CytM decreases photosynthesis under photomixotrophy Cytochrome c M decreases photosynthesis under photomixotrophy in Synechocystis

23 Photomixotrophy is a metabolic state which enables photosynthetic microorganisms to 24 simultaneously perform photosynthesis and metabolism of imported organic carbon 25 substrates. This process is complicated in cyanobacteria, since many, including 26 Synechocystis ( Synechocystis sp. PCC 6803), conduct photosynthesis and respiration in an 27 interlinked thylakoid membrane electron transport chain. Under photomixotrophy, the cell 28 must therefore tightly regulate electron fluxes from photosynthetic and respiratory 29 complexes. In this study, we demonstrate, via characterization of photosynthetic apparatus 30 and the proteome, that photomixotrophic growth results in a gradual inhibition of Q A- re- 31 oxidation in wild-type Synechocystis, which largely decreases photosynthesis over three 32 days of growth. This process is circumvented by deleting the gene encoding cytochrome c M 33 (CytM), a cryptic c -type heme protein widespread in cyanobacteria. The ΔCytM strain 34 maintained active photosynthesis over the three-day period, demonstrated by high 35 photosynthetic O 2 and CO 2 fluxes and effective yields of photosystems I and II. Overall, this 36 resulted in a higher growth rate than wild type, which was maintained by accumulation of 37 proteins involved in phosphate and metal uptake, and cofactor biosynthetic enzymes. While 38 the exact role of CytM has not been determined, a mutant deficient in the thylakoid-localised 39 respiratory terminal oxidases and CytM (ΔCox/Cyd/CytM) displayed a similar phenotype 40 under photomixotrophy to ΔCytM. This, in combination with other physiological data, 41 suggests that CytM does not transfer electrons to these complexes, which had previously 42 been hypothesized. In summary, our data suggest that CytM may have a regulatory role in 43 photomixotrophy by modulating the photosynthetic capacity of cells. These results demonstrate that during photomixotrophic growth, the electron flow at PSII 292 acceptor site gradually becomes inhibited in WT leading to drastically slower electron 293 transfer from PSII to Cyt b 6 f on the third day. Deletion of CytM circumvents this inhibition, 294 maintains PSII reaction center protein D1 amounts and a steady electron flux from PSII to 295 Cyt f .


Introduction 45
Switching between different trophic modes is an advantageous feature, which provides great 46 metabolic flexibility for cyanobacteria. For a long time, these photosynthetic prokaryotes 47 were considered as a group of predominantly photoautotrophic organisms (Smith 1983, Stal 48 and Moezelaar 1997). Lately, accumulating evidence marks the physiological and ecological 49 importance of trophic modes involving organic carbon assimilation, e.g. photomixotrophy 50 (Zubkov andTarran 2008, Moore et al 2013). Dissolved organic carbon, most notably 51 monosaccharides, including glucose and fructose, accumulates in the environment, mainly 52 during phytoplankton blooms (Teeling et al 2012, Ittekot et al 1981. During 53 photomixotrophy, photosynthetic organisms must balance the consumption of organic 54 carbon sources with photosynthesis and carbon fixation. 55 In the model cyanobacterium Synechocystis (Synechocystis sp. PCC 6803), 56 photomixotrophy is further complicated by the operation of anabolic and catabolic processes 57 occurring in the same cellular compartment and by the presence of an interlinked thylakoid 58 membrane-localised electron transport pathway involved in both photosynthesis and 59 respiration (Vermaas et al., 2001;Mullineaux, 2014;Lea-Smith et al., 2016). In 60 Synechocystis, photosynthetic linear electron flow is similar to other oxygenic 61 photoautotrophs. In photosystem (PS) II and PSI, the energy of the harvested photons 62 induces charge separation. Electrons from the PSII primary donor P680 pass via pheophytin 63 and the primary quinone Q A , to the secondary quinone, Q B . Oxidized P680 + is the strongest 64 biological oxidizing molecule, which drives water splitting on the luminal side of PSII. When 65 Q B is doubly reduced, it binds two protons from the cytosol, converting plastoquinone (PQ) to 66 plastoquinol (PQH 2 ), which then diffuses into the membrane PQ pool. Cytochrome (Cyt) b 6 f 67 receives two electrons from PQH 2 and transfers an electron to the mobile small protein, 68 plastocyanin (Pc) or cytochrome c 6 (Cyt c 6 ). An electron is subsequently transferred to PSI, 69 replacing a newly excited electron that is transferred from the PSI reaction center P700 + via 70 several co-factors to ferredoxin (Fed). Lastly, electrons are transferred from Fed to NADP + 71 by ferredoxin-NADP + reductase (FNR) to generate NADPH. In the respiratory electron 72 transfer pathway, PQ is reduced by NAD(P)H dehydrogenase-like complex I (NDH-1) and 73 succinate dehydrogenase (SDH), using electrons ultimately derived from Fed (Schuller et al.,74 2019) and succinate, respectively. Electrons from the PQ-pool can