Nuclear genes play important regulatory roles in the biogenesis of the photosynthetic apparatus of eukaryotic cells by encoding factors that control steps ranging from chloroplast gene transcription to post-translational processes. However, the identities of these genes and the mechanisms by which they govern these processes are largely unknown. By using glass bead-mediated transformation to generate insertional mutations in the nuclear genome of Chlamydomonas reinhardtii, we have generated four mutants that are defective in the accumulation of the cytochromeb 6 f complex. One of them, strain abf3, also fails to accumulate holocytochromec 6. We have isolated a gene, Ccs1, from a C. reinhardtii genomic library that complements both the cytochrome b 6 f and cytochromec 6 deficiencies in abf3. The predicted protein product displays significant identity with Ycf44 from the brown algaOdontella sinensis, the red alga Porphyra purpurea, and the cyanobacterium Synechocystis strain PCC 6803 (25–33% identity). In addition, we note limited sequence similarity with ResB of Bacillus subtilis and an open reading frame in a homologous operon in Mycobacterium leprae (11–12% identity). On the basis of the pleiotropicc-type cytochrome deficiency in the ccs1mutant, the predicted plastid localization of the protein, and its relationship to candidate cytochrome biosynthesis proteins in Gram-positive bacteria, we conclude that Ccs1 encodes a protein that is required for chloroplast c-type holocytochrome formation. Nuclear genes play important regulatory roles in the biogenesis of the photosynthetic apparatus of eukaryotic cells by encoding factors that control steps ranging from chloroplast gene transcription to post-translational processes. However, the identities of these genes and the mechanisms by which they govern these processes are largely unknown. By using glass bead-mediated transformation to generate insertional mutations in the nuclear genome of Chlamydomonas reinhardtii, we have generated four mutants that are defective in the accumulation of the cytochromeb 6 f complex. One of them, strain abf3, also fails to accumulate holocytochromec 6. We have isolated a gene, Ccs1, from a C. reinhardtii genomic library that complements both the cytochrome b 6 f and cytochromec 6 deficiencies in abf3. The predicted protein product displays significant identity with Ycf44 from the brown algaOdontella sinensis, the red alga Porphyra purpurea, and the cyanobacterium Synechocystis strain PCC 6803 (25–33% identity). In addition, we note limited sequence similarity with ResB of Bacillus subtilis and an open reading frame in a homologous operon in Mycobacterium leprae (11–12% identity). On the basis of the pleiotropicc-type cytochrome deficiency in the ccs1mutant, the predicted plastid localization of the protein, and its relationship to candidate cytochrome biosynthesis proteins in Gram-positive bacteria, we conclude that Ccs1 encodes a protein that is required for chloroplast c-type holocytochrome formation. Studies in the unicellular green alga Chlamydomonas reinhardtii and vascular plants have demonstrated that the proteins required for photosynthesis are encoded by genes that reside in two distinct cellular compartments: the nucleus and the chloroplast (reviewed in Refs. 1Rochaix J.-D. Annu. Rev. Cell Biol. 1992; 8: 1-28Crossref PubMed Scopus (123) Google Scholar, 2Gruissem W. Tonkyn J.C. Crit. Rev. Plant Sci. 1993; 12: 19-55Crossref Scopus (136) Google Scholar, 3Rochaix J.-D. Plant Mol. Biol. 1996; 32: 327-341Crossref PubMed Scopus (157) Google Scholar). The nucleus encodes both structural polypeptides and proteins that play pivotal roles in the regulation of chloroplast gene expression (4Kuchka M.R. Mayfield S.P. Rochaix J.-D. EMBO J. 1988; 7: 319-324Crossref PubMed Google Scholar, 5Rochaix J.-D. Kuchka M. Mayfield S. Schirmer-Rahire M. Girard-Bascou J. Bennoun P. EMBO J. 1989; 8: 1013-1021Crossref PubMed Scopus (148) Google Scholar, 6Goldschmidt-Clermont M. Girard-Bascou J. Choquet Y. Rochaix J.-D. Mol. Gen. Genet. 1990; 233: 417-425Crossref Scopus (111) Google Scholar, 7Schuster G. Gruissem W. EMBO J. 1991; 10: 1493-1502Crossref PubMed Scopus (152) Google Scholar, 8Danon A. Mayfield S.P.Y. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar, 9Girard-Bascou J. Pierre Y. Drapier D. Curr. Genet. 