Identification of Two Homologous Genes, chlA and chlA, That Are Differentially Involved in Isocyclic Ring Formation of Chlorophyll a in the Cyanobacterium Synechocystis sp. PCC 6803
2007; Elsevier BV; Volume: 283; Issue: 5 Linguagem: Inglês
10.1074/jbc.m708954200
ISSN1083-351X
AutoresKei Minamizaki, Tadashi Mizoguchi, Takeaki Goto, Hitoshi Tamiaki, Yuichi Fujita,
Tópico(s)Protist diversity and phylogeny
ResumoThe isocyclic ring (E-ring) is a common structural feature of chlorophylls. The E-ring is formed by two structurally unrelated Mg-protoporphyrin IX monomethylester (MPE) cyclase systems, oxygen-dependent (AcsF), and oxygen-independent (BchE) systems, which involve incorporation of an oxygen atom from molecular oxygen and water into the C-131 position of MPE, respectively. Which system operates in cyanobacteria that can thrive in a variety of anaerobic environments remains an open question. The cyanobacterium Synechocystis sp. PCC 6803 has two acsF-like genes, sll1214 (chlAI) and sll1874 (chlAII), and three bchE-like genes, slr0905, sll1242, and slr0309. Five mutants lacking one of these genes were isolated. The ΔchlAI mutant failed to grow under aerobic conditions with anomalous accumulation of a pigment with fluorescence emission peak at 595 nm, which was identified 3,8-divinyl MPE by high-performance liquid chromatography-mass spectrometry analysis. The growth defect of ΔchlAI was restored by the cultivation under oxygen-limited (micro-oxic) conditions. MPE accumulation was also detected in ΔchlAII grown under microoxic conditions, but not in any of the bchE mutants. The phenotype was consistent with the expression pattern of two chlA genes: chlAII was induced under micro-oxic conditions in contrast to the constitutive expression of chlAI. These findings suggested that ChlAI is the sole MPE cyclase system under aerobic conditions and that the induced ChlAII operates together with ChlAI under micro-oxic conditions. In addition, the accumulation of 3,8-divinyl MPE in the ΔchlA mutants suggested that the reduction of 8-vinyl group occurs after the formation of E-ring in Synechocystis sp. PCC 6803. The isocyclic ring (E-ring) is a common structural feature of chlorophylls. The E-ring is formed by two structurally unrelated Mg-protoporphyrin IX monomethylester (MPE) cyclase systems, oxygen-dependent (AcsF), and oxygen-independent (BchE) systems, which involve incorporation of an oxygen atom from molecular oxygen and water into the C-131 position of MPE, respectively. Which system operates in cyanobacteria that can thrive in a variety of anaerobic environments remains an open question. The cyanobacterium Synechocystis sp. PCC 6803 has two acsF-like genes, sll1214 (chlAI) and sll1874 (chlAII), and three bchE-like genes, slr0905, sll1242, and slr0309. Five mutants lacking one of these genes were isolated. The ΔchlAI mutant failed to grow under aerobic conditions with anomalous accumulation of a pigment with fluorescence emission peak at 595 nm, which was identified 3,8-divinyl MPE by high-performance liquid chromatography-mass spectrometry analysis. The growth defect of ΔchlAI was restored by the cultivation under oxygen-limited (micro-oxic) conditions. MPE accumulation was also detected in ΔchlAII grown under microoxic conditions, but not in any of the bchE mutants. The phenotype was consistent with the expression pattern of two chlA genes: chlAII was induced under micro-oxic conditions in contrast to the constitutive expression of chlAI. These findings suggested that ChlAI is the sole MPE cyclase system under aerobic conditions and that the induced ChlAII operates together with ChlAI under micro-oxic conditions. In addition, the accumulation of 3,8-divinyl MPE in the ΔchlA mutants suggested that the