Antifungal macrodiolide from Streptomyces sp.

Aerobic fermentation cultures of Streptomyces sp. produced an antifungal macrodiolide. This new antibiotic consists of two units of homononactic acid linked to form a cyclic diester. An unknown polypeptide was also isolated in trace quantities. The antibiotic with polypeptide complex showed high levels of antifungal activity compared with that of the macrodiolide alone. The macrodiolide also showed a stimulatory effect on some species of fungi. The production, purification, and characterization of these compounds are reported.     

A New Antifungal Antibiotic from Bacillus sp. KM5 Isolated from Rice Rhizospheric Soil

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences - Bacterial strain, Bacillus sp. KM5 was recently isolated and characterized by strong antifungal activities...     

Isolation, Purification and Partial Characterization of an Antifungal Agent Produced by Salt-tolerant Alkaliphilic Streptomyces violascens IN2-10

An actinobacterium strain designated as IN2-10 was isolated from Garhwal region. IN2-10 was subjected to polyphasic characterization. The organism produced white to cream colored aerial mycelium with brown diffusible pigment. This isolate showed growth at 11 % NaCl, pH 4–10, and a temperature range of 15–37 °C. The isolate IN2-10 was found to belong to the genus Streptomyces and it was closely related to Streptomyces violascens (98.57 %, 16S rDNA). Of the various media tested, the high yield of active metabolite for isolate was observed in glucose soyabean meal medium at pH 10, with salt concentration of 3 % (w/v) and at a temperature of 37 °C. The broad spectrum antifungal activity of metabolite was observed against yeasts (Candida albicans, C. parapsilosis, and C. tropicalis) as well as molds (Microsporum gypseum, M. canis, Trichophtyton rubrum, Aspergillus flavus, A. fumigatus, and A. niger). The active metabolite produced by the strain was partially characterized and was found to belong to the heptaene group of polyene class of antifungal agents. In view of the decline in the discovery of new lead compounds in recent years, further investigations would require to lead to some useful products.     

Isolation, Purification and Partial Characterization of an Antibacterial Agent Produced by Halotolerant Alkaliphilic Streptomyces sp. EWC 7(2)

A halotolerant alkaliphilic actinomycetes strain EWC 7(2) was isolated from earthworm castings located in Dehradun, India. It showed the morphological, chemotaxonomic and physiological characteristics of the genus Streptomyces. On the basis of 16S rDNA sequence analysis and physiological tests, this isolate was found to be most closely related with Streptomyces ghanaensis. The optimum metabolite production was recorded in Glucose soyabean meal medium which is active against both Gram-positive and Gram-negative bacteria including drug resistant bacterial pathogens. The antibacterial activity started in the beginning of the exponential phase of growth (2 days) and reached maximum at 3–8 days while the maximum dry mycelial weight was obtained by 3–5 days of fermentation. The best growth and metabolite production was observed at a salt concentration of 4 % (w/v) and above pH 8. The antibiotic was extracted with HP Dianion resin eluted with methanol and detected by bioautography on silica gel plates using Micrococcus luteus MTCC106 as test organism. The results of bioautography revealed the presence of single active compound. The antibiotic compound was further purified by semi-prep HPLC. The spectroscopic studies such as UV–Vis, infrared, and mass of the compound showed that the molecule may be different from other known antibiotics produced by most closely related species of Streptomyces EWC 7(2). The results obtained from this study are promising however; further studies are required to elucidate the structure of the active compound.     

New Polyene Antifungal Antibiotic Produced by a Species of Actinoplanes

A new species of Actinoplanes, which has been deposited with the designation NRRL 5325 at the Northern Utilization Research and Development Division of the U. S. Department of Agriculture, produces a polyene antifungal complex designated as Sch 16656. The complex, consisting of one major and three minor components, is isolated from the fermentation broth by a solvent extraction procedure and purified by precipitation methods. The major component is a heptaene and is highly active in vitro and in vivo against Candida albicans. It is active also against strains of Torulopsis and is significantly more potent orally than candicidin in mice against Candida infections.     

Antibacterial Activity of Streptomyces sp. VITMK1 Isolated from Mangrove Soil of Pichavaram,Tamil Nadu,India

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences - The aim of the present study was to isolate antibacterial compound from Streptomyces sp. VITMK1 isolated from...     

Enhanced Production of Nitrilase from Streptomyces sp. MTCC 7546 by Response Surface Method

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences - Nitrilases are enzymes with varied applications preferably used in preparation of industrially important...     