1992; 22: 47-52Crossref PubMed Scopus (55) Google Scholar, 10Hayes R. Kudla J. Schuster G. Gabay L. Maliga P. Gruissem W. EMBO J. 1996; 15: 1132-1141Crossref PubMed Scopus (145) Google Scholar) and in the maturation and assembly of the photosynthetic apparatus (11Howe G. Merchant S. EMBO J. 1992; 12: 2789-2801Crossref Scopus (63) Google Scholar, 12Barkan A. Voelker R. Mendelhartvig J. Johnson D. Walker M. Physiol. Plant. 1995; 93: 163-170Crossref Scopus (47) Google Scholar). Although biochemical and genetic studies are beginning to lead to a better understanding of the roles that nucleus-encoded regulatory proteins play in these processes (7Schuster G. Gruissem W. EMBO J. 1991; 10: 1493-1502Crossref PubMed Scopus (152) Google Scholar, 8Danon A. Mayfield S.P.Y. EMBO J. 1991; 10: 3993-4001Crossref PubMed Scopus (165) Google Scholar, 10Hayes R. Kudla J. Schuster G. Gabay L. Maliga P. Gruissem W. EMBO J. 1996; 15: 1132-1141Crossref PubMed Scopus (145) Google Scholar, 13Danon A. Mayfield S.P. Science. 1994; 266: 1717-1719Crossref PubMed Scopus (278) Google Scholar), the identities of many of these proteins and the mechanisms by which they exert their influence remain largely unknown. Defects in genes that regulate chloroplast gene expression or that are involved in the maturation or assembly of photosynthetic complexes are likely to cause a nonphotosynthetic phenotype. C. reinhardtii is an attractive organism for studying these genes, because nonphotosynthetic mutants are viable when provided with acetate as a carbon and energy source. With the ultimate goal of understanding the mechanisms by which nucleus-encoded factors regulate chloroplast gene expression and function, we have generated tagged nonphotosynthetic mutants, focusing initially on the isolation and characterization of nuclear genes required for the biogenesis of the cytochrome b 6 f complex of C. reinhardtii. The cytochrome b 6 f complex carries out photosynthetic electron transfer between photosystem (PS) 1The abbreviations used are: PS, photosystem; bp, base pair(s); kb, kilobase pair(s); ORF, open reading frame; RFLP, restriction fragment length polymorphism; SGII, Sager-Granick medium; SuIV, subunit IV. II and PS I and is composed of both chloroplast- and nucleus-encoded subunits (14Lemaire C. Girard-Bascou J. Wollman F.-A. Bennoun P. Biochim. Biophys. Acta. 1986; 851: 229-238Crossref Scopus (94) Google Scholar, 15Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Its compositional simplicity relative to the PS II and PS I complexes makes it an attractive model for studying the assembly of chloroplast energy-transducing complexes. The chloroplast-encoded subunits of the cytochrome b 6 f complex include products of petA (cytochrome f), petB(cytochrome b 6), petD (subunit IV), and two 4-kDa proteins encoded by the petG andpetL genes (15Pierre Y. Breyton C. Kramer D. Popot J.-L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 16Buschlen S. Choquet Y. Kuras R. Wollman F.-A. FEBS Lett. 1991; 284: 257-262Crossref PubMed Scopus (47) Google Scholar, 17Matsumoto T. Matsuo M. Matsuda Y. Plant Cell Physiol. 1991; 32: 863-872Google Scholar, 18Berthold D.A. Schmidt C.L. Malkin R. J. Biol. Chem. 1995; 270: 29293-29298Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 19Takahashi Y. Rahire M. Breyton C. Popot J.-L. Joliot P. Rochaix J.-D. EMBO J. 1996; 15: 3498-3506Crossref PubMed Scopus (75) Google Scholar, 20Cramer W.A. Soriano G.M. Zhang H. Ponamarev M.V. Smith J.L. Physiol. Plant. 1997; 100: 852-862Crossref Google Scholar). The nucleus-encoded components include the Rieske Fe-S protein (product of PetC; Ref. 21de Vitry C. J. Biol. Chem. 1994; 269: 7603-7609Abstract Full Text PDF PubMed Google Scholar) and a 4-kDa protein (product of PetM; Refs. 22de Vitry C. Breyton C. Pierre Y. Popot J.-L. J. Biol. Chem. 1996; 271: 10667-10671Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar and 23Ketchner S.L. Malkin R. Biochim. Biophys. Acta. 1996; 1273: 195-197Crossref PubMed Scopus (16) Google Scholar). The binding sites for the hemes and the Fe2S2center are well defined, and each cofactor binding site lies within a single polypeptide. This feature makes theb 6 f complex particularly suited for studies of cofactor-polypeptide association and assembly. Previous studies have identified a number of nuclear genes required for the biogenesis of the cytochrome b 6 fcomplex (9Girard-Bascou J. Pierre Y. Drapier D. Curr. Genet. 