Role of Type 2 NAD(P)H Dehydrogenase NdbC in Redox ... · PDF fileRole of Type 2 NAD(P)H Dehydrogenase NdbC in Redox Regulation of Carbon Allocation inSynechocystis1[OPEN] Tuomas Huokko,

Role of Type 2 NAD(P)H Dehydrogenase NdbC in RedoxRegulation of Carbon Allocation in Synechocystis1[OPEN]

Tuomas Huokko, Dorota Muth-Pawlak, Natalia Battchikova, Yagut Allahverdiyeva, and Eva-Mari Aro2

Laboratory of Molecular Plant Biology, Department of Biochemistry, University of Turku, Turku FI-20014,Finland

ORCID IDs: 0000-0001-5176-3639 (N.B.); 0000-0002-9262-1757 (Y.A.); 0000-0002-2922-1435 (E.-M.A.).

NAD(P)H dehydrogenases comprise type 1 (NDH-1) and type 2 (NDH-2s) enzymes. Even though the NDH-1 complex is a well-characterized protein complex in the thylakoid membrane of Synechocystis sp. PCC 6803 (hereafter Synechocystis), the exact rolesof different NDH-2s remain poorly understood. To elucidate this question, we studied the function of NdbC, one of the threeNDH-2s in Synechocystis, by constructing a deletion mutant (DndbC) for a corresponding protein and submitting the mutant tophysiological and biochemical characterization as well as to comprehensive proteomics analysis. We demonstrate that thedeletion of NdbC, localized to the plasma membrane, affects several metabolic pathways in Synechocystis in autotrophicgrowth conditions without prominent effects on photosynthesis. Foremost, the deletion of NdbC leads, directly or indirectly,to compromised sugar catabolism, to glycogen accumulation, and to distorted cell division. Deficiencies in several sugarcatabolic routes were supported by severe retardation of growth of the DndbC mutant under light-activated heterotrophicgrowth conditions but not under mixotrophy. Thus, NdbC has a significant function in regulating carbon allocation betweenstorage and the biosynthesis pathways. In addition, the deletion of NdbC increases the amount of cyclic electron transfer,possibly via the NDH-12 complex, and decreases the expression of several transporters in ambient CO2 growth conditions.

In cyanobacteria, both photosynthesis and respi-ration occur in the same cellular compartment. Thecyanobacterial thylakoid membrane hosts both thephotosynthetic and respiratory electron transportchains (ETCs), while some respiratory electron trans-port also has been localized to the plasma mem-brane (PM; Lea-Smith et al., 2013; Mullineaux, 2014;Ermakova et al., 2016; for review, Cooley and Vermaas,2001). These two major bioenergetic pathways shareseveral redox-active components in thylakoids, in-cluding the plastoquinone (PQ) pool, the cytochrome(Cyt) b6f complex, and soluble electron carriers inthylakoid lumen.

In respiration, a significant part of electrons derivedfrom organic molecules is transferred by bacterial pyr-idine nucleotide dehydrogenases into the PQ pool.

These enzymes fall into two classes called type 1 andtype 2 NAD(P)H dehydrogenases (NDH-1 andNDH-2, respectively; for review, see Melo et al., 2004).The NDH-1 enzymes are proton-pumping multi-subunit complexes resembling mitochondrial complexI; in cyanobacteria, they reside mostly in the thylakoidmembrane and function in cyclic electron transfer(CET), respiration, and C uptake (Battchikova et al.,2011; for review, see Peltier et al., 2016). In contrast,NDH-2s are composed of single polypeptides withmolecular masses around 50 kD, and they do notcontribute to the generation of the proton gradientacross the membrane (Yagi, 1991). The primarystructure of NDH-2 commonly includes two GXGXXGmotifs within the b-sheet-a-helix-b-sheet domains(Rossman fold), one for binding NAD(P)H andanother for FAD or FMN (Wierenga et al., 1986).Although the crystal structures of NDH-2 fromSaccharomyces cerevisiae (Ndi1; Feng et al., 2012)and the bacterial type NDH-2 from Caldalkalibacillusthermarum (Heikal et al., 2014) as well as fromStaphylococcus aureus (Sena et al., 2015) have beensolved, the actual mechanism of NADH:quinoneoxidoreduction still remains unclear despite recentlypresented models (Marreiros et al., 2017). It is gener-ally agreed that, in organisms where NADH oxidationis carried out solely by NDH-2s, the main function ofthese enzymes is to perform the respiratory chain-linked NADH turnover and the following donationof electrons to the protein complexes that produce theH+ gradient powering ATP production (for review,see Melo et al., 2004).

1 This work was supported by the Academy of Finland projectsCentre of Excellence in Molecular Biology of Primary Producers andAcademy Professor project (grant nos. 307335 and 303757) and by theDoctoral Programme in Molecular Life Sciences and the AlfredKordelin Foundation (to T.H.).

2 Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Eva-Mari Aro ([email protected]).

All authors designed the experiments; N.B. and Y.A. supervisedthe experiments; T.H. and D.M.-P. performed the experiments andanalyzed the data; all authors conceived the project and wrote thearticle; E.-M.A. supervised and complemented the writing.

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Besides heterotrophic organisms, where NDH-2s areusually located either in mitochondria (eukaryotes) orin the PM (prokaryotes; for review, see Melo et al.,2004), there are several photosynthetic organismswith genes encoding NDH-2s. Plant-type NDH-2s fallinto three distinct groups: NDA and NDB relating tofungal sequences and NDC of cyanobacterial origin(Michalecka et al., 2003). Most of these NDH-2s aretargeted to mitochondria or peroxisomes, but somealso are targeted to plastids. Of the six NDH-2s in theunicellular green alga Chlamydomonas reinhardtii, thethylakoid-localized NDA2 has been produced as a re-combinant protein and characterized for its enzymo-logical properties (Desplats et al., 2009). NDA2 wasshown to be able to reduce PQ by oxidizing bothNADH and NADPH. However, NDA2 showedhigher affinity to NADH than NADPH and had FMNas a cofactor instead of the more common FAD. An-other NDH-2 enzyme, NDA3, has been localized toC. reinhardtii chloroplasts (Terashima et al., 2010).Physcomitrella patens has six NDH-2 genes, and theproducts of three of them are targeted to chloroplasts(Xu et al., 2013). The genome of Arabidopsis (Arabi-dopsis thaliana) contains seven open reading framesencoding NDH-2 homologs, of which at least five havea mitochondrial localization (Michalecka et al., 2003).NDC1, the onlyNDH-2 of cyanobacterial origin (Michaleckaet al., 2003), has dual targeting to both mitochondriaand chloroplasts (Carrie et al., 2008), being involved inprenylquinone and vitamin K1 metabolism in the chlo-roplast (Piller et al., 2011).

All sequenced cyanobacterial species encode at leastone NDH-2 enzyme in their genomes (Marreiros et al.,2016). The thylakoid localization has been shown forNDH-2 in Anabaena variabilis (Alpes et al., 1989) andAnabaena sp. PCC 7120 (Howitt et al., 1993). Synecho-cystis sp. PCC 6803 (hereafter Synechocystis) has threegenes encoding NDH-2s: ndbA (slr0851), ndbB (slr1743),and ndbC (sll1484; Kaneko et al., 1996; Howitt et al.,1999). They all have conserved motifs for binding acofactor FAD and substrate NAD(P)H, and the se-quence of the binding motif suggests that NADH isstrongly preferred over NADPH (Howitt et al., 1999). Itwas shown that NdbB of Synechocystis (Howitt et al.,1999) and NDA2 of C. reinhardtii (Desplats et al., 2009)could functionally complement an Escherichia coli mu-tant lacking both NDH-1 and NDH-2s. It has beensuggested that the NDH-2s function as redox sensors inSynechocystis, possibly responding to the redox state ofthe PQ pool, rather than having any significant catalyticrole in respiration (Howitt et al., 1999). Regulation byNDH-2s also may be targeted to maintain the redoxbalance of NAD+/NADH, deduced from the fact thatwhen all three Synechocystis NDH-2s were deleted, re-duced NADH was strongly prevailing (Cooley andVermaas, 2001). In addition, it was shown recently thatNdbB is involved in vitamin K1 biosynthesis (Fatihiet al., 2015).

In order to get more detailed insights into the func-tion of NdbC in Synechocystis, the NdbC deletion

mutant was constructed and thoroughly characterized,including proteomics approaches. It is demonstratedhere that, upon autotrophic growth, NdbC has a sig-nificant function in regulating carbon allocation be-tween storage and the biosynthesis pathways, withdistinct effects on cell morphology and physiology.Intriguingly, the function of NdbC becomes absolutelyessential under light-activated heterotrophic growth(LAHG) conditions.

RESULTS

Construction of the ndbC Deletion Mutant (DndbC)

The interrupted ndbC gene from the DndbC mutantconstructed previously (Howitt et al., 1999) was am-plified using PCR, and the resulting product was usedin transformation (for details, see “Materials andMethods”).Segregationwas confirmed by PCR analysis (SupplementalFig. S1). The absence of theNdbCprotein in the constructedDndbC mutant was verified by western-blot analysis usingextracted total proteins and specific antibody raisedagainst NdbC (Agrisera; Fig. 1). To verify that thesll1485 and sll1486 genes, which are located immedi-ately downstream of ndbC in the Synechocystis genome,were not affected, their expression was studied withreverse transcription-quantitative PCR (SupplementalFig. S2). No decline in the expression of either sll1485or sll1486 was observed in DndbC compared with thewild type.

Growth, Morphology, and Intracellular GlycogenContent of the DndbC Mutant under Ambient andHigh-CO2 Conditions

The growth of DndbC was monitored under ambientCO2 conditions (LC) and in the presence of 3% CO2(HC) for several days. Based on optical density (OD750),DndbC grew slightly faster in LC (Fig. 2A) but clearlyfaster in HC conditions compared with the respectivewild type under the same conditions (Fig. 2B). Con-comitant inspection of wild-type and DndbC mutantcells with a light microscope (Fig. 3A) and a transmis-sion electronmicroscope (Fig. 3B) revealed an increasedcell size of DndbC compared with the wild type in both

Figure 1. Western-blot analysis with NdbC-specific antibody. Proteinswere extracted from wild-type cells and DndbC mutant cells grown atambient CO2 concentration. Samples were loaded based on total pro-tein amount: 12.5 mg for wild-type (WT) 50%, 25 mg for wild-type100%, and 25 mg for DndbC.

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LC and HC. In addition, the cell number in DndbCcultures, calculated from equal OD750 of wild-typeand mutant cultures, was almost 53% lower whencells were grown in LC and 59% lower in HC culturesas compared with the wild type (Table I). Since thecell size significantly affects light scattering in addi-tion to OD, the growth was followed by cell counting.Expressing the growth as an increase in cell numberreinforced the slow growth of the DndbC mutant inboth LC and HC (Fig. 2, C and D). Based on cellnumber, the growth of DndbC was about 50% slowercompared with the wild type in both LC and HC after100 h of inoculation. In accordance with the biggercell size, the dry weight per cell of DndbC was about52% higher in LC and 142% higher in HC comparedwith the wild type (Table I). In line with this, thechlorophyll a (Chl) amount per cell was about 109%bigger in the DndbC mutant in LC and 127% bigger inHC compared with the wild type. Similarly theamount of total proteins per cell in the DndbC mutantwas 94% higher in LC and about 115% higher in HCcompared with the wild type.Investigation of the cell ultrastructure with the

transmission electron microscope revealed an accu-mulation of dark granules, possibly glycogen, in theDndbC mutant cells (Fig. 3B). Determination of theintracellular glycogen content from the DndbC andwild-type cells revealed an accumulation of 50%more glycogen in DndbC in LC and 18% more in HCwhen glycogen amount was normalized to Chl con-centration (Fig. 3C). Nevertheless, when the glycogencontent was normalized to the cell number, oneDndbC cell contained about 320% more glycogen in

LC and 260% more in HC compared with the wildtype (Fig. 3D).

