Four Novel Genes Required for Optimal Photoautotrophic Growth of the Cyanobacterium Synechocystis sp. Strain PCC 6803 Identified by In Vitro Transposon Mutagenesis

Abstract

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.

The Malic Enzyme Is Required for Optimal Photoautotrophic Growth of Synechocystis sp. Strain PCC 6803 under Continuous Light but Not under a Diurnal Light Regimen

“…The entire genome has been sequenced and is predicted to encode a total of 3,168 proteins (9). Here we describe the characterization of a mutant designated 3WEZ, which we had isolated previously (17), that bears a transposon insertional mutation in the NADP ϩ -dependent malic enzyme. This enzyme catalyzes the oxidative decarboxylation of malate to pyruvate, concomitantly releasing CO 2.…”

“…The purified DNA was then used in restriction enzyme digestion, Southern blot hybridization, PCR, and DNA sequencing, all of which were performed by standard methods. Inverse PCR was performed as described previously (17). Complementation of the 3WEZ mutation by a cloned 2,089-bp PCR product containing the intact slr0721 gene was performed as described previously (5).…”

“…The transposon was determined to be inserted into the slr0721 gene, which encodes the NADP ϩ -dependent The transposon insertion at codon 51 generated a premature translational stop after amino acid 58. 3WEZ exhibits extremely poor photoautotrophic growth on agar plates which contain only BG-11 medium (17). Complementation analysis was performed with either control strain genomic DNA or a cloned 2,089-bp PCR product containing the wild-type slr0721 gene (Fig.…”

Isolation of cyanobacterial mutants exhibiting growth defects under microoxic conditions by transposon tagging mutagenesis of <i>Synechocystis</i> sp. PCC 6803

“…Consequently, we isolated five mutants (T-1 to T-5) exhibiting growth defects under microoxic con-ditions. Similar in vitro mutagenesis has been performed in Synechocystis 6803 to screen for mutant phenotypes, such as cell motility (Bhaya et al, 2001) and photoheterotrophic growth (Zhang et al, 2004). In these cases, cyanobacterial mutant libraries were generated by transformation of Synechocystis 6803 with plasmid libraries that were prepared by the introduction of a drug marker gene into a plasmid library prepared in E. coli by an in vitro transposon tagging method.…”

Identification of Two Genes, sll0804 and slr1306, as Putative Components of the CO 2 -Concentrating Mechanism in the Cyanobacterium Synechocystis sp. Strain PCC 6803

“…strain PCC 6803 genome indicates that nearly half of the 3,260 genes present are of unknown function (6). In our laboratory we are interested in identifying novel genes required for optimal oxygenic photosynthesis (3,18). In this study we report the apparent involvement of two genes, sll0804 and slr1306, in the CCM of Synechocystis sp.…”