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PubMed2003

Multiple pathways are co-regulated by the protein kinase Snf1 and the transcription factors Adr1 and Cat8.

ADR1 and CAT8 encode carbon source-responsive transcriptional regulators that cooperatively control expression of genes involved in ethanol utilization. These transcription factors are active only after the diauxic transition, when glucose is depleted and energy-generating metabolism has shifted to the aerobic oxidation of non-fermentable carbon sources. The Snf1 protein kinase complex is required for activation of their downstream target genes described previously. Using DNA microarrays, we determined the extent to which these three factors collaborate in regulating the expression of the yeast genome after glucose depletion. The expression of 108 genes is significantly decreased in the absence of ADR1. The importance of ADR1 during the diauxic transition is illustrated by the observation that expression of almost one-half of the 40 most highly glucose-repressed genes is ADR1-dependent. ADR1-dependent genes fall into a variety of functional classes with carbon metabolism containing the largest number of members. Most of the genes in this class are involved in the oxidation of different non-fermentable carbon sources. These microarray data show that ADR1 coordinates the biochemical pathways that generate acetyl-CoA and NADH from non-fermentable substrates. Only a small number of ADR1-dependent genes are also CAT8-dependent. However, nearly one-half of the ADR1-dependent genes are also dependent on the Snf1 protein kinase for derepression. Many more genes are SNF1-dependent than are either ADR1- or CAT8-dependent suggesting that SNF1 plays a broader role in gene expression than either ADR1 or CAT8. The largest class of SNF1-dependent genes encodes regulatory proteins that could extend SNF1 dependence to additional pathways.

Binding Sites↗
PubMed1979

Simultaneous determination of polar and non-polar solvents in air using a two-phase desorption from charcoal.

A gas chromatographic procedure is described which is capable of measuring both polar and non-polar organic solvents present simultaneously in the work environment at concentrations between 1/100 and 1 times the Threshold Limit Values (TLV). Airborne organics are collected on a single activated charcoal tube for periods of 3 to 6 hours and desorbed with a two-phase (water/carbon disulfide) desorption mixture. Organic and aqueous phases are analyzed separately on the same gas chromatographic column packed with Oronite NIW on Carbopack B. Recoveries were determined for fifteen common solvents. Most recoveries were greater than 90% with all coefficients of variation being less than +/- 10%. Breakthrough was observed only when solvents were present at very high concentrations. Examples of field sampling are also presented. This procedure is generally applicable for monitoring complex mixtures of volatile organic compounds in air. Advantages include excellent recoveries for polar solvents such as acetone and ethanol, utilization of common analytical instrumentation under a single set of operating conditions and compatibility with TWA personnel monitoring methodology.

Air↗
PubMed1999

Hippea maritima gen. nov., sp. nov., a new genus of thermophilic, sulfur-reducing bacterium from submarine hot vents.

Three strains of moderately thermophilic, sulfur-reducing bacteria were isolated from shallow-water hot vents of the Bay of Plenty (New Zealand) and Matupi Harbour (Papua New Guinea). Cells of all isolates were short, Gram-negative, motile rods with one polar flagellum. All strains were obligate anaerobes and grew optimally at pH 5.8-6.2, 52-54 degrees C and 2.5-3% (w/v) NaCl. Growth substrates were molecular hydrogen, acetate and saturated fatty acids; one of the strains, isolated from Matupi Harbour, was able to utilize ethanol. Elemental sulfur was required for growth. H2S and CO2 were the only growth products. No growth occurred in the absence of 100 mg yeast extract I-1. The G+C content of the DNA determined for the type strain MH2T was 40.4 mol%. Results of 16S rDNA sequencing indicated that these strains represent a distinct lineage most closely related to the genus Desulfurella. On the basis of the results of morphological, physiological and phylogenetic studies, a new genus, Hippea gen. nov., is proposed with the type species Hippea maritima gen. nov., sp. nov., of which the type strain is MH2T (= DSM 10411T).

Anaerobiosis↗
PubMed1987

Adaptation of the membrane fatty acid composition by growth in the presence of n-alkanols influences glycosyltransferase expression in Streptococcus salivarius.