Photosynthetic Characterization of the DndbC Mutant

The wild-type and DndbC cells were first subjectedto membrane inlet mass spectrometry (MIMS) with18O-enriched oxygen for gas-exchange investigations.The advantage of MIMS compared with traditionaloxygen electrode is the possibility to differentiatebetween the gross oxygen produced by PSII andthe oxygen uptake under illumination. In addition,the oxygen and CO2 exchanges can be monitoredconcomitantly.

There was no statistically significant difference indark respiration between the wild type and DndbC inLC conditions (Table II). In DndbC, the light-inducedoxygen uptake rate, which is defined as the differencebetween the light and dark oxygen uptake rates, wasabout 17% higher, whereas the gross and net oxygenevolution rates were 8.5% and 11% lower, respectively,compared with the wild type. The amplitude of the fastphase of CO2 uptake, which is related to the function ofcarbon-concentrating mechanisms, was almost 19%smaller in DndbC compared with the wild type, but nosignificant difference in the steady-state CO2 uptakeduring illumination was detected.

Next, the Chl fluorescence and P700 oxidoreductionmeasurements were performed with the DndbC cells us-ing the Dual-PAM fluorometer. The PSII yield, measuredfromdark-acclimated cells (Fv

D/FmD), after illuminationwith

a short far-red (FR) light (FvFR/Fm

FR), and the effective

Figure 2. Growth of the wild type (WT)and the DndbC mutant in LC and HCconditions under continuous illumina-tion of 50 mmol photons m22 s21. Growthwasmonitored byOD750 (A and B) and bycell number (C and D). In the beginningof the experiments, cells were inoculatedat OD750 = 0.1. Values are means 6 SD;n = 3. Photographs in A and B were takenat OD750 = 1.

Plant Physiol. Vol. 174, 2017 1865

The Role of NdbC in Synechocystis sp. PCC 6803



PSII yield [Y(II)], measured after illumination with ac-tinic light for 5 min, were each slightly lower in DndbCcompared with the wild type both in LC and HC (Fig.4A; Supplemental Table S1). Likewise, no differencewas observed in the maximum quantum yield of PSII(Fv/Fm), measured with 20 mM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), between the wild-type andDndbC cells grown at LC or HC (Supplemental TableS1). When the reoxidation of QA

2 in the dark wasrecorded after a single-turnover saturating flash, theDndbC mutant demonstrated slightly slower decay offluorescence compared with the wild type, indicatingmodified electron transfer at the PSII acceptor side(Supplemental Fig. S3A). In the presence of DCMU,which blocks electron transfer at the QB site, forcingQA

2 reoxidation to occur via charge recombinationwith the donor side components (Vass et al., 1999), nodifference in the decay of fluorescence was observedbetween DndbC and the wild type (Supplemental Fig.S2B). There was no significant difference in the effectiveyield of PSI [Y(I)], in the maximal amount of oxidizable

P700 (Pm), in the maximal fluorescence of dark-adaptedcells (Fm

D), or in the maximal fluorescence after FRillumination (Fm

FR) between the wild type and theDndbC mutant in LC or HC conditions (Fig. 4B;Supplemental Table S1). These results along with theMIMS data indicate that the deletion of NdbC does notstructurally alter PSII or PSI, yet the electron transferdownstream from PSII was slightly slower in theDndbCmutant compared with the wild type.

The fact that the slower electron transfer from PSIImight be due to a more reduced electron transfer chainmade us next monitor the redox state of P700 duringdark-light-dark transitions by the application of strongFR light (Fig. 4C). The kinetics of P700 oxidation wasclearly slower and rereduction was faster in the DndbCmutant, suggesting elevated CET in the mutant. Toverify this, the transient increase in fluorescence afterillumination (F0 rise) was monitored as a postillumi-nation rise in Chl fluorescence, which reflects the effi-ciency of the NDH-1-related CET (Mi et al., 1992a;Holland et al., 2015). In the DndbC mutant, the F0 rise

Figure 3. Phenotypes and glycogen accumu-lation of wild-type (WT) and DndbC cells in LCandHC growth conditions. A, Lightmicroscopeimages of wild-type and DndbC cells. B,Transmission electron microscope images ofwild-type and DndbC cells in LC. The arrowindicates putative glycogen granules (G). C andD, Intracellular glycogen content in the wildtype and the DndbC mutant based on the Chlamount (C) and on the cell number (D). Valuesare means 6 SD; n = 4. Asterisks indicate sta-tistically significant differences between thewild type and the DndbC mutant (P # 0.05).

Table I. Comparison of cell number, dry weight, Chl, and protein content between wild-type and DndbC mutant cells

The number of wild-type and DndbC cells in 1 mL at the OD of 1, dry weight (mg per OD or mg per 107 cells), Chl amount (mg Chl per OD or mgChl per 107 cells), and total protein amount (mg per OD or mg per 107 cells) are shown. Values per OD are means 6 SD; n = 3 to 4, and valuesper cell number are calculated based on these. Asterisks indicate statistically significant differences between the wild type and the DndbC mutant(P , 0.05).

Conditions Cells 107/mL Dry Weight Chl Protein Dry Weight Chl Protein

OD750 = 1 mg per OD mg per OD mg per OD mg per 107 cells mg per 107 cells mg 107 cellsWild type LC 18.4 6 1.21 5.4 6 0.25 4.2 6 0.08 0.30 6 0.00 0.29 0.23 0.016DndbC LC 8.5 6 1.01* 3.7 6 0.41* 4.1 6 0.11 0.27 6 0.01* 0.44 0.48 0.031Wild type HC 17.6 6 1.30 4.6 6 0.30 3.9 6 0.03 0.23 6 0.02 0.26 0.22 0.013DndbC HC 7.2 6 1.19* 4.5 6 0.16 3.6 6 0.04* 0.20 6 0.01 0.63 0.50 0.028


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was significantly higher and reached the maximumclearly faster compared with the wild type (Fig. 4D).

Characterization of the DndbC Mutant at the Protein Level

Since the characterization of the DndbCmutant by themethods described above did not provide any clearfunctional role for the NdbC protein, we next took aproteomics approach and asked whether the NdbCdeletion induces any major changes in the proteome.First, we performed a global label-free tandem massspectrometry (MS/MS) protein identification andquantitation based on a data-dependent analysis(DDA). We quantified (with P # 0.05) 543 proteins inHC (Supplemental Tables S2 and S4) and 699 proteinsin LC (Supplemental Tables S3 and S5) with at least twopeptides. The fold change (FC) threshold of the practi-cal significance was set to 21.3 # FC and FC $ 1.3.The deletion of NdbC resulted in the differential

regulation of a significant amount of proteins. In theHC-grown DndbC cells, 128 proteins were up-regulatedwhile 287 proteins were down-regulated in comparisonwith the wild type (Supplemental Table S4); in theLC-grown DndbC cells, the numbers were 211 and293, respectively (Supplemental Table S5). Proteinswere quantified by DDA from cells grown both in HCand LC, while selected reaction monitoring (SRM)was applied only for LC-grown cells.

Differential Protein Expression in the Wild Type andDndbC Revealed by a Global Proteomic Approach

In both LC and HC conditions, three enzymes,phosphofructokinase (PfkA), pyruvate kinase (Pyk1),and glyceraldehyde-3-phosphate dehydrogenase(Gap1), which are involved specifically in glycolysis,were down-regulated in the DndbC mutant (Table III).A similar response was observed for phosphoglyceratekinase (Pgk) functioning in glycolysis and the Calvin-Benson cycle and for two key enzymes of the oxidativepentose phosphate (OPP) pathway, Glc-6-P dehydro-genase and 6-phosphogluconolactonase, encoded byzwf and pgl, respectively. It is important to note that thedeletion of NdbC did not have a significant effect on the

expression of proteins functioning in glycogen biosyn-thesis (GlgA,C,D) or in glycogen degradation (GlgX,P;Supplemental Tables S4 and S5). Furthermore, proteinsessential to cell division, including FtsZ, ZipN, MinC,MinD, and MinE, were down-regulated in bothgrowth conditions, as were the components of the ureatransporter (UrtA,E; Table III). In addition, PII andPipX, participating in the regulation of nitrogen me-tabolism, were down-regulated (Table III), while theglobal nitrogen regulator NtcA was not affected(Supplemental Tables S4 and S5). The cyanobacteria-specific AbrB-like transcriptional regulator (cyAbrB)Sll0359 and also the DNA repair protein RecA weredown-regulated in LC and HC (Table III). In addition,the deletion of NdbC decreased, both in LC and HCconditions, the expression of proteins encoded bygenes located in extrachromosomal plasmids. Theyincluded, among others, Sll0653 to Sll0655 and Slr6012to Slr6016 from pSYSX and several proteins frompSYSA that have been annotated as CRISP-relatedproteins (Scholz et al., 2013). The latter comprisedCsm6 and Cas7 from the CRISP2 group as well asCmr6, Cmr4, and Cmr2 from CRISP3 (Table III). Incontrast, the flavodiiron proteins Flv1 and Flv3 in-volved in the Mehler-like reaction and ChlD in-volved in Chl biosynthesis were up-regulated in theDndbC mutant in both growth conditions. In addi-tion, the circadian clock proteins KaiB1 and KaiC1were up-regulated in a coordinated manner in bothgrowth conditions, but proteins involved in polysac-charide (RfbB) and amino acid (Sll0664) transport,together with a putative Mg2+ (MgtE) transporter,were down-regulated.

Growth conditions, however, also had an impact onthe expression of several proteins in the DndbCmutant.In HC, the deletion of NdbC specifically included thedown-regulation of GlgB participating in glycogen bio-synthesis and the bicarbonate transporter SbtA/B aswell as the differential regulation of some subunitsbelonging to NDH-1 (Table III). In particular, the ex-pression of NdhD3 increased in the DndbC mutant,while CupS and NdhF4 were down-regulated in HCconditions. Contrary to HC, more prominent differ-ences between the DndbC mutant and the wild typewere detected at the protein level when cells were

Table II. Gas-exchange rates of wild-type and DndbC cells grown in LC

The oxygen exchange rates are shown as mmol oxygen mg21 Chl h21 and CO2 uptake rates as mmol CO2

mg21 Chl h21. Values are means6 SD; n = 3. Asterisks indicate statistically significant differences betweenthe wild type and the DndbC mutant (P , 0.05).

Parameter Component Wild Type DndbC

Oxygen uptake Dark 2.8 6 0.2 2.3 6 0.2Light induced 17.7 6 0.5 20.6 6 0.5*Total light 20.4 6 0.4 22.9 6 0.3*

Oxygen production Gross 176.5 6 5.4 161.5 6 2.5*Net 156.1 6 4.9 138.6 6 2.2*

CO2 uptake Fast phase 754.7 6 34.5 613.7 6 40.9*Steady state 171.6 6 4.9 166.3 6 6.0











grown in LC conditions. Subunits of the ATP-bindingcassette (ABC)-type bicarbonate transporter, CmpA,B,C,D, and the carbon-concentrating mechanism regu-lator CcmR were up-regulated in a coordinatedmanner (Table III). In contrast, NrtC belonging to thenitrate/nitrate transporter (NRT) was down-regulated.Considerable down-regulation was observed for bothphosphate transporters, the low-affinity phosphatetransporter system Pst1 (PstC1, PstB1, PstB19, PstS1, andSphX) and the high-affinity transporter Pst2 (PstS2). TheIlvD protein participating in Glc metabolism (the Entner-Doudoroff pathway) was up-regulated. The expressionof several proteins involved in photosynthesis wasup-regulated in the ΔndbCmutant, including themajorsubunits of the Cyt b6 f complex (PetA,B,C,D) that wereincreased cooperatively, the PSI core subunits PsaBand PsaA, and the IsiA protein (Table III). In addition,the AbrB-like transcriptional regulator Sll0822, one ofthe subunits of the alternate respiratory terminal oxi-dase CtaCII, and the circadian clock-related response

regulator RpaA were down-regulated in LC condi-tions in the DndbCmutant as compared with the wildtype. In LC, several proteins involved in the transportof amino acids, peptides, glucosylclyserol, and lipo-polysaccharides were down-regulated in the DndbCmutant, in contrast to up-regulated Fe, Mn, and Ktransporters. Furthermore, several proteins involvedin DNA repair (Slr6097, RepA, and MutS) were down-regulated in the DndbC mutant compared with thewild type only in LC.

