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PubMed1996

The S. cerevisiae outwardly-rectifying potassium channel (DUK1) identifies a new family of channels with duplicated pore domains.

Potassium channel subunits have six or two transmembrane segments in addition to a conserved pore-forming (P) domain; four subunits come together to form a channel. A gene was identified in S. cerevisiae (J0911) encoding a protein with eight probable membrane-spanning segments and two such P regions. This protein (Duk1p) is a potassium channel because Xenopus oocytes injected with the corresponding RNA express potassium currents activated by depolarization that are not seen in control oocytes. Similar potassium currents were recorded from wildtype S. cerevisiae spheroplasts, but not from those in which the DUK1 locus had been disrupted. Cells carrying the duk1 delta 1::HIS disruption in addition to a chimeric gene comprising DUK1 behind the GAL1 promoter showed outward currents when grown in galactose, but not when grown in glucose. Additional sequences with the duplicate pore motif were found in C. elegans, suggesting that these proteins represent a novel structural family of potassium channel proteins.

Amino Acid Sequence↗
PubMedDate not supplied

[The ultrastructural characteristics of Neisseria meningitidis strains isolated in different forms of infection].

The electron microscopic study of cell populations of the first generation of several N.meningitidis strains, isolated from humans and grown on artificial culture medium (Hottinger's serum agar) has made it possible to establish that these organisms are morphologically heterogeneous. It was manifested by the presence of 7 morphological variants of cells in their cultures (different strains have different proportions of these variants): (a) "normal" and similar to normal diplococcal variants; (b) atypically dividing cells; (c) spheroplastic forms; (d) protoplastic forms (typical and atypical); (e) variably gemmating forms; (f) microforms and (g) gigantic forms. These data are of interest for the microbiological diagnosis of meningitis.

Carrier State↗
PubMed2000

The importance of ATP as a regulator of glycolytic flux in Saccharomyces cerevisiae.

The control of glycolytic flux in the yeast Saccharomyces cerevisiae was studied by using permeabilized cells. Cells were harvested from chemostat cultures and, after removal of the cell wall, nystatin was used to permeabilize the spheroplasts. By this method it is possible to study the performance and regulation of a complete and functional metabolic pathway and not only a single enzymatic step. The results showed that ATP has a strong negative effect on glycolytic activity affecting several of the glycolytic enzymes. However, the main targets for ATP inhibition was phosphofructokinase and pyruvate kinase. Phospofructokinase was inhibited by ATP concentrations starting at about 1-2 mM, while pyruvate kinase required ATP levels above 2.5 mM before any inhibition was visible. These ATP concentrations were in the same range as measured for nitrogen- and glucose-limited cells cultivated in chemostat cultures. Other potential candidates as enzymes susceptible to ATP inhibition included hexokinase and enolase. The ATP:ADP ratio, as well as trehalose-6-phosphate levels, did not seem to influence the glycolytic activity.

Adenosine Diphosphate↗
PubMed1991

Energetically distinct early and late stages of HlyB/HlyD-dependent secretion across both Escherichia coli membranes.

The alternative secretion pathway which exports hemolysin across both Escherichia coli membranes into the surrounding medium is directed by an uncleaved C-terminal targeting signal and the membrane translocator proteins HlyD and HlyB. In order to identify stages and intermediates in this unconventional secretion process we have examined the effect of inhibition of the total proton motive force (delta P) and its components during the in vivo HlyB/HlyD-dependent export of a 22.4 kDa secretion competent HlyA C-terminal peptide (Actp). Secretion of Actp was severely inhibited by the proton ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which collapses simultaneously membrane potential delta psi and the proton gradient delta pH, and also by valinomycin/K+, a potassium ionophore which disrupts delta psi. The inhibition of secretion by valinomycin/K+ was ameliorated by imposition of a pH gradient, the second component of the delta P, and selective depletion of delta pH by nigericin also blocked secretion. This indicates that, as in the secretion of beta-lactamase to the periplasm, HlyB/D-directed secretion requires delta P itself and not specifically one of its components. However, inhibition of HlyB/D-dependent secretion was only marked when CCCP, valinomycin/K+ or nigericin were present during the early stage of Actp secretion; at a later stage the secretion was not significantly inhibited. HlyB/D-dependent secretion appears therefore to share with conventional secretion across the cytoplasmic membrane an early requirement for delta P, but comprises in addition a late stage which does not require delta P, delta psi or delta pH. The translocation intermediate identified in the delta P-independent late stage of secretion was associated with the membrane fraction. Analysis of the protease accessibility of this intermediate in whole cells and spheroplasts showed that it was not in the periplasm, nor was it exposed on the cell surface or on the periplasmic faces of either the inner or outer membranes. This may reflect its close association with the inner membrane or a membrane translocation complex.

