Novel assay for screening rifamycin B-producing mutants.

A simple method that allows easy identification of rifamycin B-producing strains is described. This method involves the use of an enzyme, rifamycin oxidase, which converts inactive rifamycin B to active rifamycin S. In this method, colonies to be tested are grown in pairs. The two colonies are then transferred to two plates seeded with a sensitive strain of Staphylococcus aureus, one plate of which contains the enzyme rifamycin oxidase. All paired colonies which show a larger inhibition zone diameter on the enzyme-containing plate are identified as rifamycin B producers.     

Improvement of industry-applied rifamycin B-producing strain,Amycolatopsis mediterranei,by rational screening

An industrially applied rifamycin B-producing strain, Amycolatopsis mediterranei XC 1-02, was used for further screening. A special mutation and screening procedure was adopted to select a strain, which can alleviate the inhibition caused by both aromatic amino acid and p-hydroxybenzoic acid in the pathway of rifamycin B biosynthesis as well as enhance the production of propionate, one of the precursors of rifamycin B biosynthesis. By the above methods, a strain A. mediterranei XC 9-25 was obtained, and its rifamycin B productivity in shaking flask reaches 10 g/L, which is 2.38 times higher than that of the ancestral strain XC 1-02. The productivity of rifamycin B fed-batch fermentation in 60000 L fermentor with A. mediterranei XC 9-25 reached 19.11 g/L.     

Whole genome sequence of the rifamycin B-producing strain Amycolatopsis mediterranei S699

Amycolatopsis mediterranei S699 is an actinomycete that produces an important antibiotic, rifamycin B. Semisynthetic derivatives of rifamycin B are used for the treatment of tuberculosis, leprosy, and AIDS-related mycobacterial infections. Here, we report the complete genome sequence (10.2 Mb) of A. mediterranei S699, with 9,575 predicted coding sequences.     

Novel screening assay for the detection of phenolic acid esterases

A novel plate assay method, developed for the screening of microorganisms or enzyme preparations for phenolic acid esterases, involves incorporating ethyl cinnamate into an agar medium. After inoculation and incubation, the plate is flooded with a pH-sensitive dye to reveal yellow zones around positive cultures against a blue background. A number of yeasts (Rhodotorula spp. and Candida spp.) and fungi (Penicillium sp. and Aspergillus sp.) gave positive results, while a number of commercial enzymes, particularly pectinases, also exhibited good phenolic acid esterase.J.A. Donaghy and A.M. McKay are with the Food Microbiology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5PX, UK. A.M. McKay is also with the Department of Food Science (Microbiology), The Queen's University of Belfast, Newforge Lane, Belfast BT9 5PX, UK.     

High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants.

A rapid method for identifying compounds that are potential substrates for the drug metabolising enzyme cytochrome P450 is described. The strategy is based on the detection of a degradation product of NAD(P)H oxidation during substrate turnover by the enzyme expressed in Escherichia coli cells spontaneously lysed under the experimental conditions. The performance of the method has been tested on two known substrates of the wild-type cytochrome P450 BM3, arachidonic (AA) and lauric (LA) acids, and two substrates with environmental significance, the anionic surfactant sodium dodecyl sulfate (SDS), and the solvent 1,1,2,2-tetrachloroethane (TCE). The minimal background signal given from cells expressing cytochrome P450 BM3 in the absence of added substrate is only 3% of the signal in the presence of saturating substrate. Control experiments have proven that this method is specifically detecting NADPH oxidation by catalytic turnover of P450 BM3. The assay has been adapted to a microtitre plate format and used to screen a series of furazan derivatives as potential substrates. Three derivatives were identified as substrates. The method gave a significant different signal for two isomeric furazan derivatives. All results found on the cell lysate were verified and confirmed with the purified enzyme. This strategy opens the way to automated high throughput screening of NAD(P)H-linked enzymatic activity of molecules of pharmacological and biotechnological interest and libraries of random mutants of NAD(P)H-dependent biocatalysts.     