Targeted Proteomic Approach Strengthened and Expandedthe Scope of Proteins Affected in the DndbC Mutant

We also applied to LC-grown wild-type and DndbCcells a more precise and sensitive targeted quantifi-cation method called SRM (Anderson and Hunter,2006; Ludwig et al., 2012; Vuorijoki et al., 2016). SRMwas used in order to verify the results of DDA and,

Figure 4. Photosynthetic characterization of the wild type (WT) and the DndbCmutant. A and B, Y(II) (A) and Y(I) (B) of the wildtype (black squares in LC, blue triangles in HC) and the DndbC mutant (red circles in LC, magenta triangles in HC) under illu-mination (50 mmol photons m22 s21 actinic light). Cells were dark acclimated for 10 min before each measurement, and the Chlconcentration was adjusted to 15 mg mL21. First points were measured after dark acclimation, and second points were measuredafter 10 s of FR illumination. Values are means6 SD; n = 3. C, P700 oxidoreduction in the wild type (black line) and inDndbC (redline) in LC. P700 oxidation was monitored during illumination with strong FR light for 5 s (red bar), and rereduction was mon-itored in darkness (black bar) after illumination. D, F0 rise measured from the wild type (black line) and the DndbC mutant (redline) in LC. After dark acclimation, cells were illuminated for 1 min with red actinic light (50 mmol photons m22 s21; yellow bar[only part is shown]), and the F0 rise was monitored (black bar) after turning off the actinic light. Curves were normalized to thesame amplitude to facilitate comparison of the kinetics. Mean values from three independent experiments are shown. r.u.,Relative units.


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Table III. Differential protein expression in the DndbC mutant versus the wild type

Proteins were quantified with DDA from cells grown in HC and LC and with SRM from LC-grown cells. The practical significance FC is in italictype when up-regulated (FC $ 1.3) and boldface type when down-regulated (FC # 21.3; P # 0.05) in the DndbC mutant relative to the wild type.NA, Not analyzed; ND, not detected.

Parameter Protein Protein Symbol HC DDA FC P LC DDA FC P LC SRM FC P

Cell division Sll1633 FtsZ 21.47 3.61E-04 21.43 4.65E-03 21.54 6.36E-05Sll0169 ZipN 21.01 8.58E-01 21.40 1.02E-02 21.58 7.78E-05Sll0288 MinC 21.54 4.37E-02 21.33 1.29E-02 21.45 4.99E-02Sll0289 MinD 21.42 1.99E-02 21.61 2.98E-04 21.60 3.39E-05Ssl0546 MinE 21.56 1.01E-02 21.39 3.46E-04 21.57 8.99E-05

Circadian clock Slr0757 KaiB1 21.57 1.24E-03 21.69 2.04E-04 NA NASlr0758 KaiC1 21.58 9.53E-04 21.47 2.71E-03 NA NASlr0115 RpaA 21.08 5.97E-01 21.58 3.86E-03 NA NA

DNA repair Sll0569 RecA 21.50 3.63E-03 21.66 2.35E-04 NA NASlr6097 21.75 2.03E-01 25.16 1.41E-03 NA NASlr9101 RepA 1.50 3.50E-01 22.79 3.45E-03 NA NASll1165 MutS 21.19 9.65E-01 21.75 1.61E-02 NA NA

Transport and binding proteinsInorganic phosphate uptake Sll0681 PstC1 1.22 3.46E-01 218.67 6.27E-05 212.21 2.00E-05

Sll0683 PstB1 21.00 6.20E-01 26.85 3.06E-04 211.22 3.96E-04Sll0684 PstB1’ 1.01 6.12E-01 28.10 4.64E-03 28.26 2.19E-05Sll0680 PstS1 21.12 9.51E-01 22.78 1.61E-05 24.02 7.20E-03Sll0679 SphX 1.26 2.61E-01 21.60 3.41E-02 21.63 1.65E-03Slr1247 PstS2 1.42 1.82E-01 21.34 3.85E-03 21.45 5.12E-03Sll0540 PstS 21.35 6.69E-01 23.16 4.21E-04 23.09 8.97E-06

Nitrogen uptake Sll1451 NrtB 1.07 5.13E-01 ND ND 21.62 1.37E-03Sll1452 NrtC 21.23 2.49E-01 22.24 1.96E-02 21.58 6.15E-04Sll1453 NrtD 21.05 7.89E-01 ND ND 21.92 3.05E-03Sll0108 Amt1 21.12 5.38E-01 21.20 2.57E-02 21.30 2.93E-02Slr0447 UrtA 21.67 1.02E-05 21.91 4.61E-04 NA NASll0764 UrtD ND ND 21.71 6.98E-04 NA NASll0374 UrtE 21.49 2.79E-02 21.34 1.45E-02 NA NA

HCO32 uptake Slr0040 CmpA 21.37 4.19E-02 1.62 1.75E-03 1.54 5.19E-03

Slr0041 CmpB 1.08 4.69E-01 2.44 4.26E-05 2.20 1.70E-04Slr0043 CmpC 1.24 2.57E-01 2.28 3.25E-04 2.13 3.15E-04Slr0044 CmpD 21.03 8.89E-01 2.36 4.82E-05 1.93 2.07E-04Slr1512 SbtA 21.45 9.34E-03 1.00 9.60E-01 21.03 6.94E-01Slr1513 SbtB 22.19 3.28E-07 21.02 7.85E-01 NA NA

Others Slr0982 RfbB 21.95 4.63E-03 23.09 9.96E-06 NA NASll0064 21.61 1.88E-02 22.44 1.68E-02 NA NASlr1216 MgtE 21.99 2.25E-03 22.40 2.35E-04 NA NASlr0559 NatB 21.21 1.37E-01 21.80 1.41E-03 NA NASlr1740 AppA 21.14 4.18E-02 21.70 4.64E-04 NA NASlr0251 Ycf85 21.08 6.79E-01 21.68 1.73E-02 NA NASll1927 AppF 1.47 1.30E-01 21.64 2.89E-04 NA NASlr0747 GgtA ND ND 21.56 3.45E-03 NA NASll1762 21.50 1.50E-04 21.53 1.61E-02 NA NASll0415 1.13 4.00E-01 21.50 3.41E-02 NA NASlr2131 AcrF 21.49 5.94E-02 21.37 3.76E-02 NA NASlr0369 EnvD 21.15 1.72E-01 21.36 1.87E-03 NA NASll0224 1.30 1.20E-01 21.31 9.42E-03 NA NASll0575 RfbB 21.18 6.57E-01 21.31 4.34E-02 NA NASll1270 BgtB 1.02 9.55E-01 21.39 6.99E-03 NA NASll0672 PacL 1.18 7.37E-02 1.53 4.05E-03 NA NASlr1295 SufA 1.05 4.86E-01 1.58 6.72E-05 NA NASll1598 MntC ND ND 1.65 5.79E-04 NA NASll1481 1.82 6.22E-03 1.80 2.05E-04 NA NASlr1729 KdpB 1.07 8.41E-01 2.34 2.02E-03 NA NA

NDH-1 Slr1291 NdhD2 ND ND ND ND 4.65 2.72E-04Sll1733 NdhD3 2.37 2.68E-02 1.19 1.36E-01 1.09 3.34E-01Sll1735 CupS 21.41 4.12E-04 ND ND 1.07 1.13E-01Sll0026 NdhF4 21.36 4.78E-02 ND ND 21.57 5.11E-03Slr1302 CupB 21.09 2.08E-02 21.56 4.37E-02 21.53 4.16E-03

(Table continues on following page.)





importantly, to expand the scope of quantifiedmembrane-embedded photosynthetic and respira-tory proteins. For the SRM experiments, we selected108 proteins, including those showing differential ex-pression in DDA, plus several other targets that couldprovide complementary information on proteomicchanges of important metabolic routes in the DndbCmutant. The complete list of SRM targets is shown inSupplemental Table S6. Overall, SRM quantitationconfirmed the DDA results; in the case of PsaA andPsaB proteins, however, their up-regulation wasfound to be lower than the FC threshold (Table III).The SRM results obtained for NdbC validated thedeletion of the corresponding protein in the DndbC

mutant. On the other hand, SRM revealed severalnovel differentially regulated proteins that were notdetected in the global approach or were detected andquantified but with a high P value. Importantly, wediscovered that various NDH-1 complexes respon-ded differently to the NdbC mutation. SRM resultsshowed that NDH-11, with the specific NdhD1 sub-unit, and NDH-13, with NdhD3, NdhF3, CupA, andCupS, were not affected (Supplemental Table S6). Incontrast, NdhD2, the specific subunit of the previ-ously elusive NDH-12 complex, was up-regulatedsubstantially (Table III), while NdhF4 and CupB,belonging to the NDH-14 complex, were distinctlydown-regulated. CommonNDH-1 subunits remained

Table III. (Continued from previous page.)

Parameter Protein Protein Symbol HC DDA FC P LC DDA FC P LC SRM FC P

Central metabolism/regulationSll0745 PfkA 21.80 7.16E-02 21.47 5.05E-02 NA NASlr0884 Gap1 21.75 3.36E-06 21.49 1.82E-02 21.43 1.85E-02Slr0394 Pgk 21.46 1.22E-03 21.52 2.58E-03 21.44 2.09E-04Sll0587 Pyk1 21.47 2.26E-02 21.52 3.02E-03 21.42 3.90E-01Slr1843 Zwf 21.35 9.93E-05 21.40 1.47E-03 NA NASlr0452 IlvD 1.10 3.18E-01 1.46 1.43E-03 NA NASll1479 Pgl 21.27 2.85E-02 21.46 8.12E-04 NA NASll0158 GlgB 21.52 6.74E-06 21.29 1.45E-02 NA NASll1502 GltB ND ND 2.32 5.82E-02 NA NASll1499 GltS 21.08 2.24E-01 2.91 6.35E-02 NA NASlr0710 GdhA 1.33 2.82E-04 1.48 3.41E-02 NA NASll1689 SigE ND ND ND ND 21.67 9.22E-03Sll1594 CcmR 1.17 2.75E-01 1.38 2.81E-02 1.21 7.95E-02Ssl0707 PII 21.82 1.22E-04 21.68 4.15E-03 21.70 3.13E-05Ssl0105 PipX 21.54 1.83E-03 21.42 2.15E-03 21.40 5.72E-05Sll0822 1.15 7.33E-01 21.49 9.66E-04 21.35 9.59E-02Sll0359 21.33 6.60E-03 21.35 9.62E-04 21.34 3.16E-03

Photosynthesis and respirationSll1521 Flv1 1.88 1.55E-05 1.60 2.05E-04 1.47 4.94E-04Sll0550 Flv3 1.44 3.37E-03 1.62 3.87E-03 1.44 3.53E-04Sll1317 PetA 1.09 2.40E-01 1.51 8.92E-03 1.33 7.53E-04Slr0342 PetB 1.15 6.40E-02 1.66 1.50E-02 1.41 6.61E-03Sll1316 PetC 1.08 3.23E-01 1.56 1.84E-04 NA NASlr0343 PetD 1.14 2.26E-01 1.48 1.03E-02 1.40 1.00E-04Slr1834 PsaA 1.07 3.96E-01 1.35 1.30E-02 1.22 6.46E-03Slr1835 PsaB 1.09 3.89E-01 1.31 3.12E-02 1.20 3.13E-03Slr1055 ChlH 1.41 6.17E-03 ND ND 2.08 9.75E-03Slr1777 ChlD 1.39 1.76E-02 1.48 6.57E-03 1.26 1.11E-03Sll0247 IsiA ND ND 3.80 1.56E-02 18.70 7.04E-06sll0813 CtaCII 1.55 2.05E-01 21.36 1.82E-03 NA NASll1484 NdbC 210.51 1.05E-07 25.07 1.70E-03 2195.60 4.41E-09

Plasmid encoded Sll6053 28.17 4.50E-05 2128.52 6.57E-06 NA NASll6054 2996.22 1.74E-06 2288.89 2.12E-06 NA NASll6055 210.57 1.18E-07 221.64 1.36E-06 NA NASlr6012 21.31 1.54E-03 28.50 3.25E-06 NA NASlr6013 25.42 1.38E-07 213.27 2.44E-06 NA NASlr6014 24.49 4.61E-04 27.55 1.65E-05 NA NASlr6015 24.05 3.77E-04 27.10 6.50E-05 NA NASlr6016 24.30 6.29E-06 28.84 6.05E-05 NA NASll7062 Csm6 22.78 1.40E-05 25.13 1.17E-05 NA NASll7066 Cas7 219.76 6.19E-07 281.87 3.15E-03 NA NASll7085 Cmr6 22.91 1.97E-04 24.70 2.75E-04 NA NASll7087 Cmr4 23.57 6.95E-08 235.98 2.23E-04 NA NASll7090 Cmr2 23.08 7.95E-06 29.43 2.18E-05 NA NA


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unchanged (Supplemental Table S6). Furthermore, wediscovered the down-regulation of SigE, the group2 s-factor, a decrease of NrtB and NrtD subunits of theNRT transporter, coordinately with NrtC, and an up-regulation of ChlH involved in Chl biosynthesis (Table III).

