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Journal Article
Thyssen, G, Svab Z, Maliga P.  2012.  Cell-to-cell movement of plastids in plants. Proceedings of the National Academy of Sciences of the United States of America. 109:2439-43. AbstractWebsite
Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.
Krishnan, A, Zhang S, Liu Y, Tadmori KA, Bryant DA, Dismukes GC.  2016.  Consequences of ccmR deletion on respiration, fermentation and H2 metabolism in cyanobacterium Synechococcus sp. PCC 7002. Biotechnol Bioeng. Abstract
CcmR, a LysR-type transcriptional regulator, represses the genes encoding components of the high-affinity carbon concentration mechanism in cyanobacteria. Unexpectedly, deletion of the ccmR gene was found to alter the expression of the terminal oxidase and fermentative genes, especially the hydrogenase operon in the cyanobacterium Synechococcus sp. PCC 7002. Consistent with the transcriptomic data, the deletion strain exhibits flux increases (30-50%) in both aerobic O2 respiration and anaerobic H2 evolution. To understand how CcmR influences anaerobic metabolism, the kinetics of autofermentation were investigated following photoautotrophic growth. The autofermentative H2 yield increased by 50% in the CcmR deletion strain compared to the wild-type strain, and increased to 160% (within 20 h) upon continuous removal of H2 from the medium ("milking") to suppress uptake. Consistent with this greater reductant flux to H2 , the mutant excreted less lactate during autofermentation (NAD(P)H consuming pathway). To enhance the rate of NADH production during anaerobic metabolism, the ccmR mutant was engineered to introduce GAPDH overexpression (more NADH production) and LDH deletion (less NADH consumption). The triple mutant (ccmR deletion + GAPDH overexpression + LDH deletion) showed 6-8-fold greater H2 yield than the WT strain, achieving conversion rates of 17 nmol 108 cells-1 h-1 and yield of 0.87 H2 per glucose equivalent (8.9% theoretical maximum). Simultaneous monitoring of the intracellular NAD(P)H concentration and H2 production rate by these mutants reveals an inverse correspondence between these variables indicating hydrogenase-dependent H2 production as a major sink for consuming NAD(P)H in preference to excretion of reduced carbon as lactate during fermentation.
Wang, Q., Dooner HK.  2012.  Dynamic evolution of bz orthologous regions in the Andropogoneae and other grasses.. The Plant journal : for cell and molecular biology. 72(2):212-21. Abstract
Genome structure exhibits remarkable plasticity within Zea mays. To examine how haplotype structure has evolved within the Andropogoneae tribe, we have analyzed the bz gene-rich region of maize (Zea mays), the Zea teosintes mays ssp. mexicana, luxurians and diploperennis, Tripsacum dactyloides, Coix lacryma-jobi and Sorghum propinquum. We sequenced and annotated BAC clones from these species and re-annotated the orthologous Sorghum bicolor region. Gene colinearity in the region is well conserved within the genus Zea. However, the orthologous regions of Coix and Sorghum exhibited several micro-rearrangements relative to Zea, including addition, truncation and deletion of genes. The stc1 gene, involved in the production of a terpenoid insect defense signal, is evolving particularly fast, and its progressive disappearance from some species is occurring by microhomology-mediated recombination. LTR retrotransposons are the main contributors to the dynamic evolution of the bz region. Common transposon insertion sites occur among haplotypes from different Zea mays sub-species, but not outside the species. As in Zea, different patterns of interspersion between genes and retrotransposons are observed in Sorghum. We estimate that the mean divergence times between maize and Tripsacum, Coix and Sorghum are 8.5, 12.1 and 12.4 million years ago, respectively, and that between Coix and Sorghum is 9.3 million years ago. A comparison of the bz orthologous regions of Zea, Sorghum and Coix with those of Brachypodium, Setaria and Oryza allows us to infer how the region has evolved by addition and deletion of genes in the approximately 50 million years since these genera diverged from a common progenitor.