Development of a BRET2 screening assay using beta-arrestin 2 mutants

This study has focused on enhancing the signal generated from the interaction between a G-protein-coupled receptor (GPCR) and beta-arrestin 2 (beta-arr2), measured by the bioluminescence resonance energy transfer (BRET(2)) technology. Both class A (beta(2)-adrenergic receptor [beta(2)-AR]) and class B (neurokinin-type 1 receptor [NK1-R]) GPCRs, classified based on their internalization characteristics, have been analyzed. It was evaluated whether the BRET(2) signal can be enhanced by using (1) beta-arr2 phosphorylation-independent mutant (beta-arr2 R169E) and (2) beta-arr2 mutants deficient in their ability to interact with the components of the clathrin-coated vesicles (beta-arr2 R393E, R395E and beta-arr2 373 stop). For the class B receptor, there was no major difference in the agonist-promoted BRET(2) signal when comparing results obtained with wild-type (wt) and mutant beta-arr2. However, with the class A receptor, a more than 2-fold increase in the BRET(2) signal was observed with beta-arr2 mutants lacking the AP-2 or both AP-2 and clathrin binding sites. This set of data suggests that the inability of these beta-arr2 mutants to interact with the components of the clathrin-coated vesicle probably prevents their rapid dissociation from the receptor, thus yielding an increased and more stable BRET(2) signal. The beta-arr2 R393E, R395E mutant also enhanced the signal window with other members of the GPCR family (neuropeptide Y type 2 receptor [NPY2-R] and TG1019 receptor) and was successfully applied in full-plate BRET(2)-based agonist and antagonist screening assays.     

Novel screening method for potential skin-whitening compounds by a luciferase reporter assay

Measurement of the melanin content by using B16 melanoma cells is generally applied to find novel skin-whitening agents. However, this measurement method using B16 melanoma cells has such disadvantages, as the time taken, its sensitivity, and troublesomeness. We therefore attempted in the present study to establish a reporter assay system by measuring the tyrosinase promoter activity to use for convenient, high-throughput screening of new melanogenesis inhibitors. We first confirmed the validity of this reporter assay system by using such known skin-whitening agents, as arbutin, sulforaphane, and theaflavin 3,3'-digallate. We then compared the effect of 56 compounds on the tyrosinase promoter activity to test this reporter assay system. Carnosol, and rottlerin strongly inhibited the tyrosinase promoter activity. Moreover, carnosol and rottlerin decreased melanin synthesis and tyrosinase expression in a dose-dependent manner when using B16 melanoma cells. These results indicate this new luciferase reported assay system to be an effective and convenient method for screening potential skin-whitening compounds.     

An improved method for screening alpha-acetolactate producing mutants.

Alpha-Acetolactate-deficient Lactococcus lactis ssp. lactis biovar. diacelylactis are utilised in several industrial processes for producing diacetyl and alpha-acetolactate. They can be selected by screening after random mutagenesis. We improved a previously described screening method [Monnet, C., Schmitt, P., Diviès, C., 1997. Appl. Environ. Microbiol. 63, 793-795], which makes it possible to screen up to 1000 colonies per agar plate, whereas the previous method allowed to screen only 60 colonies per agar plate. The new screening method facilitates selection of alpha-acetolactate-deficient mutants.     

Novel screening assay for in vivo selection of Klebsiella pneumoniae genes promoting gastrointestinal colonisation

ABSTRACT: BACKGROUND: Klebsiella pneumoniae is an important opportunistic pathogen causing pneumonia, sepsis and urinary tract infections. Colonisation of the gastrointestinal (GI) tract is a key step in the development of infections; yet the specific factors important for K. pneumoniae to colonize and reside in the GI tract of the host are largely unknown. To identify K. pneumoniae genes promoting GI colonisation, a novel genomic-library-based approach was employed. RESULTS: Screening of a K. pneumoniae C3091 genomic library, expressed in E. coli strain EPI100, in a mouse model of GI colonisation led to the positive selection of five clones containing genes promoting persistent colonisation of the mouse GI tract. These included genes encoding the global response regulator ArcA; GalET of the galactose operon; and a cluster of two putative membrane-associated proteins of unknown function. Both ArcA and GalET are known to be involved in metabolic pathways in Klebsiella but may have additional biological actions beneficial to the pathogen. In support of this, GalET was found to confer decreased bile salt sensitivity to EPI100. CONCLUSIONS: The present work establishes the use of genomic-library-based in vivo screening assays as a valuable tool for identification and characterization of virulence factors in K. pneumoniae and other bacterial pathogens.     

An ultrahigh-throughput screening assay for estrogen receptor ligands.

