Publications

2011
Nagornykh, M, Zakharova M, Protsenko A, Bogdanova E, Solonin A, Severinov K.  2011.  The regulation of gene expression in the Eco29kI restriction-modification system. . Nucleic Acids Res.. 39:4653-4663.
Yuzenkova, Y, Severinov K, Zenkin N.  2011.  The role of the β’ zipper element in promoter recognition. . EMBO J.. 30:3766-3775.
Agarwal, V, Metlytskaya A, Severinov K, Nair SK.  2011.  Structural basis for Microcin C7 inactivation by the acetyltransferase domain of MccE.. J. Biol. Chem.. 286:21295-21303.
Vondenhoff, GHM, Blanchaert B, Geboers S, Kazakov TS, Severinov K, Van Aerschot A.  2011.  Synthesis and evaluation of Microcin C analogues containing various peptide chains. J. Bacteriol.. 193:3618-3623.
Pronin, SV, Martinez M, Kuznedelov K, Severinov K, Shuman HA, Kozmin SA.  2011.  Synthesis enables comprehensive biochemical and antibacterial evaluation of streptolydigin sntibiotics. J. Am. Chem. Soc.. 133:12172-12184.
Berdygulova, Z, Westblade LF, Florens L, Chait BT, Ramanculov E, Washburn MP, Darst SA, Severinov K, Minakhin L.  2011.  Temporal regulation of gene expression of the Thermus thermophilus bacteriophage P23-45. J. Mol. Biol.. 405:125-142.
Carrieri, D, Momot D, Brasg IA, Ananyev GM, Lenz O, Bryant DA, Dismukes CG.  2011.  Boosting autofermentation rates and product yields with sodium stress cycling: Application to renewable fuel production by cyanobacteria. Appl. Environ. Microbiol.. :AEM.00975-10%Uhttp://aem.asm.org/cgi/content/abstract/AEM.00975-10v1. Abstract
Sodium concentration cycling was examined as a new strategy for redistributing carbon storage products and increasing autofermentative product yields following photosynthetic carbon fixation in the cyanobacterium Arthrospira (Spirulina) maxima. The salt-tolerant hyper-carbonate strain CS-328 was grown in a medium containing 0.24 to 1.24 M sodium, resulting in increased biosynthesis of soluble carbohydrates up to 50% of the dry weight at 1.24 M sodium. Hypoionic stress during dark anaerobic metabolism (autofermentation) was induced by resuspending filaments in low sodium (bi)carbonate buffer (0.21 M), which resulted in accelerated autofermentation rates. For cells grown in 1.24 M NaCl, the fermentative yields of acetate, ethanol and formate increase substantially to 0.75, 1.56 and 1.54 mmol/(gDW*day), respectively (36, 121, and 6-fold increases in rate relative to cells grown in 0.24 M NaCl). Catabolism of endogenous carbohydrate increased by approximately 2-fold upon hypoionic stress. For cultures grown at all salt concentrations, hydrogen was produced but its yield did not correlate with increased catabolism of soluble carbohydrates. Instead, ethanol excretion becomes a preferred route for fermentative NADH reoxidation together with intraceullar accumulation of reduced products of acetyl-CoA formation when cells are hypoionically stressed. In the absence of hypoionic stress, hydrogen production is a major beneficial pathway for NAD+ regeneration without wasting carbon intermediates such as ethanol derived from acetyl-CoA. This switch presumably improves the overall cellular economy by retaining carbon within the cell until aerobic conditions return and the acetyl unit can be used for biosynthesis or oxidized via respiration for much greater energy return.
McNeely, K, Xu Y, Ananyev GM, Bennette N, Bryant DA, Dismukes CG.  2011.  Characterization of a nifJ Mutant of Synechococcus sp. strain PCC 7002 Lacking Pyruvate:Ferredoxin Oxidoreductase. Appl. Environ. Microbiol.. :AEM.02792-10. AbstractWebsite
The nifJ gene codes for pyruvate:ferredoxin oxidoreductase, which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-CoA. A nifJ knock-out mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to wild type (WT) under light-dark cycling. This is attributed to an increase in the quantum yield of PSII charge separation as measured by photosynthetic electron turnover efficiency using fast repetition rate fluorometry (Fv/Fm). During autofermentation the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT, H2 production in vivo was 1.3-fold lower than WT. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with resulting loss of reductant and a 3-fold decrease in H2 production by nifJ cells compared to WT. Continuous electrochemical detection of dissolved H2 revealed two temporally resolved phases of H2 production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin because it decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD+ ratio revealed that the reductant generated by PFOR contributing to the first phase of H2 production was not in equilibrium with bulk NADH/NAD+, while the second phase corresponded to the equilibrium NADH-mediated process.
