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Archives of Microbiology (v.173, #4)
No Title by Tobias Nübel; Christof Klughammer; Robert Huber; Günter Hauska; Michael Schütz (pp. 233-244).
The sulfide-dependent reduction of exogenous ubiquinone by membranes of the hyperthermophilic chemotrophic bacterium Aquifex aeolicus (VF5), the sulfide-dependent consumption of oxygen and the reduction of cytochromes by sulfide in membranes were studied. Sulfide reduced decyl-ubiquinone with a maximal rate of up to 3.5 µmol (mg protein)–1 min–1 at 20 °C. Rates of 220 nmol (mg protein)–1 min–1 for the sulfide-dependent consumption of oxygen and 480 nmol (mg protein)–1 min–1 for the oxidation of sulfide at 20 °C were estimated. The reactions were sensitive towards 2-n-nonyl-4-hydroxyquinoline-N-oxide, but insensitive towards cyanide. Both reduction of decyl-ubiquinone and consumption of oxygen by sulfide rapidly increased with increasing temperature. For the sulfide-dependent respiratory activity, a sulfide-to-oxygen ratio of 2.3±0.2 was measured. This indicates that sulfide was oxidized to the level of zero-valent sulfur. Reduction of cytochromes by sulfide was monitored with an LED-array spectrophotometer. Reduction of cytochrome b was stimulated by 2-n-nonyl-4-hydroxyquinoline-N-oxide in the presence of excess sulfide under oxic conditions. This "oxidant-induced reduction" of cytochrome b suggests that electron transport from sulfide to oxygen in A. aeolicus employs the cytochrome bc complex via the quinone pool. Comparison of the amino acid sequence with the sequence of the sulfide:quinone oxidoreductase from Rhodobacter capsulatus and of the flavocytochrome c from Allochromatium vinosum revealed that the sulfide:quinone oxidoreductase from A. aeolicus belongs to the glutathione reductase family of flavoproteins.
Keywords: Aquifex aeolicus Sulfide oxidation Sulfide:quinone oxidoreductase Hyperthermophiles Cytochrome bc complex
No Title by Iva Slaninová; Sergej Šesták; Augustín Svoboda; Vladimír Farkaš (pp. 245-252).
Transfer of exponentially growing cells of the yeast Saccharomyces cerevisiae to hyperosmotic growth medium containing 0.7–1 M KCl, 1 M mannitol, and/or 1 M glycerol caused cessation of yeast growth for about 2 h; thereafter, growth resumed at almost the original rate. During this time, formation of fluorescent patches on the inner surface of cell walls stained with Primulin or Calcofluor white was observed. The fluorescent patches also formed in solutions of KCl or when synthesis of the cell wall was blocked with cycloheximide and/or 2-deoxyglucose. The patches gradually disappeared as the cells resumed growth, and the new buds had smooth cell walls. Electron microscopy of freeze-etched replicas of osmotically stressed cells revealed deep plasma membrane invaginations filled from the periplasmic side with an amorphous cell wall material that appeared to correspond to the fluorescent patches on the cell surface. The rate of incorporation of d-[U-14C]glucose from the growth medium into the individual cell wall polysaccharides during osmotic shock followed the growth kinetics. No differences in cell wall composition between osmotically stressed yeast and control cells were found. Hyperosmotic shock caused changes in cytoskeletal elements, as demonstrated by the disappearance of microtubules and actin microfilaments. After 2–3 h in hyperosmotic medium, both microtubules and microfilaments regenerated to their original polarized forms and the actin patches resumed their positions at the apices of growing buds. The response of S. cerevisiae strains with mutations in the osmosensing pathway genes hog1 and pbs2 to hyperosmotic shock was similar to that of the wild-type strain. We conclude that, besides causing a temporary disassembling of the cytoskeleton, hyperosmotic shock induces a change in the organization of the cell wall, apparently resulting from the displacement of periplasmic and cell wall matrix material into invaginations of the plasma membrane created by the plasmolysis.
Keywords: Osmotic shock Cytoskeleton Actin Microtubules Microfilaments Cell wall Yeast Saccharomyces cerevisiae
No Title by Thomas Wartmann; Jana Erdmann; Irene Kunze; Gotthard Kunze (pp. 253-261).
The dimorphism of the yeast Arxula adeninivorans LS3 is regulated by cultivation temperatures. Up to 42 °C the yeast grows as budding cells, which turn to mycelia at higher temperatures. To test whether the dimorphism is exclusively induced by high temperatures or also by other conditions, mutants were selected with an altered behaviour with respect to dimorphism. After mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine, five of 25,000 colonies formed a very rough surface consisting of mycelia at 30 °C, in contrast to the wild-type. These mutants allow temperature-mediated and morphology-related effects on gene expression and protein accumulation to be distinguished. Budding cells and mycelia showed different expression of genes encoding secretory proteins at the same temperature. Mycelia secreted two-fold more protein than budding cells, including the enzymes glucoamylase and invertase. This indicated that morphology, rather than temperature, is the decisive factor in the analysed processes.
