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At least 343 records · Page 19Linked to original sources
PubMed2006

A molecular approach to the characterization of the eukaryotic communities of an extreme acidic environment: methods for DNA extraction and denaturing gradient gel electrophoresis analysis.

The diversity of the phytobenthonic community present in six acidophilic microbial mats from Río Tinto (Iberian Pyritic Belt, SW Spain) was analysed by optical microscopy and two molecular techniques, denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 18S rDNA cloned gene fragments. Sixteen DNA isolation protocols as well as two commercial DNA extraction kits were tested and their efficiency compared. Purified DNA extracts were amplified by PCR using universal eukaryotic primers and the PCR products analysed by DGGE. Bead-mill homogenization was found to be superior to the other cell lysis methodologies assayed (sonication or freeze-thawing cycles) as it allowed efficiencies of cell disruption of over 95%. The methods combining bead-mill homogenization in the presence of SDS, treatment with chemical extractants (hexadecylmethylammonium bromide or guanidine isothiocyanate) and phenol extraction resulted in DNA preparations that amplified the same number of bands when analysed by DGGE as the two commercial kits assayed. The phylogenetic affiliations of the DGGE bands were determined by a BLAST search, and nine different species related to the Chlorophyta, Ciliophora, Kinetoplastida, Ascomycota, Streptophyta and Colcochaetales taxonomical groups were identified. Similar levels of diversity were found using cloning procedures. Although not all the species observed under the microscope were detected using molecular techniques, e.g. euglenas, heliozoan, or amoebae, DGGE fingerprints showed rather well the level of diversity present in the samples analysed, with limitations similar to cloning techniques.

Animals↗
PubMed2005

How genomics has affected the concept of microbiology.

Genomics influences multiple areas of microbiology, and thus affects key microbiological concepts. Recent reports that describe the large genome and unusual coding capacity of mimivirus, the minimized fungal genomes that contain elements of bacterial metabolism, and the 'signature' eukaryotic proteins in bacteria are introducing grey shades into the black-and-white distinctions between microbial domains. The concept of the 'universal' minimal genome is being challenged, and the ability of minimal genomes to support cellular complexity is under investigation. There have been intriguing insights into microbe-microbe relationships, for example conflict mediation in competing bacteriophages that rapidly evolve survival mechanisms when cooperation is experimentally enforced. Genomics has given birth to metagenomics, but has also stimulated the development of improved cultivation techniques. Lastly, the taxonomic potential of genomics is emerging, as studies of multiple strains allow us to revise and refine the bacterial species concept as well as the idea of a static genome.

Eukaryotic Cells↗
PubMed2005

New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica - a comparison.

Yeasts combine the ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. Yeasts thus provide attractive platforms for the production of recombinant proteins. Here, four important species are presented and compared: the methylotrophic Hansenula polymorpha and Pichia pastoris, distinguished by an increasingly large track record as industrial platforms, and the dimorphic species Arxula adeninivorans and Yarrrowia lipolytica, not yet established as industrial platforms, but demonstrating promising technological potential, as discussed in this article.

Cloning, Molecular↗
PubMed1989

denV gene of bacteriophage T4 restores DNA excision repair to mei-9 and mus201 mutants of Drosophila melanogaster.

The denV gene of bacteriophage T4 was fused to a Drosophila hsp70 (70-kDa heat shock protein) promoter and introduced into the germ line of Drosophila by P-element-mediated transformation. The protein product of that gene (endonuclease V) was detected in extracts of heat-shocked transformants with both enzymological and immunoblotting procedures. That protein restores both excision repair and UV resistance to mei-9 and mus201 mutants of this organism. These results reveal that the denV gene can compensate for excision-repair defects in two very different eukaryotic mutants, in that the mus201 mutants are typical of excision-deficient mutants in other organisms, whereas the mei-9 mutants exhibit a broad pleiotropism that includes a strong meiotic deficiency. This study permits an extension of the molecular analysis of DNA repair to the germ line of higher eukaryotes. It also provides a model system for future investigations of other well-characterized microbial repair genes on DNA damage in the germ line of this metazoan organism.

