Cyanobacterial Alkanes Modulate Photosynthetic Cyclic Electron Flow to Assist Growth under Cold Stress

Abstract

All cyanobacterial membranes contain diesel-range C15-C19 hydrocarbons at concentrations similar to chlorophyll. Recently, two universal but mutually exclusive hydrocarbon production pathways in cyanobacteria were discovered. We engineered a mutant of Synechocystis sp. PCC 6803 that produces no alkanes, which grew poorly at low temperatures. We analyzed this defect by assessing the redox kinetics of PSI. The mutant exhibited enhanced cyclic electron flow (CEF), especially at low temperature. CEF raises the ATP:NADPH ratio from photosynthesis and balances reductant requirements of biosynthesis with maintaining the redox poise of the electron transport chain. We conducted in silico flux balance analysis and showed that growth rate reaches a distinct maximum for an intermediate value of CEF equivalent to recycling 1 electron in 4 from PSI to the plastoquinone pool. Based on this analysis, we conclude that the lack of membrane alkanes causes higher CEF, perhaps for maintenance of redox poise. In turn, increased CEF reduces growth by forcing the cell to use less energy-efficient pathways, lowering the quantum efficiency of photosynthesis. This study highlights the unique and universal role of medium-chain hydrocarbons in cyanobacterial thylakoid membranes: they regulate redox balance and reductant partitioning in these oxygenic photosynthetic cells under stress.

Figure 1. Cartoon of cyanobacterial photosynthetic electron transport pathways.

In the linear electron transport pathway (dotted magenta line), light is first absorbed by PSII, then excited electrons are transported inside the membrane by PQ to the cyt b₆f complex, then through the thylakoid lumen by the PC to PSI. At PSI, electrons are excited by light a second time and then reduce NADP⁺. Along the way, protons are transported to the lumen to power ATP synthesis by the ATP synthase. In the cyclic pathway (dotted blue line), electrons from PSI reenter the PQ pool. Thus, the cyclic pathway produces ATP at the expense of NADPH. Inhibitors used in this study and their sites of inhibition are also indicated in red octagons. DCMU blocks electron transfer from PSII to PQ and DBMIB prevents oxidation of plastoquinone by the cyt b₆f complex. Cyt b₆f, cytochrome b₆ f complex; PC, plastocyanin; PQ, plastoquinone; PSI, photosystem I; PSII, photosystem II.

Figure 2. Knockout mutant construction strategy (A) and confirmation by PCR (B).

We constructed a plasmid, pNOalk, containing sequences flanking the ado and far genes for the ADO-type n-heptadecane biosynthesis pathway in Synechocystis 6803, around a kanamycin resistance cassette (A). We confirmed the absence of these genes from the mutant strain via PCR with 3 different primer sets (B) using genomic DNA from the wild type or the mutant strain (NOalk), or the plasmid pNOalk as templates. Binding sites of the three primer sets (1,2,3) on the wild-type chromosome are shown in panel (A).

Figure 3. Growth and alkane production by wild type (WT) and noALK strains at various temperatures.

(A) Cell growth was monitored by measuring OD₇₃₀ daily. (B) n-Heptadecane was measured via GC-MS and normalized to chlorophyll a concentration. Error bars are ± SD for n = 3 for both (A,B). Where error bars are not seen, the error is smaller than the symbol shown.

Figure 4. P₇₀₀ redox kinetics for WT and noALK strains at 20 and 30 C.

Using a JTS-10 spectrophotometer, cell suspensions were dark-adapted and then exposed to a pulse of orange actinic light (to excite both PSII and PSI) for 5 seconds (white bar above panel (A)). The actinic light was then turned off (black bar above panel (A)). During this time-course, measuring flashes of 705 nm light probed the redox state of the P₇₀₀ reaction center of PSI. Data were collected from cells that had been grown at 30 C, then measured at 30 C (panel (A,C)) or shifted to 20 C before measurement (panel (B,D)). Panels C and D show the details of re-reduction of P₇₀₀⁺ in the dark for the experiments in panels A and B, respectively. Inhibitors of linear electron flow (10 μM DCMU, which inhibits transfer from PSII to the quinone pool), and linear + cyclic electron flow (1 μM DBMIB, which blocks cyt b₆f) were added as indicated. Each trace is an average of 3 independent experiments.

Figure 5. Strain NOalk uses a higher ratio of cyclic:linear electron transport.

Panel (A) shows the half-times for re-reduction of P₇₀₀⁺ in the dark, calculated from the traces shown in Fig. 4C,D. Panel (B) shows the percentage of electron flow to P₇₀₀⁺ that is cyclic, calculated from the data in panel (A).

Figure 6. The simulated effect of cyclic electron transport on growth rate using iSyn731.

We modeled the effect of varying the recycle rate of electrons from PSI to the PQ pool on light-limited growth of Synechocystis 6803. The recycle rate was defined as the NDH-1 catalyzed electron flux from NADPH to plastoquinone divided by the electron flux into PSI. These simulations were carried out with alternative electron flow pathways (including succinate dehydrogenase and cytochrome oxidase, see Table 1) restricted to minimal flux. See methods section for further details.

Figure 7. Alkanes impact the adaptability of cyanobacteria to environmental conditions.

The lack of alkanes constrains the thylakoid electron transport chain to a higher recycle rate of electrons from PSI to the plastoquinone pool. This inflexibility in reductant partitioning leads to a narrower range of environmental conditions (in particular temperature) in which the strain can grow optimally.