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Archives of Microbiology (v.175, #1)
No Title by Christina Schilde; Johannes Wöstemeyer; Anke Burmester (pp. 1-7).
Mucoralean fungi (Zygomycota) are used for many industrial processes and also as important model organisms for investigating basic biological problems. Their genetic analysis is severely hampered by low transformation frequencies, by their strong tendency towards autonomous replication of plasmids instead of stable integration, and by the lack of reliable genetic reporter systems. We constructed plasmids for transforming the model zygomycete Absidia glauca that carry the versatile reporter gene coding for green fluorescent protein (GFP). gfp expression is controlled either by the homologous actin promoter or the promoter for the elongation factor of translation, EF1α. These plasmids also confer neomycin resistance and carry one of two genetic elements (rag1, seg1) that improve mitotic stability of the plasmid. The gfp constructs were replicated extrachromosomally and could be recovered from retransformed Escherichia coli cells. gfp expression was monitored by epifluorescence microscopy. The gfp reporter gene plasmids presented here for the model zygomycete A. glauca constitute the first reliable system that allows the monitoring of gene expression in this important group of fungi.
Keywords: Absidia Autonomous replication gfp expression Mucor Reporter gene Zygomycota
No Title by Andrea Graentzdoerffer; Andreas Pich; Jan R. Andreesen (pp. 8-18).
A probe based on the sequence of the gene encoding selenoprotein A of glycine reductase of Clostridium sticklandii was used to obtain clones of adjacent DNA that encoded the other components of glycine reductase, proteins B and C, in addition to thioredoxin and thioredoxin reductase. The genes of the thioredoxin system and the glycine reductase were shown to be transcribed together, confirming an operon structure. In addition, a gene (grdX) encoding a 13.7-kDa protein of unknown function seemed to be associated with the reductase genes. Four potential promoters were identified by mapping the 5′-end of the mRNAs. The sequence of promoter P1 was shown to be similar to the σ70 promoter consensus sequence. The other three promoters were similar to each other, but not to known promoter consensus sequences. The transcripts starting at each of the four promoters were terminated to about 80% at a predicted loop structure downstream of grdB; the remaining transcripts continued through this structure and covered the genes encoding both subunits of protein C and bmpA, a gene that was also expressed monocistronically.
Keywords: Clostridium sticklandii Glycine reductase Thioredoxin system Selenocysteine Pyruvoyl group Transcriptional regulation
No Title by Zhemin Zhou; Naoki Takaya; Maria Antonina C. Sakairi; Hirofumi Shoun (pp. 19-25).
The effects of dioxygen (O2) on the denitrification activity of the fungus Fusarium oxysporum MT-811 in fed-batch culture in a stirred jar fermentor were examined. The results revealed that fungal denitrifying activity requires a minimal amount of O2 for induction, which is repressed by excess O2. The optimal O2 supply differed between the denitrification substrates : 690 µmol O2 h–1 (g dry cell wt.)–1 for nitrate (NO3 –) and about 250 µmol O2 h–1 (g dry cell wt.)–1 for nitrite (NO2 –). The reduction of NO3 – required more O2 than that of NO2 –. With an optimal O2 supply, 80% and 52% of nitrogen atoms in NO3 – and NO2 –, respectively, were recovered as the denitrification product N2O. These features of F. oxysporum differ from those of bacterial denitrifiers that work exclusively under anoxic conditions. The denitrification activity of F. oxysporum MT-811 mutants with impaired NO3 – assimilation was about double that of the wild-type strain, suggesting competition for the substrate between assimilatory and dissimilatory types of NO3 – reduction. These results showed that denitrification by F. oxysporum has unique features, namely, a minimal O2 requirement and competition with assimilatory NO3 –.
Keywords: Aerobic denitrification Nitrate reductase Fusarium oxysporum
No Title by Hanno Richter; Dorina Vlad; Gottfried Unden (pp. 26-31).
The heterofermentative lactic acid bacterium Oenococcus oeni requires pantothenic acid for growth. In the presence of sufficient pantothenic acid, glucose was converted by heterolactic fermentation stoichiometrically to lactate, ethanol and CO2. Under pantothenic acid limitation, substantial amounts of erythritol, acetate and glycerol were produced by growing and resting bacteria. Production of erythritol and glycerol was required to compensate for the decreasing ethanol production and to enable the synthesis of acetate. In ribose fermentation, there were no shifts in the fermentation pattern in response to pantothenate supply. In the presence of pantothenate, growing O. oeni contained at least 10.2 µM HSCoA, whereas the HSCoA content was tenfold lower after growth in pantothenate-depleted media. HSCoA and acetyl-CoA are cosubstrates of phosphotransacetylase and acetaldehyde dehydrogenase from the ethanol pathway. Both enzymes were found with activities commensurate with their function in ethanol production during heterolactic fermentation. From the kinetic data of the enzymes and the HSCoA and acetyl-CoA contents, it can be calculated that, under pantothenate limitation, phosphotransacetylase, and in particular acetaldehyde dehydrogenase activities become limiting due to low levels of the cosubstrates. Thus HSCoA deficiency represents the major limiting factor in heterolactic fermentation of glucose under pantothenate deficiency and the reason for the shift to erythritol, acetate, and glycerol fermentation.
