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Archives of Microbiology (v.169, #6)
The phylogeny of unicellular, extremely halotolerant cyanobacteria by F. Garcia-Pichel; Ulrich Nübel; Gerard Muyzer (pp. 469-482).
We examined the morphology, physiology, and 16S rRNA gene sequences of three culture collection strains and of ten novel isolates of unicellular cyanobacteria from hypersaline environments. The strains were morphologically diverse, with average cell widths ranging from 2.8 to 10.3 μm. There were single-celled, colonial, and baeocyte-forming strains. However, morphological traits were markedly variable with culture conditions. In contrast, all strains displayed extreme halotolerance (growing close to optimally at above 12% salinity); all were obligately marine, euryhaline, and moderately thermophilic; and all shared a suite of chemotaxonomic markers including phycobilins, carotenoids, and mycosporine-like amino acids. 16S rRNA gene sequence analysis indicated that the strains were related to each other. Sequence similarity analysis placed the strains in a monophyletic cluster (which we named the Halothece cluster) apart from all cultured or uncultured, not extremely halotolerant cyanobacteria whose 16S rRNA gene sequences are available in public nucleotide sequence databases. This represents the first case in which a phylogenetically coherent group of cyanobacteria can be defined on the basis of physiology. The Halothece cluster contained two subclusters that may be divergent at the generic level, one encompassing 12 strains (spanning 5% 16S rRNA gene sequence divergence and named the Euhalothece subcluster), and a single deep-branching isolate. Phenotypic characterization of the isolates, including morphological, physiological, and chemotaxonomic traits, did not distinguish these subclusters and only weakly suggested the existence of two separate clades, one encompassing strains of small cell size (cell width < 5 m) and another one encompassing strains of larger cell size.
Keywords: Key words Cyanobacteria; Halophiles; Halotolerance; Hypersaline environments; Phylogeny; Taxonomy; Microbial mats
Iron regulates transcription of the Escherichia coli ferric citrate transport genes directly and through the transcription initiation proteins by Annemarie Angerer; V. Braun (pp. 483-490).
Ferric citrate induces transcription of the ferric citrate transport genes fecABCDE in Escherichia coli by binding to the outer-membrane receptor protein FecA without entering the cell. Replete iron concentrations inhibit transcription of the fec transport system via the iron-loaded Fur repressor. Here we show that the Fur repressor activated by Mn2+ (used instead of Fe2+) binds to the promoter of the regulatory genes fecIR and to the promoter of fecABCDE. DNase I footprint analysis revealed that Mn2+–Fur (50 nM) protected 30 nucleotides of the coding strand and 24 nucleotides of the noncoding strand of the fecIR promoter. Higher amounts of Mn2+–Fur (100 nM) covered 41 nucleotides of the coding strand of the fecIR promoter and 38 nucleotides of the coding strand of the fecA promoter. The corresponding region of the noncoding strand of the fecA promoter was hypersensitive to DNase I. The results of a deletion analysis of the fecA promoter supported the previously assigned –35 and –10 regions and nucleotide position +11 for FecI–RNA polymerase interaction. Induction of fecIR transcription by iron limitation increased fecB-lacZ transcription 3.5-fold, whereas under constitutive fecIR transcription, iron limitation increased fecB-lacZ transcription twofold. The two iron-regulated sites of fec transport gene transcription suggest a fast response to sufficient intracellular iron concentrations by repression of fecABCDE transcription and a slower adaptation as the result of fecIR transcription inhibition.
Keywords: Key wordsEscherichia coli; Ferric citrate transport; Transcription regulation; Surface signaling
Exponential growth of Escherichia coli B/r during its division cycle is demonstrated by the size distribution in liquid culture by F. J. Trueba; L. J. H. Koppes (pp. 491-496).
The Collins and Richmond equation was used to analyze the growth of individual bacterial cells. Birth size was derived from the size of deeply constricted cells in the sample. The analysis was applied to normalized and pooled data from electron micrographs of Escherichia coli showing that cellular length, surface, and volume do not grow linearly as reported before. We present evidence that bacteria grow exponentially during the division cycle, which is consistent with previous proposals. Our results confirm previous incorporation studies that demonstrate basically exponential growth patterns for cell mass during the division cycle.
