**5. Experimental methods**

#### **5.1 Growth conditions and growth measurements**

Control and mutant *Synechocystis* 6803 were grown in liquid BG-11 media (Williams, 1988) at 30°C and a light intensity of 25 mol photons\*m-2\*s-1, with shaking at 200 rpm on a rotary shaker. Glucose was added, when appropriate, to a final concentration of 5 mM (photomixotrophic growth). For fluorescence measurements, the BG-11 media was supplemented with 5 mM glucose and 10 M N'-(3,4-dichlorophenyl)-N,N-dimethylurea (DCMU). Antibiotics were added to the medium to a final concentration of 10 g/ml. For growth on solid media, BG-11 media was supplemented with 1.5 % agar, 0.3 % sodium thiosulfate, and 10 mM N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid/potassium hydroxide (TES/KOH), pH 8.2, and 10 M DCMU.

For growth under chloride-limiting conditions, chloride was excluded from the media by the addition of calcium nitrate, cobalt nitrate, and manganese sulfate in place of their respective chloride salts. To prevent the leaching of chloride from glass, polycarbonate flasks and carboys were used for growing these cultures. BG-11 prepared in this manner contains approximately 30 M chloride as compared to normal media, which has a chloride concentration of 480 M (Young et al., 2002). For growth assessment, cells were grown as above and the OD730 was determined at the same time each day for 9 days.

#### **5.2 PCR and DNA sequencing**

Genomic DNA was isolated as follows: A small ball of cells was scraped from a culture grown on solid media. The cells were washed with liquid BG-11 media and pelleted by centrifugation at 3326 x *g* for 1 minute. The pelleted cells were resuspended in 400 l 5 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM EDTA. Next, 100 l of lysozyme (50 mg/ml in dH2O)

residue identified in any PSII protein that potentially provides a binding site for cytochrome c550. We have also begun preliminary investigation of mutants produced at the residue adjacent to Arg320, Asp321. Replacement of this aspartate by asparagine produced a mutant with a phenotype very similar to that of the R320S mutant (Putnam-Evans, unpublished). That is, this mutant exhibits almost normal PSII function when grown in complete BG-11 media, but fails to grow photoautotrophically under chloride-limiting conditions and

Examination of the newest crystal structure reveals that Arg320 lies at the interface between cytochrome c550 and PsbU, within 2.9 angstroms of Asn49 of cytochrome c550, close enough to form a hydrogen bond. Another potential hydrogen bonding partner in *Synechocystis* is Asn51. However, in *Thermosynechococcus*, the organism from which the x-ray structure is derived, this residue is a serine residue. Nevertheless, it is located only 3.6 angstroms from Arg320. Asp321 lies within 2.7 angstroms of Asn99. Additionally, Arg320 and Asp321 of CP43 lie within 2.8 angstroms of each other. Thus, these sites are candidates for potential ligands to cytochrome c550 and PsbU. It is tempting to speculate that Arg320 of CP43 is a central player in these interactions, perhaps via a hydrogen bonding network involving several of these and perhaps other residues. Here we report the construction and preliminary characterization of two additional mutations at site 320, R320D and R320K, towards the goal of better understanding the role of this amino acid in these potential

Control and mutant *Synechocystis* 6803 were grown in liquid BG-11 media (Williams, 1988) at 30°C and a light intensity of 25 mol photons\*m-2\*s-1, with shaking at 200 rpm on a rotary shaker. Glucose was added, when appropriate, to a final concentration of 5 mM (photomixotrophic growth). For fluorescence measurements, the BG-11 media was supplemented with 5 mM glucose and 10 M N'-(3,4-dichlorophenyl)-N,N-dimethylurea (DCMU). Antibiotics were added to the medium to a final concentration of 10 g/ml. For growth on solid media, BG-11 media was supplemented with 1.5 % agar, 0.3 % sodium thiosulfate, and 10 mM N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic

For growth under chloride-limiting conditions, chloride was excluded from the media by the addition of calcium nitrate, cobalt nitrate, and manganese sulfate in place of their respective chloride salts. To prevent the leaching of chloride from glass, polycarbonate flasks and carboys were used for growing these cultures. BG-11 prepared in this manner contains approximately 30 M chloride as compared to normal media, which has a chloride concentration of 480 M (Young et al., 2002). For growth assessment, cells were grown as

Genomic DNA was isolated as follows: A small ball of cells was scraped from a culture grown on solid media. The cells were washed with liquid BG-11 media and pelleted by centrifugation at 3326 x *g* for 1 minute. The pelleted cells were resuspended in 400 l 5 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM EDTA. Next, 100 l of lysozyme (50 mg/ml in dH2O)

exhibits a marked decrease in oxygen evolution rates under chloride depletion.

protein-protein interactions in PSII.