be transferred to a 75 thylakoid-localized respiratory terminal oxidase (RTO), cytochrome bd-quinol oxidase (Cyd), 76 or via Cyt b 6 f and Pc/Cyt c 6 to a second RTO, an aa 3 -type cytochrome-c oxidase complex 77 (Cox). How Synechocystis regulates electron input from PSII and the NDH-1 and SDH 78 complexes into the photosynthetic electron transport chain and to RTOs under 79 photomixotrophic conditions is not fully understood. Moreover, Synechocystis encodes four 80 isoforms of the flavodiiron proteins (FDPs), Flv1-4, which likely utilize NAD(P)H (Vicente et 81 al., 2002;Brown et al., 2019) or reduced Fed (Santana-Sanchez et al., 2019). These 82 proteins function in light-induced O 2 reduction as hetero-oligomers consisting of Flv1/Flv3 83 and/or Flv2/Flv4 (Helman et al., 2003;Mustila et al., 2016;Allahverdiyeva et al., 2015;84 Santana-Sanchez et al., 2019). 85 In Synechocystis, the water-soluble Cyt c 6 (formerly referred to as Cyt c 553 ) can substitute for 86 Pc under conditions of copper deprivation (Durán et al., 2004). Cyt c 6 belongs to the Cyt c 87 family, whose members are characterized by a covalently bound c-type heme cofactor. C-88 type Cyts are further classified into groups such as the Cyt c 6 -like proteins, Cyt c 555 , Cyt c 550 , 89 and CytM (Bialek et al., 2008). Apart from the well-established role of Cyt c 6 in electron 90 transfer (Kerfeld and Krogman, 1998) and the role of Cyt c 550 (PsbV) in stabilizing the PS II 91 water splitting complex (Shen and Inoue, 1993), most of the Cyt c proteins remain enigmatic. 92 Cyt c M (CytM) is conserved in nearly every sequenced cyanobacterium with the exception of 93 the obligate symbionts Candidatus acetocyanobacterium thalassa and Candidatus 94 Synechococcus spongiarum (Supplemental Fig. S1; Bialek et al., 2016). In Synechocystis,95 CytM is encoded by sll1245 (Malakhov et al., 1994). Nevertheless, its subcellular location is 96 ambiguous. An early study localised CytM to the thylakoid and plasma membranes in 97 'purified' membrane fractions (Bernroitner et al., 2009). However, cross contamination 98 between membranes was not determined, which has been an issue in studies using similar 99 separation techniques (Sonoda et al., 1997;Schultze et al., 2009). In later proteomics 100 studies, CytM has not been detected or localised using membranes purified by either two-101 phase aqueous polymer partitioning or subcellular fractionation (Baers et al., 2019). 102 However, the structure of the hydrophobic N-terminus resembles a signal peptide, which 103 suggests that CytM is targeted to a membrane. Sequence similarity to the N-terminus 104 cleavage site of Synechocystis Cyt c 6 suggests that the N-terminus is processed and the 105 mature 8.3 kDa protein is inserted into the lumen (Malakhov et al., 1994). However, 106 cleavage does not seem to occur in vivo, as the protein extracted from various 107 cyanobacterial species, including Synechocystis, Synechococcus elongatus PCC 6301, and 108 Anabaena sp. PCC 7120, was found to be around 12 kDa (Cho et al., 2000;Bernroitner et 109 al., 2009), implying that the hydrophobic N-terminus remains on the protein and serves as a 110 membrane anchor. The subcellular location of CytM and whether it is membrane anchored is 111 therefore still unknown. 112 It has been suggested that CytM may play a role in respiratory or photosynthetic electron 113 transfer (Manna and Vermaas, 1997;Bernroitner et al., 2009 photosynthesis or dark respiratory rates (Malakhov et al., 1994) under these conditions. 121 Cold, high light, and salt stress, however, induce gene expression and the stress-induced 122 co-transcriptional regulation between cytM (CytM), petJ (Cyt c 6 ), and petE (Pc) suggests a 123 stress-related role in electron transfer (Malakhov et al., 1999). 124 Besides environmental stresses, CytM has been linked to organic carbon-assimilating 125 trophic modes. A dark-adapted variant of Leptolyngbya boryana was found to grow faster 126 than wild type (WT) in heterotrophy. Genome re-sequencing revealed that the fast-growing 127 strain harboured a disrupted cytM (Hiraide et al., 2015). In line with this, the cytM deletion 128 mutant of Synechocystis demonstrated a growth advantage over the WT under dark and 129 light-activated heterotrophic conditions, and under photomixotrophic conditions (Hiraide et 130 al., 2015). Under dark heterotrophic conditions, ΔCytM had higher dark respiration and net 131 photosynthesis. However, the physiological mechanism and the functional role of CytM 132 remains entirely unknown. 