This fragment was then introduced into the Tsll0606 mutant cells by standard transformation procedures. D2 and CP47 proteins were modestly affected, the D1 and CP43 MT-DADMe-ImmA components were dramatically reduced. Analysis of two-dimensional blue native/lithium dodecyl sulfate-polyacrylamide gels indicated that no intact PS II monomer or dimers were observed in the mutant. The CP43-less PS II monomer did accumulate to detectable levels. Our results indicate that this Sll0606 protein is required for the assembly/stability of a functionally qualified Photosystem II. Keywords:Bioenergetics, Membrane Biogenesis, Membrane Energetics, Photosynthesis, Grow, Photosystem II, Cyanobacteria == Introduction == In higher plants, algae, and cyanobacteria, at least six intrinsic proteins appear to be required for oxygen evolution by PS II2(13). These are CP47, CP43, the D1 and D2 proteins, and the and subunits of cytochromeb559. Insertional inactivation or deletion of the genes for these components results in the absence of PS II complex assembly and the complete loss of oxygen evolution activity (for a review, observe Ref.4). For maximal rates of oxygen evolution in cyanobacteria, the extrinsic proteins PsbO, PsbU, PsbV, and PsbQ must also be present (5). Additionally, a large number of other intrinsic membrane components are present in PS II complexes (68), even though functions of many of these proteins remain obscure. The most recent crystal structure of the thermophilic cyanobacteriumThermosynechococcus elongatus(9) indicates that PS II contains 20 protein components (it should be noted that PsbQ, which is essential for maximum PS II activity in cyanobacteria (10), is usually missing from the current crystal structure). PS II assembly and turnover requires a variety of other protein components (for a comprehensive review, observe Ref.11). Although many of these proteins are conserved in all oxygenic organisms, a subset is present only in the cyanobacteria. These include theSynechocystissp. PCC 6803 (henceforthSynechocystis) proteins Slr0286 (12) and Slr2013 (13), which were identified during the screening of suppressor strains of various D2 mutants. The functions of these components remain poorly comprehended. The PratA protein (encoded byslr0248) is a periplasmic component that appears to be involved in D1 processing (14) and may directly interact with the C-terminal domain name of the D1 protein (15). Finally, an operon containing six genes (slr0144slr0152) has been identified, which may be involved in the assembly/stability of PS Rabbit polyclonal to AHSA1 II (16). The deletion of this operon leads to a 35% loss of MT-DADMe-ImmA oxygen evolution, slower deactivation of the higher S-states, and slower photoautotrophic growth. It should also be noted that carotenoids (as well as chlorophyll) are critically important for PS II assembly/stability. Simultaneous genetic deletion of thecrtB andcrtH genes inSynechocystisleads to a complete loss of carotenoid biosynthesis and the loss of assembly of intact, fully functional PS II reaction centers. This is the result of a markedly decreased accumulation of the CP47, CP43, and D1 proteins under either light-activated heterotrophic or continuous illumination growth conditions. The small amounts of CP47 and D1 that do accumulate are found almost exclusively in the CP43-less RC47 complex (17), with little or no assembled CP43 being observed. Consequently, carotenoids appear to be required for CP47, CP43, and D1 accumulation and integration into the functional PS II reaction center complex. In vitrotransposon mutagenesis is usually a powerful tool for identifying genes required for photoautotrophy (18). Earlier, we have used this technique to identify previously undescribed components of the cyanobacterial carbon-concentrating mechanism (19) and have also elucidated a differential role for the malic enzyme in carbon metabolism under continuousversusdiurnal illumination (20). In the current study, a cyanobacterial mutant (designated Tsll0606) bearing a transposon insertion in thesll0606 gene was MT-DADMe-ImmA isolated. This mutant exhibits drastically altered PS II characteristics and fails to integrate CP43, D1, and CP47 into functional oxygen-evolving PS II reaction centers. The characteristics of this mutant indicate that this Sll0606 protein is a cyanobacteria-specific assembly/stability factor for PS II. == MATERIALS AND METHODS == == == == == == Cyanobacterial Strains and Growth Conditions == A glucose-tolerant strain ofSynechocystissp. MT-DADMe-ImmA strain PCC 6803 (21) was used as the DNA recipient strain in this study, and the derivative kanamycin-resistant strain K3 (22) was used as a wild-type control strain. Cells of all strains were managed under photoheterotrophic growth conditions at 30 C with a light intensity of 20 mol of photons m2s1on BG-11 growth medium (23) supplemented with 10 mmTES-KOH (pH 8.2), 5 mmglucose, 10 mN-(3,4-dichlorophenyl)-N-dimethylurea (DCMU), 0.3% (w/v) sodium thiosulfate, MT-DADMe-ImmA and 1.5% (w/v) agar. Where appropriate, kanamycin.