Heterologous biosynthesis of myxobacterial lanthipeptides melittapeptins.

The myxobacteria are an attractive bioresource for bioactive compounds since the large size genome contains many biosynthetic gene clusters of secondary metabolites. The genome of the myxobacterium Melittangium boletus contains three biosynthetic gene clusters for lanthipeptide production. One of the gene clusters includes genes coding lanthipeptide precursor (melA), class II lanthipeptide synthetase (melM), and transporter (melT).

High-intensity green light potentially activates the actinorhodin biosynthetic pathway in Streptomyces coelicolor A3(2).

The development of practices that enhance the potential of actinomycetes as major antibiotic producers is a challenge in discovering new secondary metabolites. Light, an essential external stimulus for most microorganisms, could be exploited to manipulate their physiological processes. However, the effects of monochromatic green light on the production of secondary metabolites in actinomycetes have not yet been reported.

Phenomenological interpretations of the mechanism for the concentration-dependent positive effect of antibiotic lincomycin on A3(2).

The antibiotic lincomycin binds to the 23S ribosomal RNA peptidyl transferase loop region to inhibit protein synthesis. However, lincomycin can also stimulate the growth and secondary metabolism of actinomycetes in a concentration-dependent manner. In A3(2), lincomycin stimulates the production of the blue-pigmented antibiotic actinorhodin at concentrations below the minimum inhibitory concentration.

Heterologous Production and Structure Determination of a New Lanthipeptide Sinosporapeptin Using a Cryptic Gene Cluster in an Actinobacterium Sinosporangium siamense.

Lipolanthine is a subclass of lanthipeptide that has the modification of lipid moiety at the N-terminus. A cryptic biosynthetic gene cluster comprising four genes (sinA, sinKC, sinD, and sinE) involved in the biosynthesis of lipolanthine was identified in the genome of an actinobacterium Sinosporangium siamense. Heterologous coexpression of a precursor peptide coding gene sinA and lanthipeptide synthetase coding gene sinKC in the host Escherichia coli strain BL21(DE3) resulted in the synthesis of a new lanthipeptide, sinosporapeptin.

Unique Physiological and Genetic Features of Ofloxacin-Resistant Mutants.

New antimicrobial agents are urgently needed to combat the emergence and spread of multidrug-resistant bacteria. Activating the cryptic biosynthetic gene clusters for actinomycete secondary metabolites can provide essential clues for research into new antimicrobial agents. An effective method for this purpose is based on drug resistance selection.

Lincomycin-Induced Secondary Metabolism in Streptomyces lividans 66 with a Mutation in the Gene Encoding the RNA Polymerase Beta Subunit.

Activating the genetic potential of Streptomyces strains to produce secondary metabolites can improve the production of useful biologically active compounds and facilitate the discovery of novel biologically active compounds. In this study, we found that Streptomyces lividans carrying the R440H mutation in rpoB, encoding the RNA polymerase beta subunit, grown in the presence of lincomycin at concentrations below the minimum inhibitory concentration (MIC) produced abundant amounts of actinorhodin and certain cryptic secondary metabolites despite culture conditions that restrict their production by the wild-type strain. The results indicate that lincomycin at concentrations below the MIC may strongly potentiate secondary metabolite production by Streptomyces strains carrying a specific rpoB mutation.

A putative mechanism underlying secondary metabolite overproduction by Streptomyces strains with a 23S rRNA mutation conferring erythromycin resistance.

Mutations in rrn encoding ribosomal RNA (rRNA) and rRNA modification often confer resistance to ribosome-targeting antibiotics by altering the site of their interaction with the small (30S) and large (50S) subunits of the bacterial ribosome. The highly conserved central loop of domain V of 23S rRNA (nucleotides 2042-2628 in Escherichia coli; the exact position varies by species) of the 50S subunit, which is implicated in peptidyl transferase activity, is known to be important in macrolide interactions and resistance. In this study, we identified an A2302T mutation in the rrnA-23S rRNA gene and an A2281G mutation in the rrnC-23S rRNA gene that were responsible for resistance to erythromycin in the model actinomycete Streptomyces coelicolor A3(2) and its close relative Streptomyces lividans 66, respectively.

Isolation and structure determination of a new cytotoxic peptide, curacozole, from Streptomyces curacoi based on genome mining.

Using genome mining, a new cytotoxic peptide named curacozole was isolated from Streptomyces curacoi. Through ESI-MS and NMR analyses, curacozole was determined to be a macrocyclic peptide containing two isoleucine, two thiazole and three oxazole moieties. Curacozole exhibited potent cytotoxic activity against HCT116 and HOS cancer cells.