reduction of 8-vinyl group occurs after the formation of E-ring in Synechocystis sp. PCC 6803. Chlorophylls (Chls) 2The abbreviations used are: ChlchlorophyllMPEmagnesium protoporpyrin IX monomethylesterPchlideprotochlorophyllide. are a group of tetrapyrrole pigments involved in light reactions of photosynthesis. The isocyclic ring, E-ring, is a unique feature of all types of Chls, which distinguishes Chls from other tetrapyrroles such as hemes and vitamin B12. The E-ring is formed by an oxidative cyclization of C-13 methyl propionate of Mg-protoporphyrin IX monomethylester (MPE) to form 3,8-divinyl protochlorophyllide (Pchlide). The E-ring formation causes a 30-nm blue-shift of the Qy band of MPE (1Bollivar D.W. Kadish K.M. Smith K.M. Guilard R. Porphyrin Handbook. 13. Elsevier, New York2003: 49-69Google Scholar) resulting in a dramatic color change from pink to green. The E-ring formation reaction involves a 6-electron oxidation of C-13 methylpropionate with incorporation of an oxygen atom to form oxo-group at C-131 position (1Bollivar D.W. Kadish K.M. Smith K.M. Guilard R. Porphyrin Handbook. 13. Elsevier, New York2003: 49-69Google Scholar). The 131-oxo group of Chl a is derived from molecular oxygen in higher plants (2Nashrulhaq-Boyce A. Griffiths W.T. Jones O.T.G. Biochem. J. 1987; 243: 23-29Crossref PubMed Scopus (38) Google Scholar, 3Walker C.J. Mansfield K.E. Smith K.M. Castelfranco P.A. Biochem. J. 1989; 257: 599-602Crossref PubMed Scopus (54) Google Scholar) and green algae (4Bollivar D.W. Beale S.I. Photosynth. Res. 1995; 43: 113-124Crossref PubMed Scopus (20) Google Scholar), suggesting that E-ring formation is catalyzed by an oxygenase. The acsF gene encoding a protein with a monooxygenase motif has been found to be involved in the oxygen-dependent E-ring formation (5Pinta V. Picaud M. Reiss-Husson F. Astier C. J. Bacteriol. 2002; 184: 746-753Crossref PubMed Scopus (94) Google Scholar). In contrast, water is the oxygen donor for the 131-oxo group of bacteriochlorophyll a in a photosynthetic bacterium Rhodobacter sphaeroides (6Porra R.J. Schäfer W. Gad'on N. Katheder I. Drews G. Scheer H. Eur. J. Biochem. 1996; 239: 85-92Crossref PubMed Scopus (46) Google Scholar), indicating that photosynthetic bacteria produce the E-ring by a hydratase. The bchE gene has been identified as the gene responsible for the oxygen-independent E-ring formation in purple bacteria (7Bollivar D.W. Suzuki J.Y. Beatty J.T. Dobrowoski J.M. Bauer C.E. J. Mol. Biol. 1996; 237: 622-640Crossref Scopus (165) Google Scholar). The AcsF protein shows no similarity to the BchE protein, suggesting that there are two structurally unrelated E-ring formation systems. The bchE gene is found in a variety of photosynthetic bacteria such as R. capsulatus (7Bollivar D.W. Suzuki J.Y. Beatty J.T. Dobrowoski J.M. Bauer C.E. J. Mol. Biol. 1996; 237: 622-640Crossref Scopus (165) Google Scholar), R. sphaeroides (8Naylor G.W. Addlesse H.A. Gibson L.C.D. Hunter C.N. Photosynth. Res. 1999; 62: 121-139Crossref Google Scholar), Chlorobium tepidum (9Xiong J. Fischer W.M. Inoue K. Nakahara M. Bauer C.E. Science. 2000; 289: 1724-1730Crossref PubMed Scopus (389) Google Scholar), and Heliobacillus mobilis (10Xiong J. Inoue K. Bauer C.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14851-14856Crossref PubMed Scopus (142) Google Scholar). The acsF gene is distributed among photosynthetic eukaryotes such as Chlamydomonas reinhardtii (crd1 and cth1, Ref. 11Moseley J. Page M.D. Alder N.P. Eriksson M. Quinn J. Soto F. Theg S.M. Hippler M. Merchant S. Plant Cell. 2002; 14: 673-688Crossref PubMed Scopus (114) Google Scholar), Arabidopsis thaliana (chl27, Ref. 12Tottey S. Block M.A. Allen M. Westergren T. Albrieux C. Scheller H.V. Merchant S. Jensen P.E. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 16119-16124Crossref PubMed Scopus (156) Google Scholar), and barley (xantha-l, Ref. 13Rzeznicka K. Walker C.J. Westergren T. Kannangara C.G. von Wettsein D. Merchant S. Gough S.P. Hansson M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5886-5891Crossref PubMed Scopus (90) Google Scholar). Some purple bacteria such as Rubrivivax gelatinosus have both bchE and acsF genes (14Ouchane S. Steunou A-S. Picaud M. Astier C. J. Biol. Chem. 2004; 279: 6385-6394Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The distribution of acsF and bchE among extant photosynthetic organisms seems to be consistent with the oxygen levels of their natural habitats. chlorophyll magnesium protoporpyrin IX monomethylester protochlorophyllide. Cyanobacteria are prokaryotes performing oxygenic photosynthesis similar to plants. Ancient cyanobacteria are thought to have started to carry out oxygenic photosynthesis about 2.7 giga years ago (15Blankenship R.E. Molecular Mechanisms of Photosynthesis. Blackwell Science, Malden, MA2002Crossref Scopus (876) Google Scholar, 16Knoll A.H. Geobiology. 2003; 1: 3-14Crossref Google Scholar, 17Knoll A.H. Herrero A. Flores E. The Cyanobacteria: Molecular Biology, Genomics and Evolution. Caister Academic Press, Norfolk, UK2008: 1-19Google Scholar), and later one lineage of cyanobacteria to have become endosymbionts of protoeukaryotic cells leading to chloroplasts of plants (18Margulis L. Symbiosis in Cell Evolution: Microbial Communities in the Archeon and Proterozoic Eons. W. H. Freeman, San Francisco1993Google Scholar). Though current cyanobacteria are usually associated with aerobic environments, many strains thrive in environments with a variety of oxygen levels including anaerobic environments such as microbial mats, lake sediments, and soil (19Stal L. Moezelaar R. FEMS Microbiol. Rev. 1997; 21: 179-211Crossref Scopus (298) Google Scholar, 20Stal L.J. Whitton B.A. Potts M. The Ecology of Cyanobacteria: Their Diversity in Time and Space. Kluwer Academic Publishers, Dordrecht, The Netherlands2000: 61-120Google Scholar). In addition, most cyanobacteria face a diurnal light and dark cycle, and then the oxygen level in natural environments undergoes dynamic changes from anaerobic to aerobic throughout the day. Given the two structurally unrelated E-ring formation systems; the oxygen-dependent AcsF and the oxygen-independent BchE systems, it is a reasonable inference that oxygen is a key environmental factor by which Chl biosynthesis is regulated at the step of E-ring formation. However, little information is available regarding which E-ring formation system operates and how it is regulated under oxygen-fluctuated environments in cyanobacteria. Here we report the identification of two acsF-like genes, chlAI and chlAII, in the cyanobacterium Synechocystis sp. PCC 6803. We found that ChlAI is the sole E-ring formation system under aerobic conditions and that the second isoform ChlAII operates together with ChlAI under oxygen-limited (micro-oxic) conditions in Synechocystis sp. PCC 6803. Cyanobacterial Strains and Growth Conditions—Synechocystis sp. PCC 6803 (Synechocystis 6803) and its derivative strains used in this study were cultivated in BG-11 medium (21Rippka R. Deruelles J. Waterbury J.B. Herdman M. Stainer R.Y. J. Gen. Microbiol. 