133 In this study, we sought to uncover the bioenergetics of photomixotrophically grown 134 Synechocystis and physiological background behind the growth advantage of ΔCytM by 135 characterizing its photosynthetic machinery and the proteomic landscape. We demonstrate 136 gradual inhibition of Q A re-oxidation, resulting in repression of linear electron transport and 137 CO 2 fixation in Synechocystis during photomixotrophic growth. A mutant lacking CytM 138 circumvents inhibition of Q A re-oxidation during photomixotrophic growth, enabling higher 139 rates of net photosynthesis. In order to meet the substrate demand for enhanced growth, the 140 mutant retains transporter proteins, cofactor biosynthetic enzymes, and slightly adjusts 141 central carbon metabolism compared to photomixotrophic WT. Although the function of CytM 142 was previously associated with Cox, both thylakoid respiratory terminal oxidases, Cox and 143 Cyd, were found to be dispensable for the metabolic advantage conferred by deletion of 144 CytM in photomixotrophy. We conclude that when cells are exposed to high glucose 145  Malakhov et al., 1994;Hiraide et al., 2015), no growth difference was observed between 161 ΔCytM and WT under photoautotrophic conditions (Fig. 1A). 162 Next, we characterized growth under photomixotrophic conditions. To determine how 163 different starting glucose concentrations affected photomixotrophic growth (Fig. 1A, B), we 164 supplemented the medium with 5 mM and 10 mM glucose and cultivated the strains under 165 constant 50 µmol photons m −2 s −1 light. Based on optical density measurements (OD 750 ), all 166 cultures with added glucose grew substantially faster than those cultured 167 photoautotrophically (Fig. 1A, B). Deletion of cytM had no effect on cells grown at 5 mM 168 glucose. However, when cultured with 10 mM glucose, ΔCytM demonstrated 1.9±0.4 (P=6E-169 6) higher OD 750 than WT and ΔCox/Cyd/CytM demonstrated 1.9±0.6 (P=0.002) higher OD 750 170 compared to ΔCox/Cyd, after three days. In line with this, ΔCytM consumed more glucose 171 than WT ( Fig. 2A LAHG condition, ΔCytM grew faster than WT as previously reported (Hiraide et al., 2015). 180 The ΔCox/Cyd and ΔCox/Cyd/CytM mutants were unable to grow under LAHG. Previously, it 181 was reported that Cox is indispensable under this condition (Pils et al., 1997). 182 We next examined the morphology of ΔCytM and WT cells on the third day of 183 photomixotrophic growth (10 mM glucose, 50 µmol photons m −2 s −1 constant light), when the 184 highest difference in OD 750 was observed. Cell size, cell number per OD 750 , and chlorophyll 185 (chl) concentration per cell were determined. No difference was observed in cell size 186 between ΔCytM and WT (Supplemental Fig. S3), and the cell number per OD 750 was similar 187 in both strains (Fig. 2B), confirming that the difference in OD 750 reflects higher growth. 188 However, the chl a content per cell increased in ΔCytM (Fig. 2C), suggesting that the 189 photosystem content or PSII/PSI ratio has been altered in this strain. 190 Overall, the most pronounced growth advantage of ΔCytM over WT was observed when 191 cells were exposed to a light intensity of 50 µmol photons m −2 s −1 and glucose concentration 192 of 10 mM. Therefore, these conditions were used for all subsequent phenotyping 193 experiments examining cells cultured photomixotrophically. Next, we assessed photosynthetic activity by probing chl fluorescence in WT and ΔCytM 219 whole cells with multiple-turnover saturating pulses in dark, under far-red and under actinic 220 red light (Fig. 3A-D). Compared to cells cultured photoautotrophically (Supplemental Fig.  221 S4A), photomixotrophically grown WT cells demonstrated substantially higher initial 222 fluorescence (F 0 ) and slower relaxation of pulse-induced fluorescence in the dark (see F m D 223 relaxation in Fig. 3C), which suggests that the PQ pool is highly reduced. To verify this, cells 224 were exposed to far-red light, which preferentially excites PSI, resulting in oxidation of the 225 PQ-pool. If the PQ pool is highly reduced, then a lower steady-state fluorescence level ( To determine how WT builds up a highly reduced Q A over three days of photomixotrophic 253 growth, we monitored the redox kinetics of the PSII primary electron acceptor Q A (Fig. 4) by 254 firing a single-turnover saturating flash on dark-adapted cells. Relaxation of the chl 255 fluorescence yield was then recorded in the period of subsequent darkness. No difference 256 was observed between WT and ΔCytM cells cultured photoautotrophically (Supplemental 257 Fig. S9A) and on the first day of photomixotrophy, both WT and ΔCytM cells demonstrated 258 typical flash-fluorescence relaxation in the darkness. On the second day, WT cells 259 demonstrated a substantial slow-down in Q A re-oxidation reflected by slow decay kinetics 260 (Fig. 4B), while on the third day, there was a nearly complete loss of Q A -to-Q B electron 261 transfer (Fig. 4C). 262 Interestingly, the kinetics from the third day resembled a curve recorded in 263 photoautotrophically cultured WT supplemented with DCMU prior to the measurement 264 (Supplemental Fig. S9A). This supports the conclusion that Q A -to-Q B electron transfer was 265 strongly inhibited in the majority of PSII centers in WT on the third day of photomixotrophy. 266 Pre-illumination of the cells with far-red light did not accelerate Q A re-oxidation 267 (Supplemental Fig. S9B), thus supporting the idea that the inhibition is not simply due to a 268 highly reduced PQ-pool, although over-reduction of the PQ-pool cannot be excluded. conditions, confirming that electron transfer from PSII to Cyt b 6 f is inhibited. In contrast, 289 ΔCytM grown photomixotrophically ( Fig. 5B) resembled untreated WT cells subjected to 290 photoautotrophic conditions. 291 These results demonstrate that during photomixotrophic growth, the electron flow at PSII 292 acceptor site gradually becomes inhibited in WT leading to drastically slower electron 293 transfer from PSII to Cyt b 6 f on the third day. Deletion of CytM circumvents this inhibition, 294 maintains PSII reaction center protein D1 amounts and a steady electron flux from PSII to 295 Cyt f. 296

