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Archives of Microbiology (v.180, #4)
Isolation and characterization of maltokinase (ATP:maltose 1-phosphotransferase) from Actinoplanes missouriensis by Birgit Niehues; Ralf Jossek; Uwe Kramer; Anne Koch; Martin Jarling; Werner Schröder; Hermann Pape (pp. 233-239).
Crude extracts of Actinoplanes missouriensis and related strains catalyze the ATP-dependent phosphorylation of maltose to maltose 1-phosphate. The enzyme of A. missouriensis responsible for this reaction was purified and characterized. This protein has an estimated molecular mass of 57 kDa and it is most likely a monomer. The K m value was 2.6 mM for maltose and 0.54 mM for ATP. Only maltose acted effectively as phosphoryl-group acceptor, and ATP was not replaceable as phosphoryl-group donor. Tryptic peptides of the enzyme were sequenced, and the sequences of these peptides will allow construction of degenerate primers to identify the gene coding for this unique kinase.
Keywords: Actinoplanes sp. SN 223; Actinoplanes missouriensis ; Maltose; Maltose 1-phosphate; Kinase; Enzyme purification
Genetic analysis of phosphomannomutase/phosphoglucomutase from Vibrio furnissii and characterization of its role in virulence by Sun-Hoi Kim; Sun-Hee Ahn; Jong-Hee Lee; Eun-Mi Lee; Nam-Hyun Kim; Kee-Jai Park; In-Soo Kong (pp. 240-250).
The pmm gene from Vibrio furnissii, which encodes phosphomannomutase (PMM), was cloned and sequenced. The open reading frame consisted of 1,434 bp, encoding a polypeptide of 477 amino acids with a molecular mass of 53,325 Da. The predicted amino acid sequence of V. furnissii PMM showed high similarity with PMMs from other enteric bacteria, such as V. cholerae, Salmonella sp. and Escherichia coli. The PMM protein was overexpressed in E. coli as a His6-tagged recombinant protein. The estimated apparent K m and k cat values of the purified recombinant protein for mannose 1-phosphate were about 60 μM and 800 min−1, respectively. To investigate the biochemical functions and the role of pmm in the virulence of V. furnissii, a pmm knock-out mutant was constructed by homologous recombination mutation. Under the various physical conditions, cell numbers of the wild-type and the mutant did not differ. Oral introduction of bacterial suspensions to a mouse model showed that the pmm-deficient mutant decreased in viability at the intestine. Microscopy of the isolated intestines from mice revealed significant damage after 3 days in intestinal mucosa infected with the wild-type as compared with the mutant. The pmm-deficient mutant caused a reduction of virulence in mice and the loss of O-antigen polysaccharide, and showed low resistance relative to the wild-type when incubated with normal human serum.
Keywords: Vibrio furnissii ; Phosphomannomutase/phosphoglucomutase; Lipopolysaccharide; O-antigen polysaccharide; Serum resistance
Culture conditions affecting biodegradation components of the brown-rot fungus Gloeophyllum trabeum by Elisa Varela; Tunde Mester; Ming Tien (pp. 251-256).
To determine the liquid culture conditions under which the wood-degrading system of the brown-rot fungus Gloeophyllum trabeum is expressed, enzymes and metabolites from liquid and solid substrate cultures were characterized. Enzymes were analyzed by 2-D gel electrophoresis and also assayed. Growth conditions were varied by using liquid media containing: (1) low carbon, low nitrogen, (2) low carbon, high nitrogen, (3) high carbon, low nitrogen, or (4) high carbon, high nitrogen. The protein arrays expressed under the four conditions were very similar, and endo-1,4-β-glucanase (detected by 2-D gels) activity along with β-glucosidase, xylanase, and NADH/quinone oxidoreductase activities were detected. Maximal expression of the hydrolytic enzymes was observed in high carbon/high nitrogen medium, whereas the highest oxidoreductase activity was in the high carbon low nitrogen medium. Oxalate and 2,5-dimethoxybenzoquinone were detected under all culture conditions, with higher production in high carbon/low nitrogen medium. Cultures grown in this medium also yielded the highest rate of hydroxylation of p-hydroxybenzoic acid, yielding protocatechuic acid, a product of hydroxyl radical attack.
Keywords: Hydroxyl radical; Brown-rot fungus; Nutrient limitation; Gloeophyllum trabeum
Characterization of early deaths of non-Saccharomyces yeasts in mixed cultures with Saccharomyces cerevisiae by Peter Nissen; Nils Arneborg (pp. 257-263).
