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Publications

When it comes to interdisciplinary collaboration, the titles of GLBRC publications speak for themselves. Each new year of operation has seen more publications from multiple labs that span the four Research Areas, accelerating the Center's production of the basic research that generates technology to convert cellulosic biomass to advanced biofuels.

Publications

An integrated approach to reconstructing genome-scale transcriptional regulatory networks

Saheed Imam; Daniel R. Nogeura; Timothy J. Donohue

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2015

Transcriptional regulatory networks (TRNs) program cells to dynamically alter their gene expression in response to changing internal or environmental conditions. In this study, we develop a novel workflow for generating large-scale TRN models that integrates comparative genomics data, global gene expression analyses, and intrinsic properties of transcription factors (TFs). An assessment of this workflow using benchmark datasets for the well-studied γ-proteobacterium Escherichia coli showed that it outperforms expression-based inference approaches, having a significantly larger area under the precision-recall curve. Further analysis indicated that this integrated workflow captures different aspects of the E. coli TRN than expression-based approaches, potentially making them highly complementary. We leveraged this new workflow and observations to build a large-scale TRN model for the α-Proteobacterium Rhodobacter sphaeroides that comprises 120 gene clusters, 1211 genes (including 93 TFs), 1858 predicted protein-DNA interactions and 76 DNA binding motifs. We found that ~67% of the predicted gene clusters in this TRN are enriched for functions ranging from photosynthesis or central carbon metabolism to environmental stress responses. We also found that members of many of the predicted gene clusters were consistent with prior knowledge in R. sphaeroides and/or other bacteria. Experimental validation of predictions from this R. sphaeroides TRN model showed that high precision and recall was also obtained for TFs involved in photosynthesis (PpsR), carbon metabolism (RSP_0489) and iron homeostasis (RSP_3341). In addition, this integrative approach enabled generation of TRNs with increased information content relative to R. sphaeroides TRN models built via other approaches. We also show how this approach can be used to simultaneously produce TRN models for each related organism used in the comparative genomics analysis. Our results highlight the advantages of integrating comparative genomics of closely related organisms with gene expression data to assemble large-scale TRN models with high-quality predictions.

Biomass potential of switchgrass and miscanthus on the USA’s marginal lands

V. Bandaru; César Izaurralde; K. Zhao

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2015

not yet available

Cell wall composition and bioenergy potential of rice straw tissues are influenced by environment, tissue type, and genotype

Paul Tanger; Miguel E. Vega-Sánchez; Margaret Fleming; Kim Tran; Seema Singh; James B. Abrahamson; Courtney E. Jahn; Nicholas Santoro; Elizabeth B. Naredo; Marietta Baraoidan; John M.C. Danku; David E. Salt; Kenneth L. McNally; Hei Leung; Pamela C. Ronald; Daniel R. Bush; John K. McKay; Jan E. Leach

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2015

Breeding has transformed wild plant species to maximize the proportion of their photosynthetic assimilate into grain, fiber, and other products for human use. Despite progress in increasing the harvest index, much of the biomass of crop plants is not utilized.  Potential uses for these large amounts of agricultural residues that accumulate are animal fodder or bioenergy, though these may not be economically viable without additional efforts such as targeted breeding or improved processing.  We characterized leaf and stem tissue from a diverse set of rice germplasm grown in two environments (greenhouse and field) and report bioenergy-related traits across these variables.  We measured cellulose, hemicelluloses, mixed linkage glucan (MLG), lignin, ash, cell wall structural proteins (HRGPs), bulk density, as well as total glucose and xylose, and glucose and pentose yields after pretreatment of the biomass.  For cellulose, hemicelluloses, lignin, ash, total glucose, and glucose yield we find large variation between environments, irrespective of the germplasm.  We confirm previously observed positive relationships between total glucose and hemicelluloses and glucose yield, as well as negative correlations between lignin and ash with glucose yield.  Trends in our data suggest that greenhouse studies may overestimate the bioenergy potential of biomass.  Glucose yield from greenhouse grown plants predicts glucose yield in field samples and could serve in greenhouse studies as an indicator of potentially lower cost conversion of field biomass for bioenergy.  Efforts to improve bioenergy traits must examine both stem and leaf tissues as they may be under separate genetic control.