**5.1 Growth conditions and growth measurements** 

acid/potassium hydroxide (TES/KOH), pH 8.2, and 10 M DCMU.

above and the OD730 was determined at the same time each day for 9 days.

**5. Experimental methods** 

**5.2 PCR and DNA sequencing** 

was added and the mixture was incubated at 37C for 15 minutes. Then, 20 l of 500 mM EDTA, 50 l of proteinase K (10 mg/ml in 50% glycerol), and 55 l of 10% sarkosyl were added. The mixture was then incubated at 55C for 15 minutes. After 5 minutes at room temperature, 600 l of TE (5 mM Tris-HCl, pH 8.0, 5 mM EDTA) saturated phenol was added and the mixture incubated at room temperature for 10 additional minutes with gentle inversion. The mixture was then centrifuged at 18000 x *g* for 3 minutes and the aqueous phase was transferred to a new tube. Next, 100 l of 5 M NaCl, 100 l of 10% CTAB, and 600 l of chloroform were added to the sample. The tubes were then shaken on a rotary shaker for 15 minutes, centrifuged at 8300 x *g* for 3 minutes, and the aqueous phase was transferred to a new tube. Next, an equal volume of cold 100% isopropanol was added to the sample, and the tubes were gently inverted. The mixture was allowed to incubate at room temperature for 20 minutes to precipitate the DNA before centrifuging the sample at 8200 x *g* for 10 minutes at 4C to pellet the DNA. After centrifugation, the supernatant was removed and discarded, and 1 ml of cold 70% ethanol was added to the tube to wash the DNA pellet. The tubes were then centrifuged again for 10 minutes at 4C. Following this, the supernatant was quickly removed and the DNA pellet was allowed to air dry. Then, 100 l of TE buffer was added to the tube and the DNA was allowed to resuspend at room temperature overnight. Afterward, the DNA was stored at 4C.

The *psbC* gene was amplified by PCR. A typical reaction consisted of 79.7 l of sterile dH2O, 10 l of 10X PCR buffer (Invitrogen, Inc.), 4 l of 50 mM MgCl2, 0.8 l of 10 mM dNTPs, 2 l of forward primer (2 pmol/l) (Invitrogen, Inc.), 2 l of reverse primer (2 pmol/l) (Invitrogen, Inc.), 30-60 ng of genomic DNA, and 0.5 l of Taq DNA polymerase (2.5 units, recombinant). The thermal cycling routine consisted of the following steps: Step 1 – 1 cycle of 94C for 3 minutes, 60C for 45 seconds, and 72C for 2 minutes, Step 2 – 25 cycles of 94C for 1 minute, 60C for 45 seconds, and 72C for 2 minutes, Step 3 – 5 cycles of 94C for 1 minute, 60C for 45 seconds, and 72C for 2 minutes with a 5 second extension added to the elongation step each cycle, and Step 4 – 72C for 7 minutes.

Samples were cleaned using a QIAquick PCR Purification Kit (Qiagen, Inc.) and resuspended in 40 l of sterile dH2O. Following the cleanup, the samples were subjected to sequencing reactions in a PTC-100TM Programmable Thermal Cycler (MJ Research, Inc.). The components of these sequencing reactions were 5 l of sterile dH2O, 2 l of Big Dye (Applied Biosystems, Inc.), 3 l of Big Dye reaction buffer, 2 l of primer (2 pmol/l), and 30-60 ng of purified PCR product DNA. The cycling routine was as follows: 26 cycles of 94C for 1 minute, 55C for 1 minute, and 72C for 2 minutes, followed by a final elongation at 72C for 10 minutes.

After the completion of the sequencing reactions, the DNA was alcohol precipitated with ethanol and was resuspended in a 5:1 formamide:50 mM EDTA/50 mg/ml Blue Dextran mixture. Then, samples were loaded in an ABI Prism 377 DNA Sequencer (Applied Biosystems, Inc.) and the sequencer was run for 7 hours. Sequences were analyzed using the Auto Assembler DNA sequence analysis software (Applied Biosystems v.1.4.0).