Burrows, EH, Bennette NB, Carrieri D, Dixon JL, Brinker A, Frada M, Bakdassabim S N, Falkowski PG, Dismukes GC.  2012.  Dynamics of Lipid Biosynthesis and Redistribution in the Marine Diatom Phaeodactylum tricornutum under Nitrate Deprivation. Bioenerg. Res. 5:876-885. Abstract
One approach to achieve continuous overproduction of lipids in microalgal “cell factories” relies upon depletion or removal of nutrients that act as competing electron sinks (e.g., nitrate and sulfate). However, this strategy can only be effective for bioenergy applications if lipid is synthesized primarily de novo (from CO2 fixation) rather than from the breakdown and interconversion of essential cellular components. In the marine diatom, Phaeodactylum tricornutum, it was determined, using 13C-bicarbonate, that cell growth in nitrate (NO 3 − )-deprived cultures resulted predominantly in de novo lipid synthesis (60 % over 3 days), and this new lipid consisted primarily of triacylglycerides (TAGs). Nearly complete preservation of 12C occurred in all previously existing TAGs in NO 3 − -deprived cultures and thus, further TAG accumulation would not be expected from inhibition of TAG lipolysis. In contrast, both high turnover and depletion of membrane lipids, phosphatidylcholines (PCs), were observed in NO 3 − -deprived cultures (both the headgroups and fatty acid chains), while less turnover was observed in NO 3 − replete cultures. Liquid chromatography-tandem mass spectrometry mass spectra and 13C labeling patterns of PC headgroups provided insight into lipid synthesis in marine diatoms, including suggestion of an internal pool of glycine betaine that feeds choline synthesis. It was also observed that 16C fatty acid chains incorporated into TAGs and PCs contained an average of 14 13C carbons, indicating substantial incorporation of 13C-bicarbonate into fatty acid chains under both nutrient states.
Oh, H, Slattery M, Ma L, Crofts A, White KP, Mann RS, Irvine KD.  2013.  Genome-wide association of Yorkie with chromatin and chromatin-remodeling complexes. Cell reports. 3:309-18. AbstractWebsite
The Hippo pathway regulates growth through the transcriptional coactivator Yorkie, but how Yorkie promotes transcription remains poorly understood. We address this by characterizing Yorkie's association with chromatin and by identifying nuclear partners that effect transcriptional activation. Coimmunoprecipitation and mass spectrometry identify GAGA factor (GAF), the Brahma complex, and the Mediator complex as Yorkie-associated nuclear protein complexes. All three are required for Yorkie's transcriptional activation of downstream genes, and GAF and the Brahma complex subunit Moira interact directly with Yorkie. Genome-wide chromatin-binding experiments identify thousands of Yorkie sites, most of which are associated with elevated transcription, based on genome-wide analysis of messenger RNA and histone H3K4Me3 modification. Chromatin binding also supports extensive functional overlap between Yorkie and GAF. Our studies suggest a widespread role for Yorkie as a regulator of transcription and identify recruitment of the chromatin-modifying GAF protein and BRM complex as a molecular mechanism for transcriptional activation by Yorkie.
Vvedenskaya, IO, Sharp JS, Goldman SR, Kanabar PN, Livny J, Dove SL, Nickels BE.  2012.  Growth phase-dependent control of transcription start site selection and gene expression by nanoRNAs. Genes Dev. 26(13):1498-1507. Abstract
Prokaryotic and eukaryotic RNA polymerases can use 2- to ~4-nt RNAs, ‘‘nanoRNAs,’’ to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.
Vvedenskaya, IO, Sharp JS, Goldman SR, Kanabar PN, Livny J, Dove SL, Nickels BE.  2012.  Growth phase-dependent control of transcription start site selection and gene expression by nanoRNAs. Genes & development. 26:1498-507. AbstractWebsite
Prokaryotic and eukaryotic RNA polymerases can use 2- to approximately 4-nt RNAs, "nanoRNAs," to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.