McCool, NS, Robinson DM, Sheats JE, Dismukes CG.  2011.  A Co4O4 “Cubane” Water Oxidation Catalyst Inspired by Photosynthesis. Journal of the American Chemical Society. 133:11446-11449. AbstractWebsite
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Parry, JM, Singson A.  2011.  EGG molecules couple the oocyte-to-embryo transition with cell cycle progression. Results Probl Cell Differ. 53:135–151. Abstract
The oocyte-to-embryo transition is a precisely coordinated process in which an oocyte becomes fertilized and transitions to an embryonic program of events. The molecules involved in this process have not been well studied. Recently, a group of interacting molecules in C. elegans have been described as coordinating the oocyte-to-embryo transition with the advancement of the cell cycle. Genes egg-3, egg-4, and egg-5 represent a small class of regulatory molecules known as protein-tyrosine phosphase-like proteins, which can bind phosphorylated substrates and act as scaffolding molecules or inhibitors. These genes are responsible for coupling the movements and activities of regulatory kinase mbk-2 with advancement of the cell cycle during the oocyte-to-embryo transition.
Maliga, P, Svab Z.  2011.  Engineering the plastid genome of Nicotiana sylvestris, a diploid model species for plastid genetics. Methods in Molecular Biology. 701:37-50. AbstractWebsite
The plastids of higher plants have their own approximately 120-160-kb genome that is present in 1,000-10,000 copies per cell. Engineering of the plastid genome (ptDNA) is based on homologous recombination between the plastid genome and cloned ptDNA sequences in the vector. A uniform population of engineered ptDNA is obtained by selection for marker genes encoded in the vectors. Manipulations of ptDNA include (1) insertion of transgenes in intergenic regions; (2) posttransformation excision of marker genes to obtain marker-free plants; (3) gene knockouts and gene knockdowns, and (4) cotransformation with multiple plasmids to introduce nonselected genes without physical linkage to marker genes. Most experiments on plastome engineering have been carried out in the allotetraploid Nicotiana tabacum. We report here for the first time plastid transformation in Nicotiana sylvestris, a diploid ornamental species. We demonstrate that the protocols and vectors developed for plastid transformation in N. tabacum are directly applicable to N. sylvestris with the advantage that the N. sylvestris transplastomic lines are suitable for mutant screens.
Meuser, J, Boyd E, Ananyev GM, Karns D, Radakovits R, Narayana Murthy U, Ghirardi M, Dismukes G, Peters J, Posewitz M.  2011.  Evolutionary significance of an algal gene encoding an [FeFe]-hydrogenase with F-domain homology and hydrogenase activity in <i>Chlorella variabilis</i> NC64A. Planta. :1-15.Website
Geldziler, BD, Marcello MR, Shakes DC, Singson A.  2011.  The genetics and cell biology of fertilization. Methods Cell Biol.. 106:343–375. Abstract
Although the general events surrounding fertilization in many species are well described, the molecular underpinnings of fertilization are still poorly understood. Caenorhabditis elegans has emerged as a powerful model system for addressing the molecular and cell biological mechanism of fertilization. A primary advantage is the ability to isolate and propagate mutants that effect gametes and no other cells. This chapter provides conceptual guidelines for the identification, maintenance, and experimental approaches for the study fertility mutants.
Singaravelu, G, Chatterjee I, Marcello MR, Singson A.  2011.  Isolation and in vitro activation of Caenorhabditis elegans sperm. J Vis Exp. Abstract
Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm–around 300–and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for > 50% of the total cells in a typical adult worm. Therefore, isolating sperm from males is easier than from that of hermaphrodites. Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population. Males can be easily distinguished from hermaphrodites by observing the tail morphology. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures. Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids. Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm. Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes, and Nelson previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E. Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.
