Check out our New Publishers' Select for Free Articles

Archives of Microbiology (v.170, #6)

Signaling via cAMP in fungi: interconnections with mitogen-activated protein kinase pathways by J. Kronstad; Adriana De Maria; Deanna Funnell; R. David Laidlaw; Nancy Lee; Mário Moniz de Sá; Marilee Ramesh (pp. 395-404).

The cAMP signal transduction pathway controls a wide variety of processes in fungi. For example, considerable progress has been made in describing the involvement of cAMP pathway components in the control of morphogenesis in Saccharomyces cerevisiae, Ustilago maydis, and Magnaporthe grisea. These morphological processes include the establishment of filamentous growth in S. cerevisiae and U. maydis, and the differentiation of an appressorial infection structure in M. grisea. The discovery that appressorium formation requires cAMP signaling provides an immediate connection to fungal virulence. This connection may have broader implications among fungal pathogens because recent work indicates that cAMP signaling controls the expression of virulence traits in the human pathogen Cryptococcus neoformans. In this fungus, cAMP also influences mating, as has been found for Schizosaccharomyces pombe and as may occur in U. maydis. Finally, cAMP and mitogen-activated protein kinase pathways appear to function coordinately to control the response of certain fungi, e.g., Saccharomyces cerevisiae and Schizosaccharomyces pombe, to environmental stress. There are clues that interconnections between these pathways may be common in the control of many fungal processes.

Keywords: Key words Signal transduction; Fungi; Stress; Virulence; Sexual development; Mating; Appressorium; Dimorphism

Acclimation of the photosynthetic response of Chromatium vinosum to light-limiting conditions by Olga Sánchez; Hans van Gemerden; Jordi Mas (pp. 405-410).

The photosynthetic response of the purple sulfur bacterium Chromatium vinosum DSM 185 to different degrees of illumination was analyzed. The microorganism was grown in continuous culture, and samples were taken from the effluent of the culture and incubated at different irradiances to determine the specific rate of sulfur oxidation as a measure of the photosynthetic activity of the organism. The activities obtained were plotted as a function of the specific rate of light uptake, and for each set of data a photosynthesis equation was fitted, which allowed the estimation of P_max (photosynthetic capacity), q_k (the threshold irradiance for light limitation), and m (maintenance coefficient). The results indicated that cells grown under light limitation are able to achieve higher photosynthetic activities than cells grown under light saturation. The photosynthetic capacity (P_max) remained constant under all the conditions of illumination tested, while the maintenance expenses (m) were higher under light limitation. The parameter q_k, on the contrary, decreased considerably at limiting irradiances.

Keywords: Key words Purple sulfur bacteria; Light limitation; Photosynthetic response

Utilization of reducing power in light-limited cultures of Chromatiumvinosum DSM 185 by Olga Sánchez; Hans Van Gemerden; J. Mas (pp. 411-417).

This study describes how the phototrophic organism Chromatium vinosum, when grown under different degrees of light limitation, distributes the reducing power initially present in the medium as hydrogen sulfide. Under all the conditions of illumination tested, sulfur was the major store of reducing power. Glycogen, which was virtually absent under light limitation, accounted for 31.6% of the stored reducing power at saturating irradiances. Analysis of the electron budget showed that under light-limiting conditions, an important fraction of reducing power did not appear in storage products or in structural cell material. Analysis of dissolved organic carbon in the supernatant of the culture indicated the excretion of organic compounds.

Keywords: Key words Phototrophic sulfur bacteria; Reducing power; Light limitation; Storage compounds

The vhuU gene encoding a small subunit of a selenium-containing [NiFe]-hydrogenase in Methanococcus voltae appears to be essential for the cell by Matthias Pfeiffer; Helga Bestgen; A. Bürger; A. Klein (pp. 418-426).