ΔCytM has a larger pool of oxidizable PSI than WT under photomixotrophy 297
Next, we determined activity of PSI by monitoring the redox kinetics of P700, the primary 298 electron donor of PSI ( Fig. 6), which was performed simultaneously with chl fluorescence 299 measurements (Fig. 3). First, the maximal amount of oxidizable P700, P m , was determined 300 (Fig. 6A). Compared to cells cultured under photoautotrophic conditions, WT cells grown 301 photomixotrophically had 45.2±0.03% lower P m . However, the difference between ΔCytM 302 cultured under photomixotrophic and photoautotrophic conditions was negligible 303 (17.2±19.3%). Thus, under photomixotrophic conditions, ΔCytM had 132±18.7% higher 304 maximum amounts of oxidizable P700 than WT (Fig. 6A). In line with this, immunoblotting 305 revealed higher levels of PSI reaction center subunit, PsaB, in ΔCytM compared to WT 306 under photomixotrophic growth (Fig. 6B). To determine the PSI:PSII ratio, samples were 307 analysed at 77K by measuring chl fluorescence emission. No statistical difference was 308 observed between WT and ΔCytM (Supplemental Fig. S11), demonstrating that the PSII:PSI 309 ratio was similar in both strains. 310 The PSI effective yield Y(I), was also quantified, and was three times lower in 311 photomixotrophically cultured WT cells compared to those grown photoautotrophically 312 (Supplemental Fig. S5B). This is due to a strong donor side limitation of PSI Y(ND) 313 (Supplemental Fig. S5C), which demonstrates an electron shortage to P700 + . In contrast, 314 photomixotrophically cultured ΔCytM demonstrated similar Y(I) and only slightly increased 315 Y(ND) compared to photoautotrophically cultured WT and ΔCytM (Supplemental Fig. S5B, 316 C). As a result, ΔCytM had more than three times higher Y(I) than WT under 317 photomixotrophy (Supplemental Fig. S5B). 318 Next, pulse-induced P700 fast kinetics were compared between photoautotrophically and 319 photomixotrophically cultured WT (Fig. 6E) and ΔCytM (Fig. 6F). These fast kinetics reveal 320 did not exhibit the typical transient re-reduction (Fig. 6E). Importantly, P700 + relaxation after 325 the pulse (Fig. 6E) was markedly slower compared to that observed in photoautotrophically 326 cultured cells (Supplemental Fig. S12A). Collectively, these results confirm that fewer 327 electrons were transferred to P700 + , leading to higher Y(ND) in photomixotrophically grown 328 WT. Photomixotrophically cultured ΔCytM (Fig. 6F) 6F) and rapid relaxation after the pulse (Fig. 6F), 330 resembling photoautotrophically cultured ΔCytM and WT (Supplemental Fig. S12A-B). 331 Here, we have shown that the effective yield of PSI in photomixotrophically cultured WT cells 332 was considerably lower compared to photoautotrophically cultured cells, due to an electron 333 shortage at P700 + . This phenotype is eliminated by deleting cytM, as increased Y(I), higher 334 amounts of oxidizable P700 (P m ) and PsaB were observed in ΔCytM compared to WT on the 335 third day of photomixotrophy. 336

ΔCytM and ΔCox/Cyd/CytM sustain efficient net photosynthesis and CO 2 fixation 337
under photomixotrophy 338 To analyse real time gas exchange in photomixotrophically grown WT, ΔCytM, ΔCox/Cyd, 339 and ΔCox/Cyd/CytM (Fig. 7), whole cell fluxes of O 2 and CO 2 were simultaneously monitored 340 using membrane inlet mass spectrometry (MIMS Based on the assumption that during steady state photosynthesis the consumption of TC i is 353 a function of Rubisco activity (Badger et al., 1994;Sültemeyer et al., 1995), the TC i fluxes 354 represents CO 2 consumption rates. 355 In WT under 200 µmol photons m −2 s −1 white light, O 2 consumption and gross production 356 rates were similar, resulting in nearly zero net photosynthetic O 2 production. This is in line 357 with the data obtained by the O 2 electrode (Fig. 3E). Corresponding to the minor net 358 photosynthetic O 2 production observed, the rate of CO 2 consumption was negligible ( Strikingly, gross O 2 production was approximately 10 times higher compared to WT and 18 O 2 366 consumption in light followed a triphasic pattern, a characteristic trend reflecting the 367 contribution of Flv1/3 and Flv2/4 to O 2 consumption in light (Santana-Sanchez et al., 2019). 