Combined Drug Resistance Mutations Substantially Enhance Enzyme Production in Paenibacillus agaridevorans.

This study shows that sequential introduction of drug resistance mutations substantially increased enzyme production in The triple mutant YT478 ( Gln225→stop codon, K56R, and R485H), generated by screening for resistance to streptomycin and rifampin, expressed a 1,100-fold-larger amount of the extracellular enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) than the wild-type strain. These mutants were characterized by higher intracellular -adenosylmethionine concentrations during exponential phase and enhanced protein synthesis activity during stationary phase. Surprisingly, the maximal expression of CITase mRNA was similar in the wild-type and triple mutant strains, but the mutant showed greater CITase mRNA expression throughout the growth curve, resulting in enzyme overproduction.

A possible mechanism for lincomycin induction of secondary metabolism in Streptomyces coelicolor A3(2).

Lincomycin forms cross-links within the peptidyl transferase loop region of the 23S ribosomal RNA (rRNA) of the 50S subunit of the bacterial ribosome, which is the site of peptide bond formation, thereby inhibiting protein synthesis. We have previously reported that lincomycin at concentrations below the minimum inhibitory concentration potentiates the production of secondary metabolites in actinomycete strains, suggesting that activation of these strains by utilizing the dose-dependent response of lincomycin could be used to effectively induce the production of cryptic secondary metabolites. Here, we aimed to elucidate the fundamental mechanisms underlying lincomycin induction of secondary metabolism in actinomycetes.

Lincomycin at Subinhibitory Concentrations Potentiates Secondary Metabolite Production by Streptomyces spp.

Antibiotics have either bactericidal or bacteriostatic activity. However, they also induce considerable gene expression in bacteria when used at subinhibitory concentrations (below the MIC). We found that lincomycin, which inhibits protein synthesis by binding to the ribosomes of Gram-positive bacteria, was effective for inducing the expression of genes involved in secondary metabolism in Streptomyces strains when added to medium at subinhibitory concentrations.

Successful implantation after reducing matrix metalloproteinase activity in the uterine cavity.

Background: Recently, the concept of recurrent implantation failure (RIF) in assisted reproductive technology has been enlarged. Chronic uterine inflammation is a known cause of implantation failure and is associated with high matrix metalloproteinase (MMP) activity in uterine cavity flushing. MMP activity of women with RIF has been reported to be higher than that of fertile women.

New strategies for drug discovery: activation of silent or weakly expressed microbial gene clusters.

Genome sequencing of Streptomyces, myxobacteria, and fungi showed that although each strain contains genes that encode the enzymes to synthesize a plethora of potential secondary metabolites, only a fraction are expressed during fermentation. Interest has therefore grown in the activation of these cryptic pathways. We review current progress on this topic, describing concepts for activating silent genes, utilization of "natural" mutant-type RNA polymerases and rare earth elements, and the applicability of ribosome engineering to myxobacteria and fungi, the microbial groups known as excellent searching sources, as well as actinomycetes, for secondary metabolites.

Heterologous expression of a plant RelA-SpoT homologue results in increased stress tolerance in Saccharomyces cerevisiae by accumulation of the bacterial alarmone ppGpp.

The bacterial alarmone ppGpp is present only in bacteria and the chloroplasts of plants, but not in mammalian cells or eukaryotic micro-organisms such as yeasts and fungi. The importance of the ppGpp signalling system in eukaryotes has therefore been largely overlooked. Here, we demonstrated that heterologous expression of a relA-spoT homologue (Sj-RSH) isolated from the halophilic plant Suaeda japonica in the yeast Saccharomyces cerevisiae results in accumulation of ppGpp, accompanied by enhancement of tolerance against various stress stimuli, such as osmotic stress, ethanol, hydrogen peroxide, high temperature and freezing.

Rare earth elements activate the secondary metabolite-biosynthetic gene clusters in Streptomyces coelicolor A3(2).

Genome sequencing projects have revealed many biosynthesis gene clusters for the production of as-yet unknown secondary metabolites, especially in actinomycetes. Here, we report that the rare earth elements, scandium and/or lanthanum, markedly activate, ranging from 2.5- to 12-fold, the expression of nine genes belonging to nine secondary metabolite-biosynthetic gene clusters of Streptomyces coelicolor A3(2) when added to the medium at low concentrations.

Antibacterial discovery in actinomycetes strains with mutations in RNA polymerase or ribosomal protein S12.