The survival of Kluyveromyces thermotolerans and Torulaspora delbrueckii in mixed cultures with Saccharomyces cerevisiae was examined at low oxygen availability in a defined grape juice medium. In these fermentations, K. thermotolerans and T. delbrueckii died off earlier than S. cerevisiae, and K. thermotolerans and T. delbrueckii exhibited parabolic death kinetics. Furthermore, the early deaths seemed to be non-apoptotic in nature. In order to understand the mechanism causing the early deaths, various single- and mixed-culture fermentations were carried out. The early deaths could not be explained by nutrient depletion or the presence of toxic compounds. Rather, they seemed to be mediated by a cell-to-cell contact mechanism at high cell densities of S. cerevisiae, and to a lesser ability of K. thermotolerans and T. delbrueckii to compete for space, as compared to S. cerevisiae. These results contribute to an increased understanding of why K. thermotolerans and T. delbrueckii die off before S. cerevisiae in wine fermentations.
Keywords: Early death; Mixed cultures; Yeast; Cell-to-cell contact; Space limitation
Role of GacA, LasI, RhlI, Ppk, PsrA, Vfr and ClpXP in the regulation of the stationary-phase sigma factor rpoS/RpoS in Pseudomonas by Iris Bertani; Milica Ševo; Milan Kojic; Vittorio Venturi (pp. 264-271).
RpoS is the stationary phase sigma factor responsible for increased transcription of a set of genes when bacterial cells enter stationary phase and under stress conditions. In Escherichia coli, RpoS expression is modulated at the level of transcription, translation, and post-translational stability whereas in Pseudomonas, previous studies have implicated four genetic loci (psrA, gacA, lasI and rhlI) involved in rpoS transcription. In this report, the transcription, translation and proteins profiles of rpoS/RpoS were analyzed in response to growth phase of knockout genomic mutants in the P. aeruginosa transcriptional regulatory loci psrA, gacA, vfr, and in the las and rhl quorum-sensing systems. Gene expression and protein profiles were also analyzed in the ppk genomic mutant. This gene is responsible for the biosynthesis of polyphosphate, an alarmone involved in the regulation of RpoS accumulation in E. coli. Finally, the role of the ClpXP protease in RpoS regulation was also studied; in E. coli, this protease has been shown to rapidly degrade RpoS during exponential growth. These studies confirm the significant role of PsrA in rpoS transcription during the late-exponential and stationary growth phases, the probable role of Vfr in transcriptional repression during exponential phase, and the function of the ClpXP protease in RpoS degradation during exponential phase. GacA/GacS, the quorum-sensing systems, and the polyphosphate alarmone molecule were not significant in rpoS/RpoS regulation. These results demonstrate important similarities and differences with the regulation of this sigma factor in E. coli and in Pseudomonas.
Keywords: rpoS ; Regulation; Pseudomonas ; Stationary phase; Global gene regulation
Gene structure and transcriptional regulation specific to the groESL operon from the psychrophilic bacterium Colwellia maris by Seiji Yamauchi; Hidetoshi Okuyama; Eugene Hayato Morita; Hidenori Hayashi (pp. 272-278).
The groESL operon of a psychrophilic bacterium, Colwellia maris, was cloned and sequenced. The operon contains two ORFs of 291 bp and 1,650 bp separated by 210 bp. Northern blot analysis suggested that the groESL operon was transcribed as a bicistronic mRNA, and that the amount of mRNA markedly increased after the temperature was raised from 10 °C to 20 °C. Although the optimum temperatures for GroESL function are different in psychrophilic, mesophilic, and thermophilic bacteria, the deduced amino acid sequences of C. maris GroES and GroEL showed remarkably high similarity with those of GroES and GroEL from mesophilic and thermophilic bacteria. A putative promoter similar to the Escherichia coli σ32 consensus sequence was identified. One specific feature of C. maris groESL was that in the putative untranslated region the G+C content was about 24 mol%, which is much lower than that of mesophilic bacteria such as E. coli. The low G+C content may be important for maintaining transcription at low temperatures.
Keywords: Psychrophilic bacterium; Colwellia maris ; Heat shock protein; groEL gene
Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum, the maize leaf-blight fungus by Yeshitila Degefu; Mubashir Hanif (pp. 279-284).
Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium-tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) and the enhanced green fluorescent protein (EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium-tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.
Keywords: Agrobacterium-mediated transformation; Helminthosporium turcicum ; Leaf blight of maize
Fructose-1,6-bisphosphatase from Corynebacterium glutamicum: expression and deletion of the fbp gene and biochemical characterization of the enzyme by Doris Rittmann; Steffen Schaffer; Volker F. Wendisch; Hermann Sahm (pp. 285-292).