Growth of Streptococcus salivarius ATCC 25975 in the presence of n-alkanols in the series methanol to decan-1-ol led to a decrease in the unsaturated to saturated fatty acid ratio. Each member of the set of n-alkanols which was examined over a range of concentrations possessed a point at which extracellular glucosyltransferase (GTF) production was minimal; increasing the concentration of the n-alkanol past this point stimulated GTF production. This effect was greatest with hexan-1-ol although it was observed to a lesser extent with pentan-1-ol and heptan-1-ol. Reduced cell-associated fructosyltransferase activity was observed with increasing concentrations of each n-alkanol. Growth in the presence of 25 mM-propan-1-ol gave rise to a fatty acid profile in which 55% of the fatty acids were of an odd chain length. S. salivarius ATCC 25975 was shown to be able to utilize ethanol in a similar manner to propan-1-ol by growing it in the presence of 400 mM-[14C]ethanol. Analysis of the membrane lipids at the stationary phase of growth indicated that 17.6% of the carbon of the fatty acids was derived from ethanol. A leaky adh mutant, S. salivarius MJ 37501, was isolated. The leaky nature of the mutant enabled it to incorporate reduced levels of odd-chain-length fatty acids into its membrane lipids when grown in the presence of 100 mM-propan-1-ol, but not when grown in the presence of 25 mM-propan-1-ol. S. salivarius ATCC 25975 therefore metabolized propan-1-ol (and ethanol) via a constitutive alcohol dehydrogenase.

Adaptation, Biological↗
PubMed2005

Geobacter bemidjiensis sp. nov. and Geobacter psychrophilus sp. nov., two novel Fe(III)-reducing subsurface isolates.

Fe(III)-reducing isolates were recovered from two aquifers in which Fe(III) reduction is known to be important. Strain Bem(T) was enriched from subsurface sediments collected in Bemidji, MN, USA, near a site where Fe(III) reduction is important in aromatic hydrocarbon degradation. Strains P11, P35(T) and P39 were isolated from the groundwater of an aquifer in Plymouth, MA, USA, in which Fe(III) reduction is important because of long-term inputs of acetate as a highway de-icing agent to the subsurface. All four isolates were Gram-negative, slightly curved rods that grew best in freshwater media. Strains P11, P35(T) and P39 exhibited motility via means of monotrichous flagella. Analysis of the 16S rRNA and nifD genes indicated that all four strains are delta-proteobacteria and members of the Geobacter cluster of the Geobacteraceae. Differences in phenotypic and phylogenetic characteristics indicated that the four isolates represent two novel species within the genus Geobacter. All of the isolates coupled the oxidation of acetate to the reduction of Fe(III) [iron(III) citrate, amorphous iron(III) oxide, iron(III) pyrophosphate and iron(III) nitrilotriacetate]. All four strains utilized ethanol, lactate, malate, pyruvate and succinate as electron donors and malate and fumarate as electron acceptors. Strain Bem(T) grew fastest at 30 degrees C, whereas strains P11, P35(T) and P39 grew equally well at 17, 22 and 30 degrees C. In addition, strains P11, P35(T) and P39 were capable of growth at 4 degrees C. The names Geobacter bemidjiensis sp. nov. (type strain Bem(T)=ATCC BAA-1014(T)=DSM 16622(T)=JCM 12645(T)) and Geobacter psychrophilus sp. nov. (strains P11, P35(T) and P39; type strain P35(T)=ATCC BAA-1013(T)=DSM 16674(T)=JCM 12644(T)) are proposed.

Bacterial Proteins↗
PubMed2006

Tepidanaerobacter syntrophicus gen. nov., sp. nov., an anaerobic, moderately thermophilic, syntrophic alcohol- and lactate-degrading bacterium isolated from thermophilic digested sludges.

Three anaerobic, moderately thermophilic, syntrophic primary alcohol- and lactate-degrading microbes, designated strains JL(T), JE and OL, were isolated from sludges of thermophilic (55 degrees C) digesters that decomposed either municipal solid wastes or sewage sludge. The strains were strictly anaerobic organisms. All three strains grew at 25-60 degrees C and pH 5.5-8.5 and optimum growth was observed at 45-50 degrees C and pH 6.0-7.0. The three organisms grew chemo-organotrophically on a number of carbohydrates in the presence of yeast extract. In co-culture with the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus, all strains could utilize ethanol, glycerol and lactate syntrophically for growth, although these compounds were not metabolized in pure culture without additional external electron acceptors. All strains could reduce thiosulphate. Quinones were not detected. The DNA G+C contents of strains JL(T), JE and OL were 38.0, 37.3 and 37.7 mol%, respectively. Major cellular fatty acids of the strains were iso-C(15 : 0), C(16 : 0) and unsaturated species of C(15 : 1). Phylogenetic analyses based on 16S rRNA gene sequences revealed that the strains belong to a deeply branched lineage of the phylum Firmicutes; the most closely related species was Thermovenabulum ferriorganovorum (16S rRNA gene sequence similarity of 88 %). The three strains were phylogenetically very closely related to each other (99-100 % 16S rRNA gene sequence similarity) and were physiologically and chemotaxonomically similar. These genetic and phenotypic properties suggest that the strains should be classified as representatives of a novel species and genus; the name Tepidanaerobacter syntrophicus gen. nov., sp. nov. is proposed. The type strain of Tepidanaerobacter syntrophicus is strain JL(T) (=JCM 12098(T)=NBRC 100060(T)=DSM 15584(T)).

