Genes encoding two ribonucleotide reductases (RNRs) were identified in members of the genus Streptomyces. One gene, nrdJ, encoded an oligomeric protein comprising four identical subunits each with a molecular mass of " 108 kDa. The activity of this protein depended on the presence of 5'-deoxyadenosylcobalamine (coenzyme B 12 ), establishing it as a class II RNR. The Streptomyces clavuligerus nrdJ gene was cloned, using internal peptide sequences from the purified protein, and was found to encode a polypeptide of 961 aa. Molecular phylogenetic analysis showed that the S. clavuligerus class II RNR shares significant similarity with most other bacterial and archaeal class II RNRs. Two other genes, nrdA and nrdB, were initially identified in the Streptomyces coelicolor genome database in unannotated ORFs as encoding a class Ia RNR. Southern analysis demonstrated that the nrdAB genes were present in different Streptomyces spp. The S. coelicolor nrdAB genes were cloned and expressed in Escherichia coli, and the recombinant proteins were shown to represent a class I RNR. It was shown, using quantitative real-time PCR, that the S. clavuligerus class Ia and class II RNR genes were differentially transcribed during vegetative growth. The copy number of the class II nrdJ transcripts was approximately constant throughout the exponential phase of vegetative growth (3-5W10 5 copies per 400 ng total RNA after reverse transcription). In contrast, the copy number of the class Ia nrdAB transcripts was some 10-to 20-fold less than that of nrdJ in the early-exponential growth phase (28W10 4 copies), and decreased markedly at the mid-exponential (4W10 3 copies) and late-exponential phases (11W10 3 copies) of growth. A possible role for the involvement of two RNRs during vegetative growth is discussed.
The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of 13-lactam antibiotics. It consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coil thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region-Cys-Ala-Thr-Cys-and essentially the same flavin adenine dinucleotideand NADPH dinucleotide-binding sites as E. colt thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment-TrpCys-Gly-Pro-Cys-that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. colt methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (tixA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trx4 and txB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated.In a recent article, we described the characterization of a broad-range disulfide reductase from Streptomyces clavuligerus, a producer of penicillin and cephalosporin antibiotics (2). This study was prompted by the finding that the activity of isopenicillin-N-synthase, a key enzyme in the biosynthesis of these 3-lactam compounds, and its tripeptide substrate 8-(L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV) are dependent on the redox state of their cysteine amino acid residues (3, 32). Thus, in vitro conversion of ACV to isopenicillin N occurs only if both enzyme and substrate are in their reduced-thiol state. Isopenicillin-N-synthase reactions are therefore carried out in the presence of an excess of a thiol reagent such as dithiothreitol. We looked for and identified in S. clavuligerus an enzymatic system that replaces the need for dithiothreitol in isopenicillin-N-synthase reactions. That system efficiently reduces bis-ACV, the oxidized disulfide form of ACV, to its thiol state, and modulates the activity of isopenicillin-N-synthase (2). Because ACV structurally resembles glutathione, y-glutamyl-cysteinyl-glycine, the most common intracellular lowmolecular-weight thiol, we r...
Candida albicans is an opportunistic pathogen which may give rise to superficial and systemic infections. In the present study, C. albicans adhesion was studied by expression of C. albicans DNA sequences encoding adhesion functions in a nonadherent strain of Saccharomyces cerevisiae. Adherent transformant cells of S. cerevisiae harbouring a C. albicans genomic library cloned in a yeast-Escherichia coli shuttle vector were selected by using tissue culture-treated polystyrene as the attachment substratum. One transformant exhibited enhanced adhesion to treated and untreated polystyrene as well as autoaggregation, unlike control cells bearing the vector alone. Analysis of this clone revealed an insert of ca. 4.5 kb from C. albicans. Curing of the plasmid resulted in loss of adhesion and autoaggregation properties. A subclone bearing a reduced insert of 3.3 kb retained the ability to autoaggregate, to bind to treated and untreated polystyrene, and to adhere to buccal epithelial cells, unlike appropriate controls. Further subcloning of the insert to 2.7- and 1.9-kb fragments resulted in incremental decreases in adhesion and autoaggregation, whereas smaller fragments did not confer these properties. Hybridization of the 2.7-kb segment with C. albicans and S. cerevisiae DNA confirmed its origin as a single-copy sequence in the C. albicans genome as well as the absence of a homologous sequence in the genome of S. cerevisiae. The data suggest that the adhesion and aggregation phenomena of the transformant cells are related to expression of a C. albicans surface antigen encoded by the cloned DNA fragment.
Morphogenesis in the yeast Saccharomyces cerevisiae consists primarily of bud formation. Certain cell division cycle (CDC) genes, CDC3, CDC10, CDC11, CDC12, are known to be involved in events critical to the pattern of bud growth and the completion of cytokinesis. Their products are associated with the formation of a ring of neck filaments that forms at the region of the mother cell-bud junction during mitosis. Morphogenesis in Candida albicans, a major fungal pathogen of humans, consists of both budding and the formation of hyphae. The latter is thought to be related to the pathogenesis and invasiveness of C. albicans. We have isolated and characterized C. albicans homologs of the S. cerevisiae CDC3 and CDC10 genes. Both C. albicans genes are capable of complementing defects in the respective S. cerevisiae genes. RNA analysis of one of the genes suggests that it is a regulated gene, with higher overall expression levels during the hyphal phase than in the yeast phase. Not surprisingly, DNA sequence analysis reveals that the proteins share extensive homology at the amino acid level with their respective S. cerevisiae counterparts. Related genes are also found in other species of Candida and, more importantly, in filamentous fungi such as Aspergillus nidulans and Neurospora crassa. A database search revealed significant sequence similarity with two peptides, one from Drosophila and one from mouse, suggesting strong evolutionary conservation of function.
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