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Archives of Microbiology (v.168, #1)
Pressure and temperature effects on growth and viability of the hyperthermophilic archaeon Thermococcus peptonophilus by F. Canganella; J. M. Gonzalez; Miki Yanagibayashi; C. Kato; Koki Horikoshi (pp. 1-7).
We studied the effects of high temperatures and elevated hydrostatic pressures on the physiological behavior and viability of the extremely thermophilic deep-sea archaeon Thermococcus peptonophilus. Maximal growth rates were observed at 30 and 45 MPa although no significant increases in cell yields were detected. Growth at 60 MPa was slower. The optimal growth temperature shifted from 85° C at 30 MPa to 90–95° C at 45 MPa. Cell viability during the stationary phase was also enhanced under high pressure. A trend towards barophily at pressures greater than those encountered in situ at the sea floor was demonstrated at increasing growth temperatures. The viability of cells during starvation, at high temperature (90, 95° C), and at low temperature (10° C) was enhanced at 30 and 45 MPa as compared to atmospheric pressure. These results show that the extremely thermophilic archaeon T. peptonophilus is a barophile.
Keywords: Key wordsThermococcus peptonophilus; Extreme thermophiles; Large-scale cultivation; Hydrostatic; pressure; Barophiles
Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis: cloning and expression in Saccharomyces cerevisiae by Mitsuyoshi Ueda; Shin-ichi Sanuki; Hiroyuki Kawachi; Kaori Shimizu; Haruyuki Atomi; Atsuo Tanaka (pp. 8-15).
Citrate synthase, an essential enzyme of the tricarboxylic acid cycle in mitochondria, was purified from acetate-grown Candida tropicalis. Results from SDS-PAGE and gel filtration showed that this enzyme was a dimer composed of 45-kDa subunits. A citrate synthase cDNA fragment was amplified by the 5′-RACE method. Nucleotide sequence analysis of this cDNA fragment revealed that the deduced amino acid sequence contained an extended leader sequence which is suggested to be a mitochondrial targeting signal, as judged from helical wheel analysis. Using this cDNA probe, one genomic citrate synthase clone was isolated from a yeast λEMBL3 library. The nucleotide sequence of the gene encoding C. tropicalis citrate synthase, CtCIT, revealed the presence of a 79-bp intron in the N-terminal region. Sequences essential as yeast splicing motifs were present in this intron. When the CtCIT gene including its intron was introduced into Saccharomyces cerevisiae using the promoter UPR-ICL, citrate synthase activity was highly induced, which strongly indicated that this intron was correctly spliced in S. cerevisiae.
Keywords: Key wordsCandida tropicalis; Citrate synthase; Intron; Mitochondria; Yeast
Thiorhodococcus minus, gen. nov., sp. nov., a new purple sulfur bacterium isolated from coastal lagoon sediments by Rémy Guyoneaud; Robert Matheron; Werner Liesack; Johannes F. Imhoff; P. Caumette (pp. 16-23).
A new marine phototrophic purple sulfur bacterium (strain CE2203) was isolated in pure culture from a man-made coastal lagoon located on the Atlantic coast (Arcachon Bay, France). Single cells were coccus-shaped, did not contain gas vesicles, and were highly motile. Intracellular photosynthetic membranes were of the vesicular type. Bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series were present as photosynthetic pigments. Hydrogen sulfide, thiosulfate, elemental sulfur, and molecular hydrogen were used as electron donors during photolithotrophic growth under anoxic conditions, while carbon dioxide was utilized as carbon source. Acetate, propionate, lactate, glycolate, pyruvate, fumarate, succinate, fructose, sucrose, ethanol, and propanol were photoassimilated in the presence of hydrogen sulfide. During growth on sulfide, elemental sulfur globules were stored inside the cells. Chemotrophic growth under microoxic conditions in the dark was possible. The DNA base composition was 66.9 mol% G+C. Comparative sequence analysis of the 16S rRNA gene confirmed the membership of strain CE2203 in the family Chromatiaceae. Morphological characteristics of strain CE2203 indicated a close affiliation to the genera Thiocystis and Thiocapsa. However, the phylogenetic treeing revealed no closer relationship to Thiocystis spp. than to Thiocapsa roseopersicina or other known members of the Chromatiaceae. Consequently, strain CE2203 is proposed as the type strain of a new genus and species, Thiorhodococcus minus gen. nov., sp. nov.
Keywords: Key words Chromatiaceae; Thiorhodococcus; Thiocystis; Thiocapsa; Coastal lagoon; Bacteriochlorophyll a; Rhodopin; Phylogenetic; relationships
The characterization of the nv-gvpACNOFGH gene cluster involved in gas vesicle formation in Natronobacterium vacuolatum by Andrea Mayr; Felicitas Pfeifer (pp. 24-32).
