C P Wolk

Spanish National Research Council, Madrid, Madrid, Spain

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Publications (148)513.5 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: When the filamentous cyanobacterium Anabaena variabilis grows aerobically without combined nitrogen, some vegetative cells differentiate into N2-fixing heterocysts, while the other vegetative cells perform photosynthesis. Microarrays of sequences within protein-encoding genes were probed with RNA purified from extracts of vegetative cells, from isolated heterocysts, and from whole filaments to investigate transcript levels, and carbon and energy metabolism, in vegetative cells and heterocysts in phototrophic, mixotrophic, and heterotrophic cultures. Heterocysts represent only 5% to 10% of cells in the filaments. Accordingly, levels of specific transcripts in vegetative cells were with few exceptions very close to those in whole filaments and, also with few exceptions (e.g., nif1 transcripts), levels of specific transcripts in heterocysts had little effect on the overall level of those transcripts in filaments. In phototrophic, mixotrophic, and heterotrophic growth conditions, respectively, 845, 649, and 846 genes showed more than 2-fold difference (p < 0.01) in transcript levels between vegetative cells and heterocysts. Principal component analysis showed that the culture conditions tested affected transcript patterns strongly in vegetative cells but much less in heterocysts. Transcript levels of the genes involved in phycobilisome assembly, photosynthesis, and CO2 assimilation were high in vegetative cells in phototrophic conditions, and decreased when fructose was provided. Our results suggest that Gln, Glu, Ser, Gly, Cys, Thr, and Pro can be actively produced in heterocysts. Whether other protein amino acids are synthesized in heterocysts is unclear. Two possible components of a sucrose transporter were identified that were upregulated in heterocysts in two growth conditions. We consider it likely that genes with unknown function represent a larger fraction of total transcripts in heterocysts than in vegetative cells across growth conditions. This study provides the first comparison of transcript levels in heterocysts and vegetative cells from heterocyst-bearing filaments of Anabaena. Although the data presented do not give a complete picture of metabolism in either type of cell, they provide a metabolic scaffold on which to build future analyses of cell-specific processes and of the interactions of the two types of cells.
    BMC Genomics 11/2013; 14(1):759. · 4.40 Impact Factor
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    ABSTRACT: H(2) generated from renewable resources holds promise as an environmentally innocuous fuel that releases only energy and water when consumed. In biotechnology, photoautotrophic oxygenic diazotrophs could produce H(2) from water and sunlight using the cells' endogenous nitrogenases. However, nitrogenases have low turnover numbers and require large amounts of ATP. [FeFe]-hydrogenases found in other organisms can have 1000-fold higher turnover numbers and no specific requirement for ATP, but are very O(2)-sensitive. Certain filamentous cyanobacteria protect nitrogenase from O(2) by sequestering the enzyme within internally micro-oxic, differentiated cells called heterocysts. We heterologously expressed the [FeFe]-hydrogenase operon from Shewanella oneidensis MR-1 in Anabaena sp. strain PCC 7120 using the heterocyst-specific promoter P(hetN). Active [FeFe]-hydrogenase was detected in, and could be purified from, aerobically grown Anabaena sp. strain PCC 7120, but only when the organism was grown in nitrate-depleted conditions that elicited heterocyst formation. These results suggest that the heterocysts protected the [FeFe]-hydrogenase against inactivation by O(2).
    Applied and environmental microbiology 09/2012; · 3.69 Impact Factor
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    ABSTRACT: Some filamentous cyanobacteria such as Anabaena sp. strain PCC 7120 produce cells, termed heterocysts, specialized in nitrogen fixation. Heterocysts bear a thick envelope containing an inner layer of glycolipids and an outer layer of polysaccharide that restrict the diffusion of air (including O(2)) into the heterocyst. Anabaena sp. mutants impaired in production of either of those layers show a Fox(-) phenotype (requiring fixed nitrogen for growth under oxic conditions). We have characterized a set of transposon-induced Fox(-) mutants in which transposon Tn5-1063 was inserted into the Anabaena sp. chromosome open reading frame all1711 which encodes a predicted membrane protein that belongs to the major facilitator superfamily (MFS). These mutants showed higher nitrogenase activities under anoxic than under oxic conditions and altered sucrose uptake. Electron microscopy and alcian blue staining showed a lack of the heterocyst envelope polysaccharide (Hep) layer. Northern blot and primer extension analyses showed that, in a manner dependent on the nitrogen-control transcription factor NtcA, all1711 was strongly induced after nitrogen step-down. Confocal microscopy of an Anabaena sp. strain producing an All1711-green fluorescent protein (All1711-GFP) fusion protein showed induction in all cells of the filament but at higher levels in differentiating heterocysts. All1711-GFP was located in the periphery of the cells, consistent with All1711 being a cytoplasmic membrane protein. Expression of all1711 from the P(glnA) promoter in a multicopy plasmid led to production of a presumptive exopolysaccharide by vegetative cells. These results suggest that All1711, which we denote HepP, is involved in transport of glycoside(s), with a specific physiological role in production of Hep.
