Characterization of Site-Directed Mutants in the Cytochrome c-550 Protein of Photosystem II

Photosynthesis is the process by which cyanobacteria, algae, and higher plants convert light energy to chemical energy via the biosynthesis of carbohydrates. Photosystem II is a multi-protein/pigment complex embedded in the thylakoid membrane. CP43 is the product of the psbC gene, and is an intrinsic component of the photosystem II complex. CP43 is 473 amino acids long and has five hydrophilic loops connecting six transmembrane alpha helices, including a large extrinsic loop E which is exposed to the lumenal side of the thylakoid membrane. Short deletions of regions of the loop E of CP43 resulted in mutants that fail to grow photoautotrophically and are devoid of oxygen-evolving activity. A mutation was introduced in loop E that altered arginine at position 305 to a serine residue, producing a mutant with severely reduced growth and oxygen evolving activity under chloride limiting conditions. However, when grown under normal conditions, the R305S mutant showed no extreme phenotype. After isolation of photosystem II particles and chemilumininescent staining for cytochromes, it was determined that this specific mutation resulted in loss of binding of an extrinsic photosystem II protein, cytochrome c-550. Deletion of the psbV gene encoding cytochrome c-550 resulted in the loss of photoautotrophic growth in media lacking chloride and/or calcium. X-ray crystallography of photosystem II does in fact show a close proximity between cytochrome c-550 and CP43. Based on the crystal structure, two residues on cytochrome c-550 were identified that might interact with CP43 and are in close proximity with the arginine residue at position 305. These residues on cytochrome c-550 are both highly conserved asparagines, located at positions forty nine and fifty one. In this work, the asparagine residue at position fifty one on cytochrome c-550 was mutated to either an alanine or aspartic acid residue. The N51 mutations were transformed into wildtype Synechocystis, a cyanobacterial model organism that has been widely used to study photosystem II. The N51 mutants were characterized by photoautotrophic growth in complete and chloride-limiting media, oxygen evolution assays, variable florescence yield measurements and photoinactivation assays. Based on these assays, the control and N51 mutant strains exhibited similar phenotypes in complete media while the control and N51D mutant strains exhibited similar phenotypes in chloride limiting media. The N51A mutant had a small but reproducible decrease in photoautotrophic growth rate, oxygen evolution rates and enhanced sensitivity to photoinactivation in chloride limiting media. Therefore, asparagine at position fifty one on cytochrome c-550 might contribute to a weak interaction between arginine on CP43 and cytochrome c-550, which is likely to involve hydrogen bonding. However, further studies are needed to validate the proposed interaction between subunits cytochrome c-550 and CP43 of photosystem II.  M.S

Comparative Transmissibility of SARS-CoV-2 Variants Delta and Alpha in New England, USA

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant quickly rose to dominance in mid-2021, displacing other variants, including Alpha. Studies using data from the United Kingdom and India estimated that Delta was 40-80% more transmissible than Alpha, allowing Delta to become the globally dominant variant. However, it was unclear if the ostensible difference in relative transmissibility was due mostly to innate properties of Delta\u27s infectiousness or differences in the study populations. To investigate, we formed a partnership with SARS-CoV-2 genomic surveillance programs from all six New England US states. By comparing logistic growth rates, we found that Delta emerged 37-163% faster than Alpha in early 2021 (37% Massachusetts, 75% New Hampshire, 95% Maine, 98% Rhode Island, 151% Connecticut, and 163% Vermont). We next computed variant-specific effective reproductive numbers and estimated that Delta was 58-120% more transmissible than Alpha across New England (58% New Hampshire, 68% Massachusetts, 76% Connecticut, 85% Rhode Island, 98% Maine, and 120% Vermont). Finally, using RT-PCR data, we estimated that Delta infections generate on average ∼6 times more viral RNA copies per mL than Alpha infections. Overall, our evidence indicates that Delta\u27s enhanced transmissibility could be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on the underlying immunity and behavior of distinct populations

Comparative transmissibility of SARS-CoV-2 variants Delta and Alpha in New England, USA.

The SARS-CoV-2 Delta variant rose to dominance in mid-2021, likely propelled by an estimated 40%-80% increased transmissibility over Alpha. To investigate if this ostensible difference in transmissibility is uniform across populations, we partner with public health programs from all six states in New England in the United States. We compare logistic growth rates during each variant\u27s respective emergence period, finding that Delta emerged 1.37-2.63 times faster than Alpha (range across states). We compute variant-specific effective reproductive numbers, estimating that Delta is 63%-167% more transmissible than Alpha (range across states). Finally, we estimate that Delta infections generate on average 6.2 (95% CI 3.1-10.9) times more viral RNA copies per milliliter than Alpha infections during their respective emergence. Overall, our evidence suggests that Delta\u27s enhanced transmissibility can be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on underlying population attributes and sequencing data availability

Characterization of Site-Directed Mutants in the Cytochrome c-550 Protein of Photosystem II

Photosynthesis is the process by which cyanobacteria, algae, and higher plants convert light energy to chemical energy via the biosynthesis of carbohydrates. Photosystem II is a multi-protein/pigment complex embedded in the thylakoid membrane. CP43 is the product of the psbC gene, and is an intrinsic component of the photosystem II complex. CP43 is 473 amino acids long and has five hydrophilic loops connecting six transmembrane alpha helices, including a large extrinsic loop E which is exposed to the lumenal side of the thylakoid membrane. Short deletions of regions of the loop E of CP43 resulted in mutants that fail to grow photoautotrophically and are devoid of oxygen-evolving activity. A mutation was introduced in loop E that altered arginine at position 305 to a serine residue, producing a mutant with severely reduced growth and oxygen evolving activity under chloride limiting conditions. However, when grown under normal conditions, the R305S mutant showed no extreme phenotype. After isolation of photosystem II particles and chemilumininescent staining for cytochromes, it was determined that this specific mutation resulted in loss of binding of an extrinsic photosystem II protein, cytochrome c-550. Deletion of the psbV gene encoding cytochrome c-550 resulted in the loss of photoautotrophic growth in media lacking chloride and/or calcium. X-ray crystallography of photosystem II does in fact show a close proximity between cytochrome c-550 and CP43. Based on the crystal structure, two residues on cytochrome c-550 were identified that might interact with CP43 and are in close proximity with the arginine residue at position 305. These residues on cytochrome c-550 are both highly conserved asparagines, located at positions forty nine and fifty one. In this work, the asparagine residue at position fifty one on cytochrome c-550 was mutated to either an alanine or aspartic acid residue. The N51 mutations were transformed into wildtype Synechocystis, a cyanobacterial model organism that has been widely used to study photosystem II. The N51 mutants were characterized by photoautotrophic growth in complete and chloride-limiting media, oxygen evolution assays, variable florescence yield measurements and photoinactivation assays. Based on these assays, the control and N51 mutant strains exhibited similar phenotypes in complete media while the control and N51D mutant strains exhibited similar phenotypes in chloride limiting media. The N51A mutant had a small but reproducible decrease in photoautotrophic growth rate, oxygen evolution rates and enhanced sensitivity to photoinactivation in chloride limiting media. Therefore, asparagine at position fifty one on cytochrome c-550 might contribute to a weak interaction between arginine on CP43 and cytochrome c-550, which is likely to involve hydrogen bonding. However, further studies are needed to validate the proposed interaction between subunits cytochrome c-550 and CP43 of photosystem II.