2. Does transformation of psbC- Synechocystis with the recombinant plasmid and t

Question

2. Does transformation of psbC-Synechocystis with the recombinant plasmid and the wild type genomic DNA serve the same purpose? Explain.

3. Discuss the purpose and procedure of transformation of psbC-Synechocystis.

used to transform the mutant psbC- strain to revert it to wild type and to restore its ability to grow photoautotrophically Experiment Il Overview E. coli is not the sole bacterium that is amenable to molecular biology. There are many such bacteria. In this lab, one of these, the cyanobacterium Synechocystis sp. PCC6803 is introduced. This cyanobacterium has several advantages that make it a very useful organism for gene manipulations. The two main advantages are: Figure II-1. Schematic model of the photosystem Il complex from cyanobacteria cytoplasm/stroma 1. Natural transformability. In contrast to E. coli and most other bacteria, it spontaneously takes up foreign DNA that is present in the growth medium, and can integrate it into its genome through a double homologous recombination event. Double homologous recombination is appropriate to introduce gene interruptions or deletions using a construct with two regions of sequence identity with the cyanobacterial genome. D1 CP43 02 CP47 The blobs are polypeptides, the wiggles" with circles at the end represent lipids making up the bilayer 2e Pa of the membrane. Names of polypeptides and cofactors have been indicated 2. The ability to grow in many different conditions. It can grow photoautotrophically utilizing its photosynthetic system to produce sugars from CO2 and water, using light for energy. It also can grow photoheterotrophically utilizing a reduced carbon source in the growth medium. This characteristic helps Synechocystis survive under different growth conditions, and cope with a mutation in its photosynthetic system. The mutation is introduced by interrupting the psbC gene by a kanamycin resistance gene via double-homologous recombination with the pKCP43 plasmid. This plasmid contains part of the psbC gene interrupted by a kanamycin resistance gene (Figure 2). Chi Chl As most prokaryotes, Synechocystis has a double-stranded circular genome. However, in contrast to many other prokaryotes, it carries multiple (6-12) copies of the genome in a single cell. As upon transformation only a single genome copy in the cell may be altered, wild-type and mutant genomes are slowly sorted out (segregated) upon repeated cell divisions. A Synechocystis mutant means that all genome copies carry the same mutation, and that the wild-type genome copies are absent. Segregation of mutants is achieved through a steep increase in the antibiotic concentration in the growth medium. Cells that carry the mutation in every copy of the genome are at a selective advantage to cope with high concentrations of the antibiotic. MnMn Mn lumen 12kD 33 kD The doubling time of Synechocystis is 12 hours (versus 20 minutes for E. col), and it takes 7-10 days to get visible colonies of transformants on a plate 2 H20 02 +4 H This experiment consists of two parallel and eventually converging parts, which are distinguished in the experiment outline by the letters-A" and-B". Part-A" of the experiment starts by introducing a mutation in the psbC gene of wild-type Synechocystis. This gene codes for an intrinsic chlorophyll-binding protein CP43 that is essential in the assembly and function of photosystem II (Figure 1) The kanamycin-resistant phenotype of the Synechocystis psbC- mutant will be used for segregation. Complete segregation will be confirmed by performing PCR using primers upstream and downstream of the interrupted part of the psbC gene. After segregation, cells no longer have an active photosystem Il complex and depend on sugar addition for their survival In part "B" of the experiment, part of the wild-type psbC gene will be amplified and will be cloned in a plasmid. The recombinant plasmid is amplified in E. coli, sequenced, and

Explanation / Answer

Photosystem II (PSII), a large multisubunit membrane protein complex found in the thylakoid membranes of cyanobacteria, algae and plants, catalyzes light-driven oxygen evolution from water and reduction of plastoquinone.