Morsci, NS, Barr MM.  2011.  Kinesin-3 KLP-6 Regulates Intraflagellar Transport in Male-Specific Cilia of Caenorhabditis Elegans. Curr Biol. 21:1239-1244. Abstract
Cilia are cellular sensory organelles whose integrity of structure and function are important to human health [1]. All cilia are assembled and maintained by kinesin-2 motors in a process termed intraflagellar transport (IFT), but they exhibit great variety of morphology and function. This diversity is proposed to be conferred by cell-specific modulation of the core IFT by additional factors, but examples of such IFT modulators are limited [2-4]. Here we demonstrate that the cell-specific kinesin-3 KLP-6 acts as a modulator of both IFT dynamics and length in the cephalic male (CEM) cilia of Caenorhabditis elegans. Live imaging of GFP-tagged kinesins in CEM cilia shows partial uncoupling of the IFT motors of the kinesin-2 family, kinesin-II and OSM-3/KIF17, with a portion of OSM-3 moving independently of the IFT complex. KLP-6 moves independently of the kinesin-2 motors and acts to reduce the velocity of OSM-3 and IFT. Additionally, kinesin-II mutants display a novel CEM cilia elongation phenotype that is partially dependent on OSM-3 and KLP-6. Our observations illustrate modulation of the general kinesin-2-driven IFT process by a cell-specific kinesin-3 in cilia of C. elegans male neurons.
Singaravelu, G, Singson A.  2011.  New insights into the mechanism of fertilization in nematodes. Int Rev Cell Mol Biol. 289:211–238. Abstract
Fertilization results from the fusion of male and female gametes in all sexually reproducing organisms. Much of nematode fertility work was focused on Caenorhabditis elegans and Ascaris suum. The C. elegans hermaphrodite produces a limited number of sperm initially and then commits to the exclusive production of oocytes. The postmeiotic differentiation called spermiogenesis converts sessile spermatids into motile spermatozoa. The motility of spermatozoa depends on dynamic assembly and disassembly of a major sperm protein-based cytoskeleton uniquely found in nematodes. Both self-derived and male-derived spermatozoa are stored in spermatheca, the site of fertilization in hermaphrodites. The oocyte is arrested in meiotic prophase I until a sperm-derived signal relieves the inhibition allowing the meiotic maturation to occur. Oocyte undergoes meiotic maturation, enters into spermatheca, gets fertilized, completes meiosis, and exits into uterus as a zygote. This review focuses on our current understanding of the events around fertilization in nematodes.
Brimblecombe, R, Chen J, Wagner P, Buchhorn T, Dismukes CG, Spiccia L, Swiegers GF.  2011.  Photocatalytic oxygen evolution from non-potable water by a bioinspired molecular water oxidation catalyst. Journal of Molecular Catalysis A: Chemical. 338:1-6.Website
Gelfand, B, Mead J, Bruning A, Apostolopoulos N, Tadigotla V, Nagaraj V, Sengupta AM, Vershon AK.  2011.  Regulated Antisense Transcription Controls Expression of Cell-type-specific Genes in Yeast. Mol Cell Biol. 31:1701-1709. Abstract
Transcriptome profiling studies have recently uncovered a large number of noncoding RNA transcripts (ncRNAs) in eukaryotic organisms, and there is growing interest in their role in the cell. For example, in haploid Saccharomyces cerevisiae cells, the expression of an overlapping antisense ncRNA, referred to here as RME2 (Regulator of Meiosis 2), prevents IME4 expression. In diploid cells, the a1-α2 complex represses the transcription of RME2, allowing IME4 to be induced during meiosis. In this study we show that antisense transcription across the IME4 promoter region does not block transcription factors from binding and is not required for repression. Mutational analyses found that sequences within the IME4 open reading frame (ORF) are required for the repression mediated by RME2 transcription. These results support a model where transcription of RME2 blocks the elongation of the full-length IME4 transcript but not its initiation. We have found that another antisense transcript, called RME3, represses ZIP2 in a cell-type-specific manner. These results suggest that regulated antisense transcription may be a widespread mechanism for the control of gene expression and may account for the roles of some of the previously uncharacterized ncRNAs in yeast.
