Colin J. Jackson

Cytochrome p450 complement (CYPome) of the avermectin-producer Streptomyces avermitilis and comparison to that of Streptomyces coelicolor A3(2).

The genus Streptomyces produces about two-thirds of naturally occurring antibiotics and a wide array of other secondary metabolites, including antihelminthic agents, antitumor agents, antifungal agents, and herbicides. The newly completed genome sequence of the avermectin-producing bacterium Streptomyces avermitilis contains 33 cytochromes p450 (CYPs), many more than the 18 observed in Streptomyces coelicolor A3(2). Some of the likely metabolic functions are reported together with their genomic location and bioinformatic analysis. Seven entirely new CYP families were found together with close homologues of some forms observed in S. coelicolor A3(2). The presence of unusual CYP forms associated with conservons is revealed and of these, CYP157 forms in both S. avermitilis and S. coelicolor A3(2) deviate from the previously accepted rule for an EXXR motif within the K-helix of CYPs. Amongst this range of CYPs are forms associated with avermectin, filipin, geosmin, and pentalenolactone biosynthesis as well as unknown pathways of secondary metabolism.

Rapid identification of Trichophyton tonsurans by PCR-RFLP analysis of ribosomal DNA regions

BACKGROUND Culture morphology of Trichophyton (T.) tonsurans, an emerging pathogen of dermatophytosis in Japan, varies widely and species level identification is sometimes very difficult. Reliable molecular markers are expected to be introduced for their identification. OBJECTIVE The present study was conducted to evaluate the efficacy of restriction fragment length polymorphism (RFLP) analysis of PCR amplified ribosomal (r) DNA including internal transcribed spacers (ITS), as an identification tool. METHODS Total cellular DNA was extracted from 26 Japanese isolates of T. tonsurans, along with several taxa of the members in the T. mentagrophytes complex, T. rubrum, T. violaceum and Epidermophyton floccosum, using a mini-preparation method. PCR amplicons were digested with restriction enzymes Mva I or Hinf I, then electrophoresed on 5% polyacrylamide gel. RESULTS The banding profiles were observed about 8 h from initiating DNA extraction. Intraspecies polymorphism was not detected among T. tonsurans isolates, and their profiles obtained using Mva I digestion were clearly different from those of the other dermatophyte species. The restriction profiles evaluated from nucleotide sequence of the regions by a computer analysis were compatible with the electrophoresed profiles on gel. CONCLUSION PCR-RFLP analysis is a rapid and reliable tool for the identification of T. tonsurans.

Molecular Mechanisms of Itraconazole Resistance in Candida dubliniensis

ABSTRACT It has previously been shown that overexpression of the CdMDR1 gene is a major contributor to resistance in fluconazole-resistant isolates of Candida dubliniensis. However, since CdMdr1p does not mediate transport of other azole drugs such as itraconazole, we investigated the molecular mechanisms of stable resistance to itraconazole obtained in three strains of C. dubliniensis (two with nonfunctional CdCDR1 genes and one with functional CdCDR1 genes) by serial exposure to this antifungal agent in vitro. Seven derivatives that were able to grow on agar medium containing 64 μg of itraconazole per ml were selected for detailed analysis. These derivatives were resistant to itraconazole, fluconazole, and ketoconazole but were not cross resistant to inhibitors. CdMDR1 expression was unchanged in the seven resistant derivatives and their parental isolates; however, all seven derivatives exhibited increased levels of CdERG11 expression, and six of the seven derivatives exhibited increased levels of CdCDR1 expression compared to the levels of expression by their respective parental isolates. Except for one derivative, the level of rhodamine 6G efflux was decreased in the itraconazole-resistant derivatives compared to the level of efflux in their parental isolates, suggesting altered membrane properties in these derivatives. Analysis of their membrane sterol contents was consistent with a defective sterol C5,6-desaturase enzyme (CdErg3p), which was confirmed by the identification of mutations in the alleles (CdERG3) encoding this enzyme and their lack of functional complementation in a Saccharomyces cerevisiae erg3 mutant. The results of this study show that the loss of function of CdErg3p was the primary mechanism of in vitro-generated itraconazole resistance in six of the seven the C. dubliniensis derivatives. However, the mechanism(s) of itraconazole resistance in the remaining seventh derivative has yet to be determined.