Amino Acid Sequence↗
PubMed1991

Cloning and in vivo expression of the human GART gene using yeast artificial chromosomes.

Two Yeast Artificial Chromosomes (YACs) were isolated each with a full-length copy of the human gene that encodes the trifunctional protein containing phosphoribosylglycinamide synthetase (GARS), phosphoribosylglycinamide formyltransferase (GART) and phosphoribosylaminoimidazole synthetase (AIRS). The YACs were characterized by restriction mapping and by in situ hybridization of cosmid subclones containing the YAC ends to human metaphase chromosomes. One of the YACs contains co-cloned non-contiguous DNA whereas the other appears to have a single 600 kbp insert from 21q22.1, the location of the GART gene. A restriction map of the gene was obtained from two cosmid subclones which together span the 40 kb gene. The gene is functional when YAC DNA is transferred into GARS- or GARS-and-AIRS-deficient Chinese Hamster Ovary cells. The gene transfer was carried out both by lipofection using purified yeast DNA and by fusion between yeast spheroplasts and the hamster cells. Restriction analysis of DNA from cell lines whose purine auxotrophy was complemented by the YAC showed that with either method a complete and unrearranged copy of the gene can be transferred. The majority of the fusion cell lines appear to contain at least 80% of the YAC.

Acyltransferases↗
PubMed1991

Subunit III of cytochrome c oxidase is not involved in proton translocation: a site-directed mutagenesis study.

Subunit III (COIII) is one of the three core subunits of the aa3-type cytochrome c oxidase. COIII does not contain any of the redox centres and can be removed from the purified enzyme but has a function during biosynthesis of the enzyme. Dicyclohexyl carbodiimide (DCCD) modifies a conserved glutamic acid residue in COIII and abolishes the proton translocation activity of the enzyme. In this study, the invariant carboxylic acids E98 (the DCCD-binding glutamic acid) and D259 of COIII were changed by site-directed mutagenesis to study their role in proton pumping. Spectroscopy and activity measurements show that a structurally normal enzyme, which is active in electron transfer, is formed in the presence of the mutagenized COIII. Experiments with bacterial spheroplasts indicate that the mutant oxidases are fully competent in proton translocation. In the absence of the COIII gene, only a fraction of the oxidase is assembled into an enzyme with low but significant activity. This residual activity is also coupled to proton translocation. We conclude that, in contrast to numerous earlier suggestions, COIII is not an essential element of the proton pump.

Amino Acid Sequence↗
PubMed1987

Signal sequence of human preproparathyroid hormone is inactive in yeast.

The biosynthesis of human preproparathyroid hormone (hpreproPTH) and the processing to mature parathyroid hormone (hPTH) was investigated in yeast. Cells were transformed with a plasmid that carried a fusion gene made of the yeast pyruvate kinase promoter, complementary DNA (cDNA) encoding a slightly modified form of hpreproPTH and the transcription termination signal from yeast triosephosphate-isomerase. In transformed yeast cells we identified a protein that was recognized by a PTH antiserum and, on gel electrophoresis, comigrated with hpreproPTH marker. The amino-terminal sequence of the protein was consistent with that of hpreproPTH, indicating that the hormone precursor is not processed. It was localized inside the cell, when analyzed in pulse-chase experiments by trypsin accessibility in intact and lysed spheroplasts. In contrast, when mRNA from these yeast cells and from human parathyroid tissue was translated into preproPTH in a reticulocyte lysate supplemented with canine pancreatic microsomes, the preproPTHs from both mRNAs were transported and cleaved with identical efficiencies. We conclude that hpreproPTH is synthesized in yeast but not recognized and processed like a precursor of a secreted protein by the yeast secretory apparatus.

Animals↗
PubMed1984

Secretion and degradation of mutant leucine-specific binding protein molecules containing C-terminal deletions.

The leucine-specific binding protein (LS-BP), a periplasmic component of the Escherichia coli high-affinity leucine transport system, is initially synthesized in a precursor form with a 23 amino acid N-terminal leader sequence that is removed during secretion of the protein into the periplasm. Using in vitro mutagenesis, deletion mutants of the LS-BP gene have been constructed with altered or missing amino acid sequences in the C-terminal portion of the protein. These altered binding proteins exhibited normal processing and secretion but were rapidly degraded in the periplasmic space. In the presence of an uncoupler of the transmembrane potential (CCCP) the precursor forms accumulated in the membrane and were protected from degradation. The altered binding proteins also were secreted by spheroplasts of E coli, after which they were easily detected.