Chemical genomic profiling via barcode sequencing to predict compound mode of action

Jeff S. Piotrowski; S. Simpkins; S.C. Li; R. Deshpande; Sean McIlwain; Irene M. Ong; C.L. Myers; C.B. Boone; R. Andersen

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2015

Chemical genomics is an unbiased, whole-cell approach to characterizing novel compounds to determine mode of action and cellular target. Our version of this technique is built upon barcoded deletion mutants of Saccharomyces cerevisiae, and has been adapted to a high-throughput methodology using next-generation sequencing. Here we describe the steps to generate a chemical genomic profile from a compound of interest, and how to use this information to predict the cellular target.

Field production, purification and analysis of high-oleic acetyl-triacylglycerols from transgenic Camelina sativa

Jinjie Liu; Henrik Tjellstrom; Kathleen McGlew; Vincent Shaw; Adam Rice; Jeffrey Simpson; Dylan Kosma; Wei Ma; Weili Yang; Merissa Strawsine; Edgar Cahoon; Timothy P. Durrett; John Ohlrogge

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2015

A diacylglycerol acetyltransferase, EaDAcT, from Euonymus alatus, synthesizes sn-3 acetyl triacylglycerols (acetyl-TAG) when expressed in Arabidopsis, Camelina and soybean. Compared to most vegetable oils, acetyl-TAGs have reduced viscosity and improved cold temperature properties that confer advantages in applications as biodegradable lubricants, food emulsifiers, plasticizers, and ‘drop-in’ fuels for some diesel engines. A high-oleic Camelina line was engineered to express the EaDAcT gene in order to produce acetyl-TAG oils with fatty acid compositions and physiochemical properties complementary to wild-type acetyl-TAG. The accumulation of acetyl-TAGs at 70 mol% of seed TAG in field-grown high-oleic Camelina had minor or no effect on seed weight, oil content, harvest index and seed yield. The total moles of TAG increased up to 27% reflecting the ability to synthesize more acetyl-TAG from the same supply of long-chain fatty acid. Acetyl-TAG could be separated from long-chain TAG by silica column or by reverse phase chromatography. The predominant acetyl-TAG molecular species produced in high-oleic Camelina was acetyl-dioleoyl-glycerol. The crystallization temperature of high-oleic acetyl-TAG (by differential scanning calorimetry at 1.0 °C/min) was reduced by 30 °C compared to control TAG. The viscosity of high-oleic acetyl-TAG was 27% lower than TAG from the high-oleic control and the caloric content was reduced by 5%. Field production of T4 and T5 transgenic plants yielded over 250 kg seeds for oil extraction and analysis.

Integrating price feedbacks into a Great Lakes model of biomass production and environmental impact assessment

A. Egbendewe-Mondzozo; Scott M. Swinton; S Kung; W. M. Post; J.C. Binfield; W Thompson

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2015

Using subregional models of crop production choices in central Wisconsin and southwest Michigan, we predict biomass production, land use, and environmental impacts with details that are unavailable from national scale models. When biomass prices are raised exogenously, we find that the subregional models overestimate the supply, the land use, and the beneficial environmental aspects of perennial biomass crops. Multi-market price feedbacks tied to realistic policy parameters predict high threshold absolute prices for biomass to enter production and less environmental benefice from perennial biomass crops production. Also, regional specialization of biomass production in areas with lower food crop yields is observed.

