Molecular Cloning and Characterization of the ctpA Gene Encoding a ...

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A nitrofurantoin enrichment technique was used to isolate a spontaneous photosynthesis-deficient mutant strain of the unicellular cyanobacterium Synechocystis.
THE JOURNAL OF BIOLCGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 30,Issue of July 29, pp. 19354-19359, 1994 Printed in U S A .

Molecular Cloning and Characterizationof the ctpA Gene Encoding a Carboxyl-terminal Processing Protease ANALYSIS O F A SPONTANEOUS PHOTOSYSTEM II-DEFICIENT MUTANT STFWNOF THE CYANOBACTERIUM SYNECHOCYSTIS sp. PCC 6803* (Received for publication, January 24, 1994, and in revised form, May 24, 1994)

Sergey V. ShestakovSO, P. R. AnbuduraiSn, Gulshan E. StanbekovaO, Akif GadzhievO, Lisbet K. LindSII, and Himadri B. PakrasiS** From the Wepartment of Biology, Washington University, St. Louis, Missouri 63130 and the $Department of Genetics, Moscow State University, Moscow 119899, Russia

A nitrofurantoin enrichment technique was used to isolate a spontaneous photosynthesis-deficient mutant strain of the unicellular cyanobacterium Synechocystis sp. PCC 6803. This mutant, SK18, lacked any photosystem I1 (PSII) activity, but had normal photosystemI. The SK18 mutant strain could not be complemented with known genes encoding various structural proteins of PSII, but could be complemented with a recombinant plasmid pSL523 containing a1.4-kilobasepair EcoRI fragment ofthe chromosomal DNA from wild-type Synechocystis 6803 cells. Determination of the nucleotide sequence of this DNA fragment revealed a previously unidentified open reading frame (ORF) encoding a 427residue-long polypeptide. Hydrophobicity analysis of the amino acid sequence suggested that this protein is largely hydrophilic. A stretch of the first 31 amino-terminal residues of the polypeptide resembled abacterial signal peptide and may beresponsible for the translocation of this protein to the lumen space of the thylakoid membranes.The spontaneous mutation in the SK18 strain was identified to be a single nucleotide change introducing a prematureterminationcodon in thisORF. The predicted sequence of the encoded protein showed significant similarity to that of the Prc protein, a carboxyl-terminal processing protease in Escherichia coli. We suggest that the cyanobacterial protein encoded by ORF427 is a similar processing protease and name the gene ctpA (carboxylferminal processing protease).

protein complex has been identified. At least 15 polypeptides are present in PSII (see Refs. 1-3 for recent reviews). Among them, D l a n dD2, two integral membrane proteins,coordinate the P680 reaction center chlorophylls in which light-induced charge separation occurs. In addition, two other chlorophyllbinding proteins, CP47 and CP43, as well as cytochrome b,,, and PsbL proteins are known to be essential for the overall function of the PSII complex (1, 4). Other than the P680chlorophylls, PSII contains a number of inorganic and organic cofactors, e.g. plastoquinones,non-hemeiron, manganese, calcium, and chloride. Fourmanganeseatomsare critically important for the oxidation of water in thelumen-exposed part of PSII, where a 33-kDa protein (Msp) stabilizes at leastsome of these manganese atoms (1). The life cycle of the PSII complex is very interesting. PSII is damaged as an inevitable consequence of the light-mediated reduction of quinones and oxidation of water. One of the two reaction center proteins, D l , is then degraded, and a newly synthesized D l protein is incorporated in PSII (reviewed in Ref. 5). The molecular details of this process are largely unknown. The D l protein is synthesized as a precursor polypeptide, integrated into the PSII complex, and thenprocessed to its mature form by the cleavage of a carboxyl-terminal peptide (6-9). In addition, the D l protein in chloroplasts is also phosphorylated and palmitoylated (10). Anumber of other proteins of the PSII complex also undergo various post-translational modifications. None of the enzymes involved in these posttranslational events has been purified, nor do we understand the functional significance of most of these modifications. The unicellularcyanobacterium Synechocystis sp. PCC 6803 During oxygenic photosynthesis in green plants and cya- (hereafter called Synechocystis 6803) has been widely used as a nobacteria, light-induced electron transfer reactions occur pri- convenient microbial system for genetic and biochemical analmarily in large integral protein complexes in the thylakoid ysis of the PSIIcomplex. This organismis naturally transformmembrane. Among them, photosystem I1 (PSII),’ a pigment- able with exogenous DNA, and targeted modifications of variprotein complex, mediates electron transfer from water toplas- ous genescan be generated by using gene replacement toquinone, with concomitant evolution of oxygen (1).During techniques (11, 12). Moreover, PSII-deficient mutants can be recent years, a majority of the structural components of this propagated inglucose-enriched media under light.These properties of this organism have been exploited to create site-di*This work was supported by Grant GM45797 from the National rected mutations in a number of structural proteins of PSII Institutes of Health (to H. B. P.) and a grant from the Russian Foun- (1-3). Because of its natural transformability, Synechocystis dation for Basic Research (to S. V. S.). The costs of publication of this 6803 also offers a useful system for the isolation of random article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement”in accordance photosynthesis-deficient mutants and subsequent cloning of with 18 U.S.C. Section 1734 solely to indicate this fact. genes complementing such defects. A number of spontaneous 11 Current address: Dept. of Botany, The AmericanCollege, Madurai, and chemically induced PSII-deficient mutant strains of Syn625002, India. 11 Current address: Dept. of Microbiology, Umed University, S-90187 echocystis 6803 have been isolated and analyzed (13-16). In this communication, we describe the characterizationof such a Umed, Sweden. **To whom correspondence and reprint requests should be mutant strain, SK18, and the identification of a previously addressed. Tel.: 314-935-6853;Fax: 314-935-4432; E-mail: pakrasia unidentifiedgene ctpA that complements this mutant. Our biodec.wustl.edu. of CtpA, the The abbreviations usedare: PSII, photosystem 11; PSI, photosystem analysis of the predicted amino acid sequence product of this gene, suggests thatit may be a carboxyl-termiI; FeCN, &Fe(CN),; kbp, kilobase paids); ORF, open reading frame.

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ctpA Gene Encoding a C-terminal Processing Protease i n Synechocystis nal specific processing protease localized in the thylakoid lumen space. EXPERIMENTAL PROCEDURES

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TABLEI Growth, pigment composition, and photosynthetic electron transfer activities of wild-type and mutant SK18 strains Doubling times were determined by the measurementof scattering a t 730 nm.

Bacterial Strains and Culture Conditions-The wild-type strain of Wild-type SK18 Synechocystis sp. PCC 6803and the mutantstrain SK18 were grownin Doubling in time (h) 9.2 NG" B G l l medium (17) which was supplemented with 5 m filter-sterilized BGll Phycobilin content (nmoVl0' cells) 8.6 13.9 glucose, where indicated, Cultures were grown under 60 pE m-'of Chlorophyll content (nmoV10' cells) 0.9 0.7 fluorescentlight.Liquidcultureswere grown either on aplatform (nmolhmol) 9.6 19.9 shaker or withvigorous bubbling withroom air. Growth of cyanobacte- Phycobilidchlorophyll Electron transport rates (pmol0, mg rial strains wasmonitored by the measurementof scattering at 730 nm chlorophyll" h")* on a DW2000 spectrophotometer (SLM-Aminco Instruments).