Animals↗
PubMed2003

Regulation of sialic acid catabolism by the DNA binding protein NanR in Escherichia coli.

All Escherichia coli strains so far examined possess a chromosomally encoded nanATEK-yhcH operon for the catabolism of sialic acids. These unique nine-carbon sugars are synthesized primarily by higher eukaryotes and can be used as carbon, nitrogen, and energy sources by a variety of microbial pathogens or commensals. The gene nanR, located immediately upstream of the operon, encodes a protein of the FadR/GntR family that represses nan expression in trans. S1 analysis identified the nan transcriptional start, and DNA footprint analysis showed that NanR binds to a region of approximately 30 bp covering the promoter region. Native (nondenaturing) polyacrylamide gel electrophoresis, mass spectrometry, and chemical cross-linking indicated that NanR forms homodimers in solution. The region protected by NanR contains three tandem repeats of the hexameric sequence GGTATA. Gel shift analysis with purified hexahistidine-tagged or native NanR detected three retarded complexes, suggesting that NanR binds sequentially to the three repeats. Artificial operators carrying different numbers of repeats formed the corresponding number of complexes. Among the sugars tested that were predicted to be products of the nan-encoded system, only the exogenous addition of sialic acid resulted in the dramatic induction of a chromosomal nanA-lacZ fusion or displaced NanR from its operator in vitro. Titration of NanR by the nan promoter region or artificial operators carrying different numbers of the GGTATA repeat on plasmids in this fusion strain supported the binding of the regulator to target DNA in vivo. Together, the results indicate that GGTATA is important for NanR binding, but the precise mechanism remains to be determined.

Amino Acid Sequence↗
PubMed2006

Application of a wide-range yeast vector (CoMed) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts.

BACKGROUND: Yeasts provide attractive expression platforms in combining ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. However, early restriction to a single yeast platform can result in costly and time-consuming failures. It is therefore advisable to assess several selected systems in parallel for the capability to produce a particular protein in desired amounts and quality. A suitable vector must contain a targeting sequence, a promoter element and a selection marker that function in all selected organisms. These criteria are fulfilled by a wide-range integrative yeast expression vector (CoMed) system based on A. adeninivorans- and H. polymorpha-derived elements that can be introduced in a modular way. RESULTS: The vector system and a selection of modular elements for vector design are presented. Individual single vector constructs were used to transform a range of yeast species. Various successful examples are described. A vector with a combination of an rDNA sequence for genomic targeting, the E. coli-derived hph gene for selection and the A. adeninivorans-derived TEF1 promoter for expression control of a GFP (green fluorescent protein) gene was employed in a first example to transform eight different species including Hansenula polymorpha, Arxula adeninivorans and others. In a second example, a vector for the secretion of IL-6 was constructed, now using an A. adeninivorans-derived LEU2 gene for selection of recombinants in a range of auxotrophic hosts. In this example, differences in precursor processing were observed: only in A. adeninivorans processing of a MFalpha1/IL-6 fusion was performed in a faithful way. CONCLUSION: rDNA targeting provides a tool to co-integrate up to 3 different expression plasmids by a single transformation step. Thus, a versatile system is at hand that allows a comparative assessment of newly introduced metabolic pathways in several organisms or a comparative co-expression of bottleneck genes in cases where production or secretion of a certain product is impaired.

Journal Article↗
PubMed1988

Cryptoendolithic lichen and cyanobacterial communities of the Ross Desert, Antarctica.