1992; 22: 47-52Crossref PubMed Scopus (55) Google Scholar, 11Howe G. Merchant S. EMBO J. 1992; 12: 2789-2801Crossref Scopus (63) Google Scholar, 14Lemaire C. Girard-Bascou J. Wollman F.-A. Bennoun P. Biochim. Biophys. Acta. 1986; 851: 229-238Crossref Scopus (94) Google Scholar, 24Gumpel N.J. Ralley L. Girard-Bascou J. Wollman F.-A. Nugent J.H.A. Purton S. Plant Mol. Biol. 1995; 29: 921-932Crossref PubMed Scopus (50) Google Scholar, 25Bendall D.S. Sanguansermsri M. Girard-Bascou J. Bennoun P. FEBS Lett. 1986; 203: 31-35Crossref Scopus (17) Google Scholar). Among these are a distinct subset of four nuclear loci that are also required for synthesis of cytochromec 6. 2Xie, Z., Culler, D., Dreyfuss, B. W., Kuras, R., Wollman, F.-A., Girard-Bascou, J., and Merchant, S. (1998)Genetics, in press.Cytochrome c 6, a functional substitute for plastocyanin, is a soluble heme-containing protein that carries out electron transfer between the cytochromeb 6 f complex and PS I; its expression is induced under copper-deficient conditions (11Howe G. Merchant S. EMBO J. 1992; 12: 2789-2801Crossref Scopus (63) Google Scholar, 26Merchant S. Bogorad L. Mol. Cell. Biol. 1986; 6: 462-469Crossref PubMed Scopus (139) Google Scholar). Cytochromec 6 and cytochrome f arec-type cytochromes in which the heme prosthetic group is covalently attached to the apoproteins. While cytochrome fis chloroplast-encoded, cytochrome c 6 is encoded by the nuclear Cyc6 gene, synthesized in the cytosol, and imported post-translationally into the chloroplast (26Merchant S. Bogorad L. Mol. Cell. Biol. 1986; 6: 462-469Crossref PubMed Scopus (139) Google Scholar, 27Merchant S. Hill K. Howe G. EMBO J. 1991; 10: 1383-1389Crossref PubMed Scopus (69) Google Scholar). Thus, there are few biosynthetic steps that are common for cytochromec 6 and cytochrome f besides translocation across the thylakoid membrane, lumenal processing, and heme attachment. In previous work, we identified several mutants that display a cytochromeb 6 f −/cytochrome c6− phenotype, and biochemical characterization confirmed that the strains were able to translocate and process precursor and intermediate forms of cytochrome fand c 6 but were unable to convert the apocytochromes to their respective holoforms (11Howe G. Merchant S. EMBO J. 1992; 12: 2789-2801Crossref Scopus (63) Google Scholar, 28Howe G. Mets L. Merchant S. Mol. Gen. Genet. 1995; 246: 156-165Crossref PubMed Scopus (28) Google Scholar). This work defined a specific biochemical phenotype for heme attachment mutants (cytochromeb 6 f −/cytochromec 6−) and suggested that a common set of proteins is required for holocytochrome formation in the thylakoid lumen. One of these is the product of the chloroplast-encodedccsA gene, which is required for the biosynthesis and accumulation of the cytochrome b 6 fcomplex and cytochrome c 6 (29Xie Z. Merchant S. J. Biol. Chem. 1996; 271: 4632-4639Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Loss of CcsA function results in a defect at the step of heme attachment to apocytochromes c 6 and f. In the present study, we describe the isolation and characterization of a nuclear gene, Ccs1 (c -typecyt synthesis), which is required for assembly of the cytochrome b 6 f complex and cytochrome c 6. On the basis of a pleiotropicc-type cytochrome deficiency in a strain lacking functional Ccs1, we suggest that its gene product functions in the same pathway as CcsA. C. reinhardtii strain nit1–305 cw15, which is the wild type for photosynthesis, was used as a recipient for nuclear transformations. C. reinhardtii strain CC-125 (wild type) was obtained from the Chlamydomonas Genetics Center (Duke University, Durham, NC). Strain B6, isolated by Gal et al. (30Gal A. Mets L.J. Ohad I. Baltscheffsky M. Current Research in Photosynthesis. II. Kluwer Academic, The Netherlands1990: 779-781Google Scholar), does not accumulate cytochrome c 6or the cytochrome b 6 f complex due to a frameshift mutation in the chloroplast ccsA gene (11Howe G. Merchant S. EMBO J. 1992; 12: 2789-2801Crossref Scopus (63) Google Scholar, 29Xie Z. Merchant S. J. Biol. Chem. 1996; 271: 4632-4639Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar,31Howe