Passicol, an Antibacterial and Antifungal Agent Produced by Passiflora Plant Species: Preparation and Physicochemical Characteristics

An increased yield of the crude material with antibacterial and antifungal activity produced by Passiflora species was obtained by aerated bathing of cut rinds of banana passion fruit in water in rotating cylindrical bottles. The crude material was separated into several fractions by thin-layer chromatography with the use of silica gel and ethyl acetate-chloroform (2:1). The fractions showing ultraviolet absorption maxima in the vicinity of 340, 358, and 375 to 377 nm were biologically active. The data indicate that the active material, here called "Passicol" for the first time, has characteristics of the polyacetylenic group of compounds. The activity can be preserved for a long time when crude material is stored in organic solvent in the dark at 6 C. Gentle bathing of the cut rinds of Passiflora in water in the presence of air and ethylene stimulated the formation of the active principle.     

An Alkali-Halostable Endoglucanase Produced Constitutively by a Bacterium Isolated from Sambhar Lake in India with Biotechnological Potential

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences - Endoglucanase, particularly those, which can withstand extreme conditions are in demand in different...     

Ahmpatinin iBu,a new HIV-1 protease inhibitor,from Streptomyces sp. CPCC 202950

Ahmpatinin ⁱBu (1) and statinin ⁱBu (2), two new linear peptides, a novel pyrrolidine derivative, (−)-(S)-2-[3-(6-methylheptanamido)-2-oxopyrrolidin-1-yl] acetic acid (3), and three known pepstatin derivatives (4–6) along with their corresponding methanolysis artifacts (7–9) were isolated from Streptomyces sp. CPCC 202950. Their structures were elucidated on the basis of extensive spectroscopic data using Marfey''s analysis, chiral-phase HPLC, and ECD and OR calculation to determine the absolute configurations. Compound 1 contains an unusual amino acid, 4-amino-3-hydroxy-5-(4-methoxyphenyl)pentanoic acid (Ahmppa), and 3 is the first natural product with a 2-(3-amino-2-oxopyrrolidin-1-yl)acetic acid system. Compounds 1, 2, and 4–9 are HIV-1 protease inhibitors. In particular, ahmpatinin ⁱBu (1) exhibits significant inhibitory activity against HIV-1 protease with an IC₅₀ value of 1.79 nM. A preliminary structure–activity relationship is discussed.

Ahmpatinin ⁱBu and statinin ⁱBu, two new linear peptides, were isolated from Streptomyces sp. CPCC 202950. Ahmpatinin ⁱBu exhibited significant inhibitory activity against HIV-1 protease with an IC₅₀ value of 1.79 nM.     

Passicol, an Antibacterial and Antifungal Agent Produced by Passiflora Plant Species: Qualitative and Quantitative Range of Activity

Fungal molds and yeasts, an actinomycete, and gram-positive and gram-negative bacteria were tested qualitatively with an antimicrobial substance, here called "Passicol," obtained from Passiflora species. A wide range of organisms were susceptible to Passicol. Some bacteria and a yeast were tested quantitatively by a broth dilution method. Passicol was nontoxic to mice and rabbits.     

Cephamycins, a New Family of β-Lactam Antibiotics I. Production by Actinomycetes, Including Streptomyces lactamdurans sp. n

A number of actinomycetes isolated from soil were found to produce one or more members of a new family of antibiotics, the cephamycins, which are structurally related to cephalosporin C. The cephamycins were produced in submerged fermentation in a wide variety of media by one or more of eight different species of Streptomyces, including a newly described species, S. lactamdurans. These antibiotics exhibit antibacterial activity against a broad spectrum of bacteria which includes many that are resistant to the cephalosporins and penicillins.     