Bacterial Proteins↗
PubMed1986

Orthogonal-field-alternation gel electrophoresis banding patterns of DNA from yeasts.

Chromosomal DNAs from various yeast species were separated by orthogonal-field-alternation gel electrophoresis (OFAGE). To this end we developed a spheroplasting and lysis method to obtain intact DNA from both ascomycetous and basidiomycetous yeasts. The OFAGE banding patterns of 22 ascomycetous and four basidiomycetous yeast strains were compared. The strains represented species from the genera: Brettanomyces, Candida, Cryptococcus, Filobasidiella, Geotrichum, Hansenula, Kluyveromyces, Pachysolen, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Saccharomycodes, Saccharomycopsis, Schizosaccharomyces and Zygosaccharomyces. Variations occurred in the number of bands and their positions in the gel, not only among strains of different genera but also among species from the same genus and even between varieties of the same species. The ascomycetous yeasts, with the exception of Saccharomyces cerevisiae, only showed one to five bands of DNA larger than 1000 kilobase pairs (kb) in general none smaller. The patterns of the four basidiomycetous yeasts revealed also a few large DNA bands but in addition one to six bands ranging in size from 500 to 1000 kb, with the exception of a single smaller chromosome in Rhodotorula mucilaginosa. From the OFAGE banding patterns of strains studied here it appears that in Sacch. cerevisiae the partitioning of DNA over chromosomes is unique. But rather than the large number of chromosomes, the presence of four chromosomes with less than 500 kb of DNA is characteristic for Sacch. cerevisiae.

Ascomycota↗
PubMed1991

Genetic transformation of auxotrophic mutants of the filamentous yeast Trichosporon cutaneum using homologous and heterologous marker genes.

A transformation system for the filamentous yeast Trichosporon cutaneum based on auxotrophic markers is presented and techniques for the induction, isolation and characterization of mutants are described. A number of auxotrophic mutants were isolated and characterized by using biosynthetic precursors and/or inhibitors. A mutant unable to grow in the presence of ornithine could be complemented successfully by spheroplast transformation experiments using the cloned Aspergillus nidulans ornithine transcarbamoylase gene (argB gene) as selection marker with an efficiency of 5-100 transformants per microgram of DNA. In these transformants the heterologous argB gene was present in multiple tandem copies and the transforming DNA was found to remain stable after more than 50 generations in non-selective media. The same mutant could be complemented by a T. cutaneum cosmid gene library and a complementary cosmid was subsequently isolated from this library by a sib-selection strategy. This cosmid transformed T. cutaneum spheroblasts with an efficiency of 50-200 colonies per microgram of DNA. Southern blot analyses were consistent with the view that the transforming sequences became stably integrated into the host genome at the homologous site.

Arginine↗
PubMed1994

Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks.

Rearrangements within plasmid DNA are commonly observed during transformation of eukaryotic cells. One possible cause of rearrangements may be recombination between repeated sequences induced by some lesions in the plasmid. We have examined the mechanisms of transformation-associated recombination in the yeast Saccharomyces cerevisiae using a plasmid system which allowed the effects of physical state and/or extent of homology on recombination to be studied. The plasmids contain homologous or diverged (19%) repeats of the URA3 genes (from S. cerevisiae or S. carlsbergensis) separated by the genetically detectable ADE2 colour marker. Recombination during transformation for covalently closed circular plasmids was over 100-fold more frequent than during mitotic growth. The frequency of recombination is partly dependent on the method of transformation in that procedures involving lithium acetate or spheroplasting yield higher frequencies than electroporation. When present in the repeats, unique single-strand breaks that are ligatable, as well as double-strand breaks, lead to high levels of recombination between diverged and identical repeats. The transformation-associated recombination between repeat DNAs is under the influence of the RAD52 and RAD1 genes.

DNA Damage↗
PubMed1994

A homologous cell-free system for studying protein translocation across the endoplasmic reticulum membrane in fission yeast.