The haloalkaliphilic archaeon Natronobacterium vacuolatum forms cylinder-shaped gas vesicles throughout the growth cycle when grown in media containing 15–25% NaCl. Cells cultivated in media containing 13% NaCl are, however, gas-vesicle-free. The major gas vesicle structural protein, nv-GvpA, was detected by an antiserum raised against the gas vesicles of Haloferax mediterranei; the antiserum reacted with an 8.3-kDa protein in samples containing cell extracts or purified gas vesicles of N. vacuolatum. The gene encoding nv-GvpA was isolated together with six additional gvp genes; these genes are arranged consecutively in a cluster as nv-gvpACNOFGH and are cotranscribed. Transcript analysis by primer extension revealed only one start site three nucleotides upstream of the nv-gvpA reading frame. This arrangement of gvp genes differs from that of the gas-vesicle-encoding genes in Halobacterium salinarium and Hf. mediterranei. The comparison of the deduced Gvp protein sequences indicated similarities with the respective halobacterial Gvp proteins, with GvpA exhibiting the highest degree of conservation (97–100%). The second gas vesicle structural protein, nv-GvpC, was 150–250 amino acids longer than all other halobacterial GvpC proteins and was much less conserved (48–73%). The expression of the nv-gvp genes was monitored in N. vacuolatum cells cultivated in 20 or 13% salt media. Northern and Western analyses showed that despite the lack of gas vesicles in cells grown in 13% salt medium, the gvpACNOFGH gene cluster was transcribed and GvpA protein was synthesized, suggesting that the absence of gas vesicles is not due to a lack of transcription.
Keywords: Key words Gas vesicles; Natronobacterium; vacuolatum; gvp gene cluster; Gas vesicle proteins; Archaeal promoter
Evidence for an isomeric muconolactone isomerase involved in the metabolism of 4-methylmuconolactone by Alcaligenes eutrophus JMP134 by Matthias Prucha; Anke Peterseim; Dietmar Helmut Pieper (pp. 33-38).
An enzyme specifically induced during 4-methylmuconolactone metabolism by Alcaligenes eutrophus JMP 134 and that exhibited muconolactone isomerizing activity was purified to homogeneity. The enzyme, involved in the isomerization of 3-methylmuconolactone had a high degree of sequence similarity with muconolactone isomerase of Alcaligenes eutrophus JMP 134 and other previously described muconolactone isomerases of the 3-oxoadipate pathway. Kinetic analysis showed that the enzyme has a substrate spectrum and a reaction mechanism similar to those of the muconolactone isomerase, but that it has distinct kinetic properties.
Keywords: Key words Muconolactone isomerase; Methylmuconolactone; Modified ortho-cleavage; pathway
Rhodospira trueperi gen. nov., spec. nov., a new phototrophic Proteobacterium of the alpha group by Norbert Pfennig; Heinrich Lünsdorf; Jörg Süling; J. F. Imhoff (pp. 39-45).
A new phototrophic purple bacterium was isolated from a flat, laminated microbial mat in a salt marsh near Woods Hole, Mass., USA. The spiral-shaped bacterium was highly motile and had bipolar tufts of flagella and intracytoplasmic membranes of the vesicular type. The major photosynthetic pigments were identified as the carotenoid tetrahydrospirilloxanthin and bacteriochlorophyll b. The long wavelength in vivo absorption maximum of the bacteriochlorophyll was at 986 nm. The marine bacterium showed optimal growth in the presence of 2% NaCl. It utilized a number of organic substrates as carbon and energy sources and required vitamins and sulfide as a reduced sulfur source for growth. In the presence of sulfide, elemental sulfur globules were formed outside the cells. Elemental sulfur was not further oxidized to sulfate. The new isolate had a unique lipid and fatty acid composition, and according to the 16S rRNA gene sequence, it is most similar to Rhodospirillum rubrum. It is described as a new species and assigned to a new genus with the proposed name Rhodospira trueperi.
Keywords: Key words Phototrophic bacteria; Taxonomy; New; species; New genus; Rhodospira; Bacteriochlorophyll; b; Tetrahydrospirilloxanthin
Changes in the growth and enzyme level of Zymomonas mobilis under oxygen-limited conditions at low glucose concentration by Hsien Toh; H. Doelle (pp. 46-52).