We developed a general method for the site-specific deletion of gene sequences to obtain new selectable markers in the archaeon Methanococcus voltae. Using a deletion in the hisA gene, a vector was integrated into the chromosome by homologous recombination, thereby reconstituting histidine prototrophy. The vector contained the β-glucuronidase gene uidA of Escherichia coli as a reporter under the control of an M. voltae promoter that normally drives the expression of a selenium-free [NiFe]-hydrogenase after selenium deprivation. This construct has allowed us to check whether the selenium supply was sufficiently low to induce the transcription of the genes encoding the selenium-free hydrogenases. We tried to introduce a chromosomal deletion of the vhuU gene of the archaeon M. voltae by gene replacement and by keeping the cells under selenium deprivation. The gene vhuU encodes the very small, selenocysteine-containing subunit that is part of the primary reaction center of the Vhu hydrogenase. All transformants bearing the deletion also contained the vhuU wild-type gene. Therefore, the vhuU gene appears to be essential for the cell even under conditions that lead to the induction of the selenium-free homologue Vhc of the Vhu hydrogenase.

Keywords: Key words Archaea; Methanococcus voltae; Deletion; mutagenesis; Adaptation; Selenium deprivation; [NiFe]-hydrogenases; Gene replacement

Metabolism of aromatic aldehydes as cosubstrates by the acetogen Clostridium formicoaceticum by Claudia Frank; U. Schwarz; Carola Matthies; H. L. Drake (pp. 427-434).

When the acetogen Clostridium formicoaceticum was cultivated on mixtures of aromatic compounds (e.g., 4-hydroxybenzaldehyde plus vanillate), the oxidation of aromatic aldehyde groups occurred more rapidly than did O-demethylation. Likewise, when fructose and 4-hydroxybenzaldehyde were simultaneously provided as growth substrates, fructose was utilized only after the aromatic aldehyde group was oxidized to the carboxyl level. Aromatic aldehyde oxidoreductase activity was constitutive (activities approximated 0.8 U mg^–1), and when pulses of 4-hydroxybenzaldehyde were added during fructose-dependent growth, the rate at which fructose was utilized decreased until 4-hydroxybenzaldehyde was consumed. Although 4-hydroxybenzaldehyde inhibited the capacity of cells to metabolize fructose, lactate or gluconate were consumed simultaneously with 4-hydroxybenzaldehyde, and lactate or aromatic compounds lacking an aldehyde group were utilized concomitantly with fructose. These results demonstrate that (1) aromatic aldehydes can be utilized as cosubstrates and have negative effects on the homoacetogenic utilization of fructose by C. formicoaceticum, and (2) the consumption of certain substrates by this acetogen is not subject to catabolite repression by fructose.

Keywords: Key words Acetogenesis; Clostridium formicoaceticum; Aromatic aldehydes; Methoxylated aromatic; compounds; O-demethylation; Cosubstrate metabolism

The respiratory chain of the halophilic anoxygenic purple bacterium Rhodospirillum sodomense by Patrizia Bonora; Ilaria Principi; Alejandro Hochkoeppler; Roberto Borghese; D. Zannoni (pp. 435-441).

The halophilic purple nonsulfur bacterium Rhodospirillum sodomense has been previously described as an obligate phototroph that requires yeast extract and a limited number of organic compounds for photoheterotrophic growth. In this work, we report on chemoheterotrophic growth of R. sodomense in media containing either acetate or succinate supplemented with 0.3–0.5% yeast extract. Plasma membranes isolated from cells grown aerobically in the dark contained three b-type and three c-type membrane-bound cytochromes with E _m,7 of +171 ± 10, +62 ± 10 and –45 ± 13 mV (561–575 nm), and +268 ± 6, +137 ± 10 and –43 ± 12 mV (551–540 nm). A small amount of a soluble c-type cytochrome with a mol. mass of 15 kDa (E _m,7≥ +150 mV) was identified. Spectroscopic and immunological methods excluded the presence of cytochrome of the c 2 class and high-potential iron-sulfur proteins. Inhibitory studies indicated that only 60–70% of the respiratory activity was blocked by low concentrations of cyanide, antimycin A, and myxothiazol (10, 0.1, and 0.2 μM, respectively). These results were interpreted to show that the oxidative electron transport chain of R. sodomense is branched, leads to a quinol oxidase that is fully blocked by 1 mM cyanide and that is involved in light-dependent oxygen reduction, and leads to a cytochrome c oxidase that is inhibited by 10 μM cyanide. These features taken together suggest that R. sodomense differs from the closely related species Rhodospirillum salinarum and from other species of the genus Rhodospirillum in that it contains multiple membrane-bound cytochromes c.