Alcohols↗
PubMed2006

Oceanisphaera donghaensis sp. nov., a halophilic bacterium from the East Sea, Korea.

A taxonomic study was carried out on two isolates, strains BL1(T) and BL11, from marine sediment collected from the East Sea, Korea. Comparative 16S rRNA gene sequence studies showed that these isolates clearly affiliated with the Gammaproteobacteria. BL1T and BL11 were most closely related to Oceanisphaera litoralis KMM 3654T (97.6 and 97.7 % 16S rRNA gene sequence similarity, respectively). The level of 16S rRNA gene sequence similarity between strains BL1T and BL11 was 99.7 %. The two isolates were Gram-negative, aerobic, moderately halophilic, and grew in 0.5-8.0 % NaCl and at 4-42 degrees C. Strains BL1T and BL11 shared some physiological and biochemical properties with O. litoralis KMM 3654T, although they differed in that BL1T and BL11 were able to utilize ethanol, proline and alanine. The G+C contents of the genomic DNA of strains BL1T and BL11 were 56.6 and 57.1 mol%, respectively. Both strains possessed C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH, C(16 : 0) and C(18 : 1)omega7c as the major fatty acids. DNA-DNA relatedness data indicated that strains BL1T and BL11 represent a genomic species that is separate from O. litoralis KMM 3654T. On the basis of polyphasic evidence, it is proposed that strain BL1T (=KCTC 12522T = DSM 17589T) represents the type strain of a novel species, Oceanisphaera donghaensis sp. nov.

Aeromonadaceae↗
PubMed1990

Assembly of imported subunit 8 into the ATP synthase complex of isolated yeast mitochondria.

This study concerns the assembly into a multisubunit enzyme complex of a small hydrophobic protein imported into isolated mitochondria. Subunit 8 of yeast mitochondrial ATPase (normally a mitochondrial gene product) was expressed in vitro as a chimaeric precursor N9L/Y8-1, which includes an N-terminal-cleavable transit peptide to direct its import into mitochondria. Assembly into the enzyme complex of the imported subunit 8 was monitored by immunoadsorption using an immobilized anti-F1-beta monoclonal antibody. Preliminary experiments showed that N9L/Y8-1 imported into normal rho+ mitochondria, with its complement of fully assembled ATPase, did not lead to an appreciable assembly of the exogenous subunit 8. With the expectation that mitochondria previously depleted of subunit 8 could allow such assembly in vitro, target mitochondria were prepared from genetically modified yeast cells in which synthesis of subunit 8 was specifically blocked. Initially, mitochondria were prepared from strain M31, a mit- mutant completely incapable of intramitochondrial biosynthesis of subunit 8. These mit- mitochondria however were unsuitable for assembly studies because they could not import protein in vitro. A controlled depletion strategy was then evolved. An artificial nuclear gene encoding N9L/Y8-1 was brought under the control of a inducible promoter GAL1. This regulated gene construct, in a low copy number yeast expression vector, was introduced into strain M31 to generate strain YGL-1. Galactose control of the expression of N9L/Y8-1 was demonstrated by the ability of strain YGL-1 to grow vigorously on galactose as a carbon source, and by the inability to utilize ethanol alone for prolonged periods of growth. The measurement of bioenergetic parameters in mitochondria from YGL-1 cells experimentally depleted of subunit 8, by transferring growing cells from galactose to ethanol, was consistent with the presence in mitochondria of a mosaic of ATPase, namely fully assembled functional ATPase complexes and partially assembled complexes with defective F0 sectors. These mitochondria demonstrated very efficient import of N9L/Y8-1 and readily incorporated the imported processed subunit 8 protein into ATPase. Comparison of the kinetics of import and assembly of subunit 8 showed that assembly was noticeably delayed with respect to import. These findings open the way to a new systematic analysis of the assembly of imported proteins into multisubunit mitochondrial enzyme complexes.

Adenosine Triphosphatases↗
PubMed1995

Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.