Western-Blot Analysis of Selected Proteins ProvidedFurther Insights into the Consequences of ndbC Deletion

To verify the proteomic results described above,western-blot analysis of total proteins isolated from LC-grown cells was performed for several targets (Fig. 5A).The results obtained with protein-specific antibodiescorroborated conclusions made based on mass spec-trometry (MS) methods. In particular, it was confirmedthat the PsaB amount was slightly up-regulated inDndbC compared with the wild type (Fig. 5A). More-over, the amount of the small form of ferredoxin-NADP+ oxidoreductase (FNRs) was shown to be higherin DndbC compared with the wild type.

Since the proteomic results showed an interestingeffect of the NdbC deletion on NDH-1 complexes, apossible interplay between NdbC and the NDH-1 vari-antswas addressed. To this end, several NDH-1mutantswere grown in LC conditions, and total proteins wereisolated and immunoblotted with a-NdbC (Fig. 5B).Intriguingly, NdbC was not expressed in detectableamounts if the functional NDH-1 complex (DndhB/M55mutant) was missing (Fig. 5B). In line with this obser-vation, the amount of NdbC was lower compared withthe wild type when NdhD1 or NdhD2 was deleted,and the double deletion of these NDH-1 subunits de-creased the NdbC amount even more. The deletion ofNdhD3 or NdhD4 did not affect the NdbC amountcompared with the wild type.

Furthermore, western blotting was used to reveal thelocalization of NdbC in the cell. Protein fractionsrepresenting the thylakoid membrane, soluble com-partment, and the PM were analyzed with the NdbC-specific antibody. NdbC was clearly located only inthe PM, while no signal was detected from the thyla-koid membrane fraction or from the soluble proteins(Fig. 5C), in line with results obtained by proteomicstudies (Pisareva et al., 2011; Liberton et al., 2016).

Response of DndbC to Light-Activated Heterotrophy andPhotomixotrophic Conditions

Proteomics analysis of the DndbCmutant suggested aputative role for the NdbC protein in general carbonmetabolism, which prompted us to test the behavior ofthe wild type and the DndbC mutant in the presence ofan external carbon source. To this end, the wild-typeand mutant cells were first subjected to LAHG condi-tions, where the growth medium was supported with10 mM Glc and illumination was limited to 10 min(50 mmol photons m22 s21) every 24 h. Under theseconditions, the DndbCmutant practically did not grow(Fig. 6A). Next, the length of the light period was in-creased to 4 hperday (4 h of 50mmolphotonsm22 s21/20hof dark) to change the growth conditions towardmixotrophy, which restored the growth of the DndbCmutant (Fig. 6B). During days 2 to 4, the growth ofthe DndbC mutant was faster compared with thewild type. Under mixotrophic conditions (50 mmol

Figure 5. Western-blot analysis of the wild type (WT), DndbC, and dif-ferent NDH-1 mutants. A, Immunoblotting with protein-specific anti-bodies. B, Different Synechocystis NDH-1 mutants immunoblotted witha-NdbC. C, The thylakoidmembrane, PM, and soluble fractions of thewildtype immunoblotted with a-NdbC, a-PsaB, and a-allophycocyanin (APC).All the cells for protein isolation were grown in LC, and samples wereloaded based on total protein amount. Vertical lines indicate that thesamples, although run on the same gel, are not from adjacent wells.






photons m22 s21 continuous illumination + 10 mM

Glc), there was no difference in the growth based onOD between the wild type and DndbC (Fig. 6C).Nevertheless, it is important to note that, also in thepresence of Glc, the DndbC cells were considerablylarger compared with the wild type (SupplementalFig. S4), which increases OD and is in line with thephenotype observed under autotrophic growth con-ditions (Fig. 3A).

Redox State of the Electron Transport Chain in Darkness inthe DndbC Mutant

To monitor more precisely the redox state of the ETCin DndbC during incubation in darkness, the cells weregrown first in autotrophic conditions and then trans-ferred to the dark for 24 h. QA

2 reoxidation kinetics andthe fluorescence induction dynamics measured fromthe 24-h dark-adapted DndbC cells did not differ strik-ingly from those in the wild type (Fig. 7, A and B;Supplemental Fig S3, A–C). To address the possibleeffect of Glc on ETC in darkness, 24-h dark-incubatedcells were supplemented with 10 mM Glc for 15 minbefore the Chl fluorescence measurements. A shortincubation with Glc markedly slowed down QA

2

reoxidation kinetics in both the wild-type and DndbCcells, yet the kinetics became much slower in DndbC(Fig. 7C), implying a modification of the PSII acceptorside of the mutant, presumably due to the reduced PQpool. When the flash-induced fluorescence yield wasmonitored in the presence of DCMU, reflectingmore thedonor side function of PSII, no clear difference was ob-served between the wild type and DndbC (SupplementalFig. S3D). Next, the dynamic changes in the redox statusof the PQ pool in dark-incubated cells supplementedwith Glc were studied by Chl fluorescence measure-ments using a saturating pulse method (Fig. 7D). Asudden exposure of the dark-incubated wild-type cellsto steady-state actinic light induced a transient increasein the Fs level with a concomitant decrease of DF,reflecting a reduced PQ pool during the first 3 min ofillumination. The DndbC cells demonstrated a higherFs level and low Fm

D and DF values compared with thewild type (Fig. 7D). These results clearly showed thatthe addition of Glc in darkness triggers much strongerreduction of the PQ pool in DndbC than in the wildtype and that the subsequent exposure to moderatelight is capable of oxidizing the ETC.

DISCUSSION

In cyanobacteria, the physiological role of the NDH-2enzymes remains largely unknown, particularly incomparison with the much better characterizedNDH-1 complexes playing important roles in respi-ration, CET, and CO2 uptake (for review, see Peltieret al., 2016). It was hypothesized previously that, inSynechocystis, the NDH-2s do not have a metabolically

Figure 6. Growth of the wild type (WT; black squares) and the DndbCmutant (red circles) in the presence of Glc. A, Under LAHG conditions(the cells grown in darkness were exposed to 50 mmol photons m22 s21

light for 10 min once in 24 h). B, Under a 4-h photoperiod of 50 mmolphotons m22 s21/20 h of darkness. C, Under mixotrophic condi-tions (continuous illumination of 50 mmol photons m22 s21). In allcases, 10 mM Glc was added to the growth medium. Values aremeans 6 SD; n = 3.


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relevant role in respiratory energy production and,instead, were postulated to be involved in regulatoryfunctions, yet with no knowledge of specific targetsso far (Howitt et al., 1999). Here, we addressed thephysiological role of NdbC, one of the three NDH-2sencoded in the genome of Synechocystis. The deletionof NdbC (Fig. 1) revealed several significant rear-rangements in the physiology, metabolism, and mor-phology under autotrophic growth conditionswithoutaffecting respiration (Table II) and, in addition, dis-closed an indispensable function for NdbC underLAHG conditions. It is demonstrated that NdbC, lo-calized in the PM (Fig. 5C), has a significant role incarbon allocation between the storage and variousbiosynthetic pathways (for LC conditions, this is pre-sented schematically in Fig. 8).

Deletion of NdbC Distorts Cell Division

When the DndbC mutant was grown under autotro-phic conditions, in HC or LC, visible morphologicalchanges were observed. The cell size became consid-erably bigger (Fig. 3A), with increase in the content ofintracellular total protein, Chl, and dry weight com-pared with the wild type (Table I). Based on the cellnumber, DndbC demonstrated substantially slowergrowth compared with the wild type in both LC andHC (Fig. 2, C and D). This was corroborated by theproteomic examination of DndbC. The expression ofspecific cell division-related proteins (Mazouni et al.,2004) was clearly altered in DndbC, including down-regulation of FtsZ, ZipN, MinC, MinD, and MinE pro-teins in HC and LC (Table III). It is possible, however,that the influence of the NdbC deletion on cell divisionmay be a secondary effect, as some global regulators ofgene expression, such as the group 2 s-factor SigE(detected only in LC), the AbrB-like transcription factor

Sll0822 (in LC), and components of the circadian clock(KaiB and KaiC), were likewise down-regulated in theDndbC mutant. The influence of these proteins on celldivision was demonstrated previously by Osanai et al.(2013) for SigE, by Yamauchi et al. (2011) for Sll0822,and in a review by Cohen and Golden (2015) for Kaiproteins. In addition, several proteins encoded bypSYSX and pSYSA were markedly down-regulated inDndbC compared with the wild type in both HC and LCconditions (Table III). Among them are CRISPR2 andCRISPR3 (Scholz et al., 2013), which belong to theCRISPR/Cas systems responsible for resistance tohorizontal gene transfer, including phage transduc-tion, transformation, and conjugation (Marraffini andSontheimer, 2008; for review, see Sorek et al., 2013).However, the influence of NdbC deletion on these pro-teins remains unclear, since the mutant could have ex-perienced problems with plasmid replication connectedto the hindrance in cell division.

Carbon Catabolism Is Down-Regulated in the Absenceof NdbC

Measurements of the intracellular glycogen showedthat this important reserve of carbon was significantlyhigher in the DndbC mutant compared with the wildtype, both in LC and HC (Fig. 3D). The increase of in-tracellular glycogen per cell in the mutant was mark-edly more profound than the increase of the Chlamount or of the total protein content (Table I). In linewith this, the electron microscopy of DndbC cells in LCrevealed the occurrence of multiple dark spots that,probably, are glycogen granules (Fig. 3B). The accu-mulation of glycogen could be due to a misbalancebetween synthesis and degradation. It is important tonote, however, that amounts of enzymes performing

Figure 7. Fluorescence relaxation andinduction curves monitored from wild-type (WT) and DndbC mutant cells. Aand B, Cells were grown for 48 h under50mmol photonsm22 s21 in LC and thenshifted to darkness for 24 h. C and D,Cells were grown for 48 h in 50 mmolphotons m22 s21 in LC and shifted todarkness for 24. For measurements, thecell suspension was supplemented with10 mM Glc for 15 min in the dark. In theflash-induced fluorescence decay ex-periment, the values are means 6 SD;n = 3, and the fluorescence inductioncurves are representative of three bio-logical repetitions. r.u., Relative units.





glycogen synthesis (GlgA,B,C) or glycogen degradation(GlgX,P) were not coordinately altered in the DndbCmutant comparedwith the wild type either in LC orHC(Table III). Thus, the shift of the balance has to be causedby other reasons. Indeed, the proteomic analysis revealedalterations in protein contents of metabolic pathways in-volved in intracellular carbon allocation in LC and in HC;Gap1, Pgk, and Pyk1, the three enzymes catalyzing uni-directional reactions in glycolysis, were down-regulatedin the DndbC mutant (Table III).