G. Merchant S. J. Biol. Chem. 1994; 269: 5824-5832Abstract Full Text PDF PubMed Google Scholar). CC-125 and B6 were used as positive and negative control strains, respectively, for the analyses of cytochrome c 6and cytochrome f synthesis. E. coli strains XL1-Blue and DH5α were used as hosts for recombinant DNA techniques (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Cells were grown in Sager-Granick medium (33Harris E.H. The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, Inc., New York1989: 575-576Crossref Google Scholar) supplemented with ammonium nitrate (SGII-NH4) under continuous light (50 microeinsteins/m2 s) and transformed by the glass bead method (34Kindle K.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1228-1232Crossref PubMed Scopus (812) Google Scholar). 0.5 μg of plasmid pNIT1 DNA (35Sodeinde O.A. Kindle K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9199-9203Crossref PubMed Scopus (112) Google Scholar) linearized with EcoRI was used per transformation, after which the cells were plated on SGII agar plates supplemented with potassium nitrate (SGII-NO3) and grown under dim light (5 microeinsteins/m2 s) until colonies were visible (∼3 weeks). The colonies were then picked onto plates to screen for nonphotosynthetic mutants by growth under the following conditions: 1) acetate-containing SGII-NO3 plates in dim light (permissive condition), and 2) acetate-deficient SGII-NO3 plates in bright light (100 microeinsteins/m2 s; restrictive condition). Transformation experiments to demonstrate complementation of the nonphotosynthetic phenotype were performed with 5 μg of λ DNA, 1 μg of plasmid DNA, or no DNA, as described. Photosynthetic colonies were selected on acetate-free SGII-NO3 agar plates. For analyses of cellular proteins, cells were grown in high salt acetate, Tris-acetate phosphate (33Harris E.H. The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, Inc., New York1989: 575-576Crossref Google Scholar) or copper-free Tris-acetate phosphate (36Quinn J. Merchant S. Methods Enzymol. 1998; (in press)PubMed Google Scholar) media to exponential to early stationary phase (2–6 × 106cells/ml). For immunoblots of total cell extracts, cells were harvested by centrifugation and boiled for 5 min in loading buffer (0.0625m Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 1% β-mercaptoethanol, 0.00005% bromphenol blue). After centrifugation, 25 μl of the supernatant was loaded into a 12 or 15% SDS-polyacrylamide gel, and the proteins were separated by electrophoresis as described previously (27Merchant S. Hill K. Howe G. EMBO J. 1991; 10: 1383-1389Crossref PubMed Scopus (69) Google Scholar). Proteins were then transferred onto nitrocellulose paper using a Bio-Rad Transblot semidry apparatus (Bio-Rad). Antibodies against cytochrome f and cytochrome b 6 were obtained from Dr. R. Malkin (University of California, Berkeley, CA). The antibody against subunit IV (SuIV) has been described previously (37Chen X. Kindle K. Stern D. EMBO J. 1993; 12: 3627-3635Crossref PubMed Scopus (49) Google Scholar). The antibody against the D2 protein of PS II was obtained from Dr. M. Kuchka (Lehigh University, Bethlehem, PA). These primary antibodies were used at a dilution of 1:1000. The ECL chemiluminescent system (Amersham Life Science, Inc.) was used to detect immunoreactive proteins. For detection of cytochromes, cells were collected by centrifugation (3,000 × g, 5 min), washed in 10 mm sodium phosphate (pH 7.0), and resuspended to a chlorophyll concentration of 1 mg/ml in the same buffer. Cells were lysed by two cycles of slow freezing to −80 °C followed by thawing to room temperature. The soluble cell extract was separated from the insoluble membrane fraction by centrifugation (16,000 × g) at 4 °C for 10 min, and both fractions were returned to the original sample volume with buffer. The proteins were separated by nondenaturing or denaturing gel electrophoresis as described previously (11Howe G. Merchant S. EMBO J. 1992; 12: 2789-2801Crossref Scopus (63) Google Scholar), except that the denaturation buffer contained 50 mm dithiothreitol instead of β-mercaptoethanol, and the samples were denatured on ice to preserve the integrity of the heme group of the cytochromes. Proteins were then transferred to Immobilon P polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) at 4 °C, and heme-containing proteins were identified by a heme staining procedure using chemiluminescent reagents (38Vargas C. McEwan A.G. Downie J.A. Anal. Biochem. 