Dismukes, CG, McNeely K, Eng JF.  2011.  An LC-MS-based chemical and analytical method for targeted metabolite quantification in the model cyanobacterium Synechococcus sp. PCC 7002.. Analytical chemistry. 83(10):3808-16. Abstract
Herein, we detail the development of a method for the chemical isolation and tandem LC-MS/MS quantification of a targeted subset of internal metabolites from cyanobacteria. We illustrate the selection of target compounds; requirements for and optimization of mass spectral detection channels, screening, and optimization of chromatography; and development of a sampling protocol that seeks to achieve complete, representative, and stable metabolite extraction on the seconds time scale. Several key factors influencing the separation by reversed-phase ion pairing chromatography, specifically the hydrophobicity of the sample matrix and sensitivity to mobile phase acidity, are identified and resolved. We illustrate this methodology with an example from the autofermentative metabolism in the model cyanobacterium Synechococcus sp. PCC 7002, for which intracellular levels of 25 metabolites were monitored over 48 h, including intermediates in central carbon metabolism together with those involved in the cellular energy charge and redox poise. Upon removal of alternative reductant sinks (nitrate), anoxia induces autofermentation of carbohydrates with a parallel rise in the intracellular pyridine nucleotide redox poise that is specific to NAD(H) and alongside a gradual decline in the adenylate energy charge. This LC-MS/MS-based method provides for accurate time-resolved quantification of multiple metabolites in parallel, thus enabling experimental verification of the active metabolic pathways.
Grohmann, D, Nagy J, Chakraborty A, Klose D, Fielden D, Ebright RH, Michaelis J, Werner F.  2011.  The initiation factor tfe and the elongation factor Spt4/5 compete for the RNAP clamp during transcription initiation and elongation.. Molecular cell. 43(2):263-74. Abstract
TFIIE and the archaeal homolog TFE enhance DNA strand separation of eukaryotic RNAPII and the archaeal RNAP during transcription initiation by an unknown mechanism. We have developed a fluorescently labeled recombinant M. jannaschii RNAP system to probe the archaeal transcription initiation complex, consisting of promoter DNA, TBP, TFB, TFE, and RNAP. We have localized the position of the TFE winged helix (WH) and Zinc ribbon (ZR) domains on the RNAP using single-molecule FRET. The interaction sites of the TFE WH domain and the transcription elongation factor Spt4/5 overlap, and both factors compete for RNAP binding. Binding of Spt4/5 to RNAP represses promoter-directed transcription in the absence of TFE, which alleviates this effect by displacing Spt4/5 from RNAP. During elongation, Spt4/5 can displace TFE from the RNAP elongation complex and stimulate processivity. Our results identify the RNAP "clamp" region as a regulatory hot spot for both transcription initiation and transcription elongation.
Dismukes, CG, McNeely K, Robinson DM, Sheats JE.  2011.  A Co4O4 "cubane" water oxidation catalyst inspired by photosynthesis.. Journal of the American Chemical Society. 133(30):11446-9. Abstract
Herein we describe the molecular Co(4)O(4) cubane complex Co(4)O(4)(OAc)(4)(py)(4) (1), which catalyzes efficient water oxidizing activity when powered by a standard photochemical oxidation source or electrochemical oxidation. The pH dependence of catalysis, the turnover frequency, and in situ monitoring of catalytic species have revealed the intrinsic capabilities of this core type. The catalytic activity of complex 1 and analogous Mn(4)O(4) cubane complexes is attributed to the cubical core topology, which is analogous to that of nature's water oxidation catalyst, a cubical CaMn(4)O(5) cluster.
Srivastava, A, Talaue M, Liu S, Degen D, Ebright RY, Sineva E, Chakraborty A, Druzhinin SY, Chatterjee S, Mukhopadhyay J et al..  2011.  New target for inhibition of bacterial RNA polymerase: 'switch region'. Current opinion in microbiology. 14:532-43. Abstract
A new drug target - the 'switch region' - has been identified within bacterial RNA polymerase (RNAP), the enzyme that mediates bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit bacterial RNA synthesis and kill bacteria. Since the new target is present in most bacterial species, compounds that bind to the new target are active against a broad spectrum of bacterial species. Since the new target is different from targets of other antibacterial agents, compounds that bind to the new target are not cross-resistant with other antibacterial agents. Four antibiotics that function through the new target have been identified: myxopyronin, corallopyronin, ripostatin, and lipiarmycin. This review summarizes the switch region, switch-region inhibitors, and implications for antibacterial drug discovery.