Considering the first steps of the glycolytic route,the OPP pathway and the Embden-Meyerhof-Parnas(EMP) pathway were down-regulated in both studiedconditions. The amounts of two key enzymes, Glc-6-Pdehydrogenase (Zwf) and 6-phosphogluconolactonase(Gnd), specific for OPP aswell as PfkA involved in EMP

were likewise reduced in the DndbC mutant (Table III).These results are in agreement with earlier studiesshowing that the mRNA amount of ndbC follows cir-cadian rhythm peaking in the dark together with genesinvolved in sugar catabolism (Kucho et al., 2005;Layana and Diambra, 2011) as well as in the beginningof heterotrophy (Lee et al., 2007). Conversely, 6-PGhydratase (IlvD), which belongs to the recently dis-covered Entner-Doudoroff pathway in cyanobacteria(Chen et al., 2016), was up-regulated in DndbC in LC(Table III) and might partially compensate for thedown-regulation of EMP and OPP.

Overall, the observed decrease in the expressionlevels of many glycolytic enzymes suggests that theNdbC deletion caused the slowdown of sugar catabo-lism, probably resulting in the reduction of pyruvate

Figure 8. Schematic model representing the effects of NdbC deletion on protein expression in Synechocystis cells in LC con-ditions comparedwith thewild type. The up-regulated or accumulated protein complexes,metabolic routes, or compounds in theDndbC mutant are marked in red, and the down-regulated ones are marked in blue. Solid arrows represent metabolic routes oruptake, dashed arrows represent linear electron transfer, and dotted arrows represent CET. TM, Thylakoid membrane.


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and other metabolites at the consequent steps of carbonassimilation. Since NADH is utilized in glycolysis, inparticular by Gap1, which is an essential component ofthis process, the direct effect of the NdbC deletioncannot be excluded. However, other factors couldcontribute to the slowdown of sugar catabolism. Forexample, since SigE positively regulates the expressionof genes functioning in sugar catabolism (Osanai et al.,2005), its down-regulation, mentioned above, togetherwith the up-regulation of its anti-s-factor ChlH (Osanaiet al., 2009; Table III), could result in reduced amountsof glycolytic enzymes and, thus, promote glycogen ac-cumulation. In addition, the RpaA protein, the masterregulator of circadian control, was down-regulated inDndbC in LC (Table III). Its involvement in adjusting theexpression levels of enzymes participating in glycolysisand OPP has been shown in Synechococcus elongatusPCC 7942 (Markson et al., 2013). Concerning metabolicreactions downstream from glycolysis, our proteomicanalyses did not detect coordinated changes concerningenzymes in the tricarboxylic acid cycle. In contrast, GltBand GltS, two Glu synthetases of the GS-GOGATpathway, were up-regulated (in LC) at the protein leveltogether with the Gln dehydrogenase GdhA (Table III).

The Deletion of NdbC Alters the Expression of MultipleTransport and Binding Proteins Especially in LC

Even though similar changes in the proteome relatedto cell division and sugar catabolism were detectedbetween the wild type and DndbC both in HC and LC,the deletion of NdbC had an evidently more profoundeffect on the expression of a large group of trans-porters in LC compared with HC. Most of these trans-porters were reported previously to localize in the PM(Pisareva et al., 2011), in accordance with the location ofthe NdbC protein (Fig. 5C). The transport of variousnutrients, like potassium and metal ions, amino acids,and polysaccharides (Table III), demonstrated markeddown-regulation in the DndbC mutant except for theHCO3

2 transporter BCT1 (Omata et al., 1999), whichwas up-regulated in the DndbC mutant comparedwith the wild type. The strongest down-regulation wasobserved for both ABC-type inorganic phosphatetransporters (Table III), the constitutive low-affinityphosphate transporter Pst1 and the inducible, high-affinity phosphate transporter Pst2 (Pitt et al., 2010;Burut-Archanai et al., 2011). Furthermore, in the DndbCmutant, the levels of enzymes involved in nitrogenuptake, NRT transporters responsible for nitrate andnitrite uptake, as well as urea and ammonium trans-porters were down-regulated (Table III). Such coordi-nated down-regulation of different transporters in LC,when there is a shortage of terminal acceptors, unlike inHC, can be a protective response of the DndbC cellsaimed to regulate a redox homeostasis of the cells by ioninflux and efflux.In addition to transporter proteins, the expression of

several regulators of transporter functions is altered in

DndbC. While CmpR inducing BCT1 expression(Nishimura et al., 2008; Daley et al., 2012) was not af-fected (Supplemental Table S5), the Sll0822 protein,which functions as a supplementary inducer of theCmp operon (Orf et al., 2016), was down-regulated inDndbC, as was already mentioned above (Table III).Thus, the up-regulation of the Cmp operon in DndbCwas not caused by known regulating mechanisms. Theregulators of the nitrogen assimilation genes PII andPipX (Llácer et al., 2010; Espinosa et al., 2014; for re-view, see Forchhammer, 2004) were reduced in theDndbC mutant (Table III), while the global nitrogenregulator NtcA remained unchanged (SupplementalTable S5). Decreased amounts of PII and PipX, whichpromote the deactivation of NtcA (Luque et al., 2004),as well as down-regulation of Sll0822, also an inducer ofnitrogen transport (Ishii and Hihara, 2008; Yamauchiet al., 2011), could cause the low accumulation ofnitrogen transporters induced by the NdbC deletion,particularly in LC. Thus, in LC conditions, the deletionof NdbC disrupts the normal behavior of multipletransport systems, mainly of ABC-type transporterslocated in the PM.

Cross Talk between NdbC and the NDH-1 Complex: LowATP Production from Sugar Catabolism in DndbC IsCompensated by CET

Cyanobacteria can incorporate inorganic carbon (Ci)in the form of CO2 utilizing the low-CO2-induciblehigh-affinity NDH-13 complex and the constitutivelyexpressed low-affinity NDH-14 complex (for review,see Battchikova et al., 2011). MIMS measurementsrevealed that the fast phase of CO2 uptake was de-creased in the DndbC mutant in LC (Table II). Theproteomic results did not indicate changes in the ex-pression of specific subunits of NDH-13 in the DndbCmutant in LC (Table III). In contrast, NdhF4 and CupB,specific subunits of NDH-14, were coordinately down-regulated in DndbC compared with the wild type inLC (Table III), being in line with MIMS results. To getinsights into the connection between the NdbC de-letion and the expression of NDH-14, we performed awestern-blot analysis of several mutants lacking spe-cific subunits of distinct NDH-1 variants using theNdbC-specific antibody (Fig. 5B). Remarkably, NdbCwas severely down-regulated in the M55 strain, indi-cating some cross talk between this protein andNDH-1.However, it is important to note that the amount ofNdbC in the DndhD4 mutant (and in DndhD3/D4) wascomparable with that in the wild type. Thus, NdbC isinvolved in the accumulation of NDH-14, but notthe other way around. Most probably, NdbC affectsNDH-14 indirectly, since they are located in differentmembrane compartments (Liberton et al., 2016).

In the DndbC mutant, CET around PSI was clearlyelevated, as deduced from the high amplitude of F0 rise(Fig. 4D; Mi et al., 1992a; Holland et al., 2015) and fromslower oxidation and faster rereduction kinetics of P700








(Fig. 4C; Mi et al., 1992b; Fan et al., 2007). Proteomicanalysis revealed that two variants of the NDH-1 com-plex, NDH-11 and NDH-12, which are involved inCET (Mi et al., 1992a), demonstrate different responsesto NdbC deletion. NDH-11 was not affected, accordingto the levels of NdhD1 specific for this complex(Supplemental Table S6). In contrast,marked up-regulationwas observed for NdhD2 (Table III), the specificsubunit of NDH-12. The latter complex is present inSynechocystis cells inmuch smaller amounts thanNDH-11(Herranen et al., 2004), but, despite a low abundance,NDH-12 that accumulated in the DndbC mutant con-tributed to the significant increase in CET (Fig. 4, C andD). Western-blot analysis demonstrated that, in theDndhD2mutant (and in DndhD1/D2), the NdbC amountwas clearly lower comparedwith the wild type (Fig. 5B).Thus, NdbC andNDH-12 affect each other, in contrast toNDH-14.

Furthermore, according to western-blot analysis,FNRs was up-regulated in DndbC (Fig. 5A). This formof FNR oxidizes NADPH and accumulates in hetero-trophic conditions, possibly enhancing CET by trans-ferring electrons via ferredoxin to NDH-1 (Thomaset al., 2006; Korn, 2010). The elevated CET in the mu-tant was accompanied by an increase of the Cyt b6fcomplex (PetA,B,C,D; Table III). Enhanced CET andthe corresponding increase in ATP production cancompensate for lower ATP yield from glycolytic re-actions in the mutant. In addition, the up-regulation ofoxygen photoreduction (Table II), operated by theincreased Flv1 and Flv3 proteins (Table III), possiblycontributes to ATP production via a functional water-water cycle and increases the photoprotection of photo-synthetic protein complexes, especially PSI (Allahverdiyevaet al., 2015).

Even though the carbon catabolic reactions weredistorted due to the deletion of NdbC, photosynthesisin general was not compromised dramatically either inLC or in HC; no significant difference was detected inFv/Fm, in Pm, or in Y(I) between the wild type andDndbC (Fig. 4B; Supplemental Table S1). However,both the gross and net oxygen evolution (Table II), aswell as Y(II) (Fig. 4A), of DndbCwere somewhat lowercompared with the wild type, demonstrating excesselectron pressure downstream of PSII, which is inagreement with elevated CET. Cooley and Vermaas(2011) showed that the PQ pool of Synechocystis defi-cient of all three NDH-2s (DndbABC) was more oxi-dized during illumination, which is contrary to theredox status of the PQ pool in DndbC. Thus, we cannotexclude the possibility that the redox state of theNADH pool (by NDH-2) and the NADPH pool (viaNDH-1) can influence each other, at least in the case ofDndbC.

NdbC Is Indispensable in LAHG Conditions

The consequences of the blockage of the glycolyticroute, evident even in autotrophic conditions, should

become more profound when photosynthesis is limitedand all the energy for cellular needs must be derivedfrom the degradation of sugars. This was experimen-tally proven by the inability of the DndbC mutant togrow under LAHG conditions (Fig. 6A). In heterotro-phic growth conditions, 90% of supplemented Glc isdirected to OPP and only about 5% enters glycolysis(Yang et al., 2002). In DndbC, OPP is strongly down-regulated even in autotrophic conditions, and this isexpected to have serious consequences on nucleotidebiosynthesis upon LAHG conditions, due to the factthat OPP is the only source for required intermediatesin darkness (Kruger and von Schaewen, 2003). Simi-larly, DsigE, which is deficient in sugar catabolism,shows retarded growth in LAHG conditions (Osanaiet al., 2005). Fluorescence measurements further con-firmed the problems of DndbC. The ETC became se-verely and rapidly overreduced in the dark and duringthe dark-light transition upon supplementation of theculture with Glc (Fig. 7). The reason for Glc toxicitycould be a reprogramming of carbon metabolism and ahigh production of reactive oxygen species triggeringoxidative stress and preventing cell growth, eventu-ally causing cell death (Latifi et al., 2009; Narainsamyet al., 2013). In mixotrophic conditions, when the activeCalvin-Benson cycle functions together with the OPPpathway (Yang et al., 2002), the DndbCmutant was ableto grow (Fig. 6C). The up-regulated Entner-Doudoroffpathway in DndbC (in LC), which is physiologicallysignificant under mixotrophy (Chen et al., 2016), alsocould contribute to carbon flux distribution in the cells.Nevertheless, similar problems were present in celldivision, as demonstrated in autotrophic conditions(Supplemental Fig. S4). Thus, the maintenance ofDndbC growth in the medium supplemented with Glcis dependent on the regular interruption of darknesswith light periods (Fig. 6B) to efficiently oxidize theETC (Fig. 7D).