1979; 111: 1-61Crossref Google Scholar) supplemented with 10 mm HEPES-KOH, pH 8.2 at 30 °C. For culture of the gene-disrupted mutant with a kanamycin-resistant cartridge, the above medium was supplemented with 15 μg ml-1 kanamycin sulfate. Liquid cultures were bubbled with 2% CO2 in air (for aerobic conditions) under continuous illumination provided from fluorescent lamps (∼70 μmol m-2 s-1; FRL40SW, Hitachi, Japan). For agar plate culture, BG-11 liquid media were solidified with 1.5% agar (BactoAgar, Becton, Dickinson and Company, Sparks, MD). For growth under microoxic conditions, liquid cultures were bubbled with 2% CO2/N2 that were prepared by mixing pure N2 (99.999%, Nagoya Nissan, Nagoya, Japan) and CO2 with an appropriate flow rate. For cultivation on agar plates, agar plates were incubated in an anaerobic jar (BBL GasPak anaerobic systems; Becton, Dickinson and Company). Preparation of RNA and RT-PCR—Wild-type cells of Synechocystis 6803 grown under aerobic and micro-oxic conditions for 3 days were inoculated into fresh BG-11 media (50 ml) and cultivated under the same conditions for 3 days. Cells (OD730 = ∼0.7) were harvested by centrifugation at 2 °C, and cell pellets were frozen in liquid nitrogen after complete removal of culture media. Total RNA was prepared essentially as described (22Aiba H. Adhya S. de Combrugghe B. J. Biol. Chem. 1981; 256: 11905-11910Abstract Full Text PDF PubMed Google Scholar). Frozen cells were suspended in 600 μl of lysis buffer (50 mm Tris-HCl; pH8.0, 5 mm EDTA, 0.5% SDS) and mixed with phenol. After vigorous shaking, the mixture was incubated at 65 °C for 10 min with shaking at 2-min intervals. The supernatant was mixed with the same volume of phenol-chloroform (23Sambrook J. Russell D.W. Molecular Cloning, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000Google Scholar). The phenol-chloroform extraction was repeated five times, the nucleic acids were precipitated by ethanol. DNA in the nucleic acid fraction was digested with DNase I (0.1 units μl-1; RNase-free grade, Takara, Ohtsu, Japan) in the presence of RNase inhibitor (0.8 units μl-1; porcine liver; Takara) for 1.5 h at 37 °C. RNA was extracted by phenol-chloroform and concentrated by ethanol precipitation. RNA concentration was determined by absorbance at 260 nm (23Sambrook J. Russell D.W. Molecular Cloning, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000Google Scholar). The isolated total RNA (2.5 μg) was used for the synthesis of cDNA with Superscript II (10 units μl-1; Invitrogen Corp., Carlsbad, CA) and random primers according to the manufacturer's manual. After incubation at 25 °C for 10 min, at 42 °C for 50 min, and at 70 °C for 15 min, RNA was degraded by alkali treatment (0.23 n NaOH) followed by neutralization with HCl. Thus obtained cDNA fraction was used in 1:32-dilution as the template for PCR amplification with the specific primers (Supplemental Table S1). Construction of Plasmids for Gene Disruption and Transformation of Synechocystis 6803—Plasmids for gene disruption were constructed by overlap extension PCR (Supplemental Table S1; Ref. 23Sambrook J. Russell D.W. Molecular Cloning, A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000Google Scholar). DNA fragments of the upstream (f1 and r1 primers) and downstream (f2 and r2 primers) regions of the target gene were amplified by PCR from genomic DNA of Synechocystis 6803 with a standard thermal cycle (KODplus DNA polymerase; Toyobo, Osaka, Japan). A kanamycin-resistant cartridge (neo) for gene disruption was also amplified with f3 and r3 primers (Supplemental Table S1) from pMC19 (24Fujita Y. Takahashi Y. Chuganji M. Matsubara H. Plant Cell Physiol. 1992; 33: 81-92Google Scholar) as the template. A chimeric DNA fragment consisting of the upstream region of the target gene, neo and the downstream region of the target gene were amplified by overlap extension PCR and cloned into pUC118. Synechocystis 6803 was transformed with the plasmid constructed as above and the kanamycin-resistant colonies were segregated to isolate homozygous mutants (25Williams J.G.K. Methods Enzymol. 