Molecular strain typing of Trichophyton rubrum indicates multiple strain involvement in onychomycosis

Background  Trichophyton rubrum is an important cause of onychomycosis. Molecular strain typing methods have recently been developed to address questions of epidemiology and source of relapse following treatment.

Reduced Azole Susceptibility in Genotype 3 Candida dubliniensis Isolates Associated with Increased CdCDR1 and CdCDR2 Expression

ABSTRACT Candida dubliniensis is a recently identified yeast species primarily associated with oral carriage and infection in individuals infected with the human immunodeficiency virus. The species can be divided into at least four genotypes on the basis of the nucleotide sequence of the internal transcribed spacer region of the rRNA operon. Previous studies have shown that a small number of clinical isolates belonging to genotype 1 are resistant to the commonly used antifungal drug fluconazole. The aim of the present study was to investigate the molecular mechanisms responsible for reduced susceptibility to azole drugs in C. dubliniensis genotype 3 isolates obtained from a patient with fluconazole-recalcitrant oral candidiasis. Four isolates from a single clinical sample, one susceptible, the other three exhibiting reduced susceptibilities to fluconazole, itraconazole, ketoconazole, voriconazole, and posaconazole, were examined. Results showed that reduced susceptibility to azole drugs was associated with an increase in the expression of the multidrug transporters CdCDR1 and CdCDR2 which correlated with reduced intracellular accumulation of radiolabeled fluconazole and an increase in the activity of energy-dependent efflux mechanisms. In contrast to observations made in previous studies, overexpression of the multidrug transporter CdMDR1 was not observed. Despite a thorough investigation of all commonly encountered mechanisms of azole resistance, no other mechanism could be associated with reduced susceptibility to azole drugs in the clinical isolates studied. This is the first report of CdCDR2 involvement in azole resistance in C. dubliniensis.

Restriction Fragment Length Polymorphism Analysis of Ribosomal DNA Intergenic Regions Is Useful for Differentiating Strains of Trichophyton mentagrophytes

ABSTRACT Twenty isolates of Tricophyton mentagrophytes var. mentagrophytes and 47 isolates of T. mentagrophytes var. interdigitale, identified by morphological characteristics, were screened by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Sixty isolates (14 of 20 T. mentagrophytes var. mentagrophytes isolates and 46 of 47 T. mentagrophytes var. interdigitale isolates) shared an identical ITS RFLP profile and were further investigated by using a probe targeted to the rDNA nontranscribed spacer (NTS) region. Polymorphisms were observed in the NTS regions of both T. mentagrophytes var. mentagrophytes and T. mentagrophytes var. interdigitale isolates. Twenty-three individual RFLP patterns (DNA types P-1 to P-12 and A-1 to A-11) were recognized and divided into two groups depending on the presence (P) or absence (A) of a 2.5-kb band, which correlated to a large extent with the morphological variety. Eleven of 14 T. metagrophytes var. mentagrophytes isolates were A types, and all of the 46 T. mentagrophytes var. interdigitale isolates were P types. A majority of strains (23 of 60 [38.3%]) were characterized by one RFLP pattern (pattern P-1), and eight types (P-1 to P-6, P-8, and P-9) accounted for 75% (45 of 60) of all strains, including all of the T. mentagrophytes var. interdigitale isolates. The remaining 15 types were represented by one only isolate and included all of the T. mentagrophytes var. mentagrophytes isolates. We conclude that RFLP analysis of the rDNA NTS region is a valuable technique for differentiation of T. mentagrophytes strains. Furthermore, by use of this method, there appears to be a greater degree of diversity among T. mentagrophytes var. mentagrophytes isolates than among T. mentagrophytes var. interdigitale isolates.