Keywords: Key wordsEscherichia coli; Cell size distribution; Collins and Richmond; Kinetics of growth
Purification and characterization of the tetrachloroethene reductive dehalogenase of strain PCE-S by Evelyn Miller; Gert Wohlfarth; G. Diekert (pp. 497-502).
The membrane-associated tetrachloroethene reductive dehalogenase from the tetrachloroethene-reducing anaerobe, strain PCE-S, was purified 165-fold to apparent homogeneity in the presence of the detergent Triton X-100. The purified dehalogenase catalyzed the reductive dechlorination of tetrachloroethene to trichloroethene and of trichloroethene to cis-1,2-dichloroethene with reduced methyl viologen as the electron donor, showing a specific activity of 650 nkat/mg protein. The apparent K m values of the enzyme for tetrachloroethene, trichloroethene, and methyl viologen were 10 μM, 4 μM, and 0.3 mM, respectively. SDS-PAGE revealed a single protein band with an apparent molecular mass of 65 kDa. The apparent molecular mass of the native enzyme was 200 kDa as determined by gel filtration. Tetrachloroethene dehalogenase contained 0.7 ± 0.3 mol corrinoid, 1.0 ± 0.3 mol cobalt, 7.8 ± 0.5 mol iron, and 10.3 ± 2.0 mol acid-labile sulfur per mol subunit. The pH optimum was approximately 7.2, and the temperature optimum was approximately 50 °C. The dehalogenase was oxygen-sensitive with a half-life of approximately 50 min. The N-terminal amino acid sequence of the enzyme was determined, and no significant similarity was found to any part of the amino acid sequence of the tetrachloroethene (PCE) reductive dehalogenase from Dehalospirillum multivorans.
Keywords: Key words Corrinoid protein; Desulfitobacterium; Iron-sulfur protein; N-terminal amino acid sequence; PCE dehalogenase; Strain PCE-S; Tetrachloroethene; reductive dehalogenase
Purification and characterization of a catalase from the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 and its role in the oxidative stress response by Detlef P. Terzenbach; M. Blaut (pp. 503-508).
When challenged with reactive oxidants, the nonsulfur phototrophic bacterium Rhodobacter sphaeroides ATH 2.4.1 exhibited an oxidative stress response during both phototrophic and chemotrophic growth. Upon preincubation with 100 μM H2O2, catalase activity increased fivefold. Catalase was also induced by other forms of oxidative stress, heat-shock, ethanol treatment, and stationary-phase conditions. Only one band of catalase activity was detected after native and denaturing PAGE. The enzyme was purified 304-fold with a yield of 7%. The purified enzyme displayed a heterodimeric structure with subunits of 75 and 68 kDa, corresponding to a molecular mass of approximately 150 kDa for the native enzyme. The subunits had almost identical amino-terminal peptide sequences, sharing substantial similarity with other bacterial catalases. The enzyme exhibited an apparent K m of 40 mM and a V max of 285,000 U (mg protein)–1. Spectroscopic analysis indicated the presence of protoheme IX. The heme content calculated from pyridine hemochrome spectra was 0.43 mol per mol of enzyme. The enzyme had a broad pH optimum and was inhibited by cyanide, azide, hydroxylamine, 2-mercaptoethanol, and sodium dithionite. These data indicate that this catalase belongs to the class of monofunctional catalases.
Keywords: Key wordsRhodobacter sphaeroides; Catalase; Enzyme purification; Induction; Oxidative stress
Phenylacetyl-CoA:acceptor oxidoreductase, a new α-oxidizing enzyme that produces phenylglyoxylate. Assay, membrane localization, and differential production in Thauera aromatica by Sabine Schneider; G. Fuchs (pp. 509-516).
Anaerobic oxidation of phenylalanine and phenylacetate proceeds via α-oxidation of phenylacetyl-CoA to phenylglyoxylate. This four-electron oxidation system was studied in the denitrifying bacterium Thauera aromatica. It is membrane-bound and was solubilized with Triton X-100. The system used dichlorophenolindophenol as an artificial electron acceptor; a spectrophotometric assay was developed. No other products besides phenylglyoxylate and coenzyme A were observed. The enzyme was quite oxygen-insensitive and was inactivated by low concentrations of cyanide. Enzyme activity was induced under denitrifying conditions with phenylalanine and phenylacetate, it was low in cells grown with phenylglyoxylate, and it was virtually absent in cells grown with benzoate and nitrate or after aerobic growth with phenylacetate.