368 The triphasic pattern in ΔCytM was observed as an initial burst of O 2 consumption following 369 the dark-to-light transition, which faded after 1-1.5 min and continued at a relatively constant 370 rate (Fig. 7E). Accordingly, immunoblotting confirmed higher accumulation of the Flv3 371 proteins in ΔCytM. The rate of light-induced O 2 consumption in ΔCytM is comparable to the 372 reported values of photoautotrophically grown WT (Huokko et al., 2017, Santana-Sanchez et 373 al., 2019. The dark respiration rate was slightly higher in ΔCytM compared to WT, as 374 previously observed when ΔCytM was cultured under dark, heterotrophic conditions (Hiraide 375 et al 2015). 376 Similar to WT, ΔCox/Cyd (Fig. 7A,D) showed minimal photosynthetic activity on the third day 377 of photomixotrophic growth. During illumination, net O 2 production remained negative, and 378 CO 2 consumption was found to be negligible (Fig. 7A, Supplemental Fig. S13B). Only 379 residual gross O 2 production was observed and O 2 consumption was not stimulated by light 380 (Fig. 7A, D). Flv3 protein abundance in ΔCox/Cyd was comparable to WT (Fig. 7B). In sharp 381 contrast to ΔCox/Cyd, ΔCox/Cyd/CytM demonstrated high PSII activity and a net O 2 382 production rate similar to ΔCytM (Fig. 7A, F). ΔCox/Cyd/CytM displayed a triphasic O 2 383 consumption pattern under illumination (Fig. 7F) and the light-induced O 2 consumption was 384 comparable to that of ΔCytM in steady state (Fig. 7E). Compared to ΔCox/Cyd, 385 ΔCox/Cyd/CytM had higher levels of Flv3 (Fig. 7B). Notably, deleting cytM in the ΔCox/Cyd 386 mutant did not enhance dark respiration, whereas ΔCytM had higher rates compared to WT. 387 To conclude, mutants lacking CytM sustained a steady electron flux towards O 2 and CO 2 388 under photomixotrophy, reflected by substantial net O 2 production and active CO 2 389 consumption during illumination. 390

Photomixotrophically cultured ΔCytM cells accumulate transport proteins and 391
cofactor biosynthetic enzymes 392 In order to understand the metabolism of photomixotrophically grown WT and ΔCytM, we 393 analysed the total proteome by nLC-ESI-MS/MS via the data-dependent acquisition (DDA) 394 method. Samples for analysis were collected on the second day, when both WT and ΔCytM 395 cells were in late exponential phase and a substantial significant growth difference was 396 observed between the strains (Fig. 8A). 397 In total, 2,415 proteins were identified (Supplemental Dataset S1), despite the fact that the 398 dataset was slightly biased against basic (Fig. 8D) and hydrophobic proteins (Fig. 8E) Supplemental Dataset S3 shows a selection of proteins whose abundance was different in 406 ΔCytM compared to WT. The highest fold change was observed in transport proteins. 407 Among these, the constitutive low-affinity ABC-type phosphate transporters (PstA1, PstB1, 408 PstB1', PstC), periplasmic P i -binding proteins (SphX, PstS1), and extracellular lytic enzymes 409 (PhoA, NucH) are more abundant in ΔCytM. Among proteins related to C i uptake, a thylakoid 410 β-type carbonic anhydrase, EcaB, was 2.32 times (P = 7.50E-03) more abundant in ΔCytM. 411 EcaB is a CupA/B-associated protein, proposed to regulate the activity of NDH-1 3 (NDH-1 412 MS) and NDH-1 4 (NDH-1 MS') (Sun et al., 2018). NDH-1 3 facilitates inducible CO 2 -uptake, 413 whereas NDH-1 4 drives constitutive CO 2 -uptake (Ogawa, 1991). CupB is exclusively found in 414 the NDH-1 4 complex and converts CO 2 into HCO 3 − . Interestingly, no significant change was 415 observed in the level of the glucose transporter GlcP, although the growth advantage of 416 ΔCytM was observed upon exposure to glucose. 417 Chl a biosynthetic enzymes were found to accumulate in the mutant (Supplemental Dataset  418 3). ChlL, a subunit of the light-independent protochlorophyllide reductase (Wu and Vermaas 419 1995), and ChlP (4.61E-03), a geranylgeranyl reductase (Shpilyov et al., 2005), were 9.28 420 fold (P = 5.32E-03) and 1.52 fold (P = 4.61E-03) upregulated in ΔCytM, respectively. The 421 incorporation of chl into photosystems likely increases due to the elevated level of Pitt, a 422 protein contributing to the formation of photosynthetic pigments/proteins at the early stages 423 of biogenesis (Schottkowski et al., 2009 catalysing the final step in the production of biliverdin (Willows et al., 2000). Biliverdin is the 429 precursor of phycocyanobilin, which is incorporated into phycobilisomes, the light-harvesting 430 complexes of Synechocystis. 