Keywords: Key words Branched respiratory chain; Chemoheterotrophic growth; Cytochromes; Halophilic purple nonsulfur bacteria; Rhodospirillum sodomense

Sarcosine reductase of Tissierella creatinophila: purification and characterization of its components by Claudia Harms; Ute Ludwig; J. R. Andreesen (pp. 442-450).

Sarcosine reductase is the only reductase system present in Tissierella creatinophila when grown on creatinine plus formate. The acetyl-phosphate-forming component protein C was purified to homogeneity. SDS-PAGE of the purified protein revealed two protein bands with apparent mol. masses of 62 and 50 kDa. The N-terminal amino acid sequence of the two subunits was determined. Antibodies raised against each of the subunits of protein C from Eubacterium acidaminophilum cross-reacted with the corresponding protein present in T. creatinophila, Clostridium litorale and Clostridium sporogenes. The arsenate-dependent hydrolysis of acetyl phosphate catalyzed by protein C was partly inhibited by antibodies directed against the large subunit. Antibodies raised against the small subunit were twice as effective, which indicates that this subunit is the primary site of acetyl transfer from acetyl phosphate. The protein A component of the sarcosine reductase of T. creatinophila was purified to homogeneity by cochromatography with thioredoxin reductase on DEAE-Sephacel, hydroxylapatite, Q-Sepharose, and Sephacryl 100-HR. Protein A had an apparent mol. mass of 21 kDa. Its N-terminal amino acid sequence showed high similarities to that of other proteins A. Initial steps for the purification and preliminary characterization of the sarcosine-specific, substrate-binding protein B_sarcosine component of T. creatinophila indicated the involvement of a 50-kDa protein.

Keywords: Key words Tissierella creatinophila; Sarcosine; reductase; Protein C; Protein A; Protein Bsarcosine

Duplication of hyp genes involved in maturation of [NiFe] hydrogenases in Alcaligenes eutrophus H16 by Ingo Wolf; Thorsten Buhrke; Jens Dernedde; Anne Pohlmann; Bärbel Friedrich (pp. 451-459).

Alcaligenes eutrophus H16 harbors seven hyp genes (hypA, B, F, C, D, E, and X) as part of the hydrogenase gene cluster on megaplasmid pHG1. Here we demonstrate that three of the hyp genes (hypA, B, and F) are duplicated in A. eutrophus, which explains the lack of a phenotypic change in single-site mutants impaired in one of the two copies. Mutants with lesions in both copies showed clear alterations in hydrogenase activities. Deletions in hypF1 and hypF2 completely abolished activities of the soluble hydrogenase and of the membrane-bound hydrogenase, mutations in hypA1 and hypA2 totally blocked the membrane-bound hydrogenase activity, while residual soluble hydrogenase activity accounted for the extremely slow growth of the strain on H₂. Both hydrogenase activities of mutants defective in hypB1 and hypB2 were partially restored by elevating the concentration of nickel chloride in the medium. Reduction of hydrogenase activities in the double mutants correlated with varying degrees of maturation deficiency based upon the amount of unprocessed nickel-free hydrogenase precursor. Despite a high identity between the two copies of hyp gene products, substantial structural differences were identified between the two copies of hypF genes. HypF1, although functionally active, is a truncated version of HypF2, whose structure resembles HypF proteins of other organisms. Interestingly, the N-terminus of HypF2, which is missing in the HypF1 counterpart, contains a putative acylphosphatase domain in addition to a potential metal binding site.