Members of the steroid and thyroid hormone receptor superfamily (nuclear receptors) play diverse roles in mammalian physiology, in both normal and pathological states. For this reason, and because nuclear receptors are natural receptors for lipophilic small molecules, they are important therapeutic targets for the pharmaceutical industry. Here we describe a method for screening for ligands for one of these receptors, the estrogen receptor. The method is rapid, robust, and reliable, and has been used in an ultrahigh-throughput robotic screen. The receptor is crosslinked to a scintillant-containing solid support (FlashPlate) via a receptor-specific antibody. Test compounds are assayed for competition with a radiolabeled estrogen for binding to the immobilized receptor. Receptor-ligand complexes are allowed to form and receptor-bound radioactivity is detected in a scintillation counter. The assay has been designed for both isoforms of the estrogen receptor, alpha and beta, using separate antibodies for each. Given a radioactive tracer and an appropriate antibody, many of which are now commercially available, the assay could be established for any nuclear receptor.     

Simple screening method for identification of nonpleiotropic mutants for exoenzyme production.

A differential medium that distinguishes between pleiotropic and nonpleiotropic mutants for exoenzyme production has been developed for Staphylococcus simulans biovar staphylolyticus. The medium will facilitate genetic analysis of exoenzyme production by this organism. Generally useful strategies for increasing the sensitivity of indicator plates for detection of exoenzyme activities are presented.     

Automated screening for mutants affecting dopaminergic-neuron specification in C. elegans

We describe an automated method to isolate mutant Caenorhabditis elegans that do not appropriately execute cellular differentiation programs. We used a fluorescence-activated sorting mechanism implemented in the COPAS Biosort machine to isolate mutants with subtle alterations in the cellular specificity of GFP expression. This methodology is considerably more efficient than comparable manual screens and enabled us to isolate mutants in which dopamine neurons do not differentiate appropriately.     

Genetic modification of an echinocandin B-producing strain ofAspergillus nidulans to produce mutants blocked in sterigmatocystin biosynthesis

Summary The production of echinocandin B (ECB), a lipopolypeptide used for chemical manufacture of the anti-Candida agent Cilofungin^TM, was accomplished by fermentation using a strain ofAspergillus nidulans. In addition to ECB, this fermentation also produces a significant amount of sterigmatocystin (ST), a potent carcinogen structurally related to the aflatoxins. Mutants blocked in the ST biosynthetic pathway were created by genetic modification of the polyploid production strain C747. The following steps were involved: (i) reduction of the genotype to haploid by treatment with the spindle fiber poison methyl 1-(butylcarbamoyl)-2-benzimidazole carbamate (MBC), using colony morphology, conidia size, and the ability to obtain 5-fluoro-orotic acid (5-FOA)-resistant mutants as criteria for ploidy; (ii) mutagenesis of a haploid isolate using UV irradiation; and (iii) screening of mutants for inability to produce ST by thin layer chromatography. Six mutants blocked in ST production were isolated. All six remained capable of producing ECB equivalent in quantity to the haploid strain C747-GR14. One of the mutants was shown to be the result of a chromosomal translocation.     

Novel method for screening bacterial colonies for phosphatase activity.

Current methods for screening large numbers of bacterial colonies for phosphatase activity, rely heavily on the use of colorimetric assays. While such methods have been applied extensively in the laboratory, they are not without their drawbacks. We here describe a precipitating fluorescent probe that can be used to screen phosphatase activity in bacterial colonies. This probe can be incorporated directly into agar plates used to culture the organisms of interest. The approach offers several advantages over current methodologies including the ability to monitor the development of phosphatase activity with colony development, and the ability to distinguish between activity arising from cell-bound and cell-free enzyme. This enzyme probe was successfully used to detect and isolate phosphatase-producing bacteria from activated sludge.     