Qian, X, Kumaraswamy GK, Zhang S, Gates C, Ananyev GM, Bryant DA, Dismukes GC.  2015.  Inactivation of nitrate reductase alters metabolic branching of carbohydrate fermentation in the cyanobacterium Synechococcus sp. strain PCC 7002.. Biotechnol Bioeng. 113(5):979-988. Abstract
To produce cellular energy, cyanobacteria reduce nitrate as the preferred pathway over proton reduction (H2 evolution) by catabolizing glycogen under dark anaerobic conditions. This competition lowers H2 production by consuming a large fraction of the reducing equivalents (NADPH and NADH). To eliminate this competition, we constructed a knockout mutant of nitrate reductase, encoded by narB, in Synechococcus sp. PCC 7002. As expected, ΔnarB was able to take up intracellular nitrate but was unable to reduce it to nitrite or ammonia, and was unable to grow photoautotrophically on nitrate. During photoautotrophic growth on urea, ΔnarB significantly redirects biomass accumulation into glycogen at the expense of protein accumulation. During subsequent dark fermentation, metabolite concentrations-both the adenylate cellular energy charge (∼ATP) and the redox poise (NAD(P)H/NAD(P))-were independent of nitrate availability in ΔnarB, in contrast to the wild type (WT) control. The ΔnarB strain diverted more reducing equivalents from glycogen catabolism into reduced products, mainly H2 and d-lactate, by 6-fold (2.8% yield) and 2-fold (82.3% yield), respectively, than WT. Continuous removal of H2 from the fermentation medium (milking) further boosted net H2 production by 7-fold in ΔnarB, at the expense of less excreted lactate, resulting in a 49-fold combined increase in the net H2 evolution rate during 2 days of fermentation compared to the WT. The absence of nitrate reductase eliminated the inductive effect of nitrate addition on rerouting carbohydrate catabolism from glycolysis to the oxidative pentose phosphate (OPP) pathway, indicating that intracellular redox poise and not nitrate itself acts as the control switch for carbon flux branching between pathways.
Xiong, W, He L, Li Y, Dooner HK, Du C.  2013.  InsertionMapper: a pipeline tool for the identification of targeted sequences from multidimensional high throughput sequencing data.. BMC genomics. 14:679. Abstract
The advent of next-generation high-throughput technologies has revolutionized whole genome sequencing, yet some experiments require sequencing only of targeted regions of the genome from a very large number of samples. These regions can be amplified by PCR and sequenced by next-generation methods using a multidimensional pooling strategy. However, there is at present no available generalized tool for the computational analysis of target-enriched NGS data from multidimensional pools.
Vvedenskaya, IO, Vahedian-Movahed H, Bird JG, Knoblauch JG, Goldman SR, Zhang Y, Ebright RH, Nickels BE.  2014.  Interactions between RNA polymerase and the "core recognition element" counteract pausing. Science. 344(6189):1285-1289. Abstract
Transcription elongation is interrupted by sequences that inhibit nucleotide addition and cause RNA polymerase (RNAP) to pause. Here, by use of native elongating transcript sequencing (NET-seq) and a variant of NET-seq that enables analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), we analyze transcriptional pausing genome-wide in vivo in Escherichia coli. We identify a consensus pause-inducing sequence element, G₋₁₀Y₋₁G(+1) (where -1 corresponds to the position of the RNA 3' end). We demonstrate that sequence-specific interactions between RNAP core enzyme and a core recognition element (CRE) that stabilize transcription initiation complexes also occur in transcription elongation complexes and facilitate pause read-through by stabilizing RNAP in a posttranslocated register. Our findings identify key sequence determinants of transcriptional pausing and establish that RNAP-CRE interactions modulate pausing.
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.