Pseudomonas fluorescens CHA0 produces several secondary metabolites, e.g., the antibiotics pyoluteorin (Plt) and 2,4-diacetylphloroglucinol (Phl), which are important for the suppression of root diseases caused by soil-borne fungal pathogens. A Tn5 insertion mutant of strain CHA0, CHA625, does not produce Phl, shows enhanced Plt production on malt agar, and has lost part of the ability to suppress black root rot in tobacco plants and take-all in wheat. We used a rapid, two-step cloning-out procedure for isolating the wild-type genes corresponding to those inactivated by the Tn5 insertion in strain CHA625. This cloning method should be widely applicable to bacterial genes tagged with Tn5. The region cloned from P. fluorescens contained three complete open reading frames. The deduced gene products, designated PqqFAB, showed extensive similarities to proteins involved in the biosynthesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae, Acinetobacter calcoaceticus, and Methylobacterium extorquens. PQQ-negative mutants of strain CHA0 were constructed by gene replacement. They lacked glucose dehydrogenase activity, could not utilize ethanol as a carbon source, and showed a strongly enhanced production of Plt on malt agar. These effects were all reversed by complementation with pqq+ recombinant plasmids. The growth of a pqqF mutant on ethanol and normal Plt production were restored by the addition of 16 nM PQQ. However, the Phl- phenotype of strain CHA625 was due not to the pqq defect but presumably to a secondary mutation. In conclusion, a lack of PQQ markedly stimulates the production of Plt in P. fluorescens.

Amino Acid Sequence↗
PubMed1999

Molecular phylogenetic and biogeochemical studies of sulfate-reducing bacteria in the rhizosphere of spartina alterniflora

The population composition and biogeochemistry of sulfate-reducing bacteria (SRB) in the rhizosphere of the marsh grass Spartina alterniflora was investigated over two growing seasons by molecular probing, enumerations of culturable SRB, and measurements of SO42- reduction rates and geochemical parameters. SO42- reduction was rapid in marsh sediments with rates up to 3.5 &mgr;mol ml-1 day-1. Rates increased greatly when plant growth began in April and decreased again when plants flowered in late July. Results with nucleic acid probes revealed that SRB rRNA accounted for up to 43% of the rRNA from members of the domain Bacteria in marsh sediments, with the highest percentages occurring in bacteria physically associated with root surfaces. The relative abundance (RA) of SRB rRNA in whole-sediment samples compared to that of Bacteria rRNA did not vary greatly throughout the year, despite large temporal changes in SO42- reduction activity. However, the RA of root-associated SRB did increase from <10 to >30% when plants were actively growing. rRNA from members of the family Desulfobacteriaceae comprised the majority of the SRB rRNA at 3 to 34% of Bacteria rRNA, with Desulfobulbus spp. accounting for 1 to 16%. The RA of Desulfovibrio rRNA generally comprised from <1 to 3% of the Bacteria rRNA. The highest Desulfobacteriaceae RA in whole sediments was 26% and was found in the deepest sediment samples (6 to 8 cm). Culturable SRB abundance, determined by most-probable-number analyses, was high at >10(7) ml-1. Ethanol utilizers were most abundant, followed by acetate utilizers. The high numbers of culturable SRB and the high RA of SRB rRNA compared to that of Bacteria rRNA may be due to the release of SRB substrates in plant root exudates, creating a microbial food web that circumvents fermentation.

Journal Article↗
PubMed2003

Nuclear import of zinc binuclear cluster proteins proceeds through multiple, overlapping transport pathways.

In Aspergillus nidulans, the high transcriptional level of the ethanol utilization pathway genes (alc) is regulated by the specific activator AlcR. Here we have analyzed the mechanism of the nuclear import of AlcR, as well as that of other proteins belonging to the Zn(2)Cys(6) binuclear cluster family. The nuclear localization signal of AlcR maps within the N-terminal 75 amino acid residues and overlaps with its DNA-binding domain. It consists of five clusters rich in basic residues. Four of them are necessary and sufficient for nuclear targeting. The first two basic regions are crucial for both nuclear localization and recognition of AlcR-specific DNA targets. This nuclear localization signal (NLS) motif is recognized by the nuclear transport machinery of Saccharomyces cerevisiae and requires both Ran/Gsp1p activity and specific transport receptors. AlcR can be imported into nuclei via multiple transport pathways mediated by a distinct set of karyopherins composed of Kap104p, Sxm1p, and Nmd5p transport receptors. The two former karyopherins interact with the NLS of AlcR directly. Other Zn binuclear cluster proteins from S. cerevisiae, such as Gal4p and Pdr3p, also appear to be transported to the nuclei in a nonclassical, importin-alpha-independent manner and can share common importin beta receptors.