Zymomonas mobilis growing aerobically with 20 g glucose–1 (carbon-limited) in a chemostat exhibited an increase in both the molar growth yield (Yx/s) and the maximum molar growth yield (Yx/s max) and a decrease in both the specific substrate consumption rate (qs) and the maintenance energy consumption rate (me). Stepwise increase in the input oxygen partial pressure showed that anaerobic-to-aerobic transitional adaptation occurred in four stages: anaerobic (0 mm HgO2), oxygen-limited (7.6– 230 mm HgO2), intermediate (273 mm HgO2), and oxygen excess (290 mm HgO2). The steady-state biomass concentration, Yx/s, and intracellular ATP content increased between oxygen partial pressures of 7.6 and 120 mm HgO2, accompanied by a decrease in the qs and the specific acid production rate. The membrane ATPase activity decreased with increasing oxygen partial pressure and reached its lowest levels at 273 mm HgO2, which was the highest input oxygen partial pressure where steady-state conditions were possible. Glucokinase, glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and alcohol dehydrogenase activities also decreased when the oxygen partial pressure was increased above 15 mm Hg, whereas pyruvate decarboxylase was unaffected by aeration. Growth inhibition at 290 mm HgO2 was characterised by a drastic reduction in the pyruvate kinase activity and a collapse in the intracellular ATP pool. The growth and enzyme data suggest that at low glucose concentrations and oxygen-limited conditions, the increase in biomass yields is a reflection of a redirection of ATP usage rather than a net increase in energy production.
Keywords: Key wordsZymomonas mobilis; Chemostat; Anaerobic-to-aerobic transition; Maintenance energy; coefficient; Biomass yield; Metabolic rate; ATPase; activity; Energetics
The sae locus of Staphylococcus aureus controls exoprotein synthesis at the transcriptional level by A. T. Giraudo; Ambrose L. Cheung; Rosa Nagel (pp. 53-58).
Agr and sar are known regulatory loci of Staphylococcus aureus that control the production of several extracellular and cell-wall-associated proteins. A pleiotropic insertional mutation in S. aureus, designated sae, that leads to the production of drastically diminished levels of α- and β-hemolysins and coagulase and slightly reduced levels of protein A has been described. The study of the expression of the genes coding for these exoproteins in the sae::Tn551 mutant (carried out in this work by Northern blot analyses) revealed that the genes for α- and β-hemolysins (hla and hlb) and coagulase (coa) are not transcribed and that the gene for protein A (spa) is transcribed at a somewhat reduced level. These results indicate that the sae locus regulates these exoprotein genes at the transcriptional level. Northern blot analyses also show that the sae mutation does not affect the expression of agr or sar regulatory loci. An sae::Tn551 agr::tetM double mutant has been phenotypically characterized as producing reduced or null levels of α-, β-, and δ-hemolysins, coagulase, and high levels of protein A. Northern blot analyses carried out in this work with the double mutant revealed that hla, hlb, hld, and coa genes are not transcribed, while spa is transcribed at high levels. The fact that coa is not expressed in the sae agr mutant, as in the sae parental strain, while spa is expressed at the high levels characteristic of the agr parental strain, suggests that sae and agr interact in a complex way in the control of the expression of the genes of several exoproteins.
Keywords: Key wordsStaphylococcus aureus; Exoprotein; synthesis; sae mutant; agr mutant; Transcriptional; control
Properties and primary structure of a thermostable l-malate dehydrogenase from Archaeoglobus fulgidus by Anne Siri Langelandsvik; I. H. Steen; Nils-Kåre Birkeland; Torleiv Lien (pp. 59-67).
A thermostable l-malate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was isolated and characterized, and its gene was cloned and sequenced. The enzyme is a homodimer with a molecular mass of 70 kDa and catalyzes preferentially the reduction of oxaloacetic acid with NADH. A. fulgidus l-malate dehydrogenase was stable for 5 h at 90° C, and the half-life at 101° C was 80 min. Thus, A. fulgidus l-malate dehydrogenase is the most thermostable l-malate dehydrogenase characterized to date. Addition of K2HPO4 (1 M) increased the thermal stability by 40%. The primary structure shows a high similarity to l-lactate dehydrogenase from Thermotoga maritima and gram-positive bacteria, and to l-malate dehydrogenase from the archaeon Haloarcula marismortui and other l-lactate-dehydrogenase-like l-malate dehydrogenases.
Keywords: Key wordsArchaea; Archaeoglobus fulgidus; Thermophiles; Malate dehydrogenase; Lactate; dehydrogenase; Thermostable protein; mdh; Glycine motif; Lactate-dehydrogenase-like malate; dehydrogenase
Adaptive response of Haloferax mediterranei to low concentrations of NaCl (< 20%) in the growth medium by S. E. D’Souza; Wijaya Altekar; S. F. D’Souza (pp. 68-71).
Halobacteria require 20–25% NaCl for optimal growth and lyse when the salt concentration falls below 10%. The response of Haloferax mediterranei cells to low concentrations of NaCl (< 20%) in the medium was studied. The cells adapted to and grew in concentrations of NaCl as low as 10% and survived in concentrations lower than 5%. The cells synthesised a red pigment, bacterioruberin, in response to stress caused by a low concentration of NaCl (< 20%).
Keywords: Key wordsHaloferax mediterranei; Red pigment; Bacterioruberin; Osmotic stability