1988; 167: 766-778Crossref Scopus (856) Google Scholar). For the isolation of sll1214-disrupted mutant (Δsll1214), the kanamycin-resistant colonies that appeared on the first selective agar plates were picked up and cultivated under micro-oxic conditions. Complete replacement of the wild-type copy with the disrupted copy was examined by "colony PCR" (26Howitt C.A. BioTechniques. 1996; 21: 32-34Crossref PubMed Scopus (14) Google Scholar). Pigment Extraction and Spectroscopic Analysis—Pigments were extracted from cells grown under aerobic or micro-oxic conditions on agar plates for 10 days. Because Δsll1214 does not grow under aerobic conditions, it was grown under micro-oxic conditions for 3 days and incubated under aerobic conditions for 7 days to prepare the cells grown aerobically. Pigments were extracted in 90% methanol as described (24Fujita Y. Takahashi Y. Chuganji M. Matsubara H. Plant Cell Physiol. 1992; 33: 81-92Google Scholar). Chl biosynthetic intermediates without phytol were extracted by mixing the methanol extracts with a 3.5-volume of acetone and half volume of water, and phase-partitioned with a 15-volume of hexane. Fluorescence spectra of the lower acetone-methanol phase were recorded with excitation at 435 nm (model FP777w, Jasco, Hachioji, Japan). Preparation of MPE—MPE was prepared from culture medium of the R. capsulatus mutant DB575 (ΔbchE, 7) essentially as described for preparation of Pchlide (27Fujita Y. Bauer C.E. J. Biol. Chem. 2000; 275: 23583-23588Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). DB575 was grown in RCV-2/3 PY medium (80 ml in a 200-ml flask) containing 5 μg ml-1 kanamycin at 34 °C in the dark with slow shaking at 130 rpm. The culture medium was collected by centrifugation. MPE in the culture medium was extracted in one-third volume of diethyl ether. The contaminating water was removed as ice after chilling at -20 °C. The ether was then evaporated to dryness by a stream of nitrogen. The dried MPE was dissolved in Me2SO. LC/MS Analysis of Pigments—Cells grown on agar plates under aerobic (5 days) and micro-oxic (10 days) conditions were extracted with methanol. After washing with hexane, the lower methanol-phase was corrected and evaporated to almost dryness by a stream of nitrogen. The extract thus obtained was dissolved in a small amount of Me2SO for LC/MS analysis. The LC/MS was performed using a Shimadzu LCMS-2010EV system (Shimadzu, Kyoto, Japan) equipped with an electrospray ionization probe as described previously (28Nomata J. Mizoguchi T. Tamiaki H. Fujita Y. J. Biol. Chem. 2006; 281: 15021-15028Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). HPLC was performed using reverse-phase chromatography under the following conditions: column, Inertsil ODS-P (3.0 × 150 mm, GL Sciences, Tokyo, Japan); eluent, methanol/acetonitrile: 150 mm ammonium acetate (pH 5.25) = 400:50:100 (v/v/v); flow rate, 1.0 ml min-1; and detection wavelength, 415 nm. The entire absorption spectra of MPE were also recorded, during elution, using a photodiode-array detector. Preparation of Total Extracts and Immunoblot Analysis—Cells were grown under aerobic and micro-oxic conditions until OD730 reached about 1.0. Harvested cells were disrupted by sonication as described (29Yamazaki S. Nomata J. Fujita Y. Plant Physiol. 2006; 142: 911-922Crossref PubMed Scopus (72) Google Scholar). Proteins in the total extracts were separated by SDS-PAGE (a 5–20% acrylamide gradient gel, e-Pagel, ATTO, Tokyo, Japan) and electro-transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). The polyvinylidene difluoride membrane was incubated with anti-CHL27 antiserum (Agrisera, Vännäs, Sweden) in a 1000 dilution, and then with goat anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad). The specific protein signals were visualized by chemiluminescent substrate (ECL Western blotting Analysis System, GE Healthcare) with a lumino-image analyzer (LAS-3000mini, Fujifilm, Tokyo, Japan). Two acsF-like genes, sll1214 and sll1874, were found in the genome of Synechocystis sp. PCC 6803 (Synechocystis 6803) by a BLAST search with the amino acid sequences of AcsF from R. gelatinosus and CHL27 (AcsF-homolog) from A. thaliana, as pointed out previously (5Pinta V. Picaud M. Reiss-Husson F. Astier C. J. Bacteriol. 