Introduction of chemically labile substructures into Arabidopsis lignin through the use of the Cα dehydrogenase from Sphingobium sp. strain SYK-6

Y Tsuji; Ruben Vanholme; Yuki Tobimatsu; Y Ishikawa; Clifton E. Foster; N Kamimura; S Hishiyama; S Saki; A Shino; H Hara; K Sato-Izawa; P Oyarce; G Goeminne; K Morreel; i J. Kikuch; T Takano; M Fukuda; Wout Boerjan; John Ralph; E Masai; S Kajita

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2015

Bacteria-derived enzymes that can modify specific lignin substructures are potential targets to engineer plants for better biomass processability. The Gram-negative bacterium Sphingobium sp. SYK-6 possesses a Cα-dehydrogenase (LigD) enzyme that has been shown to oxidize the α-hydroxy functionalities in β–O–4-linked dimers into α-keto analogues that are more chemically labile. Here, we show that recombinant LigD can oxidize an even wider range of β–O–4-linked dimers and oligomers, including the genuine dilignols, guaiacylglycerol-β-coniferyl alcohol ether and syringylglycerol-β-sinapyl alcohol ether. We explored the possibility of using LigD for biosynthetically engineering lignin by expressing the codon-optimized ligD gene in Arabidopsis thaliana. The ligD cDNA, with or without a signal peptide for apoplast targeting, has been successfully expressed, and LigD activity could be detected in the extracts of the transgenic plants. UPLC-MS/MS-based metabolite profiling indicated that levels of oxidized guaiacyl (G) β–O–4-coupled dilignols and analogues were significantly elevated in the LigD transgenic plants regardless of the signal peptide attachment to LigD. In parallel, 2D NMR analysis revealed a 2.1- to 2.8-fold increased level of G-type α-keto-β–O–4 linkages in cellulolytic enzyme lignins isolated from the stem cell walls of the LigD transgenic plants, indicating that the transformation was capable of altering lignin structure in the desired manner.

Metabolic engineering of oilseed crops to produce high levels of novel acetyl glyceride oils with reduced viscosity, freezing point and calorific value

Jinjie Liu; Adam Rice; Kathleen McGlew; Vincent Shaw; H Park; Timothy Clemente; Michael Pollard; John Ohlrogge; Timothy P. Durrett

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2015

Seed oils have proven recalcitrant to modification for the production of industrially useful lipids. Here we demonstrate the successful metabolic engineering and subsequent field production of an oilseed crop with the highest accumulation of unusual oil achieved so far in transgenic plants. Previously, expression of the Euonymus alatus diacylglycerol acetyltransferase (Ea-DAcT) gene in wild-type Arabidopsis seeds resulted in the accumulation of 45 mol% of unusual 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) in the seed oil [Durrett et al., 2010 PNAS 107:9464]. Expression of Ea-DAcT in dgat1 mutants compromised in their ability to synthesize regular triacylglycerols increased acetyl-TAGs to 65 mol%. Camelina and soybean transformed with the EaDAcT gene accumulate acetyl-triacylglycerols (acetyl-TAGs) at up to 70 mol% of seed oil. A similar strategy of coexpression of Ea-DAcT together with RNAi suppression of DGAT1 increased acetyl-TAG levels to up to 85 mol% in field-grown transgenic Camelina. Additionally, total moles of triacylglycerol (TAG) per seed increased 20%. Analysis of the acetyl-TAG fraction revealed a 2-fold reduction in VLCFA, consistent with their displacement from the sn-3 position by acetate. Seed germination remained high and seedlings were able to metabolize the stored acetyl-TAGs as rapidly as regular triacylglycerols. Viscosity, freezing point and caloric content of the Camelina acetyl-TAG oils were reduced, enabling use of this oil in several non-food and food applications.