Keywords: Key wordsThauera aromatica; Phenylacetyl-CoA; α-Oxidation; Phenylalanine; Phenylacetyl-CoA:acceptor oxidoreductase
The two 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition by Georg Schnappauf; Markus Hartmann; M. Künzler; G. H. Braus (pp. 517-524).
Activity of the tyrosine-inhibitable 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) from Saccharomyces cerevisiae that was encoded by the ARO4 gene cloned on a high-copy-number plasmid was enhanced 64-fold as compared to the wild-type. The enzyme was purified to apparent homogeneity from the strain that harbored this recombinant plasmid. The estimated molecular weight of 42,000 of the enzyme corresponded to the calculated molecular mass of 40 kDa deduced from the DNA sequence. The enzyme could be inactivated by EDTA in a reaction that was reversed by several bivalent metal ions; presumably a metal cofactor is required for enzymatic catalysis. The Michaelis constant of the enzyme was 125 μM for phosphoenolpyruvate and 500 μM for erythrose 4-phosphate. The rate constant was calculated as 6 s–1, and kinetic data indicated a sequential mechanism of the enzymatic reaction. Tyrosine was a competitive inhibitor with phosphoenolpyruvate as substrate of the enzyme (K i of 0.9 μM) and a noncompetitive inhibitor with erythrose 4-phosphate as substrate. This is in contrast to the ARO3-encoded isoenzyme, where phenylalanine is a competitive inhibitor with erythrose 4-phosphate as a substrate of the enzyme and a noncompetitive inhibitor with phosphoenolpyruvate as substrate.
Keywords: Key wordsSaccharomyces cerevisiae; 3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase; Protein purification; Enzyme kinetics
Acetyl-CoA decarbonylase/synthase complex from Archaeoglobus fulgidus by Yao-Ren Dai; David W. Reed; Jack H. Millstein; Patricia L. Hartzell; David A. Grahame; E. DeMoll (pp. 525-529).
The acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex catalyzes the reversible cleavage and synthesis of acetyl-CoA in methanogens. This report of the enzyme complex in Archaeoglobus fulgidus demonstrates the existence of a functional ACDS complex in an organism that is not a methanogen. The A. fulgidus enzyme complex contained five subunits of 89, 72, 50, 49.5, and 18.5 kDa, and it catalyzed the overall synthesis of acetyl-CoA according to the following reaction:w CO2 + 2 Fdred(Fe2+) + 2 H+ + CH3– H4SPt + CoA ⇌ acetyl-CoA + H4SPt + 2 Fdox(Fe3+) + H2Owhere Fd is ferredoxin, and CH3–H4SPt and H4SPt denote N 5-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, respectively.
Keywords: Key words Acetyl-CoA; Sarcinapterin; Methanopterin; Multienzyme complex; Ferredoxin; Archaeoglobus; fulgidus; Methanosarcina
A temperature-sensitive DNA adenine methyltransferase mutant of Salmonella typhimurium by Rolando Brawer; Facundo D. Batista; Oscar R. Burrone; Daniel O. Sordelli; M. C. Cerquetti (pp. 530-533).
A temperature-sensitive mutant of Salmonella typhimurium was isolated earlier after transposon mutagenesis with Tn10d Tet. The mutant D220 grows well at 28 °C but has a lower growth rate and forms filaments at 37 °C. Transposon-flanking fragments of mutant D220 DNA were cloned and sequenced. The transposon was inserted in the dam gene between positions 803 and 804 (assigned allele number: dam-231 : : Tn10d Tet) and resulted in a predicted ten-amino-acid-shorter Dam protein. The insertion created a stop codon that led to a truncated Dam protein with a temperature-sensitive phenotype. The insertion dam-231 : : Tn10d Tet resulted in a dam“leaky” phenotype since methylated and unmethylated adenines in GATC sequences were present. In addition, the dam-231 : : Tn10d Tet insertion rendered dam mutants temperature-sensitive for growth depending upon the genetic background of the S. typhimurium strain. The wild-type dam gene of S. typhimurium exhibited 82% identity with the Escherichia coli dam gene.
Keywords: Key wordsSalmonella typhimurium; dam; Temperature-sensitive mutant; Insertion mutant