431 Among the photosynthetic proteins, the PSI reaction center subunit PsaB was found in equal 432 amounts in WT and ΔCytM. However, immunoblotting with an anti-PsaB antibody 433 demonstrated that ΔCytM contained higher amounts of PsaB than WT (Fig. 6B). This 434 discrepancy may be due to the fact that despite the robustness of the MS-based DDA 435 method, hydrophobic membrane proteins are prone to misquantification. Via MS analysis, 436 quantification of psbA encoded D1 was not successful. Therefore, its abundance was only 437 determined by immunoblotting (Fig. 3F), which revealed higher levels of D1 proteins in 438 ΔCytM compared to WT. Interestingly and somewhat contradictorily, the amount of PSII 439 assembly proteins encoded by the PAP-operon (Wegener et al., 2008)  show that photosynthesis was markedly decreased over three days of cultivation. This is 474 deduced from the low PSII ( b 6 f is hindered. However, this is not simply due to a highly reduced PQ pool. 495 The gradual disconnection between PSII and Cyt b 6 f and resulting decrease in 496 photosynthesis could be due to a spatial isolation of PSII via rearrangement in the thylakoid 497 to another location. Rearrangement of thylakoid-localised complexes, specifically NDH-1 and 498 SDH, has been observed in response to redox-regulated changes in the electron transport 499 chain (Liu et al 2012). Applying the same analogy to PSII, the highly reduced state of the 500 PQ-pool might trigger the complexes to arrange into a more sparse distribution during 501 photomixotrophic growth. Although cyanobacterial thylakoids are densely packed 502 membranes (Kaňa et al., 2013), lateral heterogeneity ( between PSII, Cyt b 6 f, and PSI. The rate of gross O 2 production (Fig 7A, E) was ten times 509 higher in ΔCytM than it was in WT cells cultured under photomixotrophic conditions. 510 Contrary to photomixotrophically cultured WT, ΔCytM showed a clear wave-pattern in Cyt f 511 kinetics upon dark-to-light transition and did not demonstrate slow re-reduction of Cyt f in 512 dark (Fig. 5B) or PSI donor-side limitation (Supplemental Fig. S5C). Finally, the abundance 513 of D1 (Fig. 3F), PsaB (Fig. 6B), and PetA and PetB (Supplemental Dataset S3), the core 514 subunits of PSII, PSI, and Cyt b 6 f, respectively, was higher in ΔCytM than in WT, although 515 the PSI:PSII ratio was unaltered (Supplemental Fig. S11). As a consequence, the rate of net 516 O 2 production and CO 2 consumption (Fig. 7A) was substantially higher in ΔCytM, 517 demonstrating that deletion of CytM conserves photosynthetic activity and circumvents the 518 inhibition of Q A re-oxidation in photomixotrophy. 519 The exact mechanism by which ΔCytM alleviates blockage of the electron transport pathway 520 was not elucidated in this work, nor has an exact role for this protein been determined in 521 previous studies. CytM has been suggested to play a role in transferring electrons from Cyt 522 b 6 f to Flv1/3, limiting productivity but providing a possible alternative route for safely 523 transferring electrons to O 2 (Hiraide et al., 2015). However, given the low midpoint potential 524 of CytM, a large energy barrier would have to be overcome in order for electron transfer 525 downstream of Cyt b 6 f to occur (Cho et al., 2000). Moreover, we demonstrated that the 526 absence of CytM does not decrease O 2 photoreduction driven by FDPs in ΔCox/Cyd/CytM 527 (Fig. 7A, F), thus excluding this possibility. Recently, a cyanobacterial ferredoxin, Fed2, was 528 shown to play a role in iron sensing and regulation of the IsiA antenna protein, a protein 529 which is typically expressed when cells are exposed to low-iron conditions (Schorsch et al., 530 2018). Similar to Fed2, it is possible that CytM plays a regulatory role in the cell, rather than 531 being directly involved in electron transport under photomixotrophy. 532 Under conditions when cells are exposed to glucose or other sugars, CytM may regulate 533 carbon assimilation. ΔCytM demonstrates substantial growth under dark heterotrophic 534 conditions (Hiraide et al., 2015). However, the majority of the known cyanobacteria cannot 535 grow heterotrophically, indicating that the function of CytM extends beyond the modulation of 536 heterotrophic growth (Bialek et al., 2016). Under photomixotrophic conditions, CytM likely is 537 involved in regulation of thylakoid re-arrangements or photosynthetic electron transport and 538 carbon fixation, limiting CO 2 uptake and decreasing the total amount of photosynthetic 539 proteins, which in turn reduces photosynthesis. In line with this, we observed accumulation 540 of EcaB in ΔCytM ( Fig. 9; Supplemental Dataset S3). Enhanced EcaB levels likely results in 541 greater inorganic carbon assimilation, higher carbon fixation, and increased turnover of 542 NADPH, the terminal electron acceptor in linear photosynthetic electron transport. This in 543 turn likely limits over-reduction of the photosynthetic electron transport chain. 544 Regardless of the exact role of CytM, it is clear that deletion of this protein substantially 545 increases growth of Synechocystis in photomixotrophy (Fig. 1), in line with previous studies 546 (Hiraide et al., 2015). This is possibly due to an increase in photosynthetic capacity 547 combined with efficient assimilation of glucose into central metabolism, resulting in greater 548 biomass accumulation. This resulted in increased production of proteins required for 549 enhanced growth, including those involved in phosphate uptake (PstA1, PstB1, PstB1', 550 PstC) (Supplemental Dataset S3), import of Mg 2+ (MgtE), Zn 2+ (ZiaA), and Fe 2+ (FutA2), and 551 production of chl (ChlP, ChlL) ( Fig. 9; Supplemental Dataset S3). 552 In conclusion, under long-term photomixotrophy Synechocystis cells gradually decrease 553 photosynthetic electron transport by disconnecting PSII from Cyt b 6 f. Deletion of CytM allows 554 Synechocystis to maintain efficient photosynthesis and enhanced growth under long-term 555 photomixotrophy. While we have not determined the exact function of CytM, we propose that 556 it plays a role in reducing photosynthesis under conditions when both light intensity and 557 glucose concentration fluctuate (Hieronymi and Macke, 2010;Ittekkot et al., 1985), and the 558 redox state of the intertwined photosynthetic and respiratory electron transfer rapidly 559 changes. 560 561 www.plantphysiol.org on April 21, 2020 -Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