Active Transport, Cell Nucleus↗
PubMed1973

Relationship between dimorphology and respiration in Mucor genevensis studied with chloramphenicol.

Growth of Mucor genevensis, a facultatively anaerobic dimorphic mold, in high concentrations of chloramphenicol (4 mg/ml) leads to increased numbers of yeast-like cells and small club-like mycelial forms. This change in morphology is accompanied by a threefold increase in the mass doubling time, the loss of cyanide-sensitive respiration, and the development of cyanide-insensitive respiration. Associated with these changes is the absence of cytochromes aa(3) and b and the inability of the organism to utilize ethanol; in addition, mitochondria appear more numerous and have less internal membrane. A further inhibitory action of the antibiotic, other than eliminating functional mitochondria, appears likely since microaerobic cultures which lack respiratory ability have twice the mass doubling time in the presence of the drug. Although a small inhibition of amino acid incorporation by cytoplasmic ribosomes is found with a high chloramphenicol concentration, it is insufficient to account for the effect on growth of the microaerobic culture. The nature of this additional effect of chloramphenicol remains to be determined, but it has been shown that increasing the glucose concentration can partially reverse this action of the antibiotic. The effect of the drug on the morphology of the organism is not as dramatic as that of phenethyl alcohol in producing yeast-like forms. However, in view of the action of chloramphenicol in eliminating functional mitochondria in M. genevensis the suggestion that phenethyl alcohol exerts its effect in promoting yeast-like morphology by uncoupling oxidative phosphorylation should be re-examined.

Aerobiosis↗
PubMed1995

Possible central adenosinergic modulation of ethanol-induced alterations in [14C]glucose utilization in mice.

The possible role of brain adenosine in acute ethanol-induced alteration in glucose utilization in the whole brain, as well as in the specific brain areas (cerebellum and brain stem), was investigated. Mice were killed 20-min postethanol, and the fresh tissue slices (300 microns) of brain and/or specific brain areas were incubated for 100 min in a 5.5 mM glucose medium in Warburg flasks using [6-(14)C]glucose as a tracer. Trapped 14CO2 was counted to estimate glucose utilization. Ethanol (2 g/kg, i.p.) markedly increased the glucose utilization in whole brain and in both motor areas of brain. Theophylline (50 mg/kg, i.p.), an adenosine antagonist, significantly reduced ethanol-induced increase in glucose utilization in whole brain, as well as in brain areas. However, adenosine agonist N6-cyclohexyladenosine (CHA; 0.1 mg/kg, i.p.) on the contrary, significantly accentuated ethanol-induced increase in glucose utilization in these tissues that was nearly completely blocked by theophylline pretreatment. Theophylline alone did not produce any significant change in glucose utilization, whereas CHA alone (in vivo and in vitro) significantly increased glucose utilization, as well as ethanol-induced increase in glucose utilization in an additive manner. Relevant supportive data were obtained by experiments in which adenosine deaminase (ADA), p-sulfophenyltheophylline (8-SPT), and CHA were administered in vitro to the slice preparations. Both ADA and 8-SPT were effective in almost completely blocking the ethanol-induced increase in glucose utilization, whereas CHA further enhanced the ethanol-induced increase in glucose utilization in an additive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed2006

Chemo-enzymatic production of fuel ethanol from cellulosic materials utilizing yeast expressing beta-glucosidases.

Ethanol was produced in a considerably high yield by fermenting hydrolyzates from cellulosic materials by means of a recombinant laboratory yeast expressing beta-glucosidases. Tissue paper, cotton, and sawdust were hydrolyzed by two-step sulfuric acid hydrolysis to give mixtures containing glucose, cellobiose, and higher cello-oligosaccharides. After the cellulosic material was partially hydrolyzed with 80% sulfuric acid, the hydrolysis was continued with 5% sulfuric acid. Except for non-carbohydrate components, all constituents in the hydrolyzates were fermented by the yeast that was preincubated in the medium that the plasmid encoded by the beta-glucosidases gene was kept in the multiplicated yeast. A solution containing 4% hydrolyzates from paper was fermented to give as high as 1.9% maximum ethanol concentration and 70% ethanol conversion. Cotton also gave a similar result. Sawdust was converted into ethanol in approx 22% conversion. Accordingly, it was revealed that the beta-glucosidases-expressing yeast can ferment the cello-oligosaccharides obtained by hydrolysis of cellulosic materials into ethanol. In addition, a hydrolyzate containing a high glucose proportion gave a high ethanol concentration in a short time.

Aspergillus↗