Lutz, KA, Azhagiri A, Maliga P.  2011.  Transplastomics in Arabidopsis: progress toward developing an efficient method. Methods in Molecular Biology. 774:133-47. AbstractWebsite
Protocols developed for plastome engineering in Nicotiana tabacum rely on biolistic delivery of the transforming DNA to chloroplasts in intact leaf tissue; integration of the foreign DNA into the plastid genome by homologous recombination via flanking plastid DNA (ptDNA) targeting regions; and gradual dilution of non-transformed ptDNA during cultivation in vitro. Plastid transformation in Arabidopsis was obtained by combining the tobacco leaf transformation protocol with Arabidopsis-specific tissue culture and plant regeneration protocols. Because the leaf cells in Arabidopsis are polyploid, this protocol yielded sterile plants. Meristematic cells in a shoot apex or cells of a developing embryo are diploid. Therefore, we developed a regulated embryogenic root culture system that will generate diploid tissue for plastid transformation. This embryogenic culture system is created by steroid-inducible expression of the BABY BOOM transcription factor. Plastid transformation in Arabidopsis will enable the probing of plastid gene function, and the characterization of posttranscriptional mechanisms of gene regulation and the regulatory interactions of plastid and nuclear genes.
2010
Staley, B K, Irvine KD.  2010.  Warts and yorkie mediate intestinal regeneration by influencing stem cell proliferation. Current biology : CB. 20:1580-7. AbstractWebsite
Homeostasis in the Drosophila midgut is maintained by stem cells [1, 2]. The intestinal epithelium contains two types of differentiated cells that are lost and replenished: enteroendocrine (EE) cells and enterocytes (ECs). Intestinal stem cells (ISCs) are the only cells in the adult midgut that proliferate [3, 4], and ISC divisions give rise to an ISC and an enteroblast (EB), which differentiates into an EC or an EE cell [3-5]. If the midgut epithelium is damaged, then ISC proliferation increases [6-12]. Damaged ECs express secreted ligands (Unpaired proteins) that activate Jak-Stat signaling in ISCs and EBs to promote their proliferation and differentiation [7, 9, 13, 14]. We show that the Hippo pathway components Warts and Yorkie mediate a transition from low- to high-level ISC proliferation to facilitate regeneration. The Hippo pathway regulates growth in diverse organisms and has been linked to cancer [15, 16]. Yorkie is activated in ECs in response to tissue damage or activation of the damage-sensing Jnk pathway. Activation of Yorkie promotes expression of unpaired genes and triggers a nonautonomous increase in ISC proliferation. Our observations uncover a role for Hippo pathway components in regulating stem cell proliferation and intestinal regeneration.
Abrouk, M, Murat F, Pont C, Messing J, Jackson S, Faraut T, Tannier E, Plomion C, Cooke R, Feuillet C et al..  2010.  Palaeogenomics of plants: synteny-based modelling of extinct ancestors. Trends Plant Sci. 15:479-87. AbstractWebsite
In the past ten years, international initiatives have led to the development of large sets of genomic resources that allow comparative genomic studies between plant genomes at a high level of resolution. Comparison of map-based genomic sequences revealed shared intra-genomic duplications, providing new insights into the evolution of flowering plant genomes from common ancestors. Plant genomes can be presented as concentric circles, providing a new reference for plant chromosome evolutionary relationships and an efficient tool for gene annotation and cross-genome markers development. Recent palaeogenomic data demonstrate that whole-genome duplications have provided a motor for the evolutionary success of flowering plants over the last 50-70 million years.
