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A mutation was recovered in the slr0721 gene, which encodes the decarboxylating NADP ؉ -dependent malic enzyme in the cyanobacterium Synechocystis sp. strain PCC 6803, yielding the mutant 3WEZ. Under continuous light, 3WEZ exhibits poor photoautotrophic growth while growing photoheterotrophically on glucose at rates nearly indistinguishable from wild-type rates. Interestingly, under diurnal light conditions (12 h of light and 12 h of dark), normal photoautotrophic growth of the mutant is completely restored.Cyanobacteria are photoautotrophic gram-negative eubacteria that are capable of performing oxygenic photosynthesis. Synechocystis sp. strain PCC 6803 is a naturally transformable (7) unicellular cyanobacterium and has proven to be one of the best model organisms for studying the mechanism and regulation of oxygenic photosynthesis (14); it has also been used in a variety of global gene expression (6,8,13) and metabolomic (15, 16) studies. In addition to autotrophic growth, the presence of an unidentified mutation in the Williams strain of Synechocystis (14) confers glucose tolerance to this organism. With glucose as a carbon source, this strain can be grown under mixotrophic, photoheterotrophic (continuous photosynthetic illumination at 20 to 40 mol of photons m Ϫ2 s Ϫ1 in the presence of the photosystem II inhibitor dichloromethylurea [DCMU]), and heterotrophic (nonphotosynthetic continuous illumination at Ͻ1 mol of photons m Ϫ2 s Ϫ1

Four novel Synechocystis sp. strain PCC 6803 genes (sll1495, sll0804, slr1306, and slr1125) which encode hypothetical proteins were determined by transposon mutagenesis to be required for optimal photoautotrophic growth. Mutations were also recovered in ccmK4, a carboxysome coat protein homologue, and me, the decarboxylating NADP ؉ -dependent malic enzyme. This is the first report that these known genes are required for optimal photoautotrophy.Photosynthesis is one of the most important biological processes and occurs in a very diverse set of organisms ranging from prokaryotes to eukaryotes. Recently, much effort has been directed towards understanding the structure and function of proteins involved in photosynthesis (photosystem I, photosystem II, cytochrome b 6 /f complex, Calvin-Benson cycle enzymes, etc.). While much progress has been made in the understanding of the functional organization of these proteins, relatively little is known concerning the organization of other protein components which must be involved in the regulation, assembly, and turnover of the proteins involved in photosynthesis. Cyanobacteria are photoautotrophic gram-negative eubacteria capable of performing oxygenic photosynthesis in a manner quite similar to that in eukaryotic algae and higher plants. Synechocystis sp. strain PCC 6803 is a naturally competent unicellular cyanobacterium and has proved to be one of the best model organisms for studying the mechanism and regulation of oxygenic photosynthesis (15). We are interested in identifying the genes required for oxygenic photosynthesis. In this study, we used a hyperactive Tn5-based in vitro transposition system to introduce random insertional mutations into Synechocystis and have identified a number of mutants which are incapable of undergoing optimal photoautotrophic growth. Here we describe the production, identification, and characterization of a number of these mutants. The structure and possible function of the affected genes in these mutants will also be discussed.A glucose-tolerant strain of Synechocystis sp., PCC 6803 (15), was used as a parental control and as the DNA recipient strain in the present study. Cells of both the control strain and the derivative photosynthetic mutants were maintained under photoheterotrophic growth conditions at 30°C with a light intensity of 20 mol of photons m Ϫ2 s Ϫ1 (fluorescent light) in liquid BG-11 growth medium (ATCC medium 616) supplemented with 10 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid]-KOH (pH 8.2), 5 mM glucose, and 10 M DCMU [N-(3,4-dichlorophenyl)-NЈ-dimethylurea]. Liquid cultures were bubbled continuously with air. For autotrophic cell culture, the glucose and DCMU were omitted. For cultures grown on plates, the BG-11 medium was supplemented with 1.5% agar and 0.3% sodium thiosulfate. When appropriate, kanamycin was included in the media at a final concentration of 10 g/ml.

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An insertional transposon mutation in the sll0606 gene was found to lead to a loss of photoautotrophy but not photoheterotrophy in the cyanobacterium Synechocystis sp. PCC 6803. Complementation analysis of this mutant (Tsll0606) indicated that an intact sll0606 gene could fully restore photoautotrophic growth. Gene organization in the vicinity of sll0606 indicates that it is not contained in an operon. No electron transport activity was detected in Tsll0606 using water as an electron donor and 2,6-dichlorobenzoquinone as an electron acceptor, indicating that Photosystem II (PS II) was defective. Electron transport activity using dichlorophenol indolephenol plus ascorbate as an electron donor to methyl viologen, however, was the same as observed in the control strain. This indicated that electron flow through Photosystem I was normal. Fluorescence induction and decay parameters verified that Photosystem II was highly compromised. The quantum yield for energy trapping by Photosystem II (F V /F M ) in the mutant was less than 10% of that observed in the control strain. The small variable fluorescence yield observed after a single saturating flash exhibited aberrant Q A ؊ reoxidation kinetics that were insensitive to dichloromethylurea. Immunological analysis indicated that whereas the D2 and CP47 proteins were modestly affected, the D1 and CP43 components were dramatically reduced. Analysis of twodimensional 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 the Sll0606 protein is required for the assembly/stability of a functionally competent Photosystem II.In higher plants, algae, and cyanobacteria, at least six intrinsic proteins appear to be required for oxygen evolution by PS II 2 (1-3). These are CP47, CP43, the D1 and D2 proteins, and the ␣ and ␤ subunits of cytochrome b 559 . 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, see 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 (6 -8), although the functions of many of these proteins remain obscure. The most recent crystal structure of the thermophilic cyanobacterium Thermosynechococcus 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 missing from the current crystal structure).PS II assembly and turnover requires a variety of other protein components (for a comprehensive review, see Ref. 11). Although many of these proteins are conserved in all oxygenic organisms, a subset is present only in the cyanobacteria. These include the Synechocystis sp. PC...

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Insertional transposon mutations in the sll0804 and slr1306 genes were found to lead to a loss of optimal photoautotrophy in the cyanobacterium Synechocystis sp. strain PCC 6803 grown under ambient CO 2 concentrations (350 ppm). Mutants containing these insertions (4BA2 and 3ZA12, respectively) could grow photoheterotrophically on glucose or photoautotrophically at elevated CO 2 concentrations (50,000 ppm). Both of these mutants exhibited an impaired affinity for inorganic carbon. Consequently, the Sll0804 and Slr1306 proteins appear to be putative components of the carbon-concentrating mechanism in Synechocystis sp. strain PCC 6803.