Krishnan, A, Kumaraswamy GK, Vinyard DJ, Gu H, Ananyev GM, Posewitz MZ, Dismukes GC.  2015.  Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant.. Plant J. 81(6):947-960. Abstract
Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP-glucose pyrophosphorylase. This mutant is unable to convert glucose-1-phosphate to ADP-glucose, the precursor of starch biosynthesis. During nutrient-replete culturing, sta6 does not re-direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC-MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO2) decrease in sta6 due to attenuated rates of NADPH re-oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system-wide consequences of slower NADPH re-oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high-light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient-replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress.
Wang, W, Wu Y, Messing J.  2012.  The mitochondrial genome of an aquatic plant, Spirodela polyrhiza. PloS one. 7:e46747. AbstractWebsite
BACKGROUND: Spirodela polyrhiza is a species of the order Alismatales, which represent the basal lineage of monocots with more ancestral features than the Poales. Its complete sequence of the mitochondrial (mt) genome could provide clues for the understanding of the evolution of mt genomes in plant. METHODS: Spirodela polyrhiza mt genome was sequenced from total genomic DNA without physical separation of chloroplast and nuclear DNA using the SOLiD platform. Using a genome copy number sensitive assembly algorithm, the mt genome was successfully assembled. Gap closure and accuracy was determined with PCR products sequenced with the dideoxy method. CONCLUSIONS: This is the most compact monocot mitochondrial genome with 228,493 bp. A total of 57 genes encode 35 known proteins, 3 ribosomal RNAs, and 19 tRNAs that recognize 15 amino acids. There are about 600 RNA editing sites predicted and three lineage specific protein-coding-gene losses. The mitochondrial genes, pseudogenes, and other hypothetical genes (ORFs) cover 71,783 bp (31.0%) of the genome. Imported plastid DNA accounts for an additional 9,295 bp (4.1%) of the mitochondrial DNA. Absence of transposable element sequences suggests that very few nuclear sequences have migrated into Spirodela mtDNA. Phylogenetic analysis of conserved protein-coding genes suggests that Spirodela shares the common ancestor with other monocots, but there is no obvious synteny between Spirodela and rice mtDNAs. After eliminating genes, introns, ORFs, and plastid-derived DNA, nearly four-fifths of the Spirodela mitochondrial genome is of unknown origin and function. Although it contains a similar chloroplast DNA content and range of RNA editing as other monocots, it is void of nuclear insertions, active gene loss, and comprises large regions of sequences of unknown origin in non-coding regions. Moreover, the lack of synteny with known mitochondrial genomic sequences shed new light on the early evolution of monocot mitochondrial genomes.
Vinyard, DJ, Sun JS, Gimpel J, Ananyev GM, Mayfield SP, Dismukes GC.  2016.  Natural isoforms of the Photosystem II D1 subunit differ in photoassembly efficiency of the water-oxidizing complex.. Photosynth Res.. Abstract
Oxygenic photosynthesis efficiency at increasing solar flux is limited by light-induced damage (photoinhibition) of Photosystem II (PSII), primarily targeting the D1 reaction center subunit. Some cyanobacteria contain two natural isoforms of D1 that function better under low light (D1:1) or high light (D1:2). Herein, rates and yields of photoassembly of the Mn4CaO5 water-oxidizing complex (WOC) from the free inorganic cofactors (Mn2+, Ca2+, water, electron acceptor) and apo-WOC-PSII are shown to differ significantly: D1:1 apo-WOC-PSII exhibits a 2.3-fold faster rate-limiting step of photoassembly and up to seven-fold faster rate to the first light-stable Mn3+ intermediate, IM1*, but with a much higher rate of photoinhibition than D1:2. Conversely, D1:2 apo-WOC-PSII assembles slower but has up to seven-fold higher yield, achieved by a higher quantum yield of charge separation and slower photoinhibition rate. These results confirm and extend previous observations of the two holoenzymes: D1:2-PSII has a greater quantum yield of primary charge separation, faster [P680 + Q A - ] charge recombination and less photoinhibition that results in a slower rate and higher yield of photoassembly of its apo-WOC-PSII complex. In contrast, D1:1-PSII has a lower quantum yield of primary charge separation, a slower [P680 + Q A - ] charge recombination rate, and faster photoinhibition that together result in higher rate but lower yield of photoassembly at higher light intensities. Cyanobacterial PSII reaction centers that contain the high- and low-light D1 isoforms can tailor performance to optimize photosynthesis at varying light conditions, with similar consequences on their photoassembly kinetics and yield. These different efficiencies of photoassembly versus photoinhibition impose differential costs for biosynthesis as a function of light intensity.