Construction of cytM deletion mutants 573
Unmarked mutants of Synechocystis lacking cytM were constructed via a two-step 574 homologous recombination protocol according to Lea-Smith et al., 2016. To generate 575 marked mutants approximately 1 µg of plasmid pCytM-2 was mixed with Synechocystis cells 576 for 6 hours in liquid media, followed by incubation on BG-11 agar plates for approximately 24 577 hours. An additional 3 mL of agar containing kanamycin was added to the surface of the plate 578 followed by further incubation for approximately 1-2 weeks. Transformants were subcultured 579 to allow segregation of mutant alleles. Segregation was confirmed by PCR using primers 580 CytMf and CytMr, which flank the deleted region. To remove the npt1/sacRB cassette to 581 generate unmarked mutants, mutant lines were transformed with 1 µg of the markerless 582 CytM-1 construct. Following incubation in BG-11 liquid media for 4 days and agar plates 583 containing sucrose for a further 1-2 weeks, transformants were patched on kanamycin and 584 sucrose plates. Sucrose resistant, kanamycin sensitive strains containing the unmarked 585 deletion were confirmed by PCR using primers flanking the deleted region (Supplemental 586 Fig. S2B). The ∆Cox/Cyd/CytM unmarked strain was generated via the same method in the 587 background of the unmarked ∆Cox/Cyd strain (Lea-Smith et al., 2013). 588

Cultivation 589
Cells kept in cryogenic storage were revived on BG-11 agar plates at 3% CO 2 . Pre-590 experimental cultures were inoculated at 0.1 OD 750 by transferring a patch of cells from 591 plates into 30 ml BG-11 medium buffered with 10 mM TES-KOH (pH 8.2) in 100 ml 592 Erlenmeyer flasks. Cultures were shaken at 120 rpm at 30°C and exposed to constant white 593 fluorescent light of 50 µmol photons m −2 s −1 intensity in a Sanyo Environmental Test 594 Chamber (Sanyo Co, Japan) which was saturated with 3% CO 2 . Pre-experimental cultures 595 were cultivated for three days with density typically reaching 2.5±0.5 OD 750 . 596 Experimental cultures for growth and photophysiological experiments were inoculated in 30 597 ml fresh BG-11 media at 0.1 OD 750 from harvested pre-experimental cultures. The media 598 was buffered with 10 mM TES-KOH (pH 8.2), the CO 2 concentration was atmospheric, and 599 cultures were agitated in 100 ml Erlenmeyer flasks at 120 rpm in AlgaeTRON AG130 cool- For photophysiological studies, cells were cultivated under condition (c) for three days. For 605 proteomics analysis, cells were cultivated similarly to (c), with the exception of an extra three 606 day long pre-cultivation step at atmospheric CO 2 without glucose. 607 Cell counting, cell size determination 608 Cell number was determined with a Nexcelom Cellometer X2 via the following method. 609 Sample OD 750 was adjusted to one, brightfield images were captured, and the cell number 610 was determined by the Nexcelom software. In order to exclude the visual glitches falsely 611 recognized as cells by the software, only the four most populous cell size groups were 612 averaged. Typically, three thousand cells were counted per plate. 613

Glucose determination 614
Glucose concentration of the spent media was determined spectrophotometrically with the 615 commercial High Sensitivity Glucose Assay Kit (Sigma-Aldrich, U.S.). Prior to 616 measurements, the cell suspension was centrifuged at 5000 g for 10 min and the 617 supernatant was filtered through a 0.2 µm filter. 618

MIMS measurements 619
Gas fluxes of intact cells were measured using membrane inlet mass spectrometry. The in-620 house built system consists of a DW-1 oxygen electrode chamber (Hansatech Ltd., U.K.) 621 connected to the vacuum line of a mass spectrometer (Prima PRO model, Thermo Scientific, 622 U.S.). The sample cuvette was separated from the vacuum line by a Hansatech S4 PTFE 623 membrane (Hansatech Ltd., U.K.). Samples were pelleted and re-suspended in fresh BG-11 624 supplemented by 10 mM glucose and buffered to pH 8.2 with 10 mM TES-KOH. Chl a 625 concentration was adjusted to 10 µg ml −1 . Prior to measurements, the sample was enriched 626 with 98 % 18 O 2 heavy isotope (CK Isotopes Limited, U.K.), the dissolved total inorganic 627 carbon concentration was adjusted to 1.5 mM by adding NaHCO 3 , and then 10-15 min dark 628 adaptation was applied.

