Mutants Of Microorganisms Research Articles

Complementation Assays for Co-chaperone Function.

The development of mutant microorganisms lacking J domain proteins (JDPs; formerly called Hsp40s) has enabled the development of complementation assays for testing the co-chaperone function of JDPs. In these assays, an exogenously expressed novel JDP is tested for its ability to functionally substitute for a non-expressed or nonfunctional endogenous JDP(s) by reversing a stress phenotype. For example, the in vivo functionality of prokaryotic JDPs can be tested on the basis of their ability to reverse the thermosensitivity of a dnaJ cbpA mutant strain of the bacterium Escherichia coli (OD259). Similarly, the in vivo functionality of eukaryotic JDPs can be assessed in a thermosensitive ydj1 mutant strain of the yeast Saccharomyces cerevisiae (JJ160). Here we outline the use of these thermosensitive microorganisms in complementation assays to functionally characterize a JDP from the bacterium, Agrobacterium tumefaciens (AgtDnaJ), and a JDP from the trypanosomal parasite, Trypanosoma cruzi (TcJ2).

Dissolved organic metabolite extraction from high-salt media.

We describe considerations and strategies for developing a nuclear magnetic resonance (NMR) sample preparation method to extract low molecular weight metabolites from high-salt spent media in a model coculture system of phytoplankton and marine bacteria. Phytoplankton perform half the carbon fixation and oxygen generation on Earth. A substantial fraction of fixed carbon becomes part of a metabolite pool of small molecules known as dissolved organic matter (DOM), which are taken up by marine bacteria proximate to phytoplankton. There is an urgent need to elucidate these metabolic exchanges due to widespread anthropogenic transformations on the chemical, phenotypic, and species composition of seawater. These changes are increasing water temperature and the amount of CO2 absorbed by the ocean at energetic costs to marine microorganisms. Little is known about the metabolite-mediated, structured interactions occurring between phytoplankton and associated marine bacteria, in part because of challenges in studying high-salt solutions on various analytical platforms. NMR analysis is problematic due to the high-salt content of both natural seawater and culture media for marine microbes. High-salt concentration degrades the performance of the radio frequency coil, reduces the efficiency of some pulse sequences, limits signal-to-noise, and prolongs experimental time. The method described herein can reproducibly extract low molecular weight DOM from small-volume, high-salt cultures. It is a promising tool for elucidating metabolic flux between marine microorganisms and facilitates genetic screens of mutant microorganisms.

CHEMICAL MUTAGENESIS OF THE LYSINE-PRODUCING STRAIN Brevibacterium sp. IMV B-7447

The aim of the work was to obtain a producer strain with increased lysine accumulation using the chemical mutagenesis method. Methods. To achieve the goal, we used the method of treating the lysine-producing strain with the chemical mutagen N-methyl-N-nitro-N-nitrosoguanidine, cultivating the resulting clone and determining the accumulation of lysine in flasks and a bioreactor. Results. The optimal concentrations and duration of mutagen action for the production of mutant microorganisms were found. Сlones with the maximum lysine accumulation were selected. Mutagenesis was carried out consecutively three times. As a result, lysine-producing strain Brevibacterium sp. IMV B-7796 auxotrophic regarding leucine and threonine with maximum accumulation of the target amino acid was obtained. Conclusions. The lysine producer Brevibacterium sp. IMV B-7796 was obtained, which produced 65.0 g/dm3 of lysine in a bioreactor under conditions of periodic cultivation with feeding. The Brevibacterium sp. IMV B-7796 strain was proposed as a basis for the creation of a genetically modified strain with increased accumulation of lysine for further use in industrial lysine technology.

Aerospace Technology Improves Fermentation Potential of Microorganisms.