Keywords: Oenococcus oeni Lactic acid bacteria Heterolactic fermentation Erythritol Acetate Pantothenate HSCoA
No Title by Jana Strigáčová; Peter Chovanec; Tibor Liptaj; Daniela Hudecová; Timotej Turský; Martin Šimkovič; Ľudovít Varečka (pp. 32-40).
Glutamic acid decarboxylase (GAD) activity was measured in homogenates of conidia and both submerged and aerial mycelia of Trichoderma viride. The GAD activity in conidia had a temperature optimum at 30 °C and a pH optimum at pH 4. GAD was stimulated by EDTA (2 mM) and was insensitive to treatment with calmodulin antagonists calmidazolium (10 µM) or phenothiazine neuroleptics (60 µM). Cyclosporin A (up to 300 µM) partially inhibited GAD in the homogenate, but not in the supernatant obtained after centrifuging the homogenate. Attempts to release GAD activity from the homogenate using high ionic strength, detergents, or urea failed. Freezing-thawing led to the partial increase of activity in the conidial homogenate. These results indicate that GAD is a membrane-bound enzyme. The highest specific activity of GAD was present in the mitochondrial/vacuolar organellar fraction. Germination of conidia in the submerged culture led to a temporary decrease in GAD activity. After prolonged cultivation, the activity displayed quasi-oscillatory changes. The stationary state was characterized by a high GAD activity. The presence of γ-aminobutyric acid in the submerged mycelia was demonstrated. In surface culture in the dark, GAD activity increased in a monophasic manner until conidia formation. The illumination of dark-cultivated mycelia by a white-light pulse caused a dramatic increase in GAD activity. Light-induced changes were not observed in mutants with delayed onset of conidiation. In the dark or upon illumination by light pulse, the increase of GAD activity preceded the appearance of conidia. Thus, GAD activity in T. viride is closely associated with its developmental status and may represent a link between differentiation events and energy metabolism.
Keywords: Trichoderma viride Glutamic acid decarboxylase Conidia Mycelium Development Light
No Title by Miyuki Kawano; Ryoko Abuki; Kazuei Igarashi; Yoshimi Kakinuma (pp. 41-45).
Two high-affinity K+ uptake systems, KtrI and KtrII, have been reported in Enterococcus hirae. A mutant, JEMK1, defective in these two systems did not grow at pH 10 in low-K+ medium (less than 1 mM K+), but grew well when supplemented with 10 mM KCl. In this mutant, we found an energy-dependent K+ uptake at pH 10 with a low affinity for K+ (K m of ~20 mM) and an extremely high rate [V max of 1.6 µmol min–1 (mg protein)–1]. Rb+ uptake [K m of ~40 mM and V max of 0.5 µmol min–1 (mg protein)–1], which was inhibited competitively by K+ and less prominently by Cs+, was also observed. The specificity of this transport is likely to be K+>Rb+>Cs+. This peculiar K+ transport plays a role as a salvage mechanism against defects in high-affinity systems in the K+ homeostasis of this bacterium.
Keywords: K+ transport KtrI KtrII Enterococcus hirae
No Title by Cristina Solano; Begoña Sesma; Miguel Alvarez; Elena Urdaneta; David Garcia-Ros; Alfonso Calvo; Carlos Gamazo (pp. 46-51).
To confirm the existence in nature of Salmonella enteritidis strains of different degrees of virulence and to elucidate the mechanisms underlying the effects of such strains on the epithelial barrier function, the consequences of infection of Caco-2 cells and HEp-2 cells with 15 S. enteritidis strains in a chicken infection model were examined. The more virulent strains of S. enteritidis, which are biofilm producers in adherence test medium, were able to disrupt HEp-2 and Caco-2 monolayers, as shown by transmonolayer electrical resistance and lactate dehydrogenase activity. In contrast, the low-virulence strains of S. enteritidis, which do not produce biofilms in adherence test medium, had no effect on the same cells. An avirulent rough mutant of Salmonella minnesota exhibited a pattern of behaviour similar to that of the low virulence strains of S. enteritidis, whilst a clinical Salmonella typhi strain caused rapid injury to the monolayers. The effect of supernatants of Salmonella cultures in adherence test medium on the integrity of Caco-2 cell monolayers indicated that the high-virulence S. enteritidis strains, but not the low-virulence strains, release a soluble factor when incubated under optimum biofilm-forming conditions, which enables the disruption of the integrity of Caco-2 monolayers.
Keywords: Salmonella Biofilm HEp-2 Caco-2 Virulence
No Title by Ulrike Johnsen; Martina Selig; Karina B. Xavier; Helena Santos; Peter Schönheit (pp. 52-61).
