombinant E. coli argB auxotrophic strains. Extracts of E. coli cells carrying cloned corynebacterial DNA display an ornithine acetyltransferase activity (encoded by argJ) which alleviates the acetylornithinase (encoded by argE) deficiency of the enterobacterial host. In contrast to Bacillus stearothermophilus ornithine acetyltransferase which also exhibits acetylglutamate synthase activity, C. glutamicum ornithine acetyltransferase appears monofunctional. ArgA and ArgB proteins from different sources share highly significant similarities. The evolutionary implications of these data are discussed.