It is highly possible to obtain high-quality microbial products in appreciable amounts, as aerospace technology is advancing continuously. Genome-wide genetic variations in microorganisms can be triggered by space microgravity and radiation. Mutation rate is high, mutant range is wide, and final mutant character is stable. Therefore, space microorganism breeding is growing to be a new and promising area in microbial science and has greatly propelled the development of fermentation technology. Numerous studies have discovered the following improvements of fermentation potential in microorganisms after exposure to space: (1) reduction in fermentation cycle and increase in growth rate; (2) improvement of mixed fermentation species; (3) increase in bacterial conjugation efficiency and motility; (4) improvement of the bioactivity of various key enzymes and product quality; (5) enhancement of multiple adverse stress resistance; (6) improvement of fermentation metabolites, flavor, appearance, and stability. Aerospace fermentation technology predominantly contributes to bioprocessing in a microgravity environment. Unlike terrestrial fermentation, aerospace fermentation keeps cells suspended in the fluid medium without significant shear forces. Space radiation and microgravity have physical, chemical, and biological effects on mutant microorganisms by causing alternation in fluid dynamics and genome, transcriptome, proteome, and metabolome levels.

Mutasynthesis of Medicinally Significant Natural Products Through Manipulation of Gene Governing Starter Unit

Most of the biologically active microbial natural products and their analogs bear a complex molecular architecture. The semisynthetic modifications and stereospecific diversity- oriented synthesis of these native natural products to generate analogs are difficult and time-consuming. Mutasynthesis is a powerful tool that utilizes the microorganism's genetic and metabolic engineering skills to produce derivatives of complex natural products of microbial origin. Mutasynthesis is based on the cellular uptake of chemically modified intermediates from the culture media and their addition to the secondary metabolism by mutant microorganisms. This review will describe the importance of mutasynthesis in the generation of complex microbial secondary metabolites. We have covered a literature search on mutasynthesis over the last ten years (2011-2020) in this review.

Competition for nutritional resources masks the true frequency of bacterial mutants

BackgroundIt is widely assumed that all mutant microorganisms present in a culture are able to grow and form colonies, provided that they express the features required for selection. Unlike wild-type Escherichia coli, PHO-constitutive mutants overexpress alkaline phosphatase and hence can hydrolyze glycerol-2-phosphate (G2P) to glycerol and form colonies on plates having G2P as the sole carbon source. These mutations mostly occur in the pst operon. However, the frequency of PHO-constitutive colonies on the G2P selective plate is exceptionally low.ResultsWe show that the rate in which spontaneous PHO-constitutive mutations emerge is about 8.0 × 10−6/generation, a relatively high rate, but the growth of most existing mutants is inhibited by their neighboring wild-type cells. This inhibition is elicited only by non-mutant viable bacteria that can take up and metabolize glycerol formed by the mutants. Evidence indicates that the few mutants that do form colonies derive from microclusters of mutants on the selective plate. A mathematical model that describes the fate of the wild-type and mutant populations under these circumstances supports these results.ConclusionThis scenario in which neither the wild-type nor the majority of the mutants are able to grow resembles an unavoidable “tragedy of the commons” case which results in the collapse of the majority of the population. Cooperation between rare adjacent mutants enables them to overcome the competition and eventually form mutant colonies. The inhibition of PHO-constitutive mutants provides an example of mutant frequency masked by orders of magnitude due to a competition between mutants and their ancestral wild-type cells. Similar “tragedy of the commons-like” cases may occur in other settings and should be taken into consideration while estimating true mutant frequencies and mutation rates.

Phenotypes on demand via switchable target protein degradation in multicellular organisms.

Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation.

Microbe–mineral interactions in naturally radioactive beach sands from Espirito Santo, Brazil: experiments on mutagenicity

Previous studies on naturally radioactive materials suggested that they can have a mutagenic effect on plants (growing in sands in Kerala, South West India), and on bats (dwelling in an abandoned underground mine of primary monazite ore in Namaqualand, Western Cape, South Africa). We hypothesised, based on previous theoretical work, that radioactive sands would not induce mutants in microorganisms over time scales typical of doubling times in the natural environment. The potential of exceptionally monazite (Th)-rich mineral sands collected from the coast of Espirito Santo, Brazil to induce single-point reversion in Escherichia coli cultures (both repair-competent and repair-deficient strains) was tested using the tryptophan reverse mutation assay. The results show that at least on a short-term scale (1-7 days), the monazite-rich sands did not cause an increase in reversion above background.