Guerra, LT, Xu Y, Bennette N, McNeely K, Bryant DA, Dismukes GC.  2013.  Natural osmolytes are much less effective substrates than glycogen for catabolic energy production in the marine cyanobacterium Synechococcus sp. strain PCC 7002 .. J. Biotechnol.. 166:65-75. Abstract
ADP-glucose pyrophosphorylase, encoded by glgC, catalyzes the first step of glycogen and glucosylglycer(ol/ate) biosynthesis. Here we report the construction of the first glgC null mutant of a marine cyanobacterium (Synechococcus sp. PCC 7002) and investigate its impact on dark anoxic metabolism (autofermentation). The glgC mutant had 98% lower ADP-glucose, synthesized no glycogen and produced appreciably more soluble sugars (mainly sucrose) than wild type (WT). Some glucosylglycerol was still observed, which suggests that the mutant has another, inefficient ADP-glucose synthesis pathway. In contrast, hypersaline conditions (1M NaCl) were lethal to the mutant strain, indicating that, unlike other strains, the elevated sucrose does not compensate for the reduced GG as osmolyte. In contrast to WT, nitrate limitation did not cause bleaching of N-containing pigments or carbohydrate accumulation in the glgC mutant, indicating impaired recycling of nitrogen stores. Despite the 2-fold increase in osmolytes, both the respiration and autofermentation rates of the glgC mutant were appreciably slower (2-4-fold) and correlated quantitatively with the lower fraction of insoluble carbohydrates relative to WT (85% vs. 12%). However, the remaining insoluble carbohydrates still accounted for a high fraction of the carbohydrate catabolized (38%), indicating that insoluble carbohydrates rather than osmolytes were the preferred substrate for autofermentation.
Nickels, BE.  2012.  A new way to start: nanoRNA-mediated priming of transcription initiation.. Transcription. 3(6):300-304. Abstract
A recent study provides evidence that RNA polymerase uses 2- to ~4-nt RNAs, species termed "nanoRNAs," to prime transcription initiation in Escherichia coli. Priming of transcription initiation with nanoRNAs represents a previously undocumented component of transcription start site selection and gene expression.
Berdygulova, Z, Esyunina D, Miropolskaya N, Mukhamedyarov D, Kuznedelov K, Nickels BE, Severinov K, Kulbachinskiy A, Minakhin L.  2012.  A novel phage-encoded transcription antiterminator acts by suppressing bacterial RNA polymerase pausing.. Nucleic Acids Research. Abstract
Gp39, a small protein encoded by Thermus thermophilus phage P23-45, specifically binds the host RNA polymerase (RNAP) and inhibits transcription initiation. Here, we demonstrate that gp39 also acts as an antiterminator during transcription through intrinsic terminators. The antitermination activity of gp39 relies on its ability to suppress transcription pausing at poly(U) tracks. Gp39 also accelerates transcription elongation by decreasing RNAP pausing and backtracking but does not significantly affect the rates of catalysis of individual reactions in the RNAP active center. We mapped the RNAP-gp39 interaction site to the β flap, a domain that forms a part of the RNA exit channel and is also a likely target for λ phage antiterminator proteins Q and N, and for bacterial elongation factor NusA. However, in contrast to Q and N, gp39 does not depend on NusA or other auxiliary factors for its activity. To our knowledge, gp39 is the first characterized phage-encoded transcription factor that affects every step of the transcription cycle and suppresses transcription termination through its antipausing activity.