1993; 209: 323-326Crossref PubMed Scopus (106) Google Scholar). Membranes were immersed in the Supersignal Chemiluminescent Substrate CL-HRP (Pierce) for 30 s and exposed to film (Reflection, NEN Life Science Products). Following exposure to film, membranes were rinsed with TBS buffer (40 mm Tris, pH 7.5, 150 mm NaCl) and subjected to immunoblot analysis using antibodies against plastocyanin (1:5000), cytochrome c 6 (1:1000), and cytochromef (1:5000) as a primary antibody. Bound primary antibody was detected with an alkaline phosphatase-conjugated secondary antibody for cytochrome immunoblots and a horseradish peroxidase-conjugated secondary antibody for plastocyanin immunoblots. To monitor cytochrome f synthesis, cells were radiolabeled as described previously (39Li H.H. Quinn J. Culler D. Girard-Bascou J. Merchant S. J. Biol. Chem. 1996; 271: 31283-31289Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), with the following modifications. The cells were pulse-labeled for 10 min with Na235SO4 followed by a 40-min chase in the presence of unlabeled sulfate and chloramphenicol (250 μg/ml). The initial immunoprecipitation of cytochrome f is essentially quantitative in the case of extracts from mutant strains, while for wild-type extracts, approximately 50% of total cytochromef is removed in the first immunoprecipitate. Isolation of plasmid DNA was performed either by the boiling method (40Holmes D.S. Quigley M. Anal. Biochem. 1981; 114: 193-197Crossref PubMed Scopus (2019) Google Scholar) or by a modified alkaline lysis procedure (41Lee S. Rasheed S. BioTechniques. 1990; 9: 676-679PubMed Google Scholar). C. reinhardtii DNA was isolated as described previously (42Kindle K.L. Schnell R.A. Fernandez E. Lefebvre P.A. J. Cell Biol. 1989; 109: 2589-2601Crossref PubMed Scopus (293) Google Scholar). C. reinhardtiitotal RNA was prepared either by the method of Shepherd et al. (43Shepherd H.S. Ledoigt G. Howell S.H. Cell. 1983; 32: 99-107Abstract Full Text PDF PubMed Scopus (32) Google Scholar) or as described by Merchant et al. (27Merchant S. Hill K. Howe G. EMBO J. 1991; 10: 1383-1389Crossref PubMed Scopus (69) Google Scholar), while poly(A)+ RNA was isolated as described previously (44Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1987Google Scholar). Southern blotting and hybridization of C. reinhardtii DNA to radioactive probes was performed as described previously (35Sodeinde O.A. Kindle K.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9199-9203Crossref PubMed Scopus (112) Google Scholar). RNA blot hybridization was performed as described by Hill et al.(45Hill K.L. Li H.H. Singer J. Merchant S. J. Biol. Chem. 1991; 266: 15060-15067Abstract Full Text PDF PubMed Google Scholar). petA mRNAs were identified by hybridization to a 600-bp HindIII/AccI radiolabeled fragment from plasmid pHA0.6 (16Buschlen S. Choquet Y. Kuras R. Wollman F.-A. FEBS Lett. 1991; 284: 257-262Crossref PubMed Scopus (47) Google Scholar), and mRNAs encoding cytochromec 6 (Cyc6) were identified by hybridization to a 710-bp EcoRI radiolabeled fragment from plasmid pGEM1Crc552 7A-4 (46Merchant S. Bogorad L. J. Biol. Chem. 1987; 262: 9062-9067Abstract Full Text PDF PubMed Google Scholar). The RbcS2-specific probe consisted of the 0.8-kb EcoRI cDNA insert of the plasmid p149A, which was obtained from the Chlamydomonas Genetics Center. 5′-rapid amplification of cDNA ends experiments were performed using poly (A)+ RNA from C. reinhardtii strainnit1–305 cw15, Ccs1-specific primers (5′-AAGCTCGAGAACTGGGC-3′ and 5′-ATGAAGGTGCCCAGGCC-3′), and the Superscript Preamplification System (Life Technologies, Inc.) according to the manufacturer's instructions. Genomic DNA was digested with SalI, ligated, and transformed by electroporation into E. coli (47Tam L.-W. Lefebvre P.A. Genetics. 1993; 135: 375-384Crossref PubMed Google Scholar). A fragment from the rescued plasmid was used as a probe to screen aC. reinhardtii genomic library comprising strain 21gr DNA in λFIX II (Stratagene, La Jolla, CA); this library was obtained from Dr. R. Schnell and Dr. P. Lefebvre (University of Minnesota, St. Paul, MN; Ref. 48Schnell R.A. Lefebvre P.A. Genetics. 