2002; 184: 746-753Crossref PubMed Scopus (94) Google Scholar). Sll1214 and Sll1874 showed 42 and 39% identity, respectively, to AcsF from R. gelatinosus, and 62 and 50% identity, respectively, to CHL27 from A. thaliana. They conserved the two copies of a motif, (D/E)X28–37DEXRH, which are involved in binding of a binuclear-iron cluster for various monooxygenases (Supplemental Fig. S1A, and Ref. 30Wallar B.J. Lipscomb J.D. Chem. Rev. 1996; 96: 2625-2657Crossref PubMed Scopus (1131) Google Scholar). Sequence identity between Sll1214 and Sll1874 was 57%. Three bchE-like genes, slr0905, sll1242, and slr0309, were found in the genome by a BLAST search with the amino acid sequence of BchE from R. capsulatus. The amino acid sequences of Slr0905, Sll1242, and Slr0309 show 29, 21, and 26% identity, respectively, to BchE from R. capsulatus. BchE belongs to the radical SAM family, which uses an oxygen-sensitive [4Fe-4S] cluster and S-adenosylmethionine to catalyze diverse radical reactions such as biotin synthase (BioB), lipoate synthase (LipA), and coproporphyrinogen III oxidase (HemN; 31Layer G. Heinz D.W. Jahn D. Schubert W.D. Curr. Opn. Chem. Biol. 2004; 8: 468-476Crossref PubMed Scopus (87) Google Scholar, 32Wang S.C. Frey P.A. Trends Biochem. Sci. 2007; 32: 101-110Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). They all conserve an Fe-S binding motif including three cysteine residues (CXXXCXXC), which is found in all three BchE-like proteins (Fig. S1B). Given the possible oxygen sensitivity of BchE (33Gough S.P. Petersen B.O. Duus J.Ø. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6908-6913Crossref PubMed Scopus (86) Google Scholar), cyanobacterial BchE-like proteins may operate only under anaerobic conditions as in the case of HemN (34Xu K. Elliott T. J. Bacteriol. 1994; 176: 3196-3206Crossref PubMed Google Scholar) or an oxygen-sensitive Pchlide reductase (29Yamazaki S. Nomata J. Fujita Y. Plant Physiol. 2006; 142: 911-922Crossref PubMed Scopus (72) Google Scholar). The transcript levels of the five genes in wild-type cells grown photoautotrophically under aerobic and anaerobic conditions were semiquantified by RT-PCR (Fig. 1B). The "anaerobic" condition in this work means that the gas phase to incubate cyanobacterial cells is anaerobic (2% CO2/N2; see "Experimental Procedures"). Because cyanobacterial cells still evolve oxygen by photosynthesis under this condition, the environments of the cells is more properly referred to as "micro-oxic." PCR products derived from sll1214 and slr0905 mRNAs were detected almost equally in both cDNA preparations from aerobic and micro-oxic conditions. In contrast, PCR products from sll1874, sll1242, and slr0309 mRNAs in cells grown under micro-oxic conditions were much more abundant than those in aerobically grown cells (Fig. 1B). This result suggests that sll1874, sll1242, and slr0309 genes are induced under micro-oxic conditions while sll1214 and slr0905 are constitutively expressed. Two other genes, ho2 (sl1875) and hemN1 (sll1876), are present downstream of the sll1874 gene. The ho2 gene encodes heme oxygenase (HO) that catalyzes the oxidative cleavage of heme to form biliverdin IXα, the precursor for phycocyanobilin (35Zhang X. Migita C.T. Sato M. Sasahara M. Yoshida T. FEBS J. 2005; 272: 