Dynamics of Photosynthesis in the Glycogen-Deficient glgC Mutant of Synechococcus sp. PCC 7002by Simon A. Jackson, Julian J. Eaton-Rye, Donald A. Bryant, Matthew C. Posewitz, Fiona K. Davies

Applied and Environmental Microbiology

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Year
2015
DOI
10.1128/AEM.01751-15
Subject
Biotechnology / Food Science / Ecology / Applied Microbiology and Biotechnology

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Dynamics of Photosynthesis in the Glycogen-Deficient glgC Mutant of Synechococcus sp. PCC 1 7002 2 3

Simon A. Jackson,a Julian J. Eaton-Rye,a Donald A. Bryant,b Matthew C. Posewitz,c Fiona K. 4

Daviesc# 5 6

Department of Biochemistry, University of Otago, Dunedin, New Zealanda; Department of 7

Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 8

USAb; Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, CO, 9

USAc 10 11

Running Head: Dynamics of photosynthesis in glgC mutant 12 13 #Address correspondence to Fiona K. Davies, fdavies@mines.edu 14

S.A.J. and F.K.D. contributed equally to this work. 15 16 17 18 19 20 21 22 23

AEM Accepted Manuscript Posted Online 6 July 2015

Appl. Environ. Microbiol. doi:10.1128/AEM.01751-15

Copyright © 2015, American Society for Microbiology. All Rights Reserved. 2

ABSTRACT 24

Cyanobacterial glycogen-deficient mutants display impaired degradation of light-harvesting 25 phycobilisomes under nitrogen-limiting growth conditions, and secrete a suite of organic acids as 26 a putative reductant-spilling mechanism. This genetic background, therefore, represents an 27 important platform to better understand the complex relationships between light harvesting, 28 photosynthetic electron transport, carbon fixation and carbon/nitrogen metabolisms. In this 29 study, we conducted a comprehensive analysis of the dynamics of photosynthesis as a function 30 of reductant sink manipulation in the glycogen-deficient glgC mutant of Synechococcus sp. PCC 31 7002. The glgC mutant showed increased susceptibility to photoinhibition during the initial 32 phase of nitrogen deprivation. However, after extended periods of nitrogen deprivation, glgC 33 mutant cells maintained higher levels of photosynthetic activity than the wild type, supporting 34 continuous organic acid secretion in the absence of biomass accumulation. In contrast to the wild 35 type, the glgC mutant maintained efficient energy transfer from phycobilisomes to photosystem 36

II (PSII) reaction centers, had an elevated PSII:PSI ratio as a result of reduced PSII degradation, 37 and retained a nitrogen replete-type ultrastructure, including an extensive thylakoid membrane 38 network after prolonged nitrogen deprivation. Together, these results suggest that multiple, 39 global signals for nitrogen deprivation are not activated in the glgC mutant; allowing the 40 maintenance of active photosynthetic complexes under conditions where photosynthesis would 41 normally be abolished. 42 43 44 45 46 3

INTRODUCTION 47

Nitrogen is an essential macronutrient required for the synthesis of pigments, protein and nucleic 48 acids in cyanobacteria. When nitrogen availability is limiting for cell growth and proliferation, a 49 defined sequence of stress responses is deployed in most cyanobacteria. The immediate response 50 is to increase the expression of genes associated with nitrogen uptake and assimilation (1-4). 51

This is followed within hours by the cessation of growth, and the mobilization of internal 52 nitrogen stores through the degradation of the light-harvesting phycobilisomes, which contain 53 nitrogen-rich phycobiliproteins and phycocyanin (5-7). Phycobilisome degradation also serves to 54 reduce the light-harvesting cross section of the cell, thus avoiding excessive photon absorption 55 that can result in photooxidative damage in the absence of downstream metabolic oxidation 56 reactions. Simultaneous activation of glycogen biosynthesis, degradation of thylakoid 57 membranes, and reduction in the expression of genes associated with photosynthesis, carbon 58 fixation, and de novo protein synthesis define the short-term acclimation events to nitrogen 59 limitation (1-4). The longer-term acclimation to nitrogen limitation involves a decrease in 60 metabolic activity to a minimum level that supports cell viability using energy derived from the 61 catabolism of glycogen and cyclic electron transfer around Photosystem I (PSI) (2, 3). 62

The reduction of nitrate is an energetically expensive process that requires eight 63 electrons, and can consume up to 30% of the reducing equivalents provided by the 64 photosynthetic light reactions (8, 9). Therefore, when nitrogen availability is limited, glycogen 65 biosynthesis has been proposed to provide an alternative reductant sink and mediates redox 66 homeostasis within the cell (10). Glycogen is a highly branched polysaccharide that accumulates 67 as granules in the cytoplasm between the thylakoid membranes of cyanobacterial cells, and may 68 comprise 40-60% of the dry cell biomass during conditions of nitrogen deprivation (11). Under 69 4 these conditions glycogen is also an important energy reserve that is required for cell survival by 70 maintaining basal levels of metabolism and allowing rapid commencement of growth if 71 conditions become more favorable. As a gluconeogenic end-product, glycogen uses ADP-72 glucose as a precursor, which is synthesized from glucose 1-phosphate in a reaction catalyzed by 73

ADP-glucose pyrophosphorylase (encoded by the glgC gene) (12). The inhibition of glycogen 74 biosynthesis, via genetic deletion of the glgC gene, eliminates the primary native reductant sink 75 during nitrogen-limited growth, and cyanobacterial glgC mutants have been shown to secrete a 76 suite of organic acids under these conditions (13-16). Another intriguing phenotype of 77 cyanobacterial glgC mutants is the absence of phycobilisome degradation under nitrogen-78 limiting conditions (13-15); however, the impaired signaling mechanism(s) that prevents 79 phycobilisome degradation remains to be determined. 80

Photosynthesis and carbon/nitrogen metabolisms are heavily interconnected through 81 complex, yet poorly defined, signaling networks. The tricarboxylic acid (TCA) cycle 82 intermediate α-ketoglutarate is one key metabolite that regulates the global cellular response to 83 nitrogen limitation. The carbon skeleton of α-ketoglutarate is used to assimilate nitrogen via the 84 glutamine synthetase-glutamate synthase (GS-GOGAT) cycle; therefore, the intracellular 85 concentration of α-ketoglutarate signals the carbon/nitrogen status of the cell, and is used to 86 initiate global stress responses (17, 18). The potential biotechnological applications for 87 cyanobacteria, as a platform for the production of renewable biofuels or bulk industrial 88 chemicals, means there is interest in understanding the relationships between energy and 89 carbon/nitrogen metabolisms. 90