Inactivation of antineoplastics in clinical wastewater by electrolysis

Wastewater from clinical institutions contains a considerable amount of toxic substances. Among the toxic substances, antineoplastics may induce carcinogenesis, teratogenesis, and the emergence of mutant microorganisms in the environment. Although the incineration or chemical treatments of disposed antineoplastics are recommended, a high energy during incineration and a careful quality control during chemical treatment are required. In this study, we determined the conditions for the electrolytic treatment of an antineoplastic, epirubicin hydrochloride (EH), using two platinum electrodes with a constant current of 100 mA. We analyzed the cytotoxicity, mutagenicity and antibacterial activity of electrolyzed EH and compared them with those of unelectrolyzed EH. Nearly 100% cytotoxicity, mutagenicity and antibacterial activity were eliminated and HPLC did not detect an EH molecule, in the case of electrolysis for 6 h. We also examined the biological cytotoxicities of electrolyzed irinotecan hydrochloride, vincristine sulfate, mitomycin C, paclitaxel, methotrexate and cisplatin, and found that 72.1–99.999% toxicity was eliminated by electrolysis under the same conditions. The biological toxicity of a mixture of these drugs was determined to be decreased by approximately 99% by electrolysis under the same conditions.

Pharmaceutical Discoveries Based on Ethnomedicinal Plants: 1985 to 2000 and Beyond1

Fifteen years after a symposium on economic plants projecting to the year 2000, what predictions and remarks of the speakers with reference to ethnobotany and medicinal plants proved prophetic and what others fell short? Certainly they predicted a continuation of acculturation, biodiversity loss, and technology advances. They also foresaw the need for collaborative, multidisciplinary research and major government funding, and changes in regulations governing an expanded industry in botanicals, nutriceuticals, and phytopharmaceuticals, all of which occurred. However, many technologies were in their infancy or unknown, such as gene amplification and recombinant procedures, high-throughput screening, gene chip technology, and combinatorial chemistry, and are only now being used in research and development in the discovery of new therapeutics. Likewise, who would have foreseen worldwide devastating effects of mutant microorganisms resistant to drugs of choice for treating important diseases, such as malaria and tuberculosis? Research data involving these two infections illustrate the value of finding activity based on collections specifically targeted by indigenous users (antimalaria) and those generally used medicinally together with chemotaxonomic selections (antituberculosis). A discussion of research beyond 2000 addresses the future of ethnobotanical research, acculturation and biodiversity losses, collaborative research, regulation and/or standardization of pharmaceuticals, nutriceuticals, and phytopharmaceuticals, and new technologies.

EMERGING INFECTIOUS DISEASES AND PATHOGENS

Emerging infectious diseases are caused by old, new, and mutant microorganisms. Emergence of these pathogens can be attributed to changes in the characteristics and risk factors of patients, the widespread use of antibiotics, changes in the environment, the role of xenotransplantation, and international travel. In the United States, the incidences of C. difficile, cyclosporiasis, enterohemorrhagic E. coli gastroenteritis, Hantavirus, hepatitis C virus infection, and Lyme disease have increased significantly over the past two decades. Malassezia pachydermatis, extended spectrum beta lactamase (ESBL), Gram negative bacilli, and antibiotic resistant Enterococci, S. aureus, S. pneumoniae, and M. tuberculosis have also emerged prominently. Although not yet seen in the United States, variant Creutzfeldt-Jakob disease has made a great emotional impact on this country. Identifying, treating, and controlling emerging infectious disease and pathogens have created enormous challenges.

Molecular aspects of plant biochemistry

The integration of molecular genetics, protein chemistry and biophysics has brought about significant advances in our understanding of the topography and function of the thylakoid membrane. Using the technique of expression cloning in appropriate mutants of microorganisms, cDNAs encoding numerous plant enzymes, and in particular membrane transporters, have been identified and functionally characterized. A new chapter in plant membrane transport biochemistry has thus been opened. Manipulation of the activities of these enzymes and carriers in transgenic plants is providing increasing insight into whole plant functions and their regulatory mechanisms.