Keywords: Arxula adeninivorans Dimorphism Glucoamylase Invertase Secretion Thermoresistance
No Title by Alexandra R. Fernandes; Manuel Prieto; Isabel Sá-Correia (pp. 262-268).
Plasma membrane lipid disorganization takes place in cells of Saccharomyces cerevisiae grown under copper stress, as shown by fluorescence anisotropy measurements with the lipid reporter probe 1,6-diphenyl-1,3,5-hexatriene. The extent of plasma membrane disorganization, presumably due to copper-induced lipid peroxidation, was discontinuous when measured in cells grown in media supplemented with different concentrations of CuSO4. Results suggested the existence of adaptive mechanisms that cells employ to protect themselves against the deleterious effects of copper. The adaptive mechanisms examined in this study included the coordinate increase in the activities of Cu,Zn-superoxide dismutase (up to five-fold), glutathione reductase (up to 1.7-fold), and plasma membrane H+-ATPase (up to three-fold). These enzyme activities showed maximal levels in cells grown with copper supplied at intermediate concentrations, within the range that allowed growth. Significantly, at these concentrations, plasma membrane disorganization did not increase when increasing amounts of CuSO4 were supplied. However, at copper concentrations close to the maximal that allowed growth, the capacity of the yeast cell response to cope with the deleterious effects of copper was exceeded; plasma membrane lipid organization and plasma-membrane-bound H+-ATPase activity drastically declined in response to the increased levels of copper stress and the consequences on growth kinetics were even more severe. Our results clearly suggest that modification of plasma membrane H+-ATPase activity is either part of or the result of the global response of yeast to mild or high copper stress.
Keywords: Saccharomyces cerevisiae Copper stress Response to copper Oxidative stress Plasma membrane H+-ATPase Antioxidant enzymes Plasma membrane lipid order
No Title by Michael T. Madigan; Deborah O. Jung; Carl R. Woese; Laurie A. Achenbach (pp. 269-277).
A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15–18 °C with a growth temperature range of 0–25 °C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.
Keywords: Photosynthesis Anoxygenic phototrophic bacteria Purple bacteria Rhodoferax antarcticus Psychrophile Antarctica
No Title by Stefanie H. Baker; Donna S. Williams; Henry C. Aldrich; Alisa C. Gambrell; Jessup M. Shively (pp. 278-283).
Four genes encoding carboxysome shell peptides (csoS1A, csoS1B, csoS1C, csoS2), the genes encoding the large and small subunits of RuBisCO (cbbL, cbbS), and three unidentified ORFs constitute an operon in Thiobacillus neapolitanus. An unidentified ORF 1.54 kb in size is predicted from sequence analysis to encode a protein with a molecular mass of approximately 57 kDa. When this ORF was expressed in Escherichia coli under the control of its endogenous ribosome-binding site, no peptide product was observed. In order to correlate this ORF with a carboxysome peptide, the ORF was overexpressed in E. coli by cloning it into pProExHTb, a prokaryotic expression vector containing an E. coli ribosome binding site. When antibodies raised against the recombinant protein were used to probe an immunoblot containing carboxysome peptides, a 60-kDa peptide was recognized. The peptide was subsequently named CsoS3. CsoS3 is a minor component of the carboxysome; a peptide of this size is commonly not observed or is very faint on Coomassie blue-stained SDS-polyacrylamide gels of purified carboxysomes. Immunogold labeling established CsoS3 to be a component of the carboxysome shell.
Keywords: Carboxysome Thiobacilli RuBisCO CsoS3
No Title by Kuniaki Hosono (pp. 284-287).
The polyene antibiotic nystatin, which affects fungal membrane permeability, inhibited the growth of Zygosaccharomyces rouxii grown in medium containing 15% (w/v) NaCl, whereas yeast grown in medium without NaCl were only slightly inhibited. Nystatin caused salt-stressed cells to release large amounts of glycerol and inhibited their growth, but amino acids and materials with an absorbance at 260 nm were not released from the cells. The leakage was increased by the addition of glucose, and more than 90% of the intracellular glycerol was released into the medium during a 2-h incubation with 0.11 µM nystatin and 2% (w/v) glucose. Glycerol was indispensable for the growth of Z. rouxii grown in culture medium containing 15% NaCl.
Keywords: Salt stress Polyene antibiotic Nystatin Glycerol release Salt-tolerant yeast Zygosaccharomyces rouxii
No Title by Jochen A. Müller; Bernhard Schink (pp. 288-295).