1012-1022Crossref PubMed Scopus (26) Google Scholar), and the hemN1 gene encodes coproporphyrinogen III oxidase (CPO) that catalyzes the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX in the biosynthetic pathway common to heme and Chl a. Thus, all of the contiguous three genes, sll1874-ho2-hemN1, are probably involved in tetrapyrrole biosynthesis. To confirm whether ho2 and hemN1 are also induced under micro-oxic conditions as well as sll1874, we also examined the transcript levels of ho2 and hemN1. As expected, the expression pattern of the two genes was similar to that of sll1874. There is no obvious sequence for transcriptional termination between the intergenic regions of the three genes. The expression pattern, and the sequence features suggest that the three genes form a transcriptional unit that is induced under microoxic conditions. To identify which gene(s) encode MPE cyclase in Synechocystis 6803, we isolated five mutants lacking one of these genes (Fig. 1A). Four mutants lacking sll1874, slr0905, sll1242, and slr0309 were successfully isolated by the normal segregation procedure under aerobic conditions (Fig. 1C). However, kanamycin-resistant colonies isolated for sll1214-disruption under aerobic conditions were found to carry the wild-type copy accompanied with a small proportion of the mutant copy of sll1214 (Fig. 1C). However, the kanamycin-resistant colonies isolated under micro-oxic conditions contained no detectable wild-type copy (Fig. 1C). This suggests that sll1214 is a gene essential for growth under aerobic conditions but dispensable under micro-oxic conditions. Mutants lacking sll1214, sll1874, slr0905, sll1242, and slr0309 are called Δsll1214, Δsll1874, Δslr0905, Δsll1242, and Δslr0309, respectively, hereafter. All mutants except for Δsll1214 grew photosynthetically on an agar plate under aerobic conditions in the light (70 μmol m– s–1; Fig. 2A). The mutant Δsll1214 did not grow in this condition as expected from the segregation process. In contrast, all mutants including Δsll1214 grew photosynthetically on an agar plate under micro-oxic conditions at the same light intensity (Fig. 2B). Slight growth retardation was observed in the Δsll1874 mutant. The mutant Δsll1242 grew somewhat more slowly than the wild-type showing a yellow-green color under both aerobic and micro-oxic conditions. In liquid cultures, essentially the same result was obtained (Fig. 2, panels C and D) except for Δsll1242. The mutant Δsll1242 did not grow in liquid culture irrespective of aerobic and micro-oxic conditions. The slight growth retardation of Δsll1874 observed on the agar plate was not so evident in liquid culture (Fig. 2D). Chl contents of the five mutants grown on agar plates under aerobic and micro-oxic conditions were determined (Fig. 2, panels E and F). Because the Δsll1214 mutant did not grow under aerobic conditions, it was cultivated under the micro-oxic condition followed by aerobic cultivation. The Chl content of Δsll1214 under the aerobic cultivation was drastically reduced by 67%, while that of Δsll1214 under the micro-oxic condition was about 80% of that of the wild-type. It should be noted that the Chl content of Δsll1874 grown under the micro-oxic conditions was significantly reduced by about 20%. MPE is expected to accumulate anomalously in mutants lacking functional genes involved in MPE cyclase. To examine whether MPE is accumulated in the mutant cells, we extracted the pigments with methanol from cells grown on agar plates under aerobic and micro-oxic conditions, and recorded the fluorescence emission spectra of the lower phase after phase partitioning with n-hexane (Fig. 3). MPE shows a characteristic fluorescence emission peak at 595 nm by excitation at 435 nm (1Bollivar D.W. Kadish K.M. Smith K.M. Guilard R. Porphyrin Handbook. 