    Journal of bacteriology 06/2012; 194(17):4677-87. · 3.94 Impact Factor
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    Jinjie Liu, C Peter Wolk
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    ABSTRACT: PatA resembles a response regulator protein with a defective DNA-binding domain, and PatL (All3305) is a pentapeptide repeat protein. A yeast two-hybrid library identified PatL as a protein with which PatA may interact. Heterocysts of patA and patL Anabaena sp. form nearly exclusively terminally in long filaments, further linking the genes.
    Journal of bacteriology 09/2011; 193(21):6070-4. · 3.94 Impact Factor
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    ABSTRACT: Heterocysts are specialized cells required for aerobic fixation of dinitrogen by certain filamentous cyanobacteria. Numerous genes involved in the differentiation and function of heterocysts in Anabaena sp. strain PCC 7120 have been identified by mutagenizing and screening for mutants that require fixed nitrogen for growth in the presence of oxygen. We have verified that 10 Anabaena sp. genes, all1338, all1591, alr1728, all3278, all3520, all3582, all3850, all4019, alr4311, and all4388, identified initially by transposon mutagenesis, are such genes by complementing or reconstructing the original mutation and by determining whether the mutant phenotype might be due to a polar effect of the transposon. Elucidation of the roles of these genes should enhance understanding of heterocyst biology.
    Journal of bacteriology 05/2011; 193(14):3482-9. · 3.94 Impact Factor
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    ABSTRACT: Cyanobacteria use sunlight and water to produce hydrogen gas (H₂), which is potentially useful as a clean and renewable biofuel. Photobiological H₂ arises primarily as an inevitable by-product of N₂ fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H₂ production by this strain, six nitrogenase amino acid residues predicted to reside within 5 Å of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N₂. Most of the 49 variants examined were deficient in N₂-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N₂ atmosphere significantly increased their in vivo rates of H₂ production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H₂ compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H₂ in an aerobic, nitrogen-containing environment.
    Applied and environmental microbiology 10/2010; 76(20):6741-50. · 3.69 Impact Factor
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    ABSTRACT: Anabaena sp. strain PCC 7120, widely studied, has 145 annotated transposase genes that are part of transposable elements called insertion sequences (ISs). To determine the entirety of the ISs, we aligned transposase genes and their flanking regions; identified the ISs' possible terminal inverted repeats, usually flanked by direct repeats; and compared IS-interrupted sequences with homologous sequences. We thereby determined both ends of 87 ISs bearing 110 transposase genes in eight IS families (http://www-is.biotoul.fr/) and in a cluster of unclassified ISs, and of hitherto unknown miniature inverted-repeat transposable elements. Open reading frames were then identified to which ISs contributed and others--some encoding proteins of predictable function, including protein kinases, and restriction endonucleases--that were interrupted by ISs. Anabaena sp. ISs were often more closely related to exogenous than to other endogenous ISs, suggesting that numerous variant ISs were not degraded within PCC 7120 but transferred from without. This observation leads to the expectation that further sequencing projects will extend this and similar analyses. We also propose an adaptive role for poly(A) sequences in ISs.
    Journal of bacteriology 10/2010; 192(20):5289-303. · 3.94 Impact Factor
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    ABSTRACT: Heterocyst-forming cyanobacteria simultaneously photosynthesize, producing oxygen (O2), and fix dini-trogen (N2), initially into ammonia, using nitrogenase enzymes that are rapidly inactivated by O2. These cyanobacteria enable nitrogenases to function in an oxic environment by segregating them within specialized cells, called heterocysts, in which O2 is not produced, respiration is highly active, and an envelope barrier of glycolipids greatly slows the rate of entry of O2. We will describe the chemical structure of the heterocyst-specific glycolipids (Hgls), their physiological role, and what is known of their deposition. We will then discuss the clustered genes that encode the proteins required for their biosynthesis, how the glycolipids are believed to be synthesized, and what is known of the regulation of their biosynthesis. Finally, we will examine the relationship between their biosynthetic enzymes and other polyketide synthases, with an emphasis on those from cyanobacteria.