CONCLUSION

NADH and NADPH are fundamental reducingequivalents for the maintenance of cellular metabolism.In photosynthetic organisms, the NADH/NAD+ statusis known to regulate the coordination of nitrogen as-similation and carbon metabolism by various mecha-nisms (Dutilleul et al., 2005; Kämäräinen et al., 2017;Pétriacq et al., 2017). By applying a number of physio-logical measurements and the proteomics approach, weshowed that the deletion of NdbC has a distinct effecton the metabolism of Synechocystis. We postulate thatNdbC located in the PM is an important component inthe regulation of the cytosolic NADH/NAD+ balance.In photoautotrophic growth conditions with activephotosynthetic carbon assimilation, Synechocystis cellsdo survive in the absence of NdbC. Yet, importantly,glycolytic enzymes are down-regulated in the DndbCmutant under these growth conditions (Fig. 8), ap-parently due to the elevated NADH/NAD+ ratio.


Huokko et al.






Consequently, cells need to compensate for the lackof the enzyme by the modulation of several meta-bolic routes. This provides evidence that NdbC isimportant in fine-tuning the balance between vari-ous metabolic pathways when the cytosolic redoxbalance is influenced mainly by light reactions in thethylakoid membrane. However, the function ofNdbC becomes crucial for cell viability in hetero-trophic growth conditions. This indicates an essen-tial role of NdbC in the regulation of the cytosolic redoxenvironment when glycolysis is the sole pathway to as-similate carbon for metabolic needs.

MATERIALS AND METHODS

Strains and Culture Conditions

A Glc-tolerant Synechocystis sp. PCC 6803 (wild type) was used as the ref-erence strain. The DndbC mutant was obtained by disruption of the ndbC geneby Zeocin resistance cassette (for details, see “Mutagenesis” below). Pre-experimental cultures were always grown in BG11 medium buffered with20mMHEPES-NaOH (pH 7.5) under continuouswhite light of 50mmol photonsm22 s21 at 30°C, under air enriched with 3% CO2 (HC) with agitation of150 rpm. All experimental cultures were grown in BG11medium buffered with20 mM HEPES-NaOH (pH 7.5) under continuous illumination of 50 mmolphotons m22 s21, unless mentioned otherwise, and in growth chambers withcool-white light-emitting diodes (AlgaeTron AG130 by PSI Instruments). Insome experiments, cells were grown in different dark-light cycles where illu-mination was 50 mmol photons m22 s21 during the light period. During ex-perimental cultivations, cells were grown at 30°C with agitation of 150 rpmwithout antibiotics, and OD750 was measured using the Lambda 25 UV/VISspectrometer (PerkinElmer). Glc (10 mM) was provided when mentioned. Forphysiological measurements and protein extraction, cells were harvested frompreexperimental cultures at themidlogarithmic phase, inoculated in fresh BG11medium at OD750 = 0.1, and grown at HC or inoculated to OD750 = 0.5 andshifted to ambient air (LC) for 3 d. For activity measurements, cells wereharvested and resuspended in fresh BG11 medium at the desired Chl con-centration and acclimated under the respective growth conditions before themeasurements.

Mutagenesis

To ensure the correct wild-type background, the interrupted ndbC gene(sll1484) with Zeocin resistance cassette and its surroundingswere amplified byPCR with forward primer 59-GCTGGTACGGTGGAAAGTCT-39 (in sll1483)and reverse primer 59-CTACCGATTCATACCGGGGCTA-39 (in the end ofsll1484) from the DndbC mutant constructed by Howitt et al. (1999). Theresulting PCR product was transformed into the wild-type cells using a stan-dard established protocol (Eaton-Rye, 2011). Transformantswere segregated bygradually raising the concentration of Zeocin (from 4 to 20 mgmL2 1). Completesegregation of the mutation was confirmed by PCR (Supplemental Fig. S1).

Light Microscopy and Cell Counting

Cell suspensions in BG11 were examined using the Leitz Orthoplan LargeField ResearchMicroscope and photographedwith a digital microscope camera(Leica DFC420C). Digital slide photograph brightness and contrast were opti-mized with Leica Application Suite version 4.1. and CorelDRAW X7. A Bürkercounting chamber (Marienfeld-Superior) was applied to quantify the number ofcells in a culture (normalized to OD750 = 1).

Dry Weight and Chl Determination

Twenty milliliters of cell culture was passed through a prewashed andweighed glass-fiberfilter (Millipore). Filterswere dried at 60°C for 24 h, kept in adesiccator for 24 h, weighed, and normalized to premeasured OD750. Chl wasextracted from cells with 100% methanol, and Chl concentrations were

determined by measuring OD665 and multiplying it with extinction coefficientfactor 12.7 (Meeks and Castenholz, 1971).

Transmission Electron Microscopy

Prefixation of samples was done with 5% glutaraldehyde in 0.16 M s-collidinbuffer, pH 7.4. The rest of the sample preparation and microscopy work usingthe JEM-1400 Plus Transmission Electron Microscope was done in the Labo-ratory of Electron Microscopy, University of Turku.

Glycogen Determination

Ten milliliters of cultures at OD750 = 1 was collected, and cells were lysed bysonication (BIORUPTOR; CosmoBio). Glycogen determination was performedwith the Total Starch Assay Kit (Megazyme) according to the manufacturer’sinstructions.

MIMS

MIMS measurements were performed as described by Ermakova et al. (2016).Gas-exchange kinetics and rates were determined according to Beckmannet al. (2009).

Fluorescence Measurements

The Chl fluorescence from intact cells was recorded with a pulse amplitude-modulated fluorometer (Dual-PAM-100; Walz). Before measurements, cellsuspensions at a Chl concentration of 15mgmL21 were dark adapted for 10min.Saturating pulses of 5,000 mmol photons m22 s21 (300 ms) and strong FR light(720 nm, 75 W m22) were applied to samples when required. Y(II) was calcu-lated as (Fm9 2 Fs)/Fm9. The Fm

D was recorded after the first saturating pulsewithout actinic light, the Fm

FR was recorded subsequent to 8 s of strong FR il-lumination combined with saturating pulse, and the maximal fluorescenceduring illumination (Fm9) was recorded upon saturating pulse during illumi-nation. The maximum quantum yield of PSII was calculated as Fv/Fm = (Fm 2F0)/Fm and measured in the presence of 20 mMDCMU from dark-adapted cellsupon the application of red actinic light of 200 mmol photons m22 s21 for 1 min.

The F0 rise was measured in darkness after the termination of actinic light.Cell suspensions at a Chl concentration of 15 mg mL21 were dark adapted for10 min before the measurements and exposed to 1 min of red actinic light in-tensity of 50 mmol photons m22 s21.

The kinetics of the Chl fluorescence decay after a single-turnover saturatingflash was monitored using a fluorometer (FL 3500; PSI Instruments) accordingto Vass et al. (1999). Cells were adjusted to a Chl concentration of 7.5 mg mL21

and dark adapted for 5 min before measurements. When indicated, measure-ments were performed in the presence of 20 mM DCMU.

The Maximum and Effective Yield of PSI andOxidoreduction of P700

The P700 signal was recorded with a pulse amplitude-modulated fluo-rometer (Dual-PAM-100;Walz). Beforemeasurements, cell suspensions at a Chlconcentration of 15 mg mL21 were dark adapted for 10 min. The maximalchange of P700 upon transformation of P700 from the fully reduced to the fullyoxidized state, Pm, was achieved by the application of a saturation pulse afterpreillumination with FR light. The Y(I) was calculated as Y(I) = (Pm9 – P)/Pm.

Oxidation and rereduction of P700weremonitored from the cell suspensionsat a Chl concentration of 20 mg mL21. The cells were dark adapted for 2 minbefore measurements. For P700 oxidation, cells were illuminated with strongFR light (720 nm, 75 W m22) for 5 s, and the subsequent rereduction wasrecorded in darkness.

Proteomics

Western Blotting: Protein Isolation, Electrophoresis, andImmunodetection

Total protein extracts of Synechocystis cells were isolated as describedby Zhang et al. (2009). Proteins were separated by 12% (w/v) SDS-PAGE






containing 6 M urea, transferred to a polyvinylidene difluoride membrane(Immobilon-P; Millipore), and analyzed with protein-specific antibodies.The membrane fractions to confirm the localization of NdbCwere preparedas described by Zhang et al., (2004).

Sample Preparation for MS

Total proteins were isolated from cell cultures as described by Zhang et al.(2009). An equivalent of 30 mg of total protein was mixed with 23 Laemmlibuffer (with 8 M urea) in a 1:1 ratio and loaded on a 12% (50% acrylamide and1.3% bis-acrylamide) stacking gel (0.5 M Tris-HCl, pH 6.8) containing 6 M ureaand no SDS. Gel bands containing all proteins were reduced, alkylated, anddigested according to the protocol described by Shevchenko et al. (1996, 2006).Extracted peptides were vacuum dried and further solubilized in 2% acetoni-trile (ACN) and 0.1% formic acid (FA) just before MS analysis. Samples wereprepared in four biological replicates from the wild type and the DndbCmutant.

MS Analyses

DDA

The peptide mixtures from the wild type and the DndbC mutant were ana-lyzed on a liquid chromatography-MS/MS system with the DDA method inthree biological replicates. An equivalent of 200 ng of peptideswas separated ona nano-liquid chromatography system (EasyNanoLC 1000; Thermo FisherScientific) coupled to QExactive (Thermo Fisher Scientific). The samples werefirst loaded on a trapping column and separated in line on a 15-cm C18 column(75 mm 3 15 cm, Magic 5-mm 200 Å C18; Michrom BioResources). The mobilephase consisted of water:ACN (98:2, v/v) with 0.2% FA (solvent A) orACN:water (95:5, v/v) with 0.2% FA (solvent B). To separate the peptidemixture, a 110-min gradientwas used that startedwith 2%B, increased to 20% Bin 70 min and further to 40% B in 30 min, and then reached 100% in 5 min. MSdata were acquired automatically in positive ionization mode with 2.3-kVionization potential using Thermo Xcalibur software (Thermo Fisher Scientific).The MS method combined an MS survey scan of mass range 300 to 2,000mass-to-charge ratio and MS/MS scans for up to 10 most intense peaks fromthe full scan,with charge+2 or +3. The spectrawere registeredwith a resolution of70,000 and 17,500 (at mass-to-charge ratio 200) for full scan and for fragment ions,respectively. Fragmentation of ions occurred in high collision dissociation modewith normalized collision energy of 27%. The automatic gain control was set tomaximum fill time of 100 and 250 ms and to obtain the maximum number of 3e6and 2e4 ions for MS and MS/MS scans, respectively.

Protein Identification

The raw files were searched against the Synechocystis protein database re-trieved fromCyanobase (Kaneko et al., 1996) using an in-houseMascot (version2.4) search engine (Perkins et al., 1999) and further analyzed using ProteomeDiscoverer (version 1.4) software (Thermo Scientific). The Mascot search pa-rameters were set to trypsin as an enzyme with two miscleavages allowed, Metoxidation as variable modification, and carbamidomethylation as the fixed one.Precursor mass tolerance was restricted to monoisotopic mass of 64 ppm andfragment ion to 60.02 D. For validation of the spectrum identifications, thePercolator algorithm was used with a relaxed false discovery rate of 0.05.