Mitigation of greenhouse gases in agricultural ecosystems

Ilya Gelfand; Philip Robertson

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2015

not yet available

Plant cell wall properties contributing to improved alkaline pretreatment and enzymatic hydrolysis in diverse maize lines

M Li; Marilies Heckwolf; J Crowe; D.L. Williams; Shawn M. Kaeppler; Natalia de Leon; David B. Hodge

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2015

not yet available

Regional scale cropland carbon budgets: Evaluating a geospatial agricultural modeling system using inventory data

Xuesong Zhang; Roberto C. Izaurralde; David H. Manowitz; Ritvik Sahajpal; Tristram O. West; Allison M. Thomson; Min Xu; Kaiguang Zhao; Stephen D. LeDuc; Jimmy R. Williams

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2015

Accurate quantification and clear understanding of regional scale cropland carbon (C) cycling is critical for designing effective policies and management practices that can contribute toward stabilizing atmospheric CO2 concentrations. However, extrapolating site-scale observations to regional scales represents a major challenge confronting the agricultural modeling community. This study introduces a novel geospatial agricultural modeling system (GAMS) exploring the integration of the mechanistic Environmental Policy Integrated Climate model, spatially-resolved data, surveyed management data, and supercomputing functions for cropland C budgets estimates. This modeling system creates spatially-explicit modeling units at a spatial resolution consistent with remotely-sensed crop identification and assigns cropping systems to each of them by geo-referencing surveyed crop management information at the county or state level. A parallel computing algorithm was also developed to facilitate the computationally intensive model runs and output post-processing and visualization. We evaluated GAMS against National Agricultural Statistics Service (NASS) reported crop yields and inventory estimated county-scale cropland C budgets averaged over 2000–2008. We observed good overall agreement, with spatial correlation of 0.89, 0.90, 0.41, and 0.87, for crop yields, Net Primary Production (NPP), Soil Organic C (SOC) change, and Net Ecosystem Exchange (NEE), respectively. However, we also detected notable differences in the magnitude of NPP and NEE, as well as in the spatial pattern of SOC change. By performing crop-specific annual comparisons, we discuss possible explanations for the discrepancies between GAMS and the inventory method, such as data requirements, representation of agroecosystem processes, completeness and accuracy of crop management data, and accuracy of crop area representation. Based on these analyses, we further discuss strategies to improve GAMS by updating input data and by designing more efficient parallel computing capability to quantitatively assess errors associated with the simulation of C budget components. The modularized design of the GAMS makes it flexible to be updated and adapted for different agricultural models so long as they require similar input data, and to be linked with socio-economic models to understand the effectiveness and implications of diverse C management practices and policies.

Statistical challenges in analyses of chamber-based soil CO2 and N2O emissions data

A. N. Kravchenko; Philip Robertson

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2015

Measurements of soil greenhouse gas (GHG) emissions have gained a lot of attention in an effort to potentially increase agriculture’s role in mitigating climate effects. However, it seems not well recognized that the nature of chamber-based GHG data is such that analyses require advanced statistical techniques to fully explore experimental treatment effects. Moreover, for soil GHG data some experimental design approaches can enhance while others can weaken a study’s ability to detect treatment differences. Here we identify and explore the implications of key choices in experimental design and statistical analyses relevant to chamber-based soil GHG studies. In particular, we discuss 1) relative contributions of different sources of random variability in GHG field studies; 2) relative benefits of increasing the numbers of samples at different replication levels to increase statistical power; and 3) benefits of accounting for heterogeneous variances and using repeated measures analysis in GHG studies. CO2 and N2O emissions data 23 collected from three experimental sites in Michigan demonstrated high spatial and temporal variability for CO2 and N2O fluxes. For both gases the total variability is dominated by small scale spatio-temporal variabilit sources, which constituted 55% of the total variability for CO2 and 95% for N2O fluxes. While increasing the number of replicate plots is the main route of rising statistical power, increasing the number of sub-samples (chambers and gas samples) per replicate plot can also provide substantial gains. Judicious repeated measures analysis and especially accounting for heterogeneous variances are important strategies for the efficient analysis of chamber-based GHG data.