Cryptoendolithic microbial communities in the Ross Desert (McMurdo Dry Valleys) are characterized on the basis of photosynthetic microorganisms and fungi. Two eukaryotic communities (the lichen-dominated and Hemichloris communities) and three cyanobacterial communities (the red Gloeocapsa, Hormathonema-Gloeocapsa, and Chroococcidiopsis communities) are described. Eleven coccoid, one pleurocapsoid, and five filamentous cyanobacteria occurring in these communities are characterized and illustrated. The moisture grade of the rock substrate seems to affect pH, formation of primary iron stain, and the distribution of microbial communities.

Antarctic Regions↗
PubMed2001

Fungal histidine kinases.

Eukaryotic cells predominantly use serine, threonine, and tyrosine phosphorylation in various intracellular signal transduction pathways. In contrast, prokaryotic organisms employ numerous "two-component" systems, in which signaling is achieved by transferring a phosphoryl group from phosphohistidine in the "sensor kinase" component to aspartate in the "response regulator" component. In the last several years, genetic screens and genome projects have identified sensor kinases and response regulators in lower eukaryotes and plants, revealing that eukaryotic organisms also make use of His-Asp phosphotransfer in a limited number of signaling pathways. Extensive studies in yeasts have demonstrated that a variation of the two-component system, a multistep "phosphorelay," is the prevailing mechanism among distantly related yeast species. In the budding yeast Saccharomyces cerevisiae, a His-Asp-His-Asp phosphorelay transmits osmotic stress signals to a mitogen-activated protein kinase (MAPK) cascade to induce adaptive responses. A phosphorelay in the fission yeast Schizosaccharomyces pombe, analogous to the S. cerevisiae phosphorelay, is responsible for MAPK activation in response to peroxide stress. Mammalian cells do not have any two-component or phosphorelay systems, although protein histidine kinases unrelated to the sensor kinase may be involved in cellular signaling. Because some phosphorelay proteins are essential for virulence of microbial pathogens, including the yeast fungus Candida albicans, novel antibiotics targeted to phosphorelays may be effective against eukaryotic pathogens without causing host cell damage.

Animals↗
PubMed1998

Use of a rapid throughput in vivo screen to investigate inhibitors of eukaryotic topoisomerase II enzymes.

Topoisomerase II catalyzes the passage of one DNA helix through another via a transient double-stranded break. The essential nature of this enzyme in cell proliferation and its mechanism of action make it an ideal target for cytotoxic agents. Saccharomyces cerevisiae topoisomerase II has been frequently used as a model for testing potential inhibitors of eukaryotic topoisomerase II as antitumor agents. The standard in vivo method of estimating the sensitivity of S. cerevisiae to the antitopoisomerase drugs is via inhibition or kill curves which rely on viable-cell counts and is labor intensive. We present an alternative to this, a high-throughput in vivo screen. This method makes use of a drug-permeable S. cerevisiae strain lacking endogenous topoisomerase II, which is modified to express either human topoisomerase IIalpha or IIbeta or S. cerevisiae topoisomerase II carried on plasmids. Each modified strain expresses a full-length topoisomerase II enzyme, as opposed to the more commonly used temperature-sensitive S. cerevisiae mutant expressing yeast or yeast/human hybrid enzymes. A comparison of this new method with a plating-and-counting method gave similar drug sensitivity results, with increased accuracy and reduced manual input for the new method. The information generated has highlighted the sensitivities of different topoisomerase II enzymes and isoenzymes to several different classes of topoisomerase II inhibitor.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Detection of intimins alpha, beta, gamma, and delta, four intimin derivatives expressed by attaching and effacing microbial pathogens.

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), and Citrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated alpha, beta, delta, and gamma, respectively.

Adhesins, Bacterial↗
PubMedDate not supplied

The distinct signaling mechanisms of microbial sensory rhodopsins in Archaea, Eubacteria and Eukarya.