The class II fructose-1,6-bisphosphatase gene of Corynebacterium glutamicum, fbp, was cloned and expressed with a N-terminal His-tag in Escherichia coli. Purified, His-tagged fructose-1,6-bisphosphatase from C. glutamicum was shown to be tetrameric, with a molecular mass of about 140 kDa for the homotetramer. The enzyme displayed Michaelis-Menten kinetics for the substrate fructose 1,6-bisphosphate with a K m value of about 14 µM and a V max of about 5.4 µmol min−1 mg−1 and k cat of about 3.2 s−1. Fructose-1,6-bisphosphatase activity was dependent on the divalent cations Mg2+ or Mn2+ and was inhibited by the monovalent cation Li+ with an inhibition constant of 140 µM. Fructose 6-phosphate, glycerol 3-phosphate, ribulose 1,5-bisphosphate and myo-inositol-monophosphate were not significant substrates of fructose-1,6-bisphosphatase from C. glutamicum. The enzymatic activity was inhibited by AMP and phosphoenolpyruvate and to a lesser extent by phosphate, fructose 6-phosphate, fructose 2,6-bisphosphate, and UDP. Fructose-1,6-bisphosphatase activities and protein levels varied little with respect to the carbon source. Deletion of the chromosomal fbp gene led to the absence of any detectable fructose-1,6-bisphosphatase activity in crude extracts of C. glutamicum WTΔfbp and to an inability of this strain to grow on the carbon sources acetate, citrate, glutamate, and lactate. Thus, fbp is essential for growth on gluconeogenic carbon sources and likely codes for the only fructose-1,6-bisphosphatase in C. glutamicum.
Keywords: Class II fructose-1,6-bisphosphatase; Gluconeogenesis; fbp gene; Corynebacterium glutamicum
Xylose and some non-sugar carbon sources cause catabolite repression in Saccharomyces cerevisiae by Mónica M. Belinchón; Juana M. Gancedo (pp. 293-297).
Glucose and other sugars, such as galactose or maltose, are able to cause carbon catabolite repression in Saccharomyces cerevisiae. Although glycolytic intermediates have been suggested as signal for repression, no evidence for such a control mechanism is available. The establishment of a correlation between levels of intracellular metabolites and the extent of catabolite repression may facilitate the identification of potential signal molecules in the process. To set a framework for such a study, the repression produced by xylose, glycerol and dihydroxyacetone upon genes belonging to different repressible circuits was tested, using an engineered strain of S. cerevisiae able to metabolize xylose. Xylose decreased the derepression of various enzymes in the presence of ethanol by at least 10-fold; the corresponding mRNAs were not detected in these conditions. Xylose also impaired the derepression of galactokinase and invertase. Glycerol and dihydroxyacetone decreased 2- to 3-fold the derepression observed in ethanol or galactose but did not affect invertase derepression. For yeast cells grown in media with different carbon sources, no correlation was found between repression of fructose-1,6-bisphosphatase and intracellular levels of glucose 6-phosphate or fructose 1,6-bisphosphate.
Keywords: Yeast; Catabolite repression; Gluconeogenesis; Fructose-1,6-bisphosphatase; Isocitrate lyase; Glutamate dehydrogenase; Galactokinase; Invertase; Glucose-6-phosphate
Cloning and partial characterization of the gene encoding the putative elongation factor Ts of Streptococcus suis serotype 2 by Gabriela Martinez; Josée Harel; Sonia Lacouture; Marcelo Gottschalk (pp. 298-302).
Streptococcus suis infection has a substantial impact on the swine industry. In addition, S. suis serotype 2 is recognized as a zoonotic agent. In this paper, we report the cloning and complete sequence of the gene coding for the putative elongation factor Ts (tsf-like) of S. suis. The putative tsf gene seems to be transcribed from a promoter located within the cloned DNA fragment, as its expression is not dependent on insertional orientation within the plasmid. One copy of the tsf gene was detected in the chromosome of S. suis by Southern blot analysis. Interestingly, the elongation factor Ts expressed by all reference strains of all S. suis serotypes were antigenically similar, as determined by Western blot.
Keywords: Elongation factor Ts; Antigen cloning
γ-Butyrolactone autoregulators and receptor proteins in non-Streptomyces actinomycetes producing commercially important secondary metabolites by Sun-Uk Choi; Chang-Kwon Lee; Yong-Il Hwang; Hiroshi Kinosita; Takuya Nihira (pp. 303-307).
The presence of γ-butyrolactone autoregulators and their receptor proteins were investigated in five representative strains of non-Streptomyces actinomycetes producing commercially important secondary metabolites. Ethyl acetate extracts of culture were assayed using wild-type S. virginiae for virginiae butanolide, S. lavendulae FRI-5 for IM-2, and S. griseus HH1 for A-factor. Actinoplanes teichomyceticus and Amycolatopsis mediterranei were shown to produce autoregulators. Corresponding autoregulator-binding activities were found in the crude cell-free lysates of these strains, using the binding assay with tritium-labeled autoregulator analogues as ligands, which suggests that non-Streptomyces actinomycetes might have autoregulator-dependent signaling cascades.
Keywords: γ-Butyrolactone autoregulator; Autoregulator receptor; Non-Streptomyces actinomycetes