Blue Light Overrides Repression of Asexual Sporulation by Mating Type Genes in the Basidiomycete Coprinus cinereus

Monokaryotic mycelia of the homobasidiomycete Coprinus cinereus form asexual spores (oidia) constitutively in abundant numbers. Mycelia with mutations in both mating type loci (Amut Bmut homokaryons) also produce copious oidia but only when exposed to blue light. We used such an Amut Bmut homokaryon to define environmental and inherent factors that influence the light-induced oidiation process. We show that the Amut function causes repression of oidiation in the dark and that light overrides this effect. Similarly, compatible genes from different haplotypes of the A mating type locus repress sporulation in the dark and not in the light. Compatible products of the B mating type locus reduce the outcome of light on A-mediated repression but the mutated B function present in the Amut Bmut homokaryons is not effective. In dikaryons, the coordinated regulation of asexual sporulation by compatible A and B mating type genes results in moderate oidia production in light. Copyright 1998 Academic Press.     

Immunocytochemical localization of IdiA, a protein expressed under iron or manganese limitation in the mesophilic cyanobacterium Synechococcus PCC 6301 and the thermophilic cyanobacterium Synechococcus elongatus

Iron-deficiency-induced protein A (IdiA) with a calculated molecular mass of 35 kDa has previously been shown to be essential under manganese- and iron-limiting conditions in the cyanobacteria Synechococcus PCC 6301 and PCC 7942. Studies of mutants indicated that in the absence of IdiA mainly photosystem II becomes damaged, suggesting that the major function of IdiA is in Mn and not Fe metabolism (Michel et al. 1996, Microbiology 142: 2635–2645). To further elucidate the function of IdiA, the immunocytochemical localization of IdiA in the cell was examined. These investigations provided evidence that under mild Fe deficiency IdiA is intracellularly localized and is mainly associated with the thylakoid membrane in Synechococcus PCC 6301. The protein became distributed throughout the cell under severe Fe limitation when substantial morphological changes had already occurred. For additional verification of a preferential thylakoid membrane association of IdiA, these investigations were extended to the thermophilic Synechococcus elongatus. In this cyanobacterium Mn deficiency could be obtained more rapidly than in the mesophilic Synechococcus PCC 6301 and PCC 7942, and the thylakoid membrane structure proved to be more stable under limiting growth conditions. The immunocytochemical investigations with this cyanobacterium clearly supported a thylakoid membrane association of IdiA. In addition, evidence was obtained for a localization of IdiA on the cytoplasmic side of the thylakoid membrane. All available data support a function of IdiA as an Mn-binding protein that facilitates transport of Mn via the thylakoid membrane into the lumen to provide photosystem II with Mn. A possible explanation for the observation that IdiA was not only expressed under Mn deficiency but also under Fe deficiency is given in the discussion. Received: 28 July 1997 / Accepted: 26 November 1997     

The Proteome and Lipidome of Synechocystis sp. PCC 6803 Cells Grown under Light-Activated Heterotrophic Conditions

Cyanobacteria are photoautotrophic prokaryotes with a plant-like photosynthetic machinery. Because of their short generation times, the ease of their genetic manipulation, and the limited size of their genome and proteome, cyanobacteria are popular model organisms for photosynthetic research. Although the principal mechanisms of photosynthesis are well-known, much less is known about the biogenesis of the thylakoid membrane, hosting the components of the photosynthetic, and respiratory electron transport chain in cyanobacteria. Here we present a detailed proteome analysis of the important model and host organism Synechocystis sp. PCC 6803 under light-activated heterotrophic growth conditions. Because of the mechanistic importance and severe changes in thylakoid membrane morphology under light-activated heterotrophic growth conditions, a focus was put on the analysis of the membrane proteome, which was supported by a targeted lipidome analysis. In total, 1528 proteins (24.5% membrane integral) were identified in our analysis. For 641 of these proteins quantitative information was obtained by spectral counting. Prominent changes were observed for proteins associated with oxidative stress response and protein folding. Because of the heterotrophic growth conditions, also proteins involved in carbon metabolism and C/N-balance were severely affected. Although intracellular thylakoid membranes were significantly reduced, only minor changes were observed in their protein composition. The increased proportion of the membrane-stabilizing sulfoqinovosyl diacyl lipids found in the lipidome analysis, as well as the increased content of lipids with more saturated acyl chains, are clear indications for a coordinated synthesis of proteins and lipids, resulting in stabilization of intracellular thylakoid membranes under stress conditions.Cyanobacteria are a widespread group of photoautotrophic organisms, which significantly contribute to global carbon fixation. Cyanobacteria and plant chloroplasts share a common ancestor, and thus cyanobacteria have a plant-like photosynthetic metabolism (1, 2). Consequently, they are established model organisms for studies, aiming to elucidate photosynthetic mechanisms. Both, chloroplasts and cyanobacteria, have two internal membrane systems, that is, the inner envelope and the cytoplasmic membrane (CM)¹ in chloroplasts or cyanobacteria, respectively, as well as the thylakoid membrane (TM) system, which harbors the complexes of the photosynthetic electron transfer chain (3, 4). The photosynthetic electron transfer chain typically consists of the three membrane integral protein complexes: photosystem I (PS I), photosystem II (PS II), and the cytochrome b₆f complex, as well as of the soluble electron carriers plastoquinone and plastocyanin (5, 6). In the end, reduction equivalents are produced, which are used for CO₂-fixation (7). However, besides the ability to grow photoautotrophically, some cyanobacteria are also capable to grow photoheterotrophically, where they use reduced organic compounds as carbon source, or even completely heterotrophically by using reduced organic compounds as carbon and energy source (8). The well-characterized cyanobacterium Synechocystis sp. PCC 6803 (9) (hereafter: Synechocystis) can grow in darkness under light-activated heterotrophic growth (LAHG) conditions by using glucose as carbon and energy source (10). Enhanced sugar catabolism in LAHG cultures is, for example, reflected by increased activities of enzymes involved in sugar catabolism, such as glucokinase and pyruvate kinase (11). The effects of LAHG conditions on the abundance of soluble Synechocystis proteins have been analyzed previously, although only 23 proteins with a significantly altered expression level (LAHG versus autotrophic growth) have been described. This study has e.g. indicated that under LAHG conditions glucose is mainly degraded by the oxidative pentose phosphate (OPP) pathway (12). The histidine kinase 8 (Hik8) as well as the sigma factor E (SigE), regulating the expression of sugar-degrading genes, were shown to be essential for LAHG (13, 14).Although readjustments of the cellular energy metabolism are important, the impact on the cellular membrane architecture is more striking. The ability of Synechocystis to grow under LAHG conditions has been used recently to analyze TM formation within cyanobacterial cells (15). Although dark-adapted Synechocystis cells have no active PS II complex, complete photosynthetic activity is regained within 24 h after transferring dark-adapted cells into the light. Furthermore, reappearance of photosynthetic electron transfer processes is coupled to the formation of internal TMs. However, it is essentially still completely enigmatic how the formation of internal TM is controlled, although some proteins have been suggested to be involved. These proteins include the vesicle inducing protein in plastids 1 (Vipp1), DnaK proteins, a prohibitin-like protein, as well as the YidC protein, a membrane protein integrase (16–19). Nevertheless, although some proteins have been suggested to be more directly involved in TM formation, the stability of the TM is also globally affected indirectly by pathways, which control the biogenesis of lipids and/or cofactors, and mutants defective in synthesis of chlorophyll or of the membrane lipid phosphatidylglycerol (PG) have severely reduced TM systems (20, 21).In the present work, we combined prefractioning of Synechocystis cellular membranes with a global proteome and lipidome analysis, to shift the analytical focus toward the rearrangement of the internal thylakoid membrane system observed in Synechocystis cells under LAHG conditions, with a significantly larger coverage of the proteome than in former studies. Furthermore, also the effect on Synechocystis lipids was analyzed in a targeted mass spectrometric approach, revealing significant adjustment of fatty acid saturation in response to the LAHG conditions.     