Dismukes, CG, McNeely K, Xu Y, Ananyev GM, Bennette N, Bryant DA.  2011.  Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate:ferredoxin oxidoreductase.. Applied and environmental microbiology. 77(7):2435-44. Abstract
The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout 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 the wild-type (WT) rate under conditions of light-dark cycling. This result is attributed to an increase in the quantum yield of photosystem II (PSII) charge separation as measured by photosynthetic electron turnover efficiency determined using fast-repetition-rate fluorometry (F(v)/F(m)). 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 cells, H(2) production in vivo was 1.3-fold lower than the WT level. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with a resulting loss of reductant and a 3-fold decrease in H(2) production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H(2) revealed two temporally resolved phases of H(2) production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin, because its level 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 H(2) production was not in equilibrium with bulk NADH/NAD(+) and that the second phase corresponded to the equilibrium NADH-mediated process.
Kuznedelov, K, Semenova E, Knappe T, Marahiel M, Ebright RHE, Severinov K.  2011.  Antibacterial threaded lasso-peptide capistruin is a DNA-dependent RNA polymerase inhibitor. J. Mol. Biol. 412:842-848.
Gallavotti, A, Malcomber S, Gaines C, Stanfield S, Whipple C, Kellogg E, Schmidt RJ.  2011.  BARREN STALK FASTIGIATE1 is an AT-hook Protein Required for the Formation of Maize ears. Plant Cell. 23:1756-1771. AbstractWebsite
Ears are the seed-bearing inflorescences of maize (Zea mays) plants and represent a crucial component of maize yield. The first step in the formation of ears is the initiation of axillary meristems in the axils of developing leaves. In the classic maize mutant barren stalk fastigiate1 (baf1), first discovered in the 1950s, ears either do not form or, if they do, are partially fused to the main stalk. We positionally cloned Baf1 and found that it encodes a transcriptional regulator containing an AT-hook DNA binding motif. Single coorthologs of Baf1 are found in syntenic regions of brachypodium (Brachypodium distachyon), rice (Oryza sativa), and sorghum (Sorghum bicolor), suggesting that the gene is likely present in all cereal species. Protein-protein interaction assays suggest that BAF1 is capable of forming homodimers and heterodimers with other members of the AT-hook family. Another transcriptional regulator required for ear initiation is the basic helix-loop-helix protein BARREN STALK1 (BA1). Genetic and expression analyses suggest that Baf1 is required to reach a threshold level of Ba1 expression for the initiation of maize ears. We propose that Baf1 functions in the demarcation of a boundary region essential for the specification of a stem cell niche.
Calvino, M., Bruggmann R, Messing J.  2011.  Characterization of the small RNA component of the transcriptome from grain and sweet sorghum stems. BMC Genomics. 12:356. AbstractWebsite
ABSTRACT: BACKGROUND: Sorghum belongs to the tribe of the Andropogoneae that includes potential biofuel crops like switchgrass, Miscanthus and successful biofuel crops like corn and sugarcane. However, from a genomics point of view sorghum has compared to these other species a simpler genome because it lacks the additional rounds of whole genome duplication events. Therefore, it has become possible to generate a high-quality genome sequence. Furthermore, cultivars exists that rival sugarcane in levels of stem sugar so that a genetic approach can be used to investigate which genes are differentially expressed to achieve high levels of stem sugar. RESULTS: Here, we characterized the small RNA component of the transcriptome from grain and sweet sorghum stems, and from F2 plants derived from their cross that segregated for sugar content and flowering time. We found that variation in miR172 and miR395 expression correlated with flowering time whereas variation in miR169 expression correlated with sugar content in stems. Interestingly, genotypic differences in the ratio of miR395 to miR395* were identified, with miR395* species expressed as abundantly as miR395 in sweet sorghum but not in grain sorghum. Finally, we provided experimental evidence for previously annotated miRNAs detecting the expression of 25 miRNA families from the 27 known and discovered 9 new miRNAs candidates in the sorghum genome. CONCLUSIONS: Sequencing the small RNA component of sorghum stem tissue provides us with experimental evidence for previously predicted microRNAs in the sorghum genome and microRNAs with a potential role in stem sugar accumulation and flowering time.
Severinov, K, Semenova E, Kazakov T.  2011.  Class I microcins: Their structures activities, and mechanisms of resistance. Prokaryotic Antimicrobial Peptides: from Genes to Applications. :289-308.