Ananyev, GM, Gates C, Dismukes GC.  2016.  The Oxygen quantum yield in diverse algae and cyanobacteria is controlled by partitioning of flux between linear and cyclic electron flow within photosystem II.. Biochim Biophys Acta.. 1857(9):1380-1391. Abstract
We have measured flash-induced oxygen quantum yields (O2-QYs) and primary charge separation (Chl variable fluorescence yield, Fv/Fm) in vivo among phylogenetically diverse microalgae and cyanobacteria. Higher O2-QYs can be attained in cells by releasing constraints on charge transfer at the Photosystem II (PSII) acceptor side by adding membrane-permeable benzoquinone (BQ) derivatives that oxidize plastosemiquinone QB- and QBH2. This method allows uncoupling PSII turnover from its natural regulation in living cells, without artifacts of isolating PSII complexes. This approach reveals different extents of regulation across species, controlled at the QB- acceptor site. Arthrospira maxima is confirmed as the most efficient PSII-WOC (water oxidizing complex) and exhibits the least regulation of flux. Thermosynechococcus elongatus exhibits an O2-QY of 30%, suggesting strong downregulation. WOC cycle simulations with the most accurate model (VZAD) show that a light-driven backward transition (net addition of an electron to the WOC, distinct from recombination) occurs in up to 25% of native PSIIs in the S2 and S3 states, while adding BQ prevents backward transitions and increases the lifetime of S2 and S3 by 10-fold. Backward transitions occur in PSIIs that have plastosemiquinone radicals in the QB site and are postulated to be physiologically regulated pathways for storing light energy as proton gradient through direct PSII-cyclic electron flow (PSII-CEF). PSII-CEF is independent of classical PSI/cyt-b6f-CEF and provides an alternative proton translocation pathway for energy conversion. PSII-CEF enables variable fluxes between linear and cyclic electron pathways, thus accommodating species-dependent needs for redox and ion-gradient energy sources powered by a single photosystem.
Dooner, HK, He L.  2014.  Polarized gene conversion at the bz locus of maize.. Proc Natl Acad Sci USA. 111(38):13918-23. Abstract
Nucleotide diversity is greater in maize than in most organisms studied to date, so allelic pairs in a hybrid tend to be highly polymorphic. Most recombination events between such pairs of maize polymorphic alleles are crossovers. However, intragenic recombination events not associated with flanking marker exchange, corresponding to noncrossover gene conversions, predominate between alleles derived from the same progenitor. In these dimorphic heterozygotes, the two alleles differ only at the two mutant sites between which recombination is being measured. To investigate whether gene conversion at the bz locus is polarized, two large diallel crossing matrices involving mutant sites spread across the bz gene were performed and more than 2,500 intragenic recombinants were scored. In both diallels, around 90% of recombinants could be accounted for by gene conversion. Furthermore, conversion exhibited a striking polarity, with sites located within 150 bp of the start and stop codons converting more frequently than sites located in the middle of the gene. The implications of these findings are discussed with reference to recent data from genome-wide studies in other plants.