Clark-type electrode measurements 639
Net O 2 production of intact cells was tested in the presence of 0.5 mM 2,6-dichloro-p-640 benzoquinone (DCBQ) at 30°C with a Clark-type oxygen electrode and chamber (Hansatech 641 Ltd., U.K.). Prior to the measurements, cells were resuspended in BG-11 (pH 8.2) 642 supplemented with 10 mM glucose, the chl a concentration was adjusted to 7.5 µg ml −1 , then 643 the samples were dark adapted for 1-2 min. O 2 production was initiated by 1,000 µmol 644 photons m −2 s −1 white light using a Fiber-Lite DC-950 light source. Rates of oxygen 645 production was calculated using the Hansatech software. 646

Chl fluorescence and P700 oxidoreduction measurements 647
Whole cell chl fluorescence was measured simultaneously with P700 with a pulse amplitude-648 modulated fluorometer (Dual-PAM-100, Walz, Germany). Prior to measurements, cells were 649 resuspended in BG-11 (pH 8.2) supplemented with 10 mM glucose and the chl a 650 concentration was adjusted to 15 µg ml −1 . Measurements were performed at 30°C, and 651 samples were initially incubated in darkness for 15 minutes with stirring. To determine P m , 30 652 s strong far-red light (720 nm, 40 W m −2 ) and red multiple turnover saturating pulses (MT) 653 were applied. MT pulses were set to an intensity of 5,000 µmol photons m −2 s −1 (width: 500 654 ms). Red (635 nm) actinic light was at an intensity of 50 µmol photons m −2 s −1 was used as 655 background illumination. Photosynthetic parameters were calculated as described previously 656 (Klughammer et al 2008 a,b). 657 Relaxation of flash-induced fluorescence yield was monitored using a fluorometer (FL3500,  658 PSI Instruments, Czech Republic) as outlined previously (Allahverdiyeva et al 2003). Prior to 659 the measurement, cells were resuspended in BG-11 (pH 8.2) supplemented with 10 mM 660 glucose, adjusted to 5 µg chl a ml −1 and dark adapted for 5 min. Curves were normalized to 661 F 0 and F m . 662

Measurement of cytochrome f redox kinetics 663
Cyt f redox kinetics were determined in intact cells by deconvoluting absorbance changes at 664 546, 554, 563, and 573 nm that were measured using a JTS-10 pump probe 665 spectrophotometer (BioLogic, Grenoble, France) and appropriate 10 nm FWHM interference 666 filters. BG39 filters (Schott, Mainz, Germany) were used to shield the light detectors from 667 scattered light. Deconvolution was performed with the JTS-10 software. Prior to the 668 experiments, cells were harvested and Chl a concentration was adjusted to 5 µg ml −1 by 669 resuspension in fresh BG-11 with or without 10 mM glucose. Cells were dark-adapted for 2 670 min prior to measurements with each interference filter, and then illuminated with 500 μmol 671 photons m −2 s −1 of green light for 5 s. Flashes of white detection light were administered 672 during 200 μs dark intervals in actinic illumination. When appropriate, 20 μM DCMU was 673 added to the samples before dark-adaptation. 674

Accession numbers 694
Gene/protein names and accession numbers of all genes/proteins identified in this study are 695 listed in Supplemental Dataset S1. The mass spectrometry proteomics data was deposited 696 to    photons m −2 s −1 illumination for three days, with or without 10 mM glucose, respectively. 764 Prior to measurements, cells were resuspended in BG-11 supplemented with (C, D) and 765 without (  Subsequent relaxation of fluorescence yields in the dark was measured after a single-782 turnover saturating pulse in photomixotrophically cultured cells taken on the first (A), second 783 (B), and third day (C) of cultivation. Growth conditions are described in Fig. 3. Prior to 784 measurements, the cell suspension was adjusted to 5 µg chl ml −1 , resuspended in BG-11 785 supplemented with 10 mM glucose (C, D), and dark adapted for 5 min. 786 Oxidation of Cyt f was induced by 500 μmol photons m −1 s −1 green light. When indicated, 20 789 µM DCMU was added prior the measurement. The curves were normalized to their 790 respective maximal oxidation. The kinetics are representatives of three biological replicates. 791 three biological replicates. P700 oxidoreduction slow (C, D) and fast kinetics (E, F) were 795 measured in parallel with fluorescence (Fig. 3). Fast kinetics curves (E, F) are normalized to 796 P m and referenced against their respective minimum P700 signal detected after the pulse. 797 Cultivation, sample preparation, and experimental parameters are similar to those detailed in 798 Fig. 3. P 0 , initial P700; P m D , maximum P700 in darkness; P m , maximum P700 under far-red 799 light; P m ', maximum P700 under red actinic light. 800 applied. Samples are supplemented by 1.5 mM NaHCO 3 . Kinetics are representatives of 3-6 808 biological replicates. The source data of Fig. 7A can be found in Supplemental Table S2. 809 ellipsis marking the sampling day. Cells were cultured similarly to those used in the 812 biophysics analysis, except that the cells for proteomics were pre-cultivated under 813 atmospheric CO 2 in order to fully adapt the cells to these conditions. Importantly, the extra 814 pre-culturing step did not affect the growth of the experimental cultures. Values are means ± 815 SD, n = 3 biological replicates. Relaxation of the flash-induced fluorescence yield in the dark 816 (B) was measured in the absence (closed symbols) and in the presence of 20 µM DCMU 817 (open symbols). Differentially regulated proteins in ΔCytM were grouped according to their 818 function (C). In total, 2415 proteins were identified, out of which 634 proteins were quantified 819 and 162 were differentially regulated. The practical significance of differentially regulated 820 proteins was set to fold change (FC) > 1.5 and FC < -1.5 (P<0.05). Effect of isoelectric point 821 (pI) (D) and hydrophobicity (GRAVY) (E) of the proteins on the identification rate was 822 determined. Black squares mark all of the 3507 predicted proteins in Synechocystis, lilac 823 circles mark each protein identified in WT and in ΔCytM. 824 Values are means ± SD, n = four biological replicates. Asterisks indicate statistically significant differences (* P < 0.05, ** P < 0.001). Immunoblot analysis with D1-N antibody (F) was performed on samples taken on the third day. 15 µg total protein extract was loaded per 100% lane, 50% and 200% correspond to 7.5 µg and 30 µg, respectively.