Most of the known archaeal-type microbial rhodopsins are retinal-binding ion transporters, such as bacteriorhodopsin (BR) and proteorhodopsin (PR). Their identification is the result of extensive studies of their photochemical and biophysical properties. The cells containing these pigments, however, use other microbial rhodopsins as photosensors to monitor environmental light signals. From the early studies of sensory rhodopsin I (HsSRI) in Halobacterium salinarum and sensory rhodopsin II (NpSRII) in Natronomonas pharaonis, we now know that several microbial sensory rhodopsins in the other major domain of life relay information on light intensity and quality to the cell. Three of the most studied photosensory transduction mechanisms of these microbial rhodopsins are dealt with in this review. We discuss recent progress in the understanding of genomic organization, photochemical properties and photosignaling mechanisms with respect to biological function.

Amino Acid Sequence↗
PubMed1976

Mutagenicity and DNA-modifying activity: a comparison of two microbial assays.

Aspects of the Salmonella mutagenesis and Escherichia coli DNA polymerase deficient (pol A1) assay procedures for detecting environmental mutagens are discussed. The chief limitation of the pol A1-- assay involves substances that do not diffuse rapidly in agar. This problem can be overcome by performing the test in suspension. A simple procedure for accomplishing this is described. Although the Salmonella assay is more flexible, under routine conditions it does not respond to several classes of substances which give positive responses in the pol A1- system. For optimal testing, it is recommended that the two microbial assays be used in tandem. The DNA-modifying properties of povidone-iodine for eukaryotic and prokaryotic cells are described. Even though this substance does not display mutagenic properties in the standard Salmonella assay, it does so in suspension culture. The basis of the mutagenic and DNA-modifying properties of povidone-iodine appears to involve the iodination of the cystosine residue of DNA.

Cells, Cultured↗
PubMed2006

The introduction of fluorine atoms or trifluoromethyl groups in short cationic peptides enhances their antimicrobial activity.

The effect of introducing fluorine atoms or trifluoromethyl groups in either the peptidic chain or the C-terminal end of cationic pentapeptides is reported. Three series of amide and ester peptides were synthesised and their antimicrobial properties evaluated. An enhanced activity was found in those derivatives whose structure contained fluorine, suggesting an increase in their hydrophobicity.

Anti-Bacterial Agents↗
PubMed1990

Potent and specific inhibitors of protein kinase C of microbial origin.

Potent and specific inhibitors of protein kinase C have been found in streptomyces and fungi: Staurosporine, an alkaloid from Streptomyces sp., is the most potent inhibitor of protein kinases with an IC50 in the nanomolar range. UCN-01 (7-hydroxy staurosporine), isolated from Streptomyces sp., is a selective inhibitor of protein kinase C with antitumor activity. Calphostin, isolated from the fungus Cladosporium cladosporioides, specifically inhibits protein kinase C (IC50 = 0.05 microM) without inhibiting other protein kinases. Microbial metabolites appear to be a promising source of inhibitors that target signal transduction pathways of eukaryotes.

Alkaloids↗
PubMed2003

Novel antibacterial class.

We report the discovery and characterization of a novel ribosome inhibitor (NRI) class that exhibits selective and broad-spectrum antibacterial activity. Compounds in this class inhibit growth of many gram-positive and gram-negative bacteria, including the common respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis, and are nontoxic to human cell lines. The first NRI was discovered in a high-throughput screen designed to identify inhibitors of cell-free translation in extracts from S. pneumoniae. The chemical structure of the NRI class is related to antibacterial quinolones, but, interestingly, the differences in structure are sufficient to completely alter the biochemical and intracellular mechanisms of action. Expression array studies and analysis of NRI-resistant mutants confirm this difference in intracellular mechanism and provide evidence that the NRIs inhibit bacterial protein synthesis by inhibiting ribosomes. Furthermore, compounds in the NRI series appear to inhibit bacterial ribosomes by a new mechanism, because NRI-resistant strains are not cross-resistant to other ribosome inhibitors, such as macrolides, chloramphenicol, tetracycline, aminoglycosides, or oxazolidinones. The NRIs are a promising new antibacterial class with activity against all major drug-resistant respiratory pathogens.

Amino Acyl-tRNA Synthetases↗