    07/2009: pages 179-202;
  • Liliana Cardemil, C. Peter Wolk
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    ABSTRACT: The polysaccharides from the envelopes of heterocysts of Cylindrospermum licheniforme Kütz., and of heterocysts and spores of Anabaena variabilis Kütz., like those from the differentiated cells of Anabaena cylindrica Lemm., have a 1,3-linked backbone consisting of glucosyl and mannosyl residues in a molar ratio of approximately 3:1. As is the case with A. cylindrica the polysaccharides from A. variabilis and from the heterocysts of C. licheniforme have terminal xylosyl and galactosyl residues as side branches. In addition, the polysaccharide from C. licheniforme resembles that from A. cylindrica in having terminal mannosyl residues as side branches (absent from A. variabilis). The polysaccharides from A. variabilis resemble that from A. cylindrica in having glucose-containing side branches (absent from the heterocyst polysaccharide from C. licheniforme), but in contrast to the polysaccharides from the other two species they also have terminal arabinosyl residues as side branches. All of the polysaccharides mentioned appear to be structurally related; we present tentative structures for those not previously investigated. In contrast, the envelope of spores of C. licheniforme contains only a largely 4-linked galactan. The bulk of this envelope is not polysaccharide in nature, and contains aromatic groups.
    Journal of Phycology 06/2008; 17(3):234 - 240. · 2.24 Impact Factor
  • C. Peter Wolk, Elizabeth Wojciuch
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    ABSTRACT: The nitrogenase activity of extracts from aerobically grown. Anabaena cylindrica is proportional to the cube of the concentration of extract over a wide range of activities. This relationship is changed to one of direct proportionality, approximately, when reaction mixtures are supplemented with a constant amount of extract from algae starved for molybdenum in the presence of tungstate. The time course of solubilization of nitrogenase during cavitation is reevaluated in the light of these results. The finding that solubilization occurs at first rapidly and subsequently much more slowly is discussed with reference to the hypothesis that nitrogenase is localized in heterocysts.
    Journal of Phycology 06/2008; 7(4):339 - 344. · 2.24 Impact Factor
  • Steven K. Lorch, C. Peter Wolk
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    ABSTRACT: Gas-liquid chromatography of algal extracts provides a sensitive assay for their content of 1-(O-α-d-glycopyranosyl)-3, 25-hexacosanediol, the principal glycolipid from the envelope of heterocysts of Anabaena cylindrica. Different patterns of lipids were found in the heterocysts of 5 species of Anabaena and Nostoc. Lipids with comparable chromatographic properties were detected in Gloeocapsa.
    Journal of Phycology 06/2008; 10(3):352 - 355. · 2.24 Impact Factor
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    ABSTRACT: Wild-type Anabaena sp. strain PCC 7120, a filamentous nitrogen-fixing cyanobacterium, produces single heterocysts at semi-regular intervals. asr0100 (patU5) and alr0101 (patU3) are homologous to the 5' and 3' portions of patU of Nostoc punctiforme. alr0099 (hetZ) overlaps the 5' end of patU5. hetZ, patU5 and patU3 were all upregulated, or expressed specifically, in proheterocysts and heterocysts. Mutants of hetZ showed delayed or no heterocyst differentiation. In contrast, a patU3 mutation produced a multiple contiguous heterocyst (Mch) phenotype and restored the formation of otherwise lost intercalary heterocysts in a patA background. Decreasing the expression of patU3 greatly increased the frequency of heterocysts in a mini-patS strain. Two promoter regions and two principal, corresponding transcripts were detected in the hetZ-patU5-patU3 region. Transcription of hetZ was upregulated in a hetZ mutant and downregulated in a patU3 mutant. When mutants hetZ::C.K2 and hetZ::Tn5-1087b were nitrogen-deprived, P(hetC)-gfp was very weakly expressed, and in hetZ::Tn5-1087b, P(hetR)-gfp was relatively strongly expressed in cells that had neither a regular pattern nor altered morphology. We conclude that the hetZ-patU5-patU3 cluster plays an important role in co-ordination of heterocyst differentiation and pattern formation. The presence of homologous clusters in filamentous genera without heterocysts is suggestive of a more general role.