We report the development of a homologous in vitro assay system for analysing translocation of proteins across the endoplasmic reticulum (ER) membrane of the fission yeast Schizosaccharomyces pombe. Our protocol for preparing an S. pombe extract capable of translating natural messenger RNAs was modified from a procedure previously used for Saccharomyces cerevisiae, in which cells are lysed in a bead-beater. However, we were unable to prepare fission yeast microsomes active in protein translocation using existing budding yeast protocols. Instead, our most efficient preparations were isolated by fractionating spheroplasts, followed by extensive washing and size exclusion chromatography of the crude membranes. Translocation of two ER-targeted proteins, pre-acid phosphatase from S. pombe and prepro-alpha-factor from S. cerevisiae, was monitored using two distinct assays. First, evidence that a fraction of both proteins was sequestered within membrane-enclosed vesicles was provided by resistance to exogenously added protease. Second, the protected fraction of each protein was converted to a higher molecular weight, glycosylated form; attachment of carbohydrate to the translocated proteins was confirmed by their ability to bind Concanavalin A-Sepharose. Finally, we examined whether proteins could be translocated across fission yeast microsomal membranes after their synthesis was complete. Our results indicate that S. cerevisiae prepro-alpha-factor can be post-translationally imported into the fission yeast ER, while S. pombe pre-acid phosphatase crosses the membrane only by a co-translational mechanism.

Acid Phosphatase↗
PubMed1995

Identification of a topology control domain in the tetracycline resistance protein.

Two N-terminal fusion proteins combining Escherichia coli maltose-binding protein (MBP) and the 12-transmembrane-segment pBR322 tetracycline resistance protein (Tet) have been constructed to determine the strength and location of topology control signals within the N-terminal portion of the Tet protein. The fusions contain either a secretable (wild-type) or a nonsecretable (MBP delta 2-26) MBP domain joined to the normally cytoplasmic N-terminus of the Tet protein. The effects of MBP targeting on Tet topology were investigated by analyzing the susceptibility of fusion strains to tetracycline and by proteolysis of the fusion proteins in inverted membrane vesicles and spheroplasts. The fusion protein containing MBP delta 2-26 conferred tetracycline resistance to the host strain and gave a normal pattern of Tet digestion fragments, indicating that its Tet domain is oriented and folded properly in the membrane. In contrast, the fusion containing secretable MBP was catalytically inactive apparently due to transfer of the Tet N-terminus to the periplasm with MBP. However, protease treatment of this fusion revealed that MBP secretion seems to affect only the topology of segments 1 and/or 2 of the Tet domain. Therefore, a strong topology control sequence appears to be located in the first cytoplasmic loop of the protein.

ATP-Binding Cassette Transporters↗
PubMedDate not supplied

Bilophila wadsworthia: a unique Gram-negative anaerobic rod.

Although comprising less than 0.01% of the normal human gastrointestinal microbiota, Bilophila wadsworthia is the third most common anaerobe recovered from clinical material obtained from patients with perforated and gangrenous appendicitis. Since its discovery in 1988, B. wadsworthia has been recovered from clinical specimens associated with a variety of infections, including sepsis, liver abscesses, cholecystitis, Fournier's gangrene, soft tissue abscesses, empyema, osteomyelitis, Bartholinitis, and hidradenitis suppurativa. In addition, it has been found in the saliva and vaginal fluids of asymptomatic adults and even in the periodontal pockets of dogs. The organism is a saccharolytic, fastidious, and is easily recognized by its strong catalase reaction with 15% H2O2, production of hydrogen sulfide, and growth stimulation by bile (oxgall) and pyruvate. Approximately 75% of strains are urease positive. When grown on pyruvate-containing media, > 85% of strains demonstrate beta-lactamase production. Ribosomal RNA-based phylogenetic studies show Bilophila to be a homogeneous species, most closely related to Desulfovibrio species. Both adherence to human cells and endotoxin have been observed, and preliminary work suggests that environmental iron has a role in expression of outer membrane proteins. Penicillin-binding proteins appear to mediate the organism's susceptibility to at least some beta-lactam agents, which induce spheroplast formation that results in a haze of growth on agar dilution susceptibility test plates which is difficult to interpret. Bilophilastrains are inhibited in vitro by most antibiotics.

Journal Article↗
PubMed2000

Localization of the type I restriction-modification enzyme EcoKI in the bacterial cell.

To localise the type I restriction-modification (R-M) enzyme EcoKI within the bacterial cell, the Hsd subunits present in subcellular fractions were analysed using immunoblotting techniques. The endonuclease (ENase) as well as the methylase (MTase) were found to be associated with the cytoplasmic membrane. HsdR and HsdM subunits produced individually were soluble, cytoplasmic polypeptides and only became membrane-associated when coproduced with the insoluble HsdS subunit. The release of enzyme from the membrane fraction following benzonase treatment indicated a role for DNA in this interaction. Trypsinization of spheroplasts revealed that the HsdR subunit in the assembled ENase was accessible to protease, while HsdM and HsdS, in both ENase and MTase complexes, were fully protected against digestion. We postulate that the R-M enzyme EcoKI is associated with the cytoplasmic membrane in a manner that allows access of HsdR to the periplasmic space, while the MTase components are localised on the inner side of the plasma membrane.