Liu, G, Rogers J, Murphy CT, Rongo C.  2011.  EGF signalling activates the ubiquitin proteasome system to modulate C. elegans lifespan. EMBO J. 30:2990-3003. AbstractWebsite
Epidermal growth factor (EGF) signalling regulates growth and differentiation. Here, we examine the function of EGF signalling in Caenorhabditis elegans lifespan. We find that EGF signalling regulates lifespan via the Ras-MAPK pathway and the PLZF transcription factors EOR-1 and EOR-2. As animals enter adulthood, EGF signalling upregulates the expression of genes involved in the ubiquitin proteasome system (UPS), including the Skp1-like protein SKR-5, while downregulating the expression of HSP16-type chaperones. Using reporters for global UPS activity, protein aggregation, and oxidative stress, we find that EGF signalling alters protein homoeostasis in adults by increasing UPS activity and polyubiquitination, while decreasing protein aggregation. We show that SKR-5 and the E3/E4 ligases that comprise the ubiquitin fusion degradation (UFD) complex are required for the increase in UPS activity observed in adults, and that animals that lack SKR-5 or the UFD have reduced lifespans and indications of oxidative stress. We propose that as animals enter fertile adulthood, EGF signalling switches the mechanism for maintaining protein homoeostasis from a chaperone-based approach to an approach involving protein elimination via augmented UPS activity.
Rongo, C.  2011.  Epidermal growth factor and aging: A signaling molecule reveals a new eye opening function. Aging. 3(9):1-10. AbstractWebsite
Epidermal Growth Factor (EGF) is known for its role in promoting cell division and cellular differentiation in developing animals, but we know surprising little about what EGF does in vivo in mature adult animals. Here I review EGF signaling, emphasizing several recent studies that uncovered an unexpected role for EGF in promoting longevity and healthspan in mature adult C. elegans. EGF, acting through phospholipase Cγ and the IP3 receptor signaling, maintains pharyngeal and body wall muscle function in aging adults, and delays the accumulation of lipofuscin-enriched aging pigments within intestinal cells. EGF also acts through the Ras/ERK pathway to regulate protein homeostasis by promoting the expression of antioxidant genes, stimulating the activity of the Ubiquitin Proteasome System (UPS), and repressing the expression of small heat shock protein chaperones. The effects of EGF signaling on lifespan are largely independent of Insulin/IGF-like Signaling (IIS), as the effects of EGF signaling mutants on lifespan and heathspan are not affected by mutations in the DAF-2 insulin receptor or the DAF-16 FOXO transcription factor. Nevertheless, these two signal pathways have multiple points of overlap, coordination, and cross regulation. I propose that the IIS and EGF signaling pathways respond to environment and to developmental timing, respectively, so as to coordinate the appropriate physiological strategy that cells use to maintain protein homeostasis.
Wang, W, Kerstetter R, Michael TP..  2011.  Evolution of Genome Size in Duckweeds (Lemnaceae).. Journal of Botany.
Vondenhoff, GHM, Dubiley S, Severinov K, Lescrinier E, Rozenski J, Van Aerschot A.  2011.  Extended targeting potential and improved synthesis of Microcin C analogues as antibacterials. Bioorg. & Med. Chem.. 19:5462-5467.
Marcello, MR, Singson A.  2011.  Germline determination: don't mind the P granules.. Curr Biol.. 21(4):R155-7.
Wang, W, Messing J.  2011.  High-throughput sequencing of three Lemnoideae (duckweeds) chloroplast genomes from total DNA. PLoS One. 6:e24670. AbstractWebsite
BACKGROUND: Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. METHODS: We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs) using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. CONCLUSIONS: This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.
Semenova, E, Jore MM, Datsenko KA, Semenova A, Westra ER, Wanner B, van der Oost J, Brouns SJ, K. S.  2011.  Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence.. Proc Natl Acad Sci U S A.. 108(25):10098-103.
Minakhina, S, Tan W, Steward R.  2011.  JAK/STAT and the GATA factor Pannier control hemocyte maturation and differentiation in Drosophila. Dev Biol. 352(2):308-316.
Miclaus, M, Wu Y, Xu J, Dooner HK, Messing J.  2011.  The maize high-lysine mutant opaque7 is defective in an acyl-CoA synthetase-like protein.. Genetics. 189:1271-1280.
Ghilarov, D, Serebryakova M, Shkundina I, Severinov K.  2011.  A major portion of microcin B17 undergoes an N,O-peptidyl shift during synthesis.. J. Biol. Chem.. 286:26308-26318.
Mekler, V, Minakhin L, Sheppard C, Wigneshweraraj S, Severinov K.  2011.  Molecular mechanism of transcription inhibition by phage T7 gp2 protein.. J. Mol. Biol.. 413:1016-1027.
Mekler, V, Minakhin L, Severinov K.  2011.  On the role of downstream RNA polymerase-promoter interactions in formation of transcription initiation complex.. J. Biol. Chem.. 286:22600-22608.
Reddy, BVVG, Irvine KD.  2011.  Regulation of Drosophila glial cell proliferation by Merlin-Hippo signaling.. Development. 138:5201-5212.