Kumaraswamy, GK, Guerra T, Qian X, Zhang S, Bryant DA, Dismukes GC.  2013.  Reprogramming the glycolytic pathway for increased hydrogen production in cyanobacteria: metabolic engineering of NAD+-dependent GAPDH. Energy Environ. Sci.. 6:3722-3731. AbstractWebsite
Catabolism of glycogen stored by cyanobacteria occurs during anaerobic auto-fermentation and produces a range of C1–C3 fermentation products and hydrogen via hydrogenase. We investigated both augmenting and rerouting this carbon catabolism by engineering the glycolysis pathway at the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH-1), its major regulation site at the nexus of two pathways (Oxidative Pentose Phosphate pathway, OPP, and glycolysis/gluconeogenesis). Null (gap1::aphII) and overexpression (gap1+) strains of Synechococcus sp. strain PCC 7002 were constructed in order to produce more NADPH (via rerouting carbon through OPP) and more NADH (via opening the glycolytic bottleneck), respectively. For gap1::aphII quantitative analyses after four days of dark auto-fermentation showed undiminished glycogen catabolism rate, significant increases of intracellular metabolites in both OPP and upper-glycolysis, decrease in lower-glycolysis intermediates, 5.7-fold increase in NADPH, 2.3-fold increase in hydrogen and 1.25-fold increase in CO2vs. wild type (WT). These changes demonstrate the expected outcome of redirection of carbon catabolism through the OPP pathway with significant stimulation of OPP product yields. The gap1+ strain exhibits a large 17% increase in accumulation of glycogen during the prior photoautotrophic growth stage (gluconeogenesis), in parallel with a 2-fold increase in the total [NAD+ + NADH] pool, foreshadowing an increased catabolic capacity. Indeed, the rate of glycogen catabolism during subsequent dark auto-fermentation increased significantly (58%) vs. WT, resulting in increases in both NADH (4.0-fold) and NADPH (2.9-fold) pools, and terminal fermentation products, hydrogen (3.0-fold) D-lactate (2.3-fold) and acetate (1.4-fold). The overall energy conversion yield over four days from catabolized glycogen to hydrogen increased from 0.6 mole of hydrogen per mole of glucose (WT) to 1.4 (gap1::aphII) and 1.1 (gap1+) under headspace accumulation conditions (without hydrogen milking). These findings demonstrate the significant potential of metabolic engineering for redirecting carbon pathways for carbohydrate catabolism and hydrogen production in cyanobacteria.
Gates, C, Ananyev GM, Dismukes C.  2016.  The strontium inorganic mutant of the water oxidizing center (CaMn4O5) of PSII improves WOC efficiency but slows electron flux through the terminal acceptors.. Biochim Biophys Acta.. 1857(9):1550-1560. Abstract
Herein we extend prior studies of biosynthetic strontium replacement of calcium in PSII-WOC core particles to characterize whole cells. Previous studies of Thermosynechococcus elongatus found a lower rate of light-saturated O2 from isolated PSII-WOC(Sr) cores and 5–8 × slower rate of oxygen release. We find similar properties in whole cells, and show it is due to a 20% larger Arrhenius activation barrier for O2 evolution. Cellular adaptation to the sluggish PSII-WOC(Sr) cycle occurs in which flux through the QAQB acceptor gate becomes limiting for turnover rate in vivo. Benzoquinone derivatives that bind to QB site remove this kinetic chokepoint yielding 31% greater O2 quantum yield (QY) of PSII-WOC(Sr) vs. PSII-WOC(Ca). QY and efficiency of the WOC(Sr) catalytic cycle are greatly improved at low light flux, due to fewer misses and backward transitions and 3-fold longer lifetime of the unstable S3 state, attributed to greater thermodynamic stabilization of the WOC(Sr) relative to the photoactive tyrosine YZ. More linear and less cyclic electron flow through PSII occurs per PSII-WOC(Sr). The organismal response to the more active PSII centers in Sr-grown cells at 45 °C is to lower the number of active PSII-WOC per Chl, producing comparable oxygen and energy per cell. We conclude that redox and protonic energy fluxes created by PSII are primary determinants for optimal growth rate of T. elongatus. We further conclude that the (Sr-favored) intermediate-spin S = 5/2 form of the S2 state is the active form in the catalytic cycle relative to the low-spin S = 1/2 form.
