Francisco Malpartida

The act cluster contains regulatory and antibiotic export genes, direct targets for translational control by the bldA tRNA gene of Streptomyces.

The actII region, flanked by biosynthetic genes in the 25 kb act cluster of S. coelicolor, consists of four open reading frames, including a transcriptional activator for the biosynthetic genes, and genes controlling antibiotic export. A TTA codon (extremely rare in Streptomyces) is present both in actII-ORF2 (encoding a putative transmembrane export protein) and actII-ORF4 (the transcriptional activator gene). Change of the TTA in ORF4 to TTG reverses the normal interruption of actinorhodin synthesis caused by mutation in the pleiotropic regulatory gene bldA (which encodes the cells tRNA(Leu)(UUA)). We conclude that initiation of actinorhodin synthesis via the actII-ORF4 product, and the final step in production, antibiotic export, are twin targets via which bldA exerts developmental control of actinorhodin production.

The antibacterial and antifungal activity of a soda-lime glass containing silver nanoparticles

The antibacterial and antifungal activity of a low melting point soda-lime glass powder containing silver nanoparticles has been studied. Nano-Ag sepiolite fibres containing monodispersed silver nanoparticles (d(50) approximately 11 +/- 9 nm) were used as the source of silver. This powder presents a high antibacterial (against gram-positive and gram-negative bacteria) as well as antifungal (against I. orientalis) activity. The observed high activity against yeast has been explained by considering the inhibitory effect of the Ca(2+) lixiviated from the glass on the growth of the yeast colonies.

Organisation and functions of the actV A region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor

SummarySequence analysis of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor revealed a succession of six open reading frames (ORFs), all running in the same direction and extending over 5.32 kb. The protein product of actVA-ORF1 strongly resembles that of another gene, elsewhere in the act cluster (actII-ORF2), which codes for a trans-membrane protein previously implicated in actinorhodin export from the mycelium. This suggests that the two gene products may co-operate in actinorhodin export, perhaps being sufficient for self-protection of the organism against suicide. At least four of the other five ORFs are implicated in the control of the C-6 and C-8 ring-hydroxylation reactions, lacking in actVA mutants, that occur at middle to late stages in the actinorhodin biosynthetic pathway. This conclusion was reached by genetic mapping of actVA mutants to actVA-ORF3 and-ORF5 (and perhaps -ORF4), and by the finding of strong resemblances between the protein products of actVA-ORF2 and -ORF6 and the products of genes of the oxytetracycline or tetracenomycin gene clusters that have been implicated in ring-hydroxylation reactions in the biosynthesis of these other aromatic polyketide antibiotics.

Purification of the yeast plasma membrane ATPase solubilized with a novel zwitterionic detergent

ATPases with similar kinetic properties have been identified in the plasma membranes of Neurospora crassa [ 1,2], Schizosaccharomyces pombe [3] and Saccharomyces cerevisiae [4]. In these fungal cells the active transport of nutrients is coupled to the proton gradient and therefore it has been suggested that this ATPase operates as a proton pump [571. In order to obtain direct evidence for this important physiological role it would be necessary to purify the enzyme, incorporate it into liposomes and look for ATP-driven proton transport in these structures [8]. The purification of membrane enzymes requires in most cases their solubilization with detergents and, as stated in [9], the optimal detergent for a particular membrane protein has to be found empirically. The plasma membrane ATPase of SchizosaccharomJlces pombe has been solubilized with lysolecithin [lo] but this agent. as well as many other conventional detergents were inoperative in our hands for the solubilization of the enzyme from Saccharomyces cerevisiae . Synthetic zwitterionic detergents have been largely neglected in the study of membrane proteins [9]. However. in our hands, the yeast plasma membrane ATPase could be solubilized satisfactorily only with the novel detergent 3-(tetradecyldimethyl ammonium)-1 -propanesulfonate (zwittergent TM3 14 [ 1 l] ). This detergent may also be useful for the solubilization in active form of other membrane enzymes refractory to conventional detergents. The results presented here constitute the first report to our knowledge on the utilization of a synthetic zwitterionic detergent for the purification of membrane enzymes.

Biosynthetic Engineering of Polyene Macrolides Towards Generation of Improved Antifungal and Antiparasitic Agents

Polyene macrolides are potent antifungal agents that are also active against parasites, enveloped viruses and prion diseases. They are medically important as antifungal antibiotics but their therapeutic use is limited by serious side effects. In recent years there has been considerable progress in genetic analysis and manipulation of the streptomycetes that produce nystatin, amphotericin B, candicidin, pimaricin and rimocidin/CE-108-related polyenes. This has led to engineered biosynthesis of several new polyenes that are not easily obtained as semi-synthetic derivatives. This review summarises recent advances made since the subject was last reviewed in 2003. Polyene biosynthesis generally involves assembly and cyclisation of a polyketide chain, followed by oxidative modifications and glycosylation of the macrolactone ring. New derivatives have been obtained by engineering both early and late stages of polyene biosynthetic pathways. These compounds have allowed more detailed investigations of structure-activity relationships and some are likely to show improvements in therapeutic index. The biosynthetic approach is already yielding sufficient material for testing the toxicity and activity of new compounds, thus opening possibilities for discovery of leads for development of effective and safe antifungal and antiparasitic agents.