We show that selection of drug-resistant bacterial mutants allows the discovery of antibacterial compounds. Mutant strains of a soil-isolated Streptomyces species that does not produce antibacterials synthesize a previously unknown class of antibacterial, which we name piperidamycin. Overall, 6% of non-Streptomyces actinomycetes species and 43% of Streptomyces species that do not produce antibacterials are activated to produce them.

Bacterial alarmone, guanosine 5'-diphosphate 3'-diphosphate (ppGpp), predominantly binds the beta' subunit of plastid-encoded plastid RNA polymerase in chloroplasts.

It's alarming: Bacterial alarmone guanosine 5'-diphosphate 3'-diphosphate (ppGpp), which is a key regulatory molecule that controls the stringent response, also exists in chloroplasts of plant cells. Cross-linking experiments with 6-thioguanosine 5'-diphosphate 3'-diphosphate (6-thioppGpp) and chloroplast RNA polymerase indicate that ppGpp binds the beta' subunit of plastid-encoded plastid RNA polymerase that corresponds to the Escherichia coli beta' subunit. Chloroplasts, which are thought to have originated from cyanobacteria, have their own genetic system that is similar to that of the bacteria from which they were derived.

Dramatic activation of antibiotic production in Streptomyces coelicolor by cumulative drug resistance mutations.

We recently described a new method to activate antibiotic production in bacteria by introducing a mutation conferring resistance to a drug such as streptomycin, rifampin, paromomycin, or gentamicin. This method, however, enhanced antibiotic production by only up to an order of magnitude. Working with Streptomyces coelicolor A3(2), we established a method for the dramatic activation of antibiotic production by the sequential introduction of multiple drug resistance mutations.

Identification of the RsmG methyltransferase target as 16S rRNA nucleotide G527 and characterization of Bacillus subtilis rsmG mutants.

The methyltransferase RsmG methylates the N7 position of nucleotide G535 in 16S rRNA of Bacillus subtilis (corresponding to G527 in Escherichia coli). Disruption of rsmG resulted in low-level resistance to streptomycin. A growth competition assay revealed that there are no differences in fitness between the rsmG mutant and parent strains under the various culture conditions examined.

Mutations in rsmG, encoding a 16S rRNA methyltransferase, result in low-level streptomycin resistance and antibiotic overproduction in Streptomyces coelicolor A3(2).

Certain str mutations that confer high- or low-level streptomycin resistance result in the overproduction of antibiotics by Streptomyces spp. The str mutations that confer the high-level resistance occur within rpsL, which encodes the ribosomal protein S12, while those that cause low-level resistance are not as well known. We have used comparative genome sequencing to determine that low-level resistance is caused by mutations of rsmG, which encodes an S-adenosylmethionine (SAM)-dependent 16S rRNA methyltransferase containing a SAM binding motif.

Physiological analysis of the stringent response elicited in an extreme thermophilic bacterium, Thermus thermophilus.

Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T.

Increased expression of ribosome recycling factor is responsible for the enhanced protein synthesis during the late growth phase in an antibiotic-overproducing Streptomyces coelicolor ribosomal rpsL mutant.

K88E mutation within rpsL, which encodes the S12 ribosomal protein, enhanced the protein synthetic activity of Streptomyces coelicolor during the late growth phase, resulting in overproduction of the deep blue-pigmented polyketide antibiotic actinorhodin. In vitro cross-mixing experiments using the ribosomal and S-150 fractions derived from wild-type and K88E mutant strains suggested that one or more translation factors are enriched in the mutant's S-150 fraction, while Western analysis using antibodies against various translation factors revealed ribosome recycling factor (RRF) to be one of the enriched mediators. RRF purified from overexpressing cells stimulated mRNA-directed green fluorescent protein (GFP) synthesis in an in vitro protein synthesis system.

Expression of luteinizing hormone/chorionic gonadotropin receptor in the rat pineal gland.

Luteinizing hormone (LH) influences the secretion of melatonin (N-acetyl-5-methoxytryptamine) from the pineal gland. The present study examined the possible presence of LH/chorionic gonadotropin (CG) receptor in the pineal gland of adult female rats. Reverse transcriptase-polymerase chain reaction analyses demonstrated that LH/CG receptor mRNA is expressed in the pineal gland.

EshA accentuates ppGpp accumulation and is conditionally required for antibiotic production in Streptomyces coelicolor A3(2).

Disruption of eshA, which encodes a 52-kDa protein that is produced late during the growth of Streptomyces coelicolor A3(2), resulted in elimination of actinorhodin production. In contrast, disruption of eshB, a close homologue of eshA, had no effect on antibiotic production. The eshA disruptant accumulated lower levels of ppGpp than the wild-type strain accumulated.