Sustainable biofuels: An agro-ecological perspective

Ajay K. Bhardwaj; Terenzio Zenone; Jiquan Chen

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2015

not yet available

The Cell Wall

Nicholas S. Carpita; John Ralph; Maureen McCann

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2015

Cell walls are composed of polysaccharides, proteins, and aromatic substances. The primary wall of the cell is extensible but constrains the final size and shape of every cell. Facing walls of adjacent cells adhere to each other at the middle lamella. In some cells, secondary walls are deposited on the inner surface of the primary wall after growth has stopped. Cell walls become specialized for the function of the approximately 40 cell types that plants comprise. The cellulose microfibrils form the scaffold of all cell walls and are tethered together by cross-linking glycans; this framework is embedded in a gel of pectic substances. There are at least two types of primary walls. The Type I walls of dicots and non-commelinoid monocots have xyloglucan– cellulose networks embedded in a pectin-rich matrix and can be further cross-linked with a network of structural proteins. The Type II walls of commelinoid monocots have glucuronoarabinoxylan–cellulose networks in a relatively pectin-poor matrix. Ferulate esters and other hydroxycinnamic acids and aromatic substances cross-link the Type II walls. The cell wall is born at the cell plate. Cellulose microfibrils are synthesized at the surface of the plasma membrane at terminal complexes called particle rosettes, whereas all noncellulosic cross-linking glycans and pectic substances are made at the Golgi apparatus and secreted. All cell wall sugars are synthesized de novo from interconversion of nucleotide sugars, which are the substrates for polysaccharide synthases and glycosyl-transferases. Cell enlargement depends on the activities of endoglycosidase, endotransglycosy-lase, or expansin, or some combination of these, but cell shape is largely governed by the pattern of cellulose deposition. Cell enlargement also is accompanied by numerous changes in the structure of the wall’s cross-linking glycans and pectin matrix. Termination of cell growth is accompanied by cross-linking reactions involving proteins and aromatic substances. In addition to their use in wood, paper, and textile products, cell walls are the major textural component in fresh fruits and vegetables and constitute important dietary fibers in human nutrition. Transgenic plants with altered cell wall structures will become an important factor in crop and biomass improvement.

The cytoplasmic localization of the catalytic site of CSLF6 supports a channeling model for the biosynthesis of mixed-linkage glucan

Sang-Jin Kim; Starla Zemelis; Kenneth Keegstra; Federica Brandizzi

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2015

Mixed-linkage glucan (MLG) is a significant cell wall carbohydrate in grasses and an important carbon source for human consumption and biofuel production. MLG biosynthesis depends on the biochemical activity of membrane spanning glucan synthases encoded by the CSLH and CSLF cellulose synthase-like gene families. CSLF proteins are the best characterized to date but relatively little is known about their topology with respect to the biosynthetic membranes. In this study, we report on the topology of CSLF6 protein derived from the model grass species Brachypodium distachyon (BdCSLF6) when it is expressed in heterologous systems. Using live cell imaging and immuno-electron microscopy analyses of tobacco epidermal cells expressing BdCSLF6, we demonstrate that a functional YFP fusion of BdCSLF6 is localized to the Golgi apparatus and that the Golgi localization of BdCSLF6 is sufficient for MLG biosynthesis. By implementing protease protection assays of BdCSLF6 expressed in the yeast Pichia pastoris, we also demonstrate that the catalytic domain, the N-terminus and the C- terminus of the protein are exposed in the cytosol. Furthermore, we found that BdCSLF6 is capable of producing MLG not only in tobacco cells but also in Pichia, which generally does not produce MLG. Together, these results support the conclusion that BdCSLF6 can produce both of the linkages present in the (1,3; 1,4)-β-D-glucan chain of MLG and that the product is channeled at the Golgi into the secretory pathway for deposition into the cell wall.