Molecular Cloning and Heterologous Expression of a Biosynthetic Gene Cluster for the Antitubercular Agent d-Cycloserine Produced by Streptomyces lavendulae

In the present study, we successfully cloned a 21-kb DNA fragment containing a d-cycloserine (DCS) biosynthetic gene cluster from a DCS-producing Streptomyces lavendulae strain, ATCC 11924. The putative gene cluster consists of 10 open reading frames (ORFs), designated dcsA to dcsJ. This cluster includes two ORFs encoding d-alanyl-d-alanine ligase (dcsI) and a putative membrane protein (dcsJ) as the self-resistance determinants of the producer organism, indicated by our previous work. When the 10 ORFs were introduced into DCS-nonproducing Streptomyces lividans 66 as a heterologous host cell, the transformant acquired DCS productivity. This reveals that the introduced genes are responsible for the biosynthesis of DCS. As anticipated, the disruption of dcsG, seen in the DCS biosynthetic gene cluster, made it possible for the strain ATCC 11924 to lose its DCS production. We here propose the DCS biosynthetic pathway. First, l-serine is O acetylated by a dcsE-encoded enzyme homologous to homoserine O-acetyltransferase. Second, O-acetyl-l-serine accepts hydroxyurea via an O-acetylserine sulfhydrylase homolog (dcsD product) and forms O-ureido-l-serine. The hydroxyurea must be supplied by the catalysis of a dcsB-encoded arginase homolog using the l-arginine derivative, N^G-hydroxy-l-arginine. The resulting O-ureido-l-serine is then racemized to O-ureido-d-serine by a homolog of diaminopimelate epimerase. Finally, O-ureido-d-serine is cyclized to form DCS with the release of ammonia and carbon dioxide. The cyclization must be done by the dcsG or dcsH product, which belongs to the ATP-grasp fold family of protein.The soil-dwelling genus Streptomyces undergoes a complex morphological differentiation and produces an enormous variety of bioactive secondary metabolites. Because they include clinically useful antibiotics and immunosuppressants, the genus Streptomyces occupies an important position as an industrial microorganism.d-Cycloserine (DCS), a cyclic structural analogue of d-alanine, is produced by “Streptomyces garyphalus” and Streptomyces lavendulae. This antibiotic is used as an antitubercular agent (21). Since DCS is similar to d-alanine, it prevents the action of both alanine racemase and d-alanyl-d-alanine ligase, which are necessary for the biosynthesis of a bacterial cell wall. Thus, DCS functions as an inhibitor of bacterial cell wall biosynthesis (16, 19). Although the structure of DCS is very simple, the biosynthetic genes for DCS have never been cloned until now.In general, antibiotic biosynthetic genes form a cluster and are adjacent to their self-resistance genes. We have previously cloned a gene (orfB) that confers resistance to DCS on Streptomyces lividans and Escherichia coli from DCS-producing S. garyphalus (CSH) 5-12 (17). The sequence analysis suggests that the gene may encode a membrane protein that is necessary for the excretion of the DCS outside the cell. We have found that the same gene is also present in S. lavendulae ATCC 25233 (20). We have also previously demonstrated that d-alanyl-d-alanine ligase, which is a target enzyme of DCS, functions as a self-resistance determinant in S. lavendulae ATCC 25233 (20). Interestingly, the gene encoding d-alanyl-d-alanine ligase, designated ddlS, was located just upstream of the putative membrane protein gene, orfB (20). Although the self-resistance genes in the DCS producers have been thoroughly analyzed, no attempt to clone the DCS biosynthetic genes has been carried out yet.To clone the biosynthetic genes for DCS from another DCS-producing S. lavendulae strain, ATCC 11924, we first investigated whether this strain harbors orfB and ddlS. Since both genes were found to be conserved, the flanking region was cloned from the chromosomal DNA. Here, we hypothesize about the biosynthetic pathway for DCS on the basis of our present data and previous studies by another research group (23, 24). The present study is the first report identifying the biosynthetic gene cluster for DCS.     

New cytotoxic natural products from the marine sponge-derived fungus Pestalotiopsis sp. by epigenetic modification

Four new polyketide derivatives, pestalotiopols A–D (1–4), together with seven known compounds (5–11), were isolated from a chemical-epigenetic culture of Pestalotiopsis sp. The structures and absolute configurations of the new compounds (1–4) were determined by spectroscopic analyses, Mo₂-induced CD, and electronic circular dichroism (ECD) calculations. All the isolated compounds (1–11) were tested for their cytotoxic activities. Among these compounds, compounds 1, 2, 6 and 7 exhibited cytotoxicity against four human cancer cell lines with IC₅₀ values of 16.5–56.5 μM. The structure–activity relationships of compounds (1–11) were examined. The results indicated that both the diol system of the side chain and the aldehyde group might contribute to the cytotoxic activity. The possible biosynthetic pathways for compounds (1–4) were also postulated.

Four new polyketide derivatives, pestalotiopols A–D (1–4), together with seven known compounds (5–11), were isolated from a chemical-epigenetic culture of Pestalotiopsis sp.     