Conservation and cloning of CYP51: a sterol 14α-demethylase from Mycobacterium smegmatis

The genetic locus encoding cytochrome P450 51 (CYP51; P45014DM) in Mycobacterium smegmatis is described here together with confirmation of activity in lanosterol 14α-demethylation. The protein bound azole antifungals with high affinity and the rank order based on affinity matched the ranked order for microbiological sensitivity of the organism, thus supporting a possible role for CYP51 as a target in the antimycobacterial activity of these compounds. Non-saponifiable lipids were extracted from the bacteria grown on minimal medium. Unlike a previous report using growth on complex medium, no cholesterol was detected in two strains of M. smegmatis, but a novel lipid was detected. The genetic locus of CYP51 is discussed in relation to function; it is conserved as part of a putative operon in M. smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium bovis and consists of six open-reading frames including two CYPs and a ferredoxin under a putative Tet-R regulated promoter.

Expression, purification and use of the soluble domain of Lactobacillus paracasei β-fructosidase to optimise production of bioethanol from grass fructans

Microbial inulinases find application in food, pharmaceutical and biofuel industries. Here, a novel Lactobacillus paracasei beta-fructosidase was overexpressed as truncated cytosolic protein ((t)fosEp) in Escherichia coli. Purified (t)fosEp was thermostable (10-50 degrees C) with a pH optimum of 5; it showed highest affinity for bacterial levan (beta[2-6] linked fructose) followed by nystose, chicory inulin, 1-kestose (beta[2-1] linkages) and sucrose (K(m) values of 0.5, 15, 15.6, 49 and 398 mM, respectively). Hydrolysis of polyfructose moieties in agriculturally-sourced grass juice (GJ) with (t)fosEp resulted in the release of >13 mg/ml more bioavailable fructose than was measured in untreated GJ. Bioethanol yields from fermentation experiments with Brewers yeast and GJ+(t)fosEp were >25% higher than those achieved using untreated GJ feedstock (36.5[+/-4.3] and 28.2[+/-2.7]mg ethanol/ml, respectively). This constitutes the first specific study of the potential to ferment ethanol from grass juice and the utility of a novel core domain of beta-fructosidase from L. paracasei.

Excreted/secreted Schistosoma mansoni venom allergen-like 9 (SmVAL9) modulates host extracellular matrix remodelling gene expression

Graphical abstract

The Diversity and Importance of Microbial Cytochromes P450

The cytochromes P450 (CYPs) of microbes are enormously diverse as revealed in discoveries from the era of molecular biology and as subsequently found in genomic investigations. One percent of the genes of a microbe can encode CYPs, but in stark contrast most bacteria studied so far can survive without CYPs. Microbial eukaryotes usually have at least one CYP, due to the essential requirement of most to synthesize sterol involving CYP51, sterol 14a-demethylase. The roles of the vast majority of microbial CYPs remain to be elucidated, but many already have important fundamental roles in nature, and others are important for biotechnological purposes. Some others have, of course, provided facile models for understanding CYP structure and activity, such as CYPlOl (P450(3 ĵ ) of Pseudomonas putida and CYP102A1 (P450gĵ _3) of Bacillus megaterium. The purpose of this chapter is to provide an outline of the important biomedical and environmental roles of the microbial CYPs, including many which were unsuspected when the respective microorganisms were originally studied. This includes involvement of CYP in some of the earliest metabolic alterations in the production of penicillin, some of the early biosynthetic steps allowing the production of corticosteroids, and the first application of therapeutic CYP inhibitors, the azole antifungal agents. Current and future applications involving microbial CYPs are manifestly clear. ranging from new therapeutics to biotransformations and bioremediation.