Transcriptional organization and regulation of an antibiotic export complex in the producing Streptomyces culture

SummaryThree open reading frames (ORFs) in the actII region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor A3(2), which are involved in the export of the antibiotic are carried on two divergent transcripts. A monocistronic transcript carries actII-ORF1, encoding a putative repressor protein, and a bicistronic transcript codes for actII-ORF2 and -ORF3, whose products have been postulated to form an antibiotic export complex. The actll-ORF1 and actll-ORF2/3 transcripts each have a single promoter and the promoters for the two transcripts overlap. Both promoters are most active in cultures that have developed to the stage of actinorhodin production. The promoters resemble consensus promoters of the vegetative class in Escherichia coli and Streptomyces. We also demonstrate that these promoters are expressed in E. coli and use this finding to reveal a regulatory role for the repressor, using the xy/E reporter gene on promoter-probe shuttle vectors and regulated expression of the actII-ORF1 gene under control of Plac. The actII-ORF2/3 promoter is strongly repressed by the ORF1 product and the ORF1 product also represses its own promoter. The finding that the operator/promoter arrangement, and regulatory interconnection, of an antibiotic export/repressor gene pair in Streptomyces strikingly resemble those for tetracycline resistance in bacteria of clinical importance supports the hypothesis of an evolutionary origin of such genes in an ancestral actinomycete.

Proton translocation catalyzed by the purified yeast plasma membrane ATPase reconstituted in liposomes

The plasma membrane of animal cells contains a sodiumand potassium-pumping ATPase involved in such important physiological functions as regulation of cellular volume, generation of membrane potentials and driving of the cotransport of nutrients with sodium [1 ]. This enzyme does not seem to be present in plants, algae and fungi, probably because the rigid cell wall of these cells obviates the need for osmotic regulation [2]. On the other hand, on the basis of physiological evidence it has been postulated that the plasma membrane of the eukaryotic cells contain a proton-pumping ATPase. This enzyme would explain membrane potentials, proton secretion and the cotransport of nutrients with protons observed in these cells [3]. Although there is circumstantial evidence that the fungal [4-7] and plant [8] plasma membrane ATPases operate as proton pmnps, this important point should be established by demonstrating ATPdependent proton transport in proteoliposomes reconstituted with the purified enzyme. We have reported that the purified yeast plasma membrane ATPase reconstituted in liposomes catalysed a 32Pi-ATP exchange inhibited by proton ionophores [10]. Here, we present more direct evidence for the proton-transport activity of the enzyme by demonstrating an ATPinduced quenching of acridine dye fluorescence.

Molecular genetic analysis reveals a putative bifunctional polyketide cyclase/dehydrase gene from Streptomycea coelicolor and Streptomyces violoceoruber, and a cyclase/O-methyltransferase from Streptomyces glaucescens.

Abstract Molecular genetic analysis of the actinorhodin gene cluster from Streptomyces coelicolor has revealed a putative bifunctional cyclase/dehydrase gene. Open reading frame (ORF) 4 from both the actinorhodin (act) and granaticin (gra) polyketide synthase (PKS) gene clusters were able to relieve the block in a mutant strain of S. coelicolor which produces mutactin. This compound is a shunt product ofthe actinorhodin biosynthetic pathway which results from an aberrant intramolecular aldol cyclization, and a failure to dehydrate the hydroxyl group at C-9. These results provide compelling evidence for the existence of a novel polyketide aldolase which specifies the correct cyclization of a complex oligoketide chain.

Silver-hydroxyapatite nanocomposites as bactericidal and fungicidal materials

Abstract Silver-hydroxyapatite nanocomposites containing 1 wt.% of metallic silver have been obtained by a colloidal chemical route and subsequent chemical reduction process. The silver nanoparticles are supported on the hydroxyapatite surface without a high degree of agglomeration. The bactericidal effect against common Gram-positive and Gram-negative bacteria, as well as antifungal activity against yeast have been investigated. The results indicated a high antimicrobial activity for E. coli, M. Luteus and I. Orientalis, so this material can be a promising antimicrobial biomaterial for implant and reconstructive surgery, among other applications.

A polyketide biosynthetic gene cluster from Streptomyces antibioticus includes a LysR-type transcriptional regulator

In the search for Type II polyketide synthases (PKSs) a DNA fragment was isolated from Streptomyces antibioticus ATCC 11891 (a producer of oleandomycin). DNA sequencing of the cloned fragment revealed six complete ORFs whose deduced products showed similarities to those of other genes known to be involved in polyketide biosynthesis. Several S. coelicolor strains mutated in different steps of actinorhodin biosynthesis (actI, actIII, actV(A) and actVII) were complemented by the cloned genes, suggesting that the isolated genes encode an aromatic polyketide of unknown structure and function. The cluster also contains a putative LysR-type transcriptional regulator (ORF0), which controls PKS gene expression in a heterologous host. DNA binding assays and transcriptional analysis suggest that the pathway-specific regulator for actinorhodin biosynthesis (actII-ORF4) is also involved in the expression of the cloned PKS in the host strain.