Susceptibilities of Candida Species Isolated from the Lower Gastrointestinal Tracts of High-Risk Patients to the New Semisynthetic Echinocandin LY303366 and Other Antifungal Agents

Fifty-two percent of stool specimens collected from 1,200 high-risk patients were colonized with yeasts, primarily Candida albicans (53.6%) and Candida glabrata (35.7%). Susceptibilities to all antifungal agents tested, including LY303366, were similar to those reported previously for Candida species isolated from blood.     

Comparative Study on Structure of Exopolysaccharide and Capsular Polysaccharide Produced by Southern Ocean Origin Pseudoalteromonas sp. MB-16

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences - A comparative study on the basic structural characteristics of exopolysaccharide (EPS) and capsular...     

Cloning and characterization of the pyrrolomycin biosynthetic gene clusters from Actinosporangium vitaminophilum ATCC 31673 and Streptomyces sp. strain UC 11065

The pyrrolomycins are a family of polyketide antibiotics, some of which contain a nitro group. To gain insight into the nitration mechanism associated with the formation of these antibiotics, the pyrrolomycin biosynthetic gene cluster from Actinosporangium vitaminophilum was cloned. Sequencing of ca. 56 kb of A. vitaminophilum DNA revealed 35 open reading frames (ORFs). Sequence analysis revealed a clear relationship between some of these ORFs and the biosynthetic gene cluster for pyoluteorin, a structurally related antibiotic. Since a gene transfer system could not be devised for A. vitaminophilum, additional proof for the identity of the cloned gene cluster was sought by cloning the pyrrolomycin gene cluster from Streptomyces sp. strain UC 11065, a transformable pyrrolomycin producer. Sequencing of ca. 26 kb of UC 11065 DNA revealed the presence of 17 ORFs, 15 of which exhibit strong similarity to ORFs in the A. vitaminophilum cluster as well as a nearly identical organization. Single-crossover disruption of two genes in the UC 11065 cluster abolished pyrrolomycin production in both cases. These results confirm that the genetic locus cloned from UC 11065 is essential for pyrrolomycin production, and they also confirm that the highly similar locus in A. vitaminophilum encodes pyrrolomycin biosynthetic genes. Sequence analysis revealed that both clusters contain genes encoding the two components of an assimilatory nitrate reductase. This finding suggests that nitrite is required for the formation of the nitrated pyrrolomycins. However, sequence analysis did not provide additional insights into the nitration process, suggesting the operation of a novel nitration mechanism.     

Identification of Acanthamoeba sp. in paraffin-embedded CNS tissue from an HIV+ individual by PCR

The opportunistic pathogens, Acanthamoeba and Balamuthia, are the causative agents of the fatal central nervous system (CNS) infection granulomatous amoebic encephalitis. We report an infection of Acanthamoeba in an HIV+ individual. In the present case, multiple lesions were observed in the skin, brain, lung, liver, and bone. A polymerase chain reaction (PCR) assay specific for Acanthamoeba was positive on tissue from a brain biopsy that had been embedded in paraffin. This report demonstrates the need for the consideration of Acanthamoeba infections in HIV+ individuals with skin lesions and multiple lesions throughout the body with CNS involvement. The results of the present study demonstrate that opportunistic amoebic infections can be diagnosed by PCR from paraffin-embedded biopsy material.     

Cloning, Expression and Characterization of Thermophilic and Alkalophilic N-acetylglucosaminidase from Streptomyces sp. NK52 for the Targeted Production of N-acetylglucosamine

A chitinolytic Streptomyces strain isolated from an alkaline habitat produced six different isozymes of chitinases. PCR amplification of DNA with chitinase domain specific primers yielded six amplicons of which, a 0.8 Kb fragment was cloned in pQE-30UA direct cloning vector and transformed into E. coli M15 cells (pREP4). The recombinant homodimer protein had a molecular mass of 44 kDa, and the 22 kDa monomers displayed 45 and 60 % activity in the presence of reducing agents. The size of the monomers is close to the predicted putative ORF of 17.8 kDa. The enzyme exhibits extreme pH and temperature optima of 10.0 and 70 °C respectively making it favorable for industrial applications. Its gene sequence revealed no homology with the reported N-acetylglucosaminidases suggesting that it could have novel attributes. This enzyme could be useful in the large scale production of N-acetylglucosamine which is currently having numerous therapeutic and commercial applications.