Photosynthesis, the conversion of solar energy into biomass, is one of the most fundamental processes on Earth. Only photoautotrophic organisms like cyanobacteria and plants are able to use photons to split water molecules into hydrogen that can be captured in the form of NADPH, and molecular oxygen [1]. The molecular machinery responsible for the conversion process comprises three membrane-bound pigment–protein complexes — photosystems II (PSII) and I (PSI) and the Cyt b6f complex [2]. Together with the mobile electron carriers plastoquinone and plastocyanin, these form the photosynthetic electron transport chain (PET), which is localized in the thylakoid membrane, a specific membrane subcompartment characteristic of cyanobacteria and plant chloroplasts [3,4]. PET is coupled to the synthesis of ATP which, together with NADPH, is utilized for the fixation of CO2 and the subsequent synthesis of carbohydrates. Primordial cyanobacteria “invented” the process of oxygenic photosynthesis more than 2.4 billion years ago, and initiated a dramatic change in the composition of the Earth's atmosphere by releasing the oxygen formed as a byproduct of PSII activity [5]. Furthermore, the photosynthetic activity of cyanobacteria and plants is the source of almost all the biomass on Earth, including all fossil fuels.

The large multisubunit pigment–protein complexes of the PET chain in the thylakoids are assembled in a highly coordinated fashion in both time and space. Among other things, this prevents the release of free chlorophyll, a dangerous photosensitizer that can severely harm cells [6]. The whole process is guided and synchronized by a network of auxiliary proteins [7,8]. These factors are not found in the mature protein complexes, but interact transiently with distinct subunits or co-factors to direct the formation of intermediate (sub)complexes during the assembly process. Recent work has uncovered many of the principles and molecular details of PSII biogenesis in particular, and these results have been summarized in several comprehensive reviews [7,9–14]. Besides presenting an updated overview of PSII assembly factors, we have chosen to focus here on the assembly of cyanobacterial PSII by highlighting recent methodological innovations that facilitate its analysis.

2. Structure and subunit composition of mature photosystem II

PET is initiated at PSII, which catalyzes a unique reaction in nature — the light-driven oxidation of water. A high-resolution crystal structure of cyanobacterial PSII published in 2011 provides a detailed picture of protein–cofactor interactions in the complex at the atomic level [15]. In particular, novel insights into the architecture of the water-oxidizing complex (WOC) with its unique Mn4O5Ca cluster, which forms the catalytic center of PSII, have given us a deeper understanding of the water-splitting reaction [16–19]. However, differences in Mn–Mn distances between the X-ray diffraction (XRD)-based model [15] and the results of extended X-ray absorption fine structure (EXAFS) studies [20,21] have raised the question whether X-ray-induced Mn reduction might have had a distorting impact on the XRD-derived model of the WOC. This problem was recently addressed in several studies that used femtosecond X-ray pulses from a free-electron laser (XFEL) to generate a ‘radiation-damage-free’ structure of PSII [22–26]. The XFEL structure with a resolution of 1.95 Å indicated slightly shorter Mn–Mn distances within a range 0.1 to 0.2 Å relative to the previous XRD structure [15,22]. Moreover, this powerful technique provides a means of monitoring light-induced structural changes in PSII, in particular in the WOC, by time-resolved serial femtosecond crystallography experiments [23,27].

Furthermore, crystal structures of PSII from the thermophilic cyanobacteria Thermosynechococcus elongatus and Thermosynechococcus vulcanus are available, which exhibit slight differences that may reflect interspecies variation or differences in the methods used for preparation or crystallization of the complexes. For instance, Guskov et al. reported a third plastoquinone Qc and the presence of PsbY, both of which are absent from the recent 1.9 Å XRD structure [15,28].

In summary, each monomer of the dimeric PSII protein complex consists of up to 20 protein subunits, 35 chlorophyll a molecules, 20–25 lipids, 12 ?-carotenes, 2–3 plastoquinones, 2 pheophytins, 2 hemes, the WOC (Mn4O5Ca), 4 Ca2 + ions, 3 Cl? ions and a non-heme iron. Most redox centers involved in linear electron transfer are coordinated by the central transmembrane subunits D1 and D2 (PsbA, PsbD; see Fig. 1), whereas light harvesting is mainly mediated by chlorophyll molecules that are bound to the intrinsic antenna proteins CP43 and CP47 (PsbB, PsbC; see Fig. 1). Water oxidation is catalyzed by an inorganic Mn4O5Ca cluster, which is coordinated by amino acid residues in D1, D2 and CP43, and shielded from the thylakoid lumen by the so-called extrinsic subunits PsbO, PsbV and PsbU . The large number of small (< 10 kDa) and hydrophobic (1–2 transmembrane helices) subunits in PSII – 13 in all – is quite remarkable . Some of these play a protective role (e.g. Cyt b559: PsbE, PsbF), others are located at the monomer–monomer interface (PsbL, PsbM, PsbT), but the exact functions of many of them remain elusive (e.g. PsbH, PsbI, PsbJ, PsbK, PsbX, PsbY, PsbZ, Psb30).