The glucose and fructose degradation pathways were analyzed in the halophilic archaeon Halococcus saccharolyticus by 13C-NMR labeling studies in growing cultures, comparative enzyme measurements and cell suspension experiments. H. saccharolyticus grown on complex media containing glucose or fructose specifically 13C-labeled at C1 and C3, formed acetate and small amounts of lactate. The 13C-labeling patterns, analyzed by 1H- and 13C-NMR, indicated that glucose was degraded via an Entner-Doudoroff (ED) type pathway (100%), whereas fructose was degraded almost completely via an Embden-Meyerhof (EM) type pathway (96%) and only to a small extent (4%) via an ED pathway. Glucose-grown and fructose-grown cells contained all the enzyme activities of the modified versions of the ED and EM pathways recently proposed for halophilic archaea. Glucose-grown cells showed increased activities of the ED enzymes gluconate dehydratase and 2-keto-3-deoxy-gluconate kinase, whereas fructose-grown cells contained higher activities of the key enzymes of a modified EM pathway, ketohexokinase and fructose-1-phosphate kinase. During growth of H. saccharolyticus on media containing both glucose and fructose, diauxic growth kinetics were observed. After complete consumption of glucose, fructose was degraded after a lag phase, in which fructose-1-phosphate kinase activity increased. Suspensions of glucose-grown cells consumed initially only glucose rather than fructose, those of fructose-grown cells degraded fructose rather than glucose. Upon longer incubation times, glucose- and fructose-grown cells also metabolized the alternate hexoses. The data indicate that, in the archaeon H. saccharolyticus, the isomeric hexoses glucose and fructose are degraded via inducible, functionally separated glycolytic pathways: glucose via a modified ED pathway, and fructose via a modified EM pathway.
Keywords: Halococcus saccharolyticus Archaea Modified Embden-Meyerhof pathway Modified Entner-Doudoroff pathway 13C-NMR Ketohexokinase Fructose-1-phosphate kinase Glucose dehydrogenase Gluconate dehydratase 2-Keto-3-deoxy-gluconate kinase
No Title by Nadine Fournier; Lorenzo Cerutti; Nicola Beltraminelli; Ekaterina Salimova; Viesturs Simanis (pp. 62-69).
The onset of septum formation in the fission yeast Schizosaccharomyces pombe is signaled via the spg1p GTPase-switch, which is part of the septation initiation network. This is negatively regulated by the two-component GTPase-activating protein (GAP) comprised of the products of the cdc16 and byr4 genes. Loss-of-function mutations in either of these genes result in multiple rounds of septum formation without cell cleavage. In this work, we demonstrate that attenuation of the protein kinase cdc7p can rescue the lethality of a null allele of cdc16. This observation provides the basis for selection of chromosomal mutations and multicopy suppressors that attenuate the signaling of septation. Using this screen, mutations in all the previously described septation initiation network genes were obtained, with the exception of byr4, sid4 and plo1. We also demonstrate that increased expression of the dma1 gene can rescue the lethality of a null allele of cdc16. The implications for the regulation of septum formation in fission yeast are discussed.
Keywords: Schizosaccharomyces pombe Cytokinesis Septum GTPase GTPase-activating protein Cell cycle
No Title by Paola Rappelli; Franco Carta; Giuseppe Delogu; Maria Filippa Addis; Daniele Dessì; Piero Cappuccinelli; Pier Luigi Fiori (pp. 70-74).
We recently reported that most Trichomonas vaginalis isolates cultured in vitro are infected by Mycoplasma hominis. In this work, we have characterized some aspects of the relationships between the two microorganisms. PCR, cultivation, and immunological methods revealed that the number of M. hominis organisms carried by T. vaginalis in culture varied from isolate to isolate, suggesting a specific multiplicity of infection. Moreover, infected T. vaginalis isolates were able to pass bacteria not only to M. hominis-free protozoa, but also to human-derived epithelial cells. The in vitro transmission of the bacterium from T. vaginalis to both uninfected parasite isolates and human epithelial cells suggests a role for T. vaginalis as a carrier of the M. hominis infection in vivo.
Keywords: Trichomonas vaginalis Mycoplasma hominis Symbiosis Sexually transmitted diseases
No Title by Miho Suzuki; Zong Jun Cui; Masaharu Ishii; Yasuo Igarashi (pp. 75-78).
Hydrogenobacter thermophilus strain TK-6 was observed to grow anaerobically on nitrate as an electron acceptor when molecular hydrogen was used as an energy source. Nitrite was detected as the product of a respiratory reaction. 15NO, 15N2O, and 15N2 were detected with Na15NO3 as an electron acceptor. Western immunoblot analysis showed that cell-free extracts from cells grown on nitrate reacted with antibodies against heme cd 1-type nitrite reductase from Pseudomonas aeruginosa. The positive bands, which had molecular masses similar to that of the heme cd 1-type nitrite reductase, were also stained by heme staining. These results indicate that nitrite reductase of strain TK-6 is a heme cd 1-type enzyme. Activity of ATP:citrate lyase, one of the key enzymes of the reductive TCA cycle, was detected in cell-free extract of cells cultivated on nitrate, which indicates that the cycle operates during anaerobic growth.
Keywords: Hydrogenobacter thermophilus TK-6 Anaerobic growth Nitrate reduction