Human molybdenum cofactor deficiency.

Molybdenum cofactor deficiency is an inborn error of metabolism first identified in 1980.1,2 Cofactor deficient patients exhibit combined deficiencies of three molybdoenzymes, sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase, all of which depend on the presence of a tightly bound molybdenum-molybdopterin complex for catalytic activity. The study of molybdenum cofactor deficiency has been carried out in parallel with studies defining the molecular structure of the molybdenum cofactor and those establishing the pathway of its biosynthesis in microorganisms. Thus, when the first patient was identified, very little information was available regarding even basic structural aspects of the cofactor. However, as more patients have been characterized, more information has been obtained to shed light on the structure of the pterin they lack. Many of the human studies have been greatly facilitated by study of enzymes and biosynthetic intermediates in molybdopterin mutants in microorganisms. Transfer of this knowledge to studies of the human patient population is an ongoing process which may lead ultimately to the design of effective therapeutic agents for correction or alleviation of the cofactor deficiency disease.

5104801 Mutant microorganisms useful for cleavage of organic C-S bonds: John J Kilbane assigned to Institute of Gas Technology

5104801 Mutant microorganisms useful for cleavage of organic C-S bonds: John J Kilbane assigned to Institute of Gas Technology

5002888 Mutant microorganisms useful for cleavage of organic C-S bonds: John J Kilbane assigned to Institute of Gas Technology

5002888 Mutant microorganisms useful for cleavage of organic C-S bonds: John J Kilbane assigned to Institute of Gas Technology

4806426 Mutant microorganisms containing recombinant DNA and their use in the production of amylases: Charles A Colson, Philippe Lejeune, Corinne Walon, Karine Willemot, Dion Valmont, Belgium assigned to CPC International Inc

4806426 Mutant microorganisms containing recombinant DNA and their use in the production of amylases: Charles A Colson, Philippe Lejeune, Corinne Walon, Karine Willemot, Dion Valmont, Belgium assigned to CPC International Inc

Kinetics of amino acid production by over-producer mutant microorganisms

Experimental data were modeled based on the literature for the production of nine different amino acids by ten different mutant over-producer microorganisms. Mathematical modeling helped to better understand common aspects of these fermentations, which were not stressed previously by plotting the data only. A Luedeking-Piret type of a model was found to be valid for representing all the amino acid production data. This result indicated the similarity among the amino acid production patterns of all the mutant microorganisms concerned. The results also indicated that amino acid production by the mutant microorganisms was mostly non-growth associated, and that the substrate was allocated by all the microorganisms, mainly for biomass and amino acid production. The amino acid production pattern of the microorganisms did not change when the microorganisms reached the stationary phase, but the depletion of the substrate stopped production immediately.

4654303 Construction of novel mutant microorganisms: Scott Hagedorn assigned to Celanese Corporation

4654303 Construction of novel mutant microorganisms: Scott Hagedorn assigned to Celanese Corporation

Effect of a single amino acid substitution on stability of conformation of a protein

THE role of individual amino acid residues in stabilising protein molecules is of interest, especially in connection with the mechanism of thermoresistance of thermophilic bacteria1 and in connection with the physicochemical basis of temperature sensitive mutants used widely in molecular biology studies. This problem has been approached mainly with the use of synthetic polypeptide2, chemical modification of natural proteins3, or homologous proteins4–9 from different species. Many mutants of microorganisms are available from genetic studies, however, in which the mutant proteins differ from wild type only at a single amino acid position. Such proteins often differ markedly in stability. We describe here a study of the α-subunit of tryptophan synthetase of Escherichia coli. The data suggest that the stability of a protein may be highly correlated with the hydrophobicity of the amino acid residues at some suitable positions. Further studies of this type may clarify the role of individual amino acid residues in stabilising proteins.

A suggested phenotypic classification and terminology for enzyme mutants in micro-organisms.

A Suggested Phenotypic Classification and Terminology for Enzyme Mutants in Micro-Organisms