The roles of CyanoQ and CyanoP, the cyanobacterial homologs of PsbQ and PsbP in plants, are still unclear. Neither protein can be discerned in the PSII crystal structures based on material isolated from T. elongatusor T. vulcanus[15,28]. In addition, biochemical and mass spectrometric analyses of various tagged or untagged PSII preparations from these strains have failed to detect either protein [32–37]. Recently, in a detailed investigation of this issue, PSII complexes isolated from T. elongatuswere probed with antibodies against CyanoQ and CyanoP [38]. The CyanoQ:PSII monomer and CyanoP:PSII monomer ratios obtained were 0.4:1 and < 0.01:1, respectively. SDS-PAGE analysis permitted no clear assignment of a CyanoQ-related band, perhaps because the protein may comigrate with PsbV [38]. In contrast, PSII complexes isolated from the cyanobacterium Synechocystis sp. PCC 6803 (hereafter: Synechocystis) were found to contain CyanoQ in stoichiometric amounts [39,40]. These CyanoQ containing PSII complexes resemble mainly a fully assembled PSII population with highest activity and stability [39]. Moreover, up to eight copies per PSII dimer may be present in a recently postulated intermediate PSII complex that lacks PsbV and PsbU [41]. In contrast, CyanoP is only detectable in substoichiometric quantities in Synechocystis [42]. A comparative study of several CyanoP disruption mutants from different laboratories came to the conclusion that it might stabilize PSII charge separation and plays a constitutive role in maintenance of PSII activity but the results differ considerably between the tested strains so that a general conclusion might be questionable [43]. This study reveals also the inherent problem of the analysis of cyanobacterial mutant strains, as the used wild type background might influence the results and in cyanobacteria secondary mutations frequently occur that might also alter the phenotype of the desired mutation [43,44].

It was recently shown that recombinant CyanoP binds preferentially to a Psb27-containing PSII assembly/repair intermediate from T. elongatus at a position that is usually occupied by PsbO in the mature complex (Fig. 1and [36,45]). The observation that CyanoP binds preferentially to the free D1 C-terminus led to the hypothesis that CyanoP might facilitate the incorporation of manganese during PSII assembly [45]. A function for CyanoP as a potential PSII assembly factor has been considered earlier [42,46,47]. A recent study analyzed a ?cyanoP?ycf48 double deletion mutant of Synechocystis and concluded that CyanoP impedes the assembly of PSII in the absence of Ycf48 (Fig. 1 and [48]). Interestingly, PPL1, the closest homolog of CyanoP in the plant PsbP family, is also required for efficient PSII assembly in Arabidopsis thaliana[49,50]. Thus several lines of evidence suggest that CyanoP may act as a PSII assembly factor rather than as a structural component of the mature complex in cyanobacteria.

3. Photosynthetic biomass production rapidly declines in mesophilic cyanobacteria grown above their physiological temperatures largely due to the imbalance between degradation and repair of the D1 protein subunit of the heat susceptible Photosystem II reaction centers (PSIIRC). Here we show that simultaneous replacement of two conserved residues in the D1 protein of the mesophilic Synechocystis sp. PCC 6803, by the analogue residues present in the thermophilic Thermosynechococcus elongatus, enables photosynthetic growth, extensive biomass production and markedly enhanced stability and repair rate of PSIIRC for seven days even at 43°C but only at elevated CO2 (1%). Under the same conditions, the Synechocystiscontrol strain initially presented very slow growth followed by a decline after 3 days. Change in the thylakoid membrane lipids, namely the saturation of the fatty acids is observed upon incubation for the different strains, but only the double mutant shows a concomitant major change of the enthalpy and entropy for the light activated QA??QB electron transfer, rendering them similar to those of the thermophilic strain. Following these findings, computational chemistry and protein dynamics simulations we propose that the D1 double mutation increases the folding stability of the PSIIRC at elevated temperatures. This, together with the decreased impairment of D1 protein repair under increased CO2 concentrations result in the observed photothermal tolerance of the photosynthetic machinery in the double mutant.

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