1993; 134: 737-747Crossref PubMed Google Scholar). A C. reinhardtii cDNA library made from RNA isolated from vegetative cells of strain CC-621 in λZAP II (Stratagene) was obtained from Dr. J. Woessner and Dr. U. Goodenough (Washington University, St. Louis, MO; Ref. 49Waffenschmidt S. Woessner J.P. Beer K. Goodenough U.W. Plant Cell. 1993; 5: 809-820PubMed Google Scholar). The library (8 × 104 independent plaques) was screened with a mixture of probes made from the 0.45-kb SphI and 1.5-kbSphI/KpnI fragments (probes 2 and 3, Fig. 4) of pCcs1–1. Since we were unable to convert the cDNA insert from the single positive plaque into a plasmid by superinfection with a helper, the cDNA insert was polymerase chain reaction-amplified (30 cycles of the following: 92 °C for 30 s; 45 °C for 1 min; 72 °C for 2 min) using T3 and T7 primers. The fragment was digested withEcoRI/ScaI and XhoI/ScaI to produce 0.5-kb ScaI/EcoRI and 2.0-kbXhoI/ScaI fragments, which were then subcloned into pBluescript KSII− (the ScaI site is located within the cDNA, while the terminal EcoRI andXhoI sites are derived from the λZAP II vector). Plasmid pCcs1–1 was digested withSacI and religated to make the plasmid pCcs1–4 in which the 5′-region of genomic DNA in pCcs1–1 was deleted. pCcs1–4 was used as a template for genomic sequencing. Nested deletions of plasmids containing cDNA fragments were made using the Erase-a-Base kit (Promega Corp., Madison, WI). Plasmids were sequenced using the Silver Sequence DNA sequencing system (Promega) or by dye terminator cycle sequencing using 3′ dye-labeled dideoxynucleotide triphosphates and run on an ABI PRISM 377 DNA sequencer (Perkin-Elmer). The DNA sequence was derived from sequencing the entire length of both strands of the cDNA and genomic clones. Nuclear transformation was used to generate insertion mutations in genes required for biogenesis of the cytochromeb 6 f complex of C. reinhardtii. Approximately 2500 independent transformants were grown on acetate-supplemented plates and subsequently screened for mutants that had lost the ability to grow phototrophically. Eight nonphotosynthetic transformants were identified and screened further for defects in accumulation of the cytochromeb 6 f complex by immunoblot analysis using an antibody against subunit IV (SuIV) to probe total cell extracts. Since strains that carry mutations affecting the accumulation of cytochrome f, cytochrome b 6, or SuIV also fail to accumulate the other subunits of the complex (14Lemaire C. Girard-Bascou J. Wollman F.-A. Bennoun P. Biochim. Biophys. Acta. 1986; 851: 229-238Crossref Scopus (94) Google Scholar, 37Chen X. Kindle K. Stern D. EMBO J. 1993; 12: 3627-3635Crossref PubMed Scopus (49) Google Scholar,50Kuras R. Wollman F.-A. EMBO J. 1994; 13: 1019-1027Crossref PubMed Scopus (155) Google Scholar), the abundance of SuIV is diagnostic of the entire cytochromeb 6 f complex. Four of the eight strains accumulated drastically reduced amounts of SuIV (strains 3, 9, 30, and 34; Fig. 1). These strains were designated as abf (accumulation of the b 6 f complex) mutants. The abf mutants did not accumulate cytochromeb 6 or cytochrome f, as expected (Fig.1). All abf mutants exhibited wild-type levels of the D2 protein of PS II, which suggests that these mutations do not affect the accumulation of cytochrome b 559, which is an integral component of PS II. We next performed a Southern blot analysis to determine whether the mutants exhibited an RFLP that might indicate an insertion in eitherPetC and PetM, the two known nuclear genes that encode subunits of the complex. Since no PetC orPetM RFLPs were observed (data not shown), we concluded that the lesions in the abf mutants most likely affected 1) a previously unidentified nucleus-encoded subunit of the complex, 2) a regulatory gene required for the expression of structural genes of the complex, or 3) a gene required for the maturation or assembly of the complex. Nuclear mutations have defined four loci,CCS1–CCS4, involved specifically in the biogenesis of chloroplast c-type cytochromes.2 These strains exhibit a pleiotropic c-type cytochrome deficiency that is characteristic of a defect at the post-translational step of heme attachment. To determine whether any of the abf