Tracking synthesis and turnover of triacylglycerol in leaves

Henrik Tjellstrom; Merissa Strawsine; John B. Ohlrogge

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2015

Triacylglycerol (TAG) represents less than 1% of leaf glycerolipids but can accumulate under stress and other conditions or if leaves are supplied with fatty acids or in plants transformed with regulators or enzymes of lipid metabolism. To better understand the metabolism of TAG in leaves, pulse-chase radiolabeling experiments were designed to probe its synthesis and turnover. When Arabidopsis leaves were incubated with 14C-lauric acid (12:0), a major initial product was 14C-TAG. Thus, despite low steady-state levels, leaves possess substantial TAG biosynthetic capacity. The contributions of diacylglycerol acyltransferase1 and phospholipid:diacylglycerol acyltransferase1 to leaf TAG synthesis were examined by labeling of dgat1 and pdat1 mutants. The dgat1 mutant displayed a major (75%) reduction in 14C-TAG accumulation whereas pdat1 TAG labeling was only slightly reduced. Thus, DGAT1 has a principal role in TAG biosynthesis in young leaves. During a four-hour chase period, radioactivity in TAG declined 70%, whereas turnover of 14C-acyl chains of PC and other polar lipids were much lower. 60% of 14C-12:0 was directly incorporated into glycerolipids without modification whereas 40% was elongated and desaturated to 16:0 and 18:1 by plastids. The unmodified 14C-12:0 and the plastid products of 14C-12:0 metabolism entered different pathways. Although plastid-modified 14C-products accumulated in MGDG, PC, PE, and DAG, there was almost no accumulation of 14C-16:0 and 18:1 in TAG. Because DAG and acyl-CoA are direct precursors of TAG, the differential labeling of polar glycerolipids and TAG by 14C-12:0 and its plastid-modified products provides evidence for multiple subcellular pools of both acyl-CoA and DAG.

Arabidopsis thaliana  IRX10 and two related proteins from psyllium and Physcomitrella patens  are xylan xylosyltransferases

Jacob Krüger Jensen; Nathan Robert Johnson; Curtis Gene Wilkerson

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2014

The enzymatic mechanism that governs the synthesis of the xylan backbone polymer, a linear chain of xylose residues connected by beta-1,4 glycosidic linkages, has remained elusive. Xylan is a major constituent of many kinds of plant cell walls, and genetic studies have identified multiple genes that affect xylan formation. In this study, we investigate several homologs of one of these previously identified xylan-related genes, IRX10 from Arabidopsis thaliana, by heterologous expression and in vitro xylan xylosyltransferase assay. We find that an IRX10 homolog from the moss Physcomitrella patens displays robust activity, and we show that the xylosidic linkage formed is a beta-1,4 linkage, establishing this protein as a xylan beta-1,4-xylosyltransferase. We also find lower but reproducible xylan xylosyltransferase activity with A. thaliana IRX10 and with a homolog from the dicot plant Plantago ovata, showing that xylan xylosyltransferase activity is conserved over large evolutionary distance for these proteins.

A Rhodobacter sphaeroides  protein mechanistically similar to Escherichia coli  DskA regulates photosynthetic growth

Christopher W. Lennon; Kimberly C. Lemmer; Jessica L. Irons; Max I. Sellman; Timothy J. Donohue; Richard L. Gourse; Wilma Ross

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2014

DksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the "stringent response" to nutrient starvation in the gammaproteobacterium Escherichia coli and for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly related Rhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length to E. coli DksA but lacks the Zn finger motif of the E. coli DksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of an E. coli strain lacking the dksA gene and modulates transcription in vitro with E. coli RNA polymerase (RNAP) similarly to E. coli DksA. RSP2654 reduces RNAP-promoter complex stability in vitro with RNAPs from E. coli or R. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity to E. coli DksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsp has distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp. IMPORTANCE The role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis in Rhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

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