Novel redox potential-based screening strategy for rapid isolation of Klebsiella pneumoniae mutants with enhanced 1,3-propanediol-producing capability

This report describes a novel redox potential (oxidoreduction potential [ORP])-based screening strategy for the isolation of mutants of Klebsiella pneumoniae which have an increased ability to produce 1,3-propanediol (1,3-PD). This method can be described as follows: first, to determine an ORP range which is preferred for the wild-type strain to grow and to produce 1,3-PD; second, to subject a chemically mutagenized culture to a reduced ORP level which is deleterious for the wild-type strain. Colonies that showed high specific growth rates at deleterious ORP levels were selected, and their abilities to produce 1,3-PD were investigated. In an ORP-based screening experiment where the ORP was controlled at -280 mV, 4 out of 11 isolated strains were recognized as positive mutant strains. A mutant which is capable of producing higher concentrations of 1,3-PD was subjected to fed-batch fermentations for further characterization. Its preferred ORP level (-280 mV) was significantly lower than that of its parent (-190 mV). The highest 1,3-PD concentration of the mutant was 915 mmol liter(-1), which was 63.1% higher than that of the parent. Metabolic-flux analysis suggested that the intracellular reductive branch of the mutant was strengthened, which improved 1,3-PD biosynthesis. The procedure and results presented here provide a novel method of screening for strains with improved product formation.     

Novel inhibitors of bacterial cytokinesis identified by a cell-based antibiotic screening assay

The continuous emergence of antibiotic resistance demands that novel classes of antibiotics continue to be developed. The division machinery of bacteria is an attractive target because it comprises seven or more essential proteins that are conserved almost throughout the bacteria but are absent from humans. We describe the development of a cell-based assay for inhibitors of cell division and its use to isolate a new inhibitor of FtsZ protein, a key player in the division machinery. Biochemical, cytological, and genetic data are presented that demonstrate that FtsZ is the specific target for the compound. We also describe the effects of more potent analogues of the original hit compound that act on important pathogens, again at the level of cell division. The assay and the compounds have the potential to provide novel antibiotics with no pool of pre-existing resistance. They have provided new insight into cytokinesis in bacteria and offer important reagents for further studies of the cell division machinery.     

Novel ionizing radiation-sensitive mutants of Deinococcus radiodurans.

Two new loci, irrB and irrI, have been identified in Deinococcus radiodurans. Inactivation of either locus results in a partial loss of resistance to ionizing radiation. The magnitude of this loss is locus specific and differentially affected by inactivation of the uvrA gene product. An irrB uvrA double mutant is more sensitive to ionizing radiation than is an irrB mutant. In contrast, the irrI uvrA double mutant and the irrI mutant are equally sensitive to ionizing radiation. The irrB and irrI mutations also reduce D. radiodurans resistance to UV radiation, this effect being most pronounced in uvrA+ backgrounds. Subclones of each gene have been isolated, and the loci have been mapped relative to each other. The irrB and irrI genes are separated by approximately 20 kb of intervening sequence that encodes the uvrA and pol genes.     

Homogeneous screening assay for human tankyrase

Tankyrase, a member of human PARP protein superfamily, catalyzes a covalent post-translational modification of substrate proteins. This modification, poly(ADP-ribos)ylation, leads to changes in protein interactions and modifies downstream signaling events. Tankyrase 1 is a potential drug target due to its functions in telomere homeostasis and in Wnt signaling. We describe here optimization and application of an activity-based homogenous assay for tankyrase inhibitors in a high-throughput screening format. The method measures the consumption of substrate by the chemical conversion of the remaining NAD(+) into a stable fluorescent condensation product. Conditions were optimized to measure the enzymatic auto-modification of a recombinant catalytic fragment of tankyrase 1. The fluorescence assay is inexpensive, operationally easy and performs well according to the statistical analysis (Z'= 0.7). A validatory screen with a natural product library confirmed suitability of the assay for finding new tankyrase inhibitors. Flavone was the most potent (IC(50)=325 nM) hit from the natural compounds. A flavone derivative, apigenin, and isopropyl gallate showed potency on the micromolar range, but displayed over 30-fold selectivity for tankyrase over the studied isoenzymes PARP1 and PARP2. The assay is robust and will be useful for screening new tankyrase inhibitors.     

Method for the rapid screening of cellulolytic streptomycetes and their mutants.

A method for the rapid screening of cellulolytic streptomycetes and their mutants is reported. The technique consists of a plate assay on media containing filter paper fibres as cellulose substrate. The cellulolytic activity is detected and measured by the formation of clearing zones around the streptomycete colonies. The sensitivity of the method is increased considerably by subjecting the plates to an additional incubation period at 43 degrees C in the presence of a buffer at pH 5.3. by replicating these colonies on other Petri plates containing the appropriate media, it is possible to assess rapidly, not only the degree of catabolic repression of the cellulase production by glucose, but also, in a semiquantitative way, the amount of enzymes produced.