Deibert, BJ, Zhang J, Smith PF, Chapman KW, Rangan S, Banerjee D, Tan K, Wang H, Pasguale N, Chen F et al..  2015.  Surface and Structural Investigation of a MnOx Birnessite-Type Water Oxidation Catalyst Formed under Photocatalytic Conditions. Chemistry. 21(40):14218-14228. Abstract
Catalytically active MnOx species have been reported to form in situ from various Mn-complexes during electrocatalytic and solution-based water oxidation when employing cerium(IV) ammonium ammonium nitrate (CAN) oxidant as a sacrificial reagent. The full structural characterization of these oxides may be complicated by the presence of support material and lack of a pure bulk phase. For the first time, we show that highly active MnOx catalysts form without supports in situ under photocatalytic conditions. Our most active (4)MnOx catalyst (∼0.84 mmol O2  mol Mn(-1) s(-1)) forms from a Mn4O4 bearing a metal-organic framework. (4)MnOx is characterized by pair distribution function analysis (PDF), Raman spectroscopy, and HR-TEM as a disordered, layered Mn-oxide with high surface area (216 m(2) g(-1)) and small regions of crystallinity and layer flexibility. In contrast, the (S)MnOx formed from Mn(2+) salt gives an amorphous species of lower surface area (80 m(2) g(-1)) and lower activity (∼0.15 mmol O2  mol Mn(-1) s(-1)). We compare these catalysts to crystalline hexagonal birnessite, which activates under the same conditions. Full deconvolution of the XPS Mn2p3/2 core levels detects enriched Mn(3+) and Mn(2+) content on the surfaces, which indicates possible disproportionation/comproportionation surface equilibria.
Li, Y, Harris L, Dooner HK.  2013.  TED, an autonomous and rare maize transposon of the mutator superfamily with a high gametophytic excision frequency.. The Plant cell. 25(9):3251-65. Abstract
Mutator (Mu) elements, one of the most diverse superfamilies of DNA transposons, are found in all eukaryotic kingdoms, but are particularly numerous in plants. Most of the present knowledge on the transposition behavior of this superfamily comes from studies of the maize (Zea mays) Mu elements, whose transposition is mediated by the autonomous Mutator-Don Robertson (MuDR) element. Here, we describe the maize element TED (for Transposon Ellen Dempsey), an autonomous cousin that differs significantly from MuDR. Element excision and reinsertion appear to require both proteins encoded by MuDR, but only the single protein encoded by TED. Germinal excisions, rare with MuDR, are common with TED, but arise in one of the mitotic divisions of the gametophyte, rather than at meiosis. Instead, transposition-deficient elements arise at meiosis, suggesting that the double-strand breaks produced by element excision are repaired differently in mitosis and meiosis. Unlike MuDR, TED is a very low-copy transposon whose number and activity do not undergo dramatic changes upon inbreeding or outcrossing. Like MuDR, TED transposes mostly to unlinked sites and can form circular transposition products. Sequences closer to TED than to MuDR were detected only in the grasses, suggesting a rather recent evolutionary split from a common ancestor.
Hawkins, JS, Delgado V, Feng L, Carlise M, Dooner HK, Bennetzen JL.  2014.  Variation in allelic expression associated with a recombination hotspot in Zea mays.. The Plant Journal, DOI: 10.1111/tpj.12537. Abstract
Gene expression is a complex process, requiring precise spatial and temporal regulation of transcription factor activity; however, modifications of individual cis- and trans-acting modules can be molded by natural selection to create a sizeable number of novel phenotypes. Results from decades of research indicate that developmental and phenotypic divergence among eukaryotic organisms is driven primarily by variation in levels of gene expression that are dictated by mutations either in structural or regulatory regions of genes. The relative contributions and interplay of cis- and trans-acting regulatory factors to this evolutionary process, however, remain poorly understood. Analysis of 8 genes in the Bz1-Sh1 interval of maize indicates significant allele-specific expression biases in at least one tissue for all genes, ranging from 1.3-fold to 36-fold. All detected effects were cis-regulatory in nature, although genetic background may also influence the level of expression bias and tissue specificity for some allelic combinations. Most allelic pairs exhibited the same direction and approximate intensity of bias across all four tissues; however, a subset of allelic pairs show alternating dominance across different tissue types or variation in the degree of bias in different tissues. In addition, the genes showing the most striking levels of allelic bias co-localize with a previously described recombination hotspot in this region, suggesting a naturally occurring genetic mechanism for creating regulatory variability for a subset of plant genes that may ultimately lead to evolutionary diversification.