Paleopedology and geochemistry of Late Mississippian (Chesterian) Pennington Formation paleosols at Pound Gap,Kentucky, USA: Implications for high-frequency climate variations

Climate during the Late Mississippian (late Chesterian) in the southern Appalachian Basin was characterized by alternating periods of aridity and humidity. Pennington Formation paleosols at Pound Gap record climate and ecological changes for latest Chesterian time, ending at the Mississippian–Pennsylvanian systemic boundary. Forty paleosols were identified, described, and assigned to seven pedotypes. Inferred soil orders considered as analogs include Histosols, Entisols, Inceptisols, Alfisols and Oxisols, but are dominated by Vertisols. Classification of an Oxisol was determined by field and geochemical evidence of intense leaching and kaolinite-dominated clay mineralogy. Field and micromorphologicial evidence suggests a polygenetic character of the Vertisols, resulting from changing soil drainage through time. Variations in soil drainage are quantified using proxy estimates of inferred soil processes such as base loss, leaching, lessivage, and oxidation. Using the CIA-K proxy, mean annual precipitation (MAP) estimates range from 519 to 1361 mm/yr. Changes in MAP correspond with variation in inferred soil processes. The flora of this time period, in response to variations in precipitation and soil drainage, also changes through time as evidenced by changes in abundance and depth of root traces. Reconstructed ecosystems range from sparse vegetative cover with shallow, tabular root systems in early Pennington soil development, to dense, deeply penetrating root systems suggestive of arborescent floral associations at the top of the succession approaching the Mississippian–Pennsylvanian boundary. This study provides greater resolution of changing climate and pedogenic processes than what is provided in previous studies of Late Mississippian climate, and suggests that the documented variability in paleosol types and soil drainage might represent the record of high-frequency climate changes likely associated with expansion and contraction of the paleo-Intertropical Convergence Zone (ITCZ).     

Determining the molecular basis for the pH-dependent interaction between the link module of human TSG-6 and hyaluronan

TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His(4)) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pK(a) values of charged residues within the Link module were determined using NMR. These data indicated that His(4) makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp(89)), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr(12)). This molecular network transmits the change in ionization state of His(4) to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His(45), is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments.     

Crystallization of the DNA-binding Escherichia coli protein FIS

The specific DNA-binding protein FIS (factor for inversion stimulation), which stimulates site-specific DNA inversion by interaction with an enhancer sequence, was purified from an Escherichia coli strain overproducing the protein. FIS was crystallized at room temperature by microdialysis against 1.2 to 1.5 M-sodium/potassium phosphate containing 10 mM-Tris.HCl, 0.5 to 1 M-NaCl and 1 mM-NaN3 at pH 8.0 to 8.2. The crystals are stout prisms and suitable for X-ray diffraction study beyond 2.5 A resolution. They belong to the orthorhombic space group P2(1)2(1)2(1). The unit cell has dimensions a = 47.57(4) A, b = 51.13(4) A, c = 79.83(6) A and contains one FIS dimer in the asymmetric unit.