DDA Quantification

Global label-free quantification for DDA data was performed using Pro-genesis QI for proteomics, LC-MS 4.0 (Nonlinear Dynamics). Peptide ions wereidentified with identification results from the DDA experiment. Proteinswere identified and quantified with at least two peptides. Protein abun-dances were estimated based on volumes of the peaks representing itspeptides detected in three biological replicates. Relative protein quantifi-cation was performed using all identified with 610 ppm mass error forprecursor mass, with nonconflicting peptides per protein. The statisticalsignificance threshold in ANOVA was set to P , 0.05, and the practicalsignificance threshold for FC was set to 61.3.

Targeted Quantification

For targeted quantification with SRM, a spectral library was created inSkyline (MacLean et al., 2010) based on the DDA results. SRM analysis wasperformed on wild-type and DndbC mutant samples injected with four

biological replicates according to the protocol described by Vuorijoki et al.(2016), with several modifications. The samples were injected with two tran-sition lists, comprising assays for 109 proteins (106 targets and three house-keeping proteins), to ensure dwell time of at least 20 ms with cycle time of 2.5 s.The mobile phases used in chromatographic separation were 0.1% FA (A) and80% ACN/0.1% FA (B). The 55-min gradient started with 8% B, which in-creased to 26% in 35 min and then to 43% in 15 min. The SRM assays for Syn-echocystis proteins were loaded from the Panorama Public (Sharma et al., 2014)data repository, and the assays for the missing targets were developedaccording to the published protocol. Data analysis was performed inRStudio with MSstats algorithm version 3.5.1 (Choi et al., 2014). Data werenormalized with global standard normalization with the following housekeepingpeptides: sll1818, FSLEPLDR and SYTDQPQIGR; slr0638, GVIAVTER; andsll0145, AISLSDLGLTPNNDGK.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. PCR analysis to verify full segregation of theDndbC mutant.

Supplemental Figure S2. Reverse transcription-quantitative PCR analysisof sll1485 and sll1486 expression in the wild type and the DndbC mutant.

Supplemental Figure S3. Relaxation of the flash-induced fluorescenceyield in darkness from the wild type and the DndbC mutant in differentconditions.

Supplemental Figure S4. Light microscope images from wild-type andDndbC cells grown under 4 h of 50 mmol photons m22 s21/20 h ofdark + 10 mM Glc and under mixotrophic conditions.

Supplemental Table S1. Photosynthetic parameters of the wild type andthe DndbC mutant grown in LC and HC conditions.

Supplemental Table S2. Identification of proteins in the wild type and theDndbC mutant grown under HC conditions with DDA.

Supplemental Table S3. Identification of proteins in the wild type and theDndbC mutant grown under LC conditions with DDA.

Supplemental Table S4. Results of global protein quantification for theDndbC mutant versus the control (wild type) grown under HC condi-tions (P # 0.05).

Supplemental Table S5. Results of global protein quantification for theDndbCmutant versus the control (wild type) grown under LC conditions(P # 0.05).

Supplemental Table S6. Results of SRM protein quantification for theDndbC mutant versus the control (wild type) grown under LC conditions.

ACKNOWLEDGMENTS

We thank Prof. Teruo Ogawa and Prof. Wim Vermaas for sharingSynechocystis mutants as well as Anita Santana Sanchez for helping withMIMS measurements.

Received March 23, 2017; accepted May 19, 2017; published May 22, 2017.

LITERATURE CITED

Allahverdiyeva Y, Isojärvi J, Zhang P, Aro EM (2015) Cyanobacterial oxy-genic photosynthesis is protected by flavodiiron proteins. Life (Basel) 5:716–743

Alpes I, Scherer S, Böger P (1989) The respiratory NADH dehydrogenaseof the cyanobacterium Anabaena variabilis: purification and characteri-zation. Biochim Biophys Acta 973: 41–46

Anderson L, Hunter CL (2006) Quantitative mass spectrometric multiplereaction monitoring assays for major plasma proteins. Mol Cell Pro-teomics 5: 573–588

Battchikova N, Eisenhut M, Aro EM (2011) Cyanobacterial NDH-1 com-plexes: novel insights and remaining puzzles. Biochim Biophys Acta1807: 935–944


Huokko et al.













Beckmann K, Messinger J, Badger MR, Wydrzynski T, Hillier W (2009)On-line mass spectrometry: membrane inlet sampling. Photosynth Res102: 511–522

Burut-Archanai S, Eaton-Rye JJ, Incharoensakdi A (2011) Na+-stimulatedphosphate uptake system in Synechocystis sp. PCC 6803 with Pst1 as amain transporter. BMC Microbiol 11: 225

Carrie C, Murcha MW, Kuehn K, Duncan O, Barthet M, Smith PM, EubelH, Meyer E, Day DA, Millar AH, et al (2008) Type II NAD(P)H dehy-drogenases are targeted to mitochondria and chloroplasts or peroxisomesin Arabidopsis thaliana. FEBS Lett 582: 3073–3079

Chen X, Schreiber K, Appel J, Makowka A, Fähnrich B, Roettger M,Hajirezaei MR, Sönnichsen FD, Schönheit P, Martin WF, et al (2016)The Entner-Doudoroff pathway is an overlooked glycolytic route incyanobacteria and plants. Proc Natl Acad Sci USA 113: 5441–5446

Choi M, Chang CY, Clough T, Broudy D, Killeen T, MacLean B, Vitek O(2014) MSstats: an R package for statistical analysis of quantitative massspectrometry-based proteomic experiments. Bioinformatics 30: 2524–2526

Cohen SE, Golden SS (2015) Circadian rhythms in cyanobacteria. Micro-biol Mol Biol Rev 79: 373–385

Cooley JW, Vermaas WF (2001) Succinate dehydrogenase and other res-piratory pathways in thylakoid membranes of Synechocystis sp. strainPCC 6803: capacity comparisons and physiological function. J Bacteriol183: 4251–4258

Daley SM, Kappell AD, Carrick MJ, Burnap RL (2012) Regulation of thecyanobacterial CO2-concentrating mechanism involves internal sensingof NADP+ and a-ketogutarate levels by transcription factor CcmR. PLoSONE 7: e41286

Desplats C, Mus F, Cuiné S, Billon E, Cournac L, Peltier G (2009) Char-acterization of Nda2, a plastoquinone-reducing type II NAD(P)H de-hydrogenase in Chlamydomonas chloroplasts. J Biol Chem 284: 4148–4157

Dutilleul C, Lelarge C, Prioul JL, De Paepe R, Foyer CH, Noctor G (2005)Mitochondria-driven changes in leaf NAD status exert a crucial influ-ence on the control of nitrate assimilation and the integration of carbonand nitrogen metabolism. Plant Physiol 139: 64–78

Eaton-Rye JJ (2011) Construction of gene interruptions and gene deletionsin the cyanobacterium Synechocystis sp. strain PCC 6803. Methods MolBiol 684: 295–312

Ermakova M, Huokko T, Richaud P, Bersanini L, Howe CJ, Lea-Smith DJ,Peltier G, Allahverdiyeva Y (2016) Distinguishing the roles of thylakoidrespiratory terminal oxidases in the cyanobacterium Synechocystis sp.PCC 6803. Plant Physiol 171: 1307–1319

Espinosa J, Rodríguez-Mateos F, Salinas P, Lanza VF, Dixon R, de la CruzF, Contreras A (2014) PipX, the coactivator of NtcA, is a global regulatorin cyanobacteria. Proc Natl Acad Sci USA 111: E2423–E2430

Fan DY, Nie Q, Hope AB, Hillier W, Pogson BJ, Chow WS (2007) Quan-tification of cyclic electron flow around photosystem I in spinach leavesduring photosynthetic induction. Photosynth Res 94: 347–357

Fatihi A, Latimer S, Schmollinger S, Block A, Dussault PH, Vermaas WF,Merchant SS, Basset GJ (2015) A dedicated type II NADPH dehydro-genase performs the penultimate step in the biosynthesis of vitamin K1in Synechocystis and Arabidopsis. Plant Cell 27: 1730–1741

Feng Y, Li W, Li J, Wang J, Ge J, Xu D, Liu Y, Wu K, Zeng Q, Wu JW, et al(2012) Structural insight into the type-II mitochondrial NADH dehy-drogenases. Nature 491: 478–482

Forchhammer K (2004) Global carbon/nitrogen control by PII signaltransduction in cyanobacteria: from signals to targets. FEMS MicrobiolRev 28: 319–333

Heikal A, Nakatani Y, Dunn E, Weimar MR, Day CL, Baker EN, Lott JS,Sazanov LA, Cook GM (2014) Structure of the bacterial type II NADHdehydrogenase: a monotopic membrane protein with an essential role inenergy generation. Mol Microbiol 91: 950–964

Herranen M, Battchikova N, Zhang P, Graf A, Sirpiö S, Paakkarinen V,Aro EM (2004) Towards functional proteomics of membrane proteincomplexes in Synechocystis sp. PCC 6803. Plant Physiol 134: 470–481

Holland SC, Kappell AD, Burnap RL (2015) Redox changes accompanyinginorganic carbon limitation in Synechocystis sp. PCC 6803. Biochim Bio-phys Acta 1847: 355–363

Howitt C, Smith G, Day D (1993) Cyanide-insensitive oxygen uptake andpyridine nucleotide dehydrogenases in the cyanobacterium AnabaenaPCC 7120. Biochim Biophys Acta 1141: 313–320

Howitt CA, Udall PK, Vermaas WF (1999) Type 2 NADH dehydrogenasesin the cyanobacterium Synechocystis sp. strain PCC 6803 are involved inregulation rather than respiration. J Bacteriol 181: 3994–4003

Ishii A, Hihara Y (2008) An AbrB-like transcriptional regulator, Sll0822, isessential for the activation of nitrogen-regulated genes in Synechocystissp. PCC 6803. Plant Physiol 148: 660–670

Kämäräinen J, Huokko T, Kreula S, Jones PR, Aro EM, Kallio P (2017)Pyridine nucleotide transhydrogenase PntAB is essential for optimalgrowth and photosynthetic integrity under low-light mixotrophic con-ditions in Synechocystis sp. PCC 6803. New Phytol 214: 194–204

Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y, MiyajimaN, Hirosawa M, Sugiura M, Sasamoto S, et al (1996) Sequence analysisof the genome of the unicellular cyanobacterium Synechocystis sp.strain PCC6803. II. Sequence determination of the entire genome andassignment of potential protein-coding regions. DNA Res (Suppl) 3:185–209

Korn A (2010) Respective roles of the ferredoxin:NADP-oxidoreductaseisoforms in the cyanobacterium Synechocystis sp. PCC 6803. PhD thesis.Universite Paris Sud, Paris

Kruger NJ, von Schaewen A (2003) The oxidative pentose phosphatepathway: structure and organisation. Curr Opin Plant Biol 6: 236–246

Kucho K, Okamoto K, Tsuchiya Y, Nomura S, Nango M, Kanehisa M,Ishiura M (2005) Global analysis of circadian expression in the cyano-bacterium Synechocystis sp. strain PCC 6803. J Bacteriol 187: 2190–2199

Latifi A, Ruiz M, Zhang CC (2009) Oxidative stress in cyanobacteria. FEMSMicrobiol Rev 33: 258–278

Layana C, Diambra L (2011) Time-course analysis of cyanobacteriumtranscriptome: detecting oscillatory genes. PLoS ONE 6: e26291

Lea-Smith DJ, Ross N, Zori M, Bendall DS, Dennis JS, Scott SA, SmithAG, Howe CJ (2013) Thylakoid terminal oxidases are essential for thecyanobacterium Synechocystis sp. PCC 6803 to survive rapidly changinglight intensities. Plant Physiol 162: 484–495

Lee S, Ryu JY, Kim SY, Jeon JH, Song JY, Cho HT, Choi SB, Choi D, deMarsac NT, Park YI (2007) Transcriptional regulation of the respiratorygenes in the cyanobacterium Synechocystis sp. PCC 6803 during the earlyresponse to glucose feeding. Plant Physiol 145: 1018–1030

