Characterization of site-directed mutants in manganese-stabilizing protein (MSP) of Synechocystis sp. PCC6803 unable to grow photoautotrophically in the absence of cytochrome c-550.
To investigate the interaction between the manganese-stabilizing protein (MSP) and cytochrome c-550 (cyt. c-550) of the photosystem II (PSII) complex in the cyanobacterium Synechocystis sp. PCC6803, three site-directed amino acid substitution mutants in MSP (MSP-D159N, MSP-R163L, MSP-D 159N/R 163L) were created by single and double amino acid substitution mutagenesis. The modified psbO genes encoding the mutants forms of MSP were used to transform a single-deletion mutant deltapsO strain lacking MSP as well as a double-deletion strain deltapsbO:deltapsbV lacking both MSP and cyt. c-550. The mutant forms of MSP were expressed in each case and all permitted autotrophic growth in strains expressing cyt. c-550. However, when the MSP mutations were introduced into a strain which lacks cyt. c-550 (deltapsbV), the two single amino acid substitution mutants (deltapsbV:MSP-D159N and deltapsbV:MSP-R 163L) failed to grow photoautotrophically. These strains exhibited coupled O2-evolving activity of 68-77% compared to the wild-type control using CO2 as an electron acceptor and maximal uncoupled O2-evolution rates of 42-57% using 2,6-dichloro-p-benzoquinone (DCBQ) as an artificial electron acceptor. Interestingly, when the two amino acid substitutions were together in the absence of cyt. c-550 (deltapsbV:MSP-D159N/R163L), the mutant grew photoautotrophically and the oxygen-evolving activities were higher than in the single mutants. This indicates that the MSP-D159N mutant suppresses the non-autotrophic phenotype of MSP-R163L (or vice versa) in the absence of cyt. c-550. The possibilities of a direct (ionic) or indirect interaction between D159 and R163 of MSP are discussed.
Al-Khaldi
S F
SF
Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater 74078, USA.
Coker
J
J
Shen
J R
JR
Burnap
R L
RL
D000595:Amino Acid Sequence; D002455:Cell Division / Q000235:genetics; D000458:Cyanobacteria / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D045332:Photosystem II Protein Complex; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D003574:Cytochrome c Group; D045332:Photosystem II Protein Complex; D011506:Proteins; C054266:photosystem II manganese-stabilizing protein; C031732:cytochrome C-550; D010100:Oxygen