Synthesis of fused glycoprotein D of herpes simplex virus type 1 but not type 2 inhibits Escherichia coli hosts.

Glycoprotein D from either Herpes simplex virus type 1 (gD-1) or type 2 (gD-2) has been expressed in Escherichia coli as a series of chimeric proteins. The expression vector used in this study, pJS413, was derived from pBR322 and contains several cloning sites between the lacZ promoter-operator and the phage lambda cro gene. Plasmids containing fusions between the cro gene, gD-related sequences and lacZ was constructed and shown to direct the synthesis of 160-kDa proteins. The accumulation of fusion protein could be visualized as inclusion bodies when the cells were examined by dark phase-contrast or transmission electron microscopy. None of the plasmids that encoded cro::gD gene fusions yielded significant amounts of material upon induction with isopropyl-beta-D-thiogalactopyranoside. In addition, certain plasmids produced a form of Cro-gD-1 fusion protein which resulted in severe growth inhibition of E. coli. These inhibitory effects were attributed to the presence of specific gD-1 sequences, i.e., the transmembrane and cytoplasmic anchor region of the protein.

Marine microorganisms as a source of new natural products.

Over the past decade, marine microorganisms have become recognized as an important and untapped resource for novel bioactive compounds. The oceans cover greater than 70% of the earth's surface and, taking this into account by volume, represent better than 95% of the biosphere. Given this fact, the oceans present themselves as an unexplored area of opportunity for the discovery of pharmacologically active compounds. In this review, data have been presented to illustrate the diversity of microorganisms living in the sea and the plethora of chemical compounds that have been discovered from them. However, it is important to pursue basic research on the marine environment in order to permit the continued isolation of unique microorganisms. There is still limited knowledge of the physiological requirements of most marine microorganisms, and a greater understanding of their conditions for growth will offer new insights into the complex world of marine microbiology. Clearly, a greater investment in the development of marine biotechnology will produce novel compounds that may contribute significantly toward drug development over the next decade.

Phenotypes conferred by the Bacillus subtilis recM13 mutation and the din23 fusion.

The din23 fusion encodes a B. subtilis SOS-inducible regulatory region fused to the E. coli lacZ gene (Love et al., 1985). A strain encoding the din23 fusion and a recM13 allele showed low-level constitutive beta-galactosidase expression, was induced for beta-galactosidase production by DNA gyrase inhibitors but not by DNA-damaging agents, and was slightly induced by a variety of agents which do not normally induce the SOS regulon. The din23 fusion itself resulted in high levels of spontaneous prophage induction in wild-type, recM- and recA-hosts, despite the fact that the din23recM13 strain was not induced for beta-galactosidase production by DNA-damaging agents. The results suggest that the recM gene may be involved with the regulation of the RecA protease-mediated SOS response, while the din23 gene may be involved with the regulation of an alternative function which results in the cleavage of prophage repressor.

An assay for the detection of bacterial DNA gyrase inhibitors.

In summary, we have developed a sensitive detection system for inhibitors of bacterial DNA gyrase. The use of B. subtilis as the host organism confers the advantage that it is sensitive to both gyrase subunit A and B inhibitors, whereas E. coli is relatively insensitive to B subunit inhibitors in vivo. Using this assay, we identified a new DNA gyrase inhibitor with a novel structure.

The stimulation of bioluminescence in Photobacterium leiognathi as a potential prescreen for antitumor agents.

The stimulation of bioluminescence in Photobacterium leiognathi has previously been described as a test for genotoxic compounds. An adaptation of this procedure has been developed which uses a dim variant of P. leiognathi and permits the prescreening of microbial fermentation broths for potential antitumor agents. Bioluminescence in this organism was stimulated by compounds which bind to DNA or affect DNA synthesis. Antibiotics with target sites such as protein, cell wall or RNA synthesis, did not alter bioluminescence. Fermentation broths from over 5,000 soil isolates were prescreened in this assay and 95 (1.6%) were defined as active. Further analysis of selected cultures suggested that about half produced compound(s) with DNA-binding activity. These results suggest that the photobacterium induction assay (PIA) may be useful as a prescreen for potential antitumor agents. The assay is rapid, simple and requires only microgram quantities of material for testing.

Bioxalomycins, new antibiotics produced by the marine Streptomyces sp. LL-31F508: taxonomy and fermentation.