Keywords: Key words [NiFe] hydrogenase; Metal center; assembly; hyp genes

hoxX (hypX) is a functional member of the Alcaligenes eutrophus hyp gene cluster by Thorsten Buhrke; Bärbel Friedrich (pp. 460-463).

The role of HoxX in hydrogenase biosynthesis of Alcaligenes eutrophus H16 was re-examined. The previously characterized hoxX deletion mutant HF344 and a newly constructed second hoxX mutant carrying a smaller in-frame deletion were studied. The second mutant was impaired in the activity of both the soluble and the membrane-bound hydrogenase. The two hydrogenase activities were reduced by approximately 50% due to delayed processing of the active-site-containing large subunits, while hydrogenase gene expression was not affected. We conclude that the mutation in mutant HF344 causes polarity resulting in the observed regulatory phenotype of this mutant. The data presented in this report point to an enhancing function of HoxX in the conversion of the soluble hydrogenase and of the membrane-bound hydrogenase large-subunit precursor. Thus, hoxX encodes a member of the Hyp proteins that are required for the formation of active hydrogenase and was accordingly renamed hypX.

Keywords: Key words HypX; HoxX; Alcaligenes eutrophus; [NiFe] hydrogenase

The biotin protein MadF of the malonate decarboxylase from Malonomonas rubra by Michael Berg; P. Dimroth (pp. 464-468).

The gene for the biotin protein MadF of the Na⁺-pumping malonate decarboxylase from Malonomonas rubra was expressed in Escherichia coli together with the gene for the biotin ligase birA. MadF was partially purified from cell lysates by ammonium sulfate precipitation. Almost pure biotin protein was obtained by subsequent gel chromatography. With recombinant MadF, malonate decarboxylase activity of M. rubra cell extracts previously inactivated by avidin was recovered. Thus, the biological activity of recombinant MadF was proven. Despite the coexpression of birA, MadF was poorly biotinylated. This effect was not caused by an insufficient cofactor supply due to elevated protein levels at constant biotin uptake rates. Attempts to improve the cofactor incorporation were made by site-directed mutagenesis, by coexpression of madK, and by N-terminal elongation of MadF. These measures improved the fraction of MadF containing biotin to maximally 5%. These results might indicate the existence of a biotin ligase in M. rubra with an altered substrate specificity different from that of BirA.

Activation and thermostabilization effects of cyclic 2,3-diphosphoglycerate on enzymes from the hyperthermophilic Methanopyrus kandleri by Seigo Shima; David A. Hérault; Albrecht Berkessel; R. K. Thauer (pp. 469-472).

Enzymes involved in methane formation from carbon dioxide and dihydrogen in Methanopyrus kandleri require high concentrations (> 1 M) of lyotropic salts such as K₂HPO₄/KH₂PO₄ or (NH₄)₂SO₄ for activity and for thermostability. The requirement correlates with high intracellular concentrations of cyclic 2,3-diphosphoglycerate (cDPG; ≈ 1 M) in this hyperthermophilic organism. We report here on the effects of potassium cDPG on the activity and thermostability of the two methanogenic enzymes cyclohydrolase and formyltransferase and show that at cDPG concentrations prevailing in the cells the investigated enzymes are highly active and completely thermostable. At molar concentrations also the potassium salts of phosphate and of 2,3-bisphosphoglycerate, the biosynthetic precursor of cDPG, were found to confer activity and thermostability to the enzymes. Thermodynamic arguments are discussed as to why cDPG, rather than these salts, is present in high concentrations in the cells of Mp. kandleri.

Keywords: Key words Methanogenic archaea; Formyltransferase; Cyclohydrolase; N5; N10-Methenyltetrahydromethanopterin; Formylmethanofuran; Halophilic enzymes; Thermophilic enzymes; 2-Phosphoglycerate kinase; Cyclic diphosphoglycerate; synthase

Sections

Personal tools

Archives of Microbiology (v.170, #6)

Sections

Personal tools

Local resources

Archives of Microbiology (v.170, #6)