Murat, F, Xu JH, Tannier E, Abrouk M, Guilhot N, Pont C, Messing J, Salse J.  2010.  Ancestral grass karyotype reconstruction unravels new mechanisms of genome shuffling as a source of plant evolution. Genome Res. 20:1545-57. AbstractWebsite
The comparison of the chromosome numbers of today's species with common reconstructed paleo-ancestors has led to intense speculation of how chromosomes have been rearranged over time in mammals. However, similar studies in plants with respect to genome evolution as well as molecular mechanisms leading to mosaic synteny blocks have been lacking due to relevant examples of evolutionary zooms from genomic sequences. Such studies require genomes of species that belong to the same family but are diverged to fall into different subfamilies. Our most important crops belong to the family of the grasses, where a number of genomes have now been sequenced. Based on detailed paleogenomics, using inference from n = 5-12 grass ancestral karyotypes (AGKs) in terms of gene content and order, we delineated sequence intervals comprising a complete set of junction break points of orthologous regions from rice, maize, sorghum, and Brachypodium genomes, representing three different subfamilies and different polyploidization events. By focusing on these sequence intervals, we could show that the chromosome number variation/reduction from the n = 12 common paleo-ancestor was driven by nonrandom centric double-strand break repair events. It appeared that the centromeric/telomeric illegitimate recombination between nonhomologous chromosomes led to nested chromosome fusions (NCFs) and synteny break points (SBPs). When intervals comprising NCFs were compared in their structure, we concluded that SBPs (1) were meiotic recombination hotspots, (2) corresponded to high sequence turnover loci through repeat invasion, and (3) might be considered as hotspots of evolutionary novelty that could act as a reservoir for producing adaptive phenotypes.
Simon, MA, Xu A, Ishikawa HO, Irvine KD.  2010.  Modulation of Fat:Dachsous binding by the cadherin domain kinase four-jointed. Current biology : CB. 20:811-7. AbstractWebsite
In addition to quantitative differences in morphogen signaling specifying cell fates, the vector and slope of morphogen gradients influence planar cell polarity (PCP) and growth. The cadherin Fat plays a central role in this process. Fat regulates PCP and growth through distinct downstream pathways, each involving the establishment of molecular polarity within cells. Fat is regulated by the cadherin Dachsous (Ds) and the protein kinase Four-jointed (Fj), which are expressed in gradients in many tissues. Previous studies have implied that Fat is regulated by the vector and slope of these expression gradients. Here, we characterize how cells interpret the Fj gradient. We demonstrate that Fj both promotes the ability of Fat to bind to its ligand Ds and inhibits the ability of Ds to bind Fat. Consequently, the juxtaposition of cells with differing Fj expression results in asymmetric Fat:Ds binding. We also show that the influence of Fj on Fat is a direct consequence of Fat phosphorylation and identify a phosphorylation site important for the stimulation of Fat:Ds binding by Fj. Our results define a molecular mechanism by which a morphogen gradient can drive the polarization of Fat activity to influence PCP and growth.
Wu, Y, Messing J.  2010.  RNA interference-mediated change in protein body morphology and seed opacity through loss of different zein proteins. Plant Physiol. 153:337-47. AbstractWebsite
Opaque or nonvitreous phenotypes relate to the seed architecture of maize (Zea mays) and are linked to loci that control the accumulation and proper deposition of storage proteins, called zeins, into specialized organelles in the endosperm, called protein bodies. However, in the absence of null mutants of each type of zein (i.e. alpha, beta, gamma, and delta), the molecular contribution of these proteins to seed architecture remains unclear. Here, a double null mutant for the delta-zeins, the 22-kD alpha-zein, the beta-zein, and the gamma-zein RNA interference (RNAi; designated as z1CRNAi, betaRNAi, and gammaRNAi, respectively) and their combinations have been examined. While the delta-zein double null mutant had negligible effects on protein body formation, the betaRNAi and gammaRNAi alone only cause slight changes. Substantial loss of the 22-kD alpha-zeins by z1CRNAi resulted in protein body budding structures, indicating that a sufficient amount of the 22-kD zeins is necessary for maintenance of a normal protein body shape. Among different mutant combinations, only the combined betaRNAi and gammaRNAi resulted in drastic morphological changes, while other combinations did not. Overexpression of alpha-kafirins, the homologues of the maize 22-kD alpha-zeins in sorghum (Sorghum bicolor), in the beta/gammaRNAi mutant failed to offset the morphological alterations, indicating that beta- and gamma-zeins have redundant and unique functions in the stabilization of protein bodies. Indeed, opacity of the beta/gammaRNAi mutant was caused by incomplete embedding of the starch granules rather than by reducing the vitreous zone.