An actinomycete strain designated LL-31F508 was isolated from an intertidal sediment sample collected in Key West, Florida. Culture LL-31F508 was assigned to the Streptomyces genus based on the presence of LL-diaminopimelic acid (DAP) in the cell wall and observations of spiny spores using scanning electron microscopy (SEM). Excellent antimicrobial activity against Staphylococcus and Enterococcus spp. were detected in both the supernatant and cell extract samples from fermentations of culture LL-31F508. Production of antibiotic activity peaked at 48-50 hours and closely paralleled cell growth, during which time glucose was more rapidly assimilated than dextrin. A series of new antibiotics called the bioxalomycins was identified as the antibacterial products from fermentations of this culture. Fermentation conditions for production of bioxalomycin alpha differed substantially from those required for production of a related compound, naphthyridinomycin, by the reference culture Streptomyces lusitanus NRRL 8034.

Isolation, characterization and structure of a new allenic polyine antibiotic produced by fungus LL-07F275.

Antibiotic 07F275 (1), produced by submerged fermentations of fungal culture LL-07F275, was isolated and characterized despite its inherent instability. Its UV spectrum was identical with that of nemotin, a member of the allenic polyacetylene family, but a molecular weight of 218 daltons indicated a new compound. Structure 1 was determined on the basis of spectroscopic evidence, particularly NMR. Since 1 is a thirteen carbon-containing allenic diyne, it is closely related to mycomycin.

Pyrroindomycins, novel antibiotics produced by Streptomyces rugosporus LL-42D005. II. Biological activities.

The pyrroindomycins, a complex of novel antibiotics identified in fermentation broths of "Streptomyces rugosporus" LL-42D005, demonstrated excellent in vitro activity against Gram-positive bacteria. The semisynthetic diacetyl derivative of pyrroindomycin B (pyrroindomycin B-Ac2) was bactericidal for exponential-phase cells, but not for stationary-phase cells. This compound also exhibited marginal protection against a lethal Staphylococcus aureus challenge in mice. The poor in vivo activity of this antibiotic complex may be related to binding to blood components, as suggested by elevated MICs observed in blood-containing media. Incorporation of radiolabeled precursors into DNA, RNA, and protein was inhibited in an exponential-phase culture of Bacillus subtilis within ten minutes of exposure to pyrroindomycin B-Ac2. Microscopic examinations of drug-treated cells revealed lysis within the same ten minute period. These data are consistent with an effect of pyrroindomycin B-Ac2 on the integrity of the bacterial membrane.

Pyrroindomycins, novel antibiotics produced by Streptomyces rugosporus sp. LL-42D005. I. Isolation and structure determination.

Pyrroindomycins A and B were isolated from fermentations of culture LL-42D005, a strain of Streptomyces rugosporus. Pyrroindomycins possess potent antimicrobial activities against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci. Their structures have been determined by using 1- and 2-D NMR, mass spectroscopy and chemical degradations. Pyrroindomycins are the first natural products that contain the highly unsaturated pyrroloindole moiety.

Glycothiohexide alpha, a novel antibiotic produced by "Sebekia" sp., LL-14E605. II. Isolation and physical-chemical characterization.

Glycothiohexide alpha was recovered from the fermentation broth of a "Sebekia" sp. by mixed solvent extraction, selective precipitation and adsorption chromatography on Diaion HP-20. The amount of glycothiohexide alpha present in the crude preparation was enriched by photolysis. Purification of glycothiohexide alpha was accomplished by repetitive countercurrent chromatography.

The effect of methylation inhibitors on citreamicin biosynthesis in Micromonospora citrea.

When the citreamicin-producing organism Micromonospora citrea NRRL 189351 was incubated in the presence of the methylation inhibitors sinefungin or aminopterin, biosynthesis of the zeta component was stimulated approximately 20 to 200-fold above the level normally produced. Inhibition of a second methylation reaction, which is superficially very similar to the first, was not detected. Other known methylation inhibitors failed to yield any change in the natural pattern of citreamicins produced. This approach is an excellent route for preparing citreamicin zeta, which can be used as a substrate for semi-synthesis or for further biosynthetic studies.

LL-E19085 alpha, a novel antibiotic from Micromonospora citrea: taxonomy, fermentation and biological activity.

A new antibacterial antibiotic, designated LL-E19085 alpha, was isolated from the fermentation broth of an actinomycete strain. Based on cultural, physiological, morphological and chemical characteristics, culture LL-E19085 was identified as a new subspecies of Micromonospora citrea. Antibiotic LL-E19085 alpha demonstrated potent activity against a spectrum of Gram-positive aerobic and anaerobic bacteria.

Biological activity of guanacastepene, a novel diterpenoid antibiotic produced by an unidentified fungus CR115.