mutants harbored lesions of this type, the mutants were screened for cytochromec 6 accumulation by performing immunoblots, which assess polypeptide abundance, and heme staining, which assesses holocytochrome abundance. Cell extracts were isolated from cells grown in copper-deficient medium to induce transcription of Cyc6, which is repressed by copper (27Merchant S. Hill K. Howe G. EMBO J. 1991; 10: 1383-1389Crossref PubMed Scopus (69) Google Scholar, 51Quinn J.M. Merchant S. Plant Cell. 1995; 7: 623-628PubMed Google Scholar). The B6 mutant of C. reinhardtii, which contains a mutation in the chloroplastccsA gene and does not accumulate cytochrome f or cytochrome c 6 (29Xie Z. Merchant S. J. Biol. Chem. 1996; 271: 4632-4639Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), was included as a control. As shown in Fig. 2, A andB, holocytochromes c 6 andf do not accumulate in either abf3 or B6. To confirm that the cells were copper-deficient and hence under conditions allowing expression of the Cyc6 gene, the extracts were tested for plastocyanin content, since cells accumulate plastocyanin only when the medium contains copper. Since little plastocyanin was detected under copper-deficient conditions (Fig. 2 B, bottom; compare lanes 2, 4, and 6 withlanes 1, 3, and 5), the cells were indeed copper-deficient. Moreover, Cyc6 transcripts accumulated under these conditions (Fig. 2 C). Hence, the cytochrome c 6 deficiency could not result simply from lack of Cyc6 expression due to copper contamination in the medium. The mutation did not affect accumulation of petAmRNA (Fig. 2 C), and pulse-chase labeling experiments demonstrated that abf3, like B6 and previously described heme attachment mutants (11Howe G. Merchant S. EMBO J. 1992; 12: 2789-2801Crossref Scopus (63) Google Scholar), synthesizes but is unable to accumulate cytochrome f (Fig. 2 D). The presence of plastocyanin and the PS II-associated Oee1 protein (data not shown) in extracts of cells grown in the presence of copper confirms that the synthesis of other lumenal proteins is not affected in these strains. Thus, it is unlikely that abf3 is defective in some component of the chloroplast-SecA thylakoid transport pathway (52Yuan J. Henry R. McCaffery M. Cline K. Science. 1994; 266: 796-798Crossref PubMed Scopus (143) Google Scholar, 53Voelker R. Barkan A. EMBO J. 1995; 14: 3905-3914Crossref PubMed Scopus (130) Google Scholar). Together, these data suggest that abf3 most likely harbors a mutation(s) in a gene(s) required for the attachment of heme to chloroplast c-type cytochromes. A Southern blot analysis of DNA isolated from abf3 that was hybridized with a Nit1-specific probe revealed that the mutant harbored a single insertion of pNIT1 (data not shown). Since the mutation in abf3 was presumably a consequence of this plasmid DNA insertion, we attempted plasmid rescue to isolate the DNA that flanked the insertion site in abf3 (47Tam L.-W. Lefebvre P.A. Genetics. 1993; 135: 375-384Crossref PubMed Google Scholar). The recovered plasmid contained a 1.3-kb C. reinhardtii DNA fragment. When a genomic DNA blot was probed with this fragment, an RFLP was detected between wild type and abf3 (Fig.3 A, lanes 1 and2). If the nonphotosynthetic phenotype was indeed due to the insertion, the RFLP should cosegregate with the mutant phenotype in genetic crosses. However, crosses between abf3 and an appropriate marker strain did not yield viable progeny. Theref
Nuclear transformation of the unicellular green alga Chlamydomonas reinhardtii has thus far been characterized by integration of the introduced DNA into nonhomologous sites. In this study, the occurrence of homologous recombination events during transformation was investigated with the intent of developing strategies for gene targeting and gene disruption. Homologous recombination was monitored by using nonfunctional 5' and 3' deletion derivatives of the wild-type C. reinhardtii nit1 gene (encodes nitrate reductase) as selectable markers (p5' delta and p3' delta respectively) and the low reverting nit1-305 strain as the transformation recipient. After introduction of the DNA into the cell, intermolecular recombination between p5' delta and p3' delta occurs at a high frequency to restore a functional nit1 gene, indicating the presence of homologous recombination machinery in mitotic cells. Gene-targeting events at the nit1 locus were selected by restoring nit1-305 cells to prototrophy after transformation with only p5' delta and were confirmed by analysis of genomic DNA. By comparing the number of transformants obtained after transformation with p5' delta to the number obtained after transformation with a functional nit1 gene, the frequency of homologous-to-random integration events ranged between 1:1000 after glass bead-mediated transformation and 1:24 after bombardment with DNA-coated tungsten microprojectiles.