The anaerobic bacterium Sporotomaculum hydroxybenzoicum ferments 3-hydroxybenzoate to acetate, butyrate, and CO2. 3-Hydroxybenzoate was activated to 3-hydroxybenzoyl-CoA in a CoA-transferase reaction with acetyl-CoA or butyryl-CoA as CoA donors. 3-Hydroxybenzoyl-CoA was reductively dehydroxylated, forming benzoyl-CoA. This reaction was measured in cell-free extracts with cob(I)alamin as low-potential electron donor. No evidence was obtained that cob(I)alamin is the physiological electron donor; however, inhibitor studies indicated involvement of a strong nucleophile in the reaction. Benzoate was degraded by dense cell suspensions without a lag phase until an in situ ΔG′ value <–25 kJ mol–1 was reached. Benzoyl-CoA reductase was not detected. Enzyme activities for all reaction steps from glutaryl-CoA to butyryl-CoA, and ATP formation via acetate kinase were detected in cell-free extracts. Glutaconyl-CoA decarboxylase is likely to act as a primary sodium ion pump.
Keywords: Anaerobic degradation Aromatic compounds 3-Hydroxybenzoate Benzoyl-CoA pathway Reductive dehydroxylation Fermenting bacteria Sporotomaculum hydroxybenzoicum
No Title by Oussama Ahrazem; Begoña Gómez-Miranda; Alicia Prieto; Manuel Bernabé; J. Antonio Leal (pp. 296-302).
The polysaccharides obtained from the alkali-extractable, water-soluble fraction (F1SS) from the cell wall of Myrothecium verrucaria and Myrothecium atroviride were shown to be composed of β-(1→6)-galactofuranose fully substituted at O-2 by terminal residues of α-glucopyranose and α-glucuronic acid. Glucuronic acid was substituted at O-4 by glucopyranose in the Myrothecium species M. inundatum, M. setiramosum, M. prestonii, M. tongaense and M. roridum. The acidic polysaccharides from Phaeostilbella atra (=Myrothecium atrum) and Myrothecium gramineum lacked the backbone of 2,6 di-O-substituted galactofuranose and contained a high amount of O-5-substituted β-galactofuranose. The structures of the polysaccharides isolated from Myrothecium cinctum and Myrothecium penicilloides were unrelated to each other and to the polysaccharides from the other species analysed. The usefulness of these polysaccharides as a characteristic for delimitation of the genus Myrothecium is discussed.
Keywords: Myrothecium Cell-wall polysaccharides Taxonomy
No Title by Rolf Müller; Klaus Gerth; Petra Brandt; Helmut Blöcker; Stefan Beyer (pp. 303-306).
During a screening program intended to identify genes encoding enzymes typical for secondary metabolism in Sorangium cellulosum So ce90, an aromatic amino acid decarboxylase gene (ddc) was detected. Expression of ddc in Escherichia coli and subsequent enzyme assays with cell-free extracts confirmed the proposed function derived from amino acid sequence comparisons. In contrast to other aromatic amino acid decarboxylases of eukaryotic origin, the S. cellulosum Ddc converted only l-dihydroxy phenylalanine. This is the first report of a gene encoding an l-dihydroxy phenylalanine decarboxylase in bacteria.
Keywords: Myxobacteria Sorangium cellulosum Tyrosine l-Dopa Aromatic amino acid decarboxylase
No Title by Sun N. Wai; Yoshimitsu Mizunoe; Akemi Takade; Shin-ichi Yoshida (pp. 307-310).
Like many other gram-negative bacteria, starved cells of Aeromonas hydrophila can be induced into a viable but nonculturable (VBNC) state by incubation at low temperature, as shown here by using various bacterial enumeration methods. Starved A. hydrophila strain HR7 cells at 4 °C reached the nonculturable stage in about 45 days. The cells were resuscitated by either a solid medium resuscitation method, using solid agar amended with H2O2-degrading agents, catalase or sodium pyruvate, or a liquid medium resuscitation method, by incubating nonculturable cells in liquid media containing these compounds before spreading onto plates. The liquid medium resuscitation method using catalase resulted in nearly complete recovery of nonculturable cells.
Keywords: Aeromonas hydrophila Nonculturable cells Resuscitation Catalase Sodium pyruvate
No Title by Yoichi Ogawa; Keiko Ishikawa; Masahiro Mii (pp. 311-315).
A wild-type Agrobacterium tumefaciens strain CNI5 isolated from crown gall of chrysanthemum (Dendranthema grandiflora Tzvelev) was characterized. Strain CNI5 was classified into biovar 1, based on physiological and biochemical characteristics, and was resistant to ampicillin. Strain CNI5 induced tumors at a higher frequency and on a larger area of explants in most tested plant species, especially in chrysanthemum cultivars, than the octopine-type strain C58C1cmr (pTiB6S3). Agropine and mannopine were detected in tumors induced by strain CNI5 and were specifically catabolized by this strain. Strain CNI5 harbored five plasmids including one plasmid that shared sequence similarity to Tl-DNA of the octopine-type Ti plasmid and four cryptic plasmids.
Keywords: Agrobacterium tumefaciens Chrysanthemum Crown gall tumor Ti plasmid Virulence Agropine