13. Elsevier, New York2003: 49-69Google Scholar). Only the extract from Δsll1214 of the five mutants grown aerobically exhibited a 595-nm emission peak, suggesting the accumulation of MPE in the Δsll1214 cells under the aerobic condition (Fig. 3A). MPE accumulation was also detected in Δsll1214 and Δsll1874 mutant cells grown under the microoxic conditions (Fig. 3B). This anomalous accumulation of MPE in Δsll1214 and Δsll1874 grown under micro-oxic conditions suggested that Chl biosynthesis is limited at the step of MPE cyclization, resulting in reduced Chl contents (Fig. 2F). The aerobically grown Δsll1874 mutant cells did not accumulate MPE, which is consistent with the result of RT-PCR. The expression level of sll1874 under aerobic conditions is much lower than that under micro-oxic conditions (Fig. 1B). No significant accumulation of MPE was detected in any three bchE-mutants including Δsll1242 under any conditions examined as well as the wild-type cells. No significant MPE accumulation was detected in aerobically grown Δsll1242 cells even though the Chl content was reduced significantly (Fig. 2, E and F). Mg-protoporphyrin IX, the direct precursor of MPE, shows fluorescence spectra identical to MPE (1Bollivar D.W. Kadish K.M. Smith K.M. Guilard R. Porphyrin Handbook. 13. Elsevier, New York2003: 49-69Google Scholar). To confirm that the pigment accumulated in Δsll1214 and Δsll1874 is MPE not Mg-protoporphyrin IX, a series of LC/MS analysis was carried out (Fig. 4). The standard MPE sample isolated from the R. capsulatus mutant contained two MPE-like pigments eluted at 18.6 min and 25.5 min (Fig. 4A, trace 2, peaks a and b, respectively). Their absorption spectra were very similar but the Soret peaks of the 18.6-min and 25.5-min pigments were 410 nm and 415 nm, respectively (Fig. 4C, traces 1 and 2). The m/z values of the 18.6-min and 25.5-min pigments were 600.3 and 598.3 (Fig. 4D, traces 1 and 2), which matched the calculated molecular mass of 3-vinyl 8-ethyl MPE (calculated for 600.26) and 3,8-divinyl MPE (calculated for 598.24), respectively. Detection of an adduct with methanol (633.3 and 631.2) originating from the eluent supported the identification of the pigments. Thus, we concluded that the 18.6-min and 25.5-min pigments are 3-vinyl 8-ethyl MPE and 3,8-divinyl MPE, respectively. The Δsll1214 cells grown in the aerobic and micro-oxic conditions and the Δsll1874 cells grown in the micro-oxic condition accumulated commonly the 25.5-min pigment (Fig. 4A). The 25.5-min pigment accumulated in Δsll1214 and Δsll1874 commonly showed characteristic absorption spectra (Fig. 4C) and the m/z values 598.1 (Fig. 4D), indicating that the accumulated pigment in these cells is 3,8-divinyl MPE not Mg-protoporphyrin IX. It should be noted that MPE was not detected in any of the three bchE mutants, confirming the result of the fluorescence emission spectra (Fig. 3). These results suggested that none of the bchE-like genes are involved in the E-ring formation. To detect Sll1214 and Sll1874 proteins immunologically in cyanobacterial cells, we carried out immunoblot analysis using an antiserum against CHL27 protein from A. thaliana (Fig. 5). Total extracts of wild-type, Δsll1214 and Δsll1874 cells grown under aerobic and micro-oxic conditions (except for Δsll1214 grown aerobically) were prepared and probed with the antiserum. The cross-reacting polypeptide with an apparent molecular mass of 38 kDa was specifically detected in wild-type and Δsll1874 cells grown under both conditions. Since the 38-kDa polypeptide was not detected in Δsll1214 grown under the micro-oxic condition and the apparent molecular mass is in good agreement with the calculated m
Referência(s)