The related family of virulence plasmids found in the three major pathogens of the genus Yersinia all have the ability to encode a set of outer membrane proteins. In Y. enterocolitica and Y. pseudotuberculosis, these proteins are major constituents of the outer membrane when their synthesis is fully induced. In contrast, they have been difficult to detect in Y. pestis. It has recently been established that Y. pestis does synthesize these proteins, but that they are rapidly degraded due to some activity determined by the 9.5-kilobase plasmid commonly found in Y. pestis strains. We show that mutations in the pla gene of this plasmid, which encodes both the plasminogen activator and coagulase activities, blocked this degradation. A cloned 1.4-kilobase DNA fragment carrying pla was also sufficient to cause degradation in the absence of the 9.5-kilobase plasmid.
A 9.5-kilobase plasmid of Yersinia pestis , the causative agent of plague, is required for high virulence when mice are inoculated with the bacterium by subcutaneous injection. Inactivation of the plasmid gene pla , which encodes a surface protease, increased the median lethal dose of the bacteria for mice by a millionfold. Moreover, cloned pla was sufficient to restore segregants lacking the entire pla - bearing plasmid to full virulence. Both pla + strains injected subcutaneously and pla - mutants injected intravenously reached high titers in liver and spleen of infected mice, whereas pla - mutants injected subcutaneously failed to do so even though they establish a sustained local infection at the injection site. More inflammatory cells accumulated in lesions caused by the pla - mutants than in lesions produced by the pla + parent. The Pla protease was shown to be a plasminogen activator with unusual kinetic properties. It can also cleave complement C3 at a specific site.
The 9.5-kilobase plasmid of Yersinia pestis determines plasminogen activator, coagulase, pesticin, and pesticin immunity activities. We have mapped and cloned the loci encoding these activities and demonstrated that both plasminogen activator and coagulase were determined by the same gene, designated pla. The primary translation product of this gene (38 kilodaltons [kDa]) was processed in two sequential steps to produce peptides of 37 and 35 kDa. The first step in this processing occurred rapidly and probably cotranslationally and was blocked when protein export was inhibited. The second step was much slower and resulted in the presence of both the 37- and 35-kDa species in significant quantities. We also showed that the plasmid had a polA-dependent replicon and identified the region that contained its origin of replication and incompatibility functions.
We have determined the nucleotide sequence of the 1.4-kilobase DNA fragment containing the plasminogen activator gene (pla) of Yersinia pestis, which determines both plasminogen activator and coagulase activities of the species. The sequence revealed the presence of a 936-base-pair open reading frame that constitutes the pla gene. This reading frame encodes a 312-amino-acid protein of 34.6 kilodaltons and containing a putative 20-amino-acid signal sequence. The presence of a single large open reading frame is consistent with our previous conclusion that the two Pla proteins which appear in the outer membrane of pla+ Y. pestis are derived from a common precursor. The deduced amino acid sequence of Pla revealed that it possesses a high degree of homology to the products of gene E of Salmonella typhimurium and ompT of Escherichia coli but does not possess significant homology to other plasminogen activators of known sequence. We also identified a transcription unit that resides on the complimentary strand and overlaps the pla gene.