    Molecular Microbiology 01/2008; 66(6):1429-43. · 4.96 Impact Factor
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    ABSTRACT: The clones generated in a sequencing project represent a resource for subsequent analysis of the organism whose genome has been sequenced. We describe an interrelated group of cloning vectors that either integrate into the genome or replicate, and that enhance the utility, for developmental and other studies, of the clones used to determine the genomic sequence of the cyanobacterium, Anabaena sp. strain PCC 7120. One integrating vector is a mobilizable BAC vector that was used both to generate bridging clones and to complement transposon mutations. Upon addition of a cassette that permits mobilization and selection, pUC-based sequencing clones can also integrate into the genome and thereupon complement transposon mutations. The replicating vectors are based on cyanobacterial plasmid pDU1, whose sequence we report, and on broad-host-range plasmid RSF1010. The RSF1010- and pDU1-based vectors provide the opportunity to express different genes from either cell-type-specific or -generalist promoters, simultaneously from different plasmids in the same cyanobacterial cells. We show that pDU1 ORF4 and its upstream region play an essential role in the replication and copy number of pDU1, and that ORFs alr2887 and alr3546 (hetF A) of Anabaena sp. are required specifically for fixation of dinitrogen under oxic conditions.
    Archives of Microbiology 01/2008; 188(6):551-63. · 1.91 Impact Factor
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    ABSTRACT: During maturation, heterocysts form an envelope layer of polysaccharide, called heterocyst envelope polysaccharide (HEP), whose synthesis depends on a cluster of genes, the HEP island, and on an additional, distant gene, hepB, or a gene immediately downstream from hepB. We show that HEP formation depends upon the predicted glycosyl transferase genes all4160 at a third locus and alr3699, which is adjacent to hepB and is cotranscribed with it. Mutations in the histidine kinase genes hepN and hepK appear to silence the promoter of hepB and incompletely down-regulate all4160.
    Journal of Bacteriology 08/2007; 189(14):5372-8. · 3.19 Impact Factor
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    ABSTRACT: Heterocysts, formed when filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, are grown in the absence of combined nitrogen, are cells that are specialized in fixing atmospheric nitrogen (N(2)) under oxic conditions and that transfer fixed nitrogen to the vegetative cells of the filament. Anabaena sp. mutants whose sepJ gene (open reading frame alr2338 of the Anabaena sp. genome) was affected showed filament fragmentation and arrested heterocyst differentiation at an early stage. In a sepJ insertional mutant, a layer similar to a heterocyst polysaccharide layer was formed, but the heterocyst-specific glycolipids were not synthesized. The sepJ mutant did not exhibit nitrogenase activity even when assayed under anoxic conditions. In contrast to proheterocysts produced in the wild type, those produced in the sepJ mutant still divided. SepJ is a multidomain protein whose N-terminal region is predicted to be periplasmic and whose C-terminal domain resembles an export permease. Using a green fluorescent protein translationally fused to the carboxyl terminus of SepJ, we observed that in mature heterocysts and vegetative cells, the protein is localized at the intercellular septa, and when cell division starts, it is localized in a ring whose position is similar to that of a Z ring. SepJ is a novel composite protein needed for filament integrity, proper heterocyst development, and diazotrophic growth.
    Journal of Bacteriology 06/2007; 189(10):3884-90. · 3.19 Impact Factor
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    ABSTRACT: Fur (ferric uptake regulator) proteins are principally responsible for maintaining iron homeostasis in prokaryotes. Iron is usually a scarce resource. Its limitation reduces photosynthetic rates and cell growth in cyanobacteria in general and especially in cyanobacteria that are fixing dinitrogen, a process that requires the synthesis of numerous proteins with a high content of iron. This paper shows that in the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120, levels of furA mRNA and FurA protein increase significantly in response to nitrogen deprivation, and that furA up-regulation takes place specifically in proheterocysts and mature heterocysts. Great differences in a Northern blot, probed with furA, of RNA from an ntcA mutant relative to wild-type Anabaena sp. were attributable to binding of NtcA, a global regulator of nitrogen metabolism, to the promoter of furA and to the promoter of the furA antisense transcript alr1690-alpha-furA.