Liberton M, Saha R, Jacobs JM, Nguyen AY, Gritsenko MA, Smith RD,Koppenaal DW, Pakrasi HB (2016) Global proteomic analysis reveals anexclusive role of thylakoid membranes in bioenergetics of a model cy-anobacterium. Mol Cell Proteomics 15: 2021–2032

Llácer JL, Espinosa J, Castells MA, Contreras A, Forchhammer K, RubioV (2010) Structural basis for the regulation of NtcA-dependent tran-scription by proteins PipX and PII. Proc Natl Acad Sci USA 107: 15397–15402

Ludwig C, Claassen M, Schmidt A, Aebersold R (2012) Estimation ofabsolute protein quantities of unlabeled samples by selected reactionmonitoring mass spectrometry. Mol Cell Proteomics 11: M111.013987

Luque I, Vázquez-Bermúdez MF, Paz-Yepes J, Flores E, Herrero A (2004)In vivo activity of the nitrogen control transcription factor NtcA issubjected to metabolic regulation in Synechococcus sp. strain PCC 7942.FEMS Microbiol Lett 236: 47–52

MacLean B, Tomazela DM, Shulman N, Chambers M, Finney GL, FrewenB, Kern R, Tabb DL, Liebler DC, MacCoss MJ (2010) Skyline: an opensource document editor for creating and analyzing targeted proteomicsexperiments. Bioinformatics 26: 966–968

Markson JS, Piechura JR, Puszynska AM, O’Shea EK (2013) Circadiancontrol of global gene expression by the cyanobacterial master regulatorRpaA. Cell 155: 1396–1408

Marraffini LA, Sontheimer EJ (2008) CRISPR interference limits horizontalgene transfer in staphylococci by targeting DNA. Science 322: 1843–1845

Marreiros BC, Sena FV, Sousa FM, Batista AP, Pereira MM (2016) Type IINADH:quinone oxidoreductase family: phylogenetic distribution,structural diversity and evolutionary divergences. Environ Micro-biol 18: 4697–4709

Marreiros BC, Sena FV, Sousa FM, Oliveira AS, Soares CM, Batista AP,Pereira MM (2017) Structural and functional insights into the catalyticmechanism of the type II NADH:quinone oxidoreductase family. Sci Rep7: 42303

Mazouni K, Domain F, Cassier-Chauvat C, Chauvat F (2004) Molecularanalysis of the key cytokinetic components of cyanobacteria: FtsZ, ZipNand MinCDE. Mol Microbiol 52: 1145–1158

Meeks JC, Castenholz RW (1971) Growth and photosynthesis in an ex-treme thermophile, Synechococcus lividus (Cyanophyta). Arch Mikrobiol78: 25–41

Melo AM, Bandeiras TM, Teixeira M (2004) New insights into type IINAD(P)H:quinone oxidoreductases. Microbiol Mol Biol Rev 68: 603–616





Mi H, Endo T, Ogawa T, Kozi A (1992a) Thylakoid membrane-boundNADPH-specific pyridine nucleotide dehydrogenase complex medi-ates cyclic electron transport in the cyanobacterium Synechocystis sp.PCC 6803. Plant Cell Physiol 36: 661–668

Mi H, Endo T, Schreiber U, Asada K (1992b) Donation of electrons fromcytosolic components to the intersystem chain in the cyanobacteriumSynechococcus sp. PCC 7002 as determined by the reduction of P700+.Plant Cell Physiol 33: 1099–1105

Michalecka AM, Svensson AS, Johansson FI, Agius SC, Johanson U,Brennicke A, Binder S, Rasmusson AG (2003) Arabidopsis genes en-coding mitochondrial type II NAD(P)H dehydrogenases have differentevolutionary origin and show distinct responses to light. Plant Physiol133: 642–652

Mullineaux CW (2014) Co-existence of photosynthetic and respiratory ac-tivities in cyanobacterial thylakoid membranes. Biochim Biophys Acta1837: 503–511

Narainsamy K, Cassier-Chauvat C, Junot C, Chauvat F (2013) High per-formance analysis of the cyanobacterial metabolism via liquid chroma-tography coupled to a LTQ-Orbitrap mass spectrometer: evidence thatglucose reprograms the whole carbon metabolism and triggers oxidativestress. Metabolomics 9: 21–32

Nishimura T, Takahashi Y, Yamaguchi O, Suzuki H, Maeda S, Omata T(2008) Mechanism of low CO2-induced activation of the cmp bicarbonatetransporter operon by a LysR family protein in the cyanobacteriumSynechococcus elongatus strain PCC 7942. Mol Microbiol 68: 98–109

Omata T, Price GD, Badger MR, Okamura M, Gohta S, Ogawa T (1999)Identification of an ATP-binding cassette transporter involved in bi-carbonate uptake in the cyanobacterium Synechococcus sp. strain PCC7942. Proc Natl Acad Sci USA 96: 13571–13576

Orf I, Schwarz D, Kaplan A, Kopka J, Hess WR, Hagemann M, Klähn S(2016) CyAbrB2 contributes to the transcriptional regulation of low CO2acclimation in Synechocystis sp. PCC 6803. Plant Cell Physiol 57: 2232–2243

Osanai T, Imashimizu M, Seki A, Sato S, Tabata S, Imamura S, AsayamaM, Ikeuchi M, Tanaka K (2009) ChlH, the H subunit of theMg-chelatase, is an anti-sigma factor for SigE in Synechocystis sp. PCC6803. Proc Natl Acad Sci USA 106: 6860–6865

Osanai T, Kanesaki Y, Nakano T, Takahashi H, Asayama M, Shirai M,Kanehisa M, Suzuki I, Murata N, Tanaka K (2005) Positive regulationof sugar catabolic pathways in the cyanobacterium Synechocystis sp.PCC 6803 by the group 2 sigma factor sigE. J Biol Chem 280: 30653–30659

Osanai T, Kuwahara A, Iijima H, Toyooka K, Sato M, Tanaka K, IkeuchiM, Saito K, Hirai MY (2013) Pleiotropic effect of sigE over-expression oncell morphology, photosynthesis and hydrogen production in Synecho-cystis sp. PCC 6803. Plant J 76: 456–465

Peltier G, Aro EM, Shikanai T (2016) NDH-1 and NDH-2 plastoquinonereductases in oxygenic photosynthesis. Annu Rev Plant Biol 67: 55–80

Perkins DN, Pappin DJ, Creasy DM, Cottrell JS (1999) Probability-basedprotein identification by searching sequence databases using massspectrometry data. Electrophoresis 20: 3551–3567

Pétriacq P, de Bont L, Genestout L, Hao J, Laureau C, Florez-Sarasa I,Rzigui T, Queval G, Gilard F, Mauve C, et al (2017) Photoperiod affectsthe phenotype of mitochondrial complex I mutants. Plant Physiol 173:434–455

Piller LE, Besagni C, Ksas B, Rumeau D, Bréhélin C, Glauser G, KesslerF, Havaux M (2011) Chloroplast lipid droplet type II NAD(P)H quinoneoxidoreductase is essential for prenylquinone metabolism and vitaminK1 accumulation. Proc Natl Acad Sci USA 108: 14354–14359

Pisareva T, Kwon J, Oh J, Kim S, Ge C, Wieslander A, Choi JS, Norling B(2011) Model for membrane organization and protein sorting in the

cyanobacterium Synechocystis sp. PCC 6803 inferred from proteomicsand multivariate sequence analyses. J Proteome Res 10: 3617–3631

Pitt FD, Mazard S, Humphreys L, Scanlan DJ (2010) Functional charac-terization of Synechocystis sp. strain PCC 6803 pst1 and pst2 gene clustersreveals a novel strategy for phosphate uptake in a freshwater cyano-bacterium. J Bacteriol 192: 3512–3523

Scholz I, Lange SJ, Hein S, Hess WR, Backofen R (2013) CRISPR-Cassystems in the cyanobacterium Synechocystis sp. PCC6803 exhibit dis-tinct processing pathways involving at least two Cas6 and a Cmr2protein. PLoS ONE 8: e56470

Sena FV, Batista AP, Catarino T, Brito JA, Archer M, Viertler M, Madl T,Cabrita EJ, Pereira MM (2015) Type-II NADH:quinone oxidoreductasefrom Staphylococcus aureus has two distinct binding sites and is ratelimited by quinone reduction. Mol Microbiol 98: 272–288

Sharma V, Eckels J, Taylor GK, Shulman NJ, Stergachis AB, Joyner SA,Yan P, Whiteaker JR, Halusa GN, Schilling B, et al (2014) Panorama: atargeted proteomics knowledge base. J Proteome Res 13: 4205–4210

Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel di-gestion for mass spectrometric characterization of proteins and pro-teomes. Nat Protoc 1: 2856–2860

Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometricsequencing of proteins silver-stained polyacrylamide gels. Anal Chem68: 850–858

Sorek R, Lawrence CM, Wiedenheft B (2013) CRISPR-mediated adaptiveimmune systems in bacteria and archaea. Annu Rev Biochem 82: 237–266

Terashima M, Specht M, Naumann B, Hippler M (2010) Characterizingthe anaerobic response of Chlamydomonas reinhardtii by quantitativeproteomics. Mol Cell Proteomics 9: 1514–1532

Thomas JC, Ughy B, Lagoutte B, Ajlani G (2006) A second isoform of theferredoxin:NADP oxidoreductase generated by an in-frame initiation oftranslation. Proc Natl Acad Sci USA 103: 18368–18373

Vass I, Kirilovsky D, Etienne AL (1999) UV-B radiation-induced donor-and acceptor-side modifications of photosystem II in the cyanobacte-rium Synechocystis sp. PCC 6803. Biochemistry 38: 12786–12794

Vermaas W (2001) Photosynthesis and respiration in cyanobacteria. In eLS,John Wiley & Sons, London, United Kingdom

Vuorijoki L, Isojärvi J, Kallio P, Kouvonen P, Aro EM, Corthals GL, JonesPR, Muth-Pawlak D (2016) Development of a quantitative SRM-basedproteomics method to study iron metabolism of Synechocystis sp. PCC6803. J Proteome Res 15: 266–279

Wierenga RK, Terpstra P, Hol WG (1986) Prediction of the occurrence ofthe ADP-binding beta alpha beta-fold in proteins, using an amino acidsequence fingerprint. J Mol Biol 187: 101–107

Xu L, Law SR, Murcha MW, Whelan J, Carrie C (2013) The dual targetingability of type II NAD(P)H dehydrogenases arose early in land plantevolution. BMC Plant Biol 13: 100

Yagi T (1991) Bacterial NADH-quinone oxidoreductases. J Bioenerg Bio-membr 23: 211–225

Yamauchi Y, Kaniya Y, Kaneko Y, Hihara Y (2011) Physiological roles ofthe cyAbrB transcriptional regulator pair Sll0822 and Sll0359 inSynechocystis sp. strain PCC 6803. J Bacteriol 193: 3702–3709

Yang C, Hua Q, Shimizu K (2002) Metabolic flux analysis in Synechocystis usingisotope distribution from 13C-labeled glucose. Metab Eng 4: 202–216

Zhang P, Allahverdiyeva Y, Eisenhut M, Aro EM (2009) Flavodiironproteins in oxygenic photosynthetic organisms: photoprotection of pho-tosystem II by Flv2 and Flv4 in Synechocystis sp. PCC 6803. PLoS ONE 4:e5331

Zhang P, Battchikova N, Jansen T, Appel J, Ogawa T, Aro EM (2004)Expression and functional roles of the two distinct NDH-1 complexesand the carbon acquisition complex NdhD3/NdhF3/CupA/Sll1735 inSynechocystis sp PCC 6803. Plant Cell 16: 3326–3340


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Role of Type 2 NAD(P)H Dehydrogenase NdbC in Redox ... · PDF fileRole of Type 2 NAD(P)H Dehydrogenase NdbC in Redox Regulation of Carbon Allocation inSynechocystis1[OPEN] Tuomas Huokko,