Fermentation extracts of culture CR115, an unknown plant endophyte originally isolated from Costa Rica, were found to be active against antibiotic-resistant bacteria. The metabolite responsible for activity was identified as a novel diterpenoid antibiotic guanacastepene (mol. wt. 374.47 and mol. formula C22H30O5). Mechanistic studies done in an E. coli imp strain suggested membrane damage as the primary mode of bactericidal action. This compound also lysed human RBCs and caused leakage of intracellular potassium from E. coli imp.

Simaomicin (LL-D42067), a novel antibiotic from Actinomadura madurae. I. Taxonomy, fermentation and biological activity.

A new antibacterial antibiotic, designated simaomicin alpha (LL-D42067 alpha) was isolated from the fermentation broth of an actinomycete strain. Based on cultural, physiological, morphological and chemical characteristics, culture LL-D42067 was identified as a new subspecies of Actinomadura madurae. Simaomicin alpha demonstrated potent activity against Gram-positive bacteria and was active in vivo against a variety of Eimeria species causing coccidiosis in chickens.

LL-D49194 antibiotics, a novel family of antitumor agents: taxonomy, fermentation and biological properties.

A novel family of antitumor antibiotics, designated LL-D49194, was isolated from the fermentation broth of an actinomycete strain identified as Streptomyces vinaceus-drappus. LL-D49194 alpha 1 and beta 2 were active against Gram-positive and inactive against Gram-negative bacteria in vitro. The beta 1 component was not active against either Gram-positive or Gram-negative bacteria. These antibiotics exhibited significant in vivo activities against several murine tumors, albeit with differing potencies.

LL-E19020 alpha and beta, animal growth promoting antibiotics: taxonomy, fermentation and biological activity.

Antibacterial antibiotics LL-E19020 alpha and beta were isolated from the fermentation broth of an actinomycete strain. Based on cultural and physiological characteristics, culture LL-E19020 was identified as a new subspecies of Streptomyces lydicus. The LL-E19020 alpha and beta antibiotics were found to possess a very narrow antibacterial spectrum against human pathogens. In studies in chickens, LL-E19020 alpha demonstrated excellent growth promoting activity.

Calicheamicins, a novel family of antitumor antibiotics: taxonomy, fermentation and biological properties.

A novel family of antitumor antibiotics, the calicheamicins, were isolated from the fermentation broth of Micromonospora echinospora subsp. calichensis. These antibiotics exhibited significant activity against Gram-positive and Gram-negative bacteria in vitro. Calicheamicin gamma 1I demonstrated antitumor activity against P388 leukemia and B16 melanoma in vivo.

Martinomycin, a new polyether antibiotic produced by Streptomyces salvialis. I. Taxonomy, fermentation and biological activity.

Actinomycete culture LL-D37187 has been found to produce the new polyether antibiotic martinomycin. Taxonomic studies, including morphological, physiological, and cell wall chemistry analyses, revealed that culture LL-D37187 is a novel streptomycete species, and the proposed name is Streptomyces salvialis. Martinomycin exhibits activity against the Southern Army Worm (Spodoptera eridania) and Gram-positive bacteria.

Glycothiohexides, novel antibiotics produced by "Sebekia" sp. LL-14E605. I. Taxonomy, fermentation and biological evaluation.

The new glycothiohexide antibiotics, which are related to nosiheptide, were identified in fermentations of an actinomycete belonging to the genus "Sebekia". Strain LL-14E605 was classified as a "Sebekia" based on the presence of both mesodiaminopimelic acid and madurose in the cell wall and the presence of pseudosporangia encasing the spores. Culture LL-14E605 was successfully fermented in 10 to 3,000 liters of a complex medium. Antibiotic activity closely followed cell mass accumulation and usually peaked after 4 to 5 days of incubation. Glycothiohexide alpha demonstrated excellent in vitro activity against Gram-positive bacteria with MICs of 0.03 to 0.06 microgram/ml against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. However, glycothiohexide alpha failed to protect mice against a lethal challenge with Staphylococcus aureus Smith unless it was administered prior to challenge.

Phytochemical, morphological, and biological investigations of propolis from Central Chile.

Propolis from central Chile was investigated for its plant origin by microscopical analysis of pollen grains and leaf fragments found in the sample. The pollen grains that appear with significant higher frequency in the sample corresponded to four native and two introduced species, whereas leaf fragments corresponded to four native species. Seventeen phenolic compounds that belong to the phenylpropane, benzaldehyde, dihydrobenzofuran, or benzopyran classes, were isolated from an organic extract that was found to have a moderate growth inhibitory activity against Mycobacterium avium, M. tuberculosis, and two strains of Staphylococcus aureus. The components responsible for activity were determined.