    Microbiology 02/2007; 153(Pt 1):42-50. · 2.85 Impact Factor
  • Koichiro Awai, C Peter Wolk
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    ABSTRACT: Nitrogenase is oxygen-labile. Cyanobacterial heterocysts can fix N(2) in an oxic milieu because their interior is micro-oxic, for which the glycolipid layer of the heterocyst envelope is required. ORF all5341 of the Anabaena sp. genome predicts a glycosyl transferase. An insertional mutant of all5341 synthesized only a nonglycosylated form of heterocyst envelope glycolipid, and lacked a glycolipid layer. All5341 appears to be the transferase required to glycosylate the glycolipid aglycone.
    FEMS Microbiology Letters 02/2007; 266(1):98-102. · 2.05 Impact Factor
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    ABSTRACT: Regulatory genes hepK, hepN, henR, and hepS are required for heterocyst maturation in Anabaena sp. strain PCC 7120. They presumptively encode two histidine kinases, a response regulator, and a serine/threonine kinase, respectively. To identify relationships between those genes, we compared global patterns of gene expression, at 14 h after nitrogen step-down, in corresponding mutants and in the wild-type strain. Heterocyst envelopes of mutants affected in any of those genes lack a homogeneous, polysaccharide layer. Those of a henR mutant also lack a glycolipid layer. patA, which encodes a positive effector of heterocyst differentiation, was up-regulated in all mutants except the hepK mutant, suggesting that patA expression may be inhibited by products related to heterocyst development. hepS and hepK were up-regulated if mutated and so appear to be negatively autoregulated. HepS and HenR regulated a common set of genes and so appear to belong to one regulatory system. Some nontranscriptional mechanism may account for the observation that henR mutants lack, and hepS mutants possess, a glycolipid layer, even though both mutations down-regulated genes involved in formation of the glycolipid layer. HepK and HepN also affected transcription of a common set of genes and therefore appear to share a regulatory pathway. However, the transcript abundance of other genes differed very significantly from expression in the wild-type strain in either the hepK or hepN mutant while differing very little from wild-type expression in the other of those two mutants. Therefore, hepK and hepN appear to participate also in separate pathways.
    Journal of Bacteriology 12/2006; 188(21):7387-95. · 3.19 Impact Factor
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    ABSTRACT: Filamentous, heterocyst-forming cyanobacteria are multicellular organisms in which individual cells exchange nutrients and, presumably, regulatory molecules. Unknown mechanisms underlie this exchange. Classical electron microscopy shows that filamentous cyanobacteria bear a Gram-negative cell wall comprising a peptidoglycan layer and an outer membrane that are external to the cytoplasmic membrane, and that the outer membrane appears to be continuous along the filament of cells. This implies that the periplasmic space between the cytoplasmic and outer membranes might also be continuous. We propose that a continuous periplasm could constitute a communication conduit for the transfer of compounds, which is essential for the performance of these bacteria as multicellular organisms.
    Trends in Microbiology 11/2006; 14(10):439-43. · 8.43 Impact Factor
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    ABSTRACT: How heterocyst differentiation is regulated, once particular cells start to differentiate, remains largely unknown. Using near-saturation transposon mutagenesis and testing of transposon-tagged loci, we identified three presumptive regulatory genes not previously recognized as being required specifically for normal heterocyst maturation. One of these genes has a hitherto unreported mutant phenotype. Two previously identified regulatory genes were further characterized.
    Journal of Bacteriology 10/2006; 188(18):6688-93. · 3.19 Impact Factor

Publication Stats

5k Citations
513.50 Total Impact Points

Institutions

  • 2012
    • Spanish National Research Council
      • Institute of Plant Biochemistry and Photosynthesis
      Madrid, Madrid, Spain
  • 1968–2012
    • Michigan State University
      • • MSU-DOE Plant Research Laboratory
      • • Department of Plant Biology
      • • Department of Biochemistry and Molecular Biology
      • • National Superconducting Cyclotron Laboratory (NSCL)
      East Lansing, Michigan, United States
  • 1998–2006
    • Universidad Autónoma de Madrid
      • Departamento de Biología
      Madrid, Madrid, Spain
  • 1992–1994
    • Universidad de Sevilla
      • Facultad De Biología
      Sevilla, Andalusia, Spain
  • 1978–1991
    • Universität Regensburg
      Ratisbon, Bavaria, Germany
  • 1983
    • University of Missouri - St. Louis
      • Department of Biology
      Saint Louis, Michigan, United States
    • Ben-Gurion University of the Negev
      Be'er Sheva`, Southern District, Israel
  • 1982
    • Washington State University
      Pullman, Washington, United States
  • 1972
    • University of Rochester
      Rochester, New York, United States