Bacterial Proteins↗
PubMed2001

Mutant of LolA, a lipoprotein-specific molecular chaperone of Escherichia coli, defective in the transfer of lipoproteins to LolB.

The outer membrane-specific lipoproteins of Escherichia coli are released from the inner membrane as a water-soluble complex with LolA and then transferred to the outer membrane receptor, LolB. LolA thus plays a critical role in the sorting and outer membrane localization of lipoproteins. To dissect the LolA function, the highly conserved residues were subjected to random mutagenesis, followed by selection for a growth defect. LolA(R43L), one of mutants thus constructed, possessed Leu in place of Arg at position 43 and caused accumulation of the LolA(R43L)-lipoprotein complex in the periplasm. LolA(R43L) was as active as wild-type LolA as to the release of lipoproteins from spheroplasts. In marked contrast, the transfer of lipoproteins from LolA(R43L) to LolB was completely inhibited, indicating that Arg at position 43 of LolA is involved in the lipoprotein transfer reaction.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and expression of DNA encoding ovine interleukin 2.

We have generated DNA encoding the mature form of ovine interleukin 2 (IL-2) by polymerase chain reaction (PCR) using primers complementary to sequences at the 5' and 3' ends of human, murine and bovine IL-2 cDNA. The predicted PCR product of 400 bp was ligated into the yeast Ty-P1 galactose-inducible expression vector pOGS40 which was used to transform yeast spheroplasts. The fusion protein, with a Factor Xa proteolytic cleavage site between ovine IL-2 and the P1 fusion partner, was expressed from galactose-induced transformed yeast. P1:IL-2 fusion protein, which self-assembles into virus-like particles (VLPs) due to the interaction of the P1 protein, was purified from lysates of mechanically disrupted yeast by centrifugation on a discontinuous sucrose gradient. Fusion protein was detected in Western blot analysis with polyclonal antisera raised to recombinant bovine IL-2. Soluble recombinant ovine IL-2 was released from the P1 fusion protein by cleavage with Factor Xa enzyme. After purification recombinant ovine IL-2 was functionally active as shown by its ability to support the proliferation of Con A-activated T cells and was capable of generating maedi visna virus-specific cytotoxic T cells from primed precursor cells. The availability of recombinant ovine IL-2 will greatly help the analysis of the specificity of pathogen-specific cells in the sheep.

Amino Acid Sequence↗
PubMed1995

(1,3)-beta-D-glucan synthase activity in mycelial and cell wall-less phenotypes of the fz, sg, os-1 ("slime") mutant strain of Neurospora crassa.

The cell wall-less fz, sg, os-1 ("slime") triple mutant of Neurospora crassa lacks (1,3)-beta-D-glucan synthase activity. fz, sg, os-1 segregants from slime x wild-type crosses initially germinate as a plasmodium (slime-like), but develop hyphae in a few hours and acquire a stable mycelial phenotype (mycelial intermediate). The cell wall-less phenotype (stable slime) can be reisolated from mycelial intermediates by filtration-enrichment selection in medium of high osmolarity. Pairs of mycelial intermediate and stable slime obtained from a single slime-like segregant were comparatively studied. Mycelial intermediate strains synthesize a cell wall with normal amounts of (1,3)-beta-glucan, chitin, and other polysaccharides and possess (1,3)-beta-glucan synthase activity with apparently normal properties (i.e., association with membranes, stability, Km app, Vmax, stimulation by GTP). The enzyme was dissociated by treatment with Tergitol NP-40 and NaCl into a membrane-bound catalytic center and a soluble factor which activates the enzyme in the presence of GTP. Heterologous reconstitution assays demonstrated that stable slime spheroplasts had normal activity of the soluble activating factor, but were severely deficient in membrane-bound activity. The genetic composition of the viable progeny of stable slime or mycelial intermediate x wild-type crosses failed to show differences between the two extreme phenotypes of slime. However, the analysis of heterokaryons demonstrated that the stable slime homokaryotic progeny of stable slime/wild-type heterokaryons were not viable. In contrast, the behavior of mycelial intermediate/wild-type heterokaryons was normal. Apparently, stable slime strains differed from the original mycelial intermediate in a mutation(s) which arose spontaneously during the filtration-enrichment selection applied to mycelial intermediates in order to obtain the cell wall-less phenotype. This new trait impaired conidial germination and might be the actual cause of the loss of (1,3)-beta-glucan synthase activity and cell wall.

Cell Fractionation↗