DNA RESEARCH 3, (1996) Short Communication

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1 DNA RESEARCH 3, (1996) Short Communication Organization and Transcription of a Putative Gene Cluster Encoding Ribosomal Protein S14 and an Oligopeptide Permease-like Protein in the Cyanobacterium Synechococcus sp. Strain PCC 6301 Tsuneo FUJISHIRO, Takakazu KANEKOJ Masahiro SUGIURA, and Mamoru SUGITA* Center for Gene Research, Nagoya University, Nagoya , Japan (Received 31 May, 1996) Abstract A 2.0-kbp Pst I DNA fragment of the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 genome contains two open reading frames (ORFs). The first ORF of 100 codons potentially encodes a polypeptide having 47% amino acid identity to Escherichia coli ribosomal protein S14, suggesting it as a ribosomal protein S14 gene (rpsl4)- The second ORF of 351 codons is located 81 bp downstream of rpsl4 and its deduced amino acid sequence is in part similar to that of the Salmonella typhimurium oligopeptide permease membrane protein OppC. Northern blot analysis showed that rpsl4 is expressed as a 0.48-kb transcript whereas no transcript was detected from ORF351. Pulsed-field electrophoresis and blot hybridization analysis revealed that rpsl4 is a single-copy gene and is found within a 165-kbp region located upstream of rrna on the circular genome. Key words: Synechococcus sp. PCC 6301; ribosomal protein S14; transcript; oligopeptide permeaselike protein; gene map Cyanobacteria are prokaryotes carrying out plant-type oxygenic photosynthesis, and are evolutionary related to chloroplasts. 1 ' 2 Therefore, cyanobacteria are excellent model organisims for studying the molecular mechanisms of photosynthesis of plants. Cyanobacterial ribosomes are 70 S in size, and their RNA and protein components show structural similarities with those of chloroplasts and Escherichia coli. 3 About one-third of the chloroplast r-protein species are encoded by the chloroplast genome and the remainder by the nuclear genome in plants. 4 All 21 chloroplast-encoded and about half of the nuclear-encoded r-protein genes in higher plant chloroplasts have so far been characterized. 5 By contrast, only limited numbers of r-protein genes have so far been reported in cyanobacteria: genes for SI, 6 S2, 7 S7, 8 ' 9 S12, 8 * 9 S21, 10 LI, 11 L9, 12 L10, 11 Lll and L A recent genomic sequencing project of Synechocystis PCC 6803 disclosed several additional putative r-protein genes. 14 During the course of screening a gene encoding a singlestranded DNA-binding (SSB) protein isolated from Synechococcus PCC 6301 cells, a genomic DNA fragment potentially encoding r-protein S14 (rpsl4) was obtained. In E. coli, rpsl4 is present within the spc operon containing Communicated by Mituru Takanami * To whom correspondence should be addressed. Tel , Fax f Present address: Kazusa DNA Research Institute, Kisarazu 292, Japan. the genes for L14, L24, L5, S14, S8, L6, L18, S5, L30, L15, SecY and L36 in this order 15 whereas chloroplast rpsl4 is located separately from the SlO/spc-like operon in the chloroplast genome. 3 ' 4 Mapping and transcription pattern of the cyanobacterial rpsl4 indicate that it is not clustered with any other r-protein genes. This suggests that the gene arrangement of this r-protein gene(s) in cyanobacteria is similar to that in chloroplasts. 1. Putative Genes for Ribosomal Protein S14 and Oligopeptide Permease-like Protein A Synechococcus PCC 6301 genomic DNA library constructed in AEMBL4 8 was screened by plaque hybridization with a degenerate oligonucleotide, 5' AACGTIGTIAAC(T/C)TIGTIGG(T/C)CG(G/C)GTI- GG(T/C)CG(G/C)GA(T/C) CCIGA 3', which was designed based on the N-terminal amino acid sequence (NVVNLVGRVGRDPE, positions 4-17) determined for a 16-kDa SSB protein from Synechococcus PCC 6301 (Fujishiro et al., unpublished). Hybridization, washing and detection of positive plaques were performed according to the procedure of ECL 3'-oligolabelling and detection systems (Amersham). A positive clone ATF17 containing a 15.3-kbp genomic DNA fragment was isolated. A 2.5- kbp Hindlll fragment that hybridized to the probe and an overlapping 2.0-kbp Pst I fragment were subcloned. The Pst I fragment was sequenced and found to contain

2 166 rpsl4 and oppc Homologues of Synechococcus PCC 6301 [Vol. 3. Pst I.... Stu I. CTGCAGTTTGGCTGACCATTGCAGCACCTTTCGCCG^ACAGCCCGAAAGGCCTCTGGTAn^GGAGAATGCTGTCTAAATTCCAAAGCATCTCTTCAAG ORF100 (rpsi4) M A K K A M V E R D R K R K K L V E K Y A A K R E A L K E Q F HindiII A A A T S Q S E R L E L H R K L Q Q L P R N S A P N R V R N R C W V T G R P R G Y Y R D F G L C R N V L R E M A H Q G L L P G V V K stu I ORF351 CGAGCTGGTAAGTCAGCCGCGATCGCCTCATTCAAGTTTTAAAGTCAAAGGGGAGTCCAATGGGCTCCCTTTTATT'rrGGAGGCCTGCGGGATGACTGAG 500 S S W * ^_ M T E R R S P L P R S W T S W G V G L L L L L Y L G A L G A D C L A P Y R Q G P I D L E T G Q R E V I T D W N A P S P L R I W V V G D P Y R V L S W K G D R H L L G T T G P G K L H L L G T D D Q G R D Q F S R L L Y G S R I S L S V G L V G V I L T F P L G I L V G G I A G Y W G G W V D A L I M R L V E V L M T I P S L Y L L V A L A A V L P P G L S S A E R F L L I V A I T S L I N W A G L A R V I R G Q V L S L R E R E Y V Q A S Q V M G A S R L Y L L I R H I L P Q T L T Y V I I A A T L A V P S F I V A E S V L S L V G L G I Q Q P D P S W G N M L S L A T N A S M L L L N P W L I W P P A L L I V V A V L A F N V V G D G L R D R L D P R R * TAAATTTTTAGATAATTTACTAATAGCTATGTTCCTGAAATCCCTCTTTCAGAAAGGATTTTTCAGATCGAGCTTTCGTTTCTTGAAGTCAAAAAGACAA 1800 EcdRl ORF-X M N F A Pst I ACGGACTTGTGGACTCGCCCTCGCTGCTTGTTTGGCCGGCGGCGCCGTACTCGAAGCGCAGCCCGCTGCAG 1971 R T C G L A L A A C L A G G A V L E A Q P A A Figure 1. Nucleotide and deduced amino acid sequences of the 2.0-kbp Pst I region in the ATF17 insert. Putative ribosome-binding sequences (SD) and promoter sequences ( 10, 35) are underlined. Palindromic sequences are indicated by inverted arrows. ORF-X does not correspond to any known polypeptides deposited in protein sequence databases (Swiss-Prot. Rel. 32 and NBRF-PDB Rel. 46). The sequence has been deposited in DDBJ, EMBL and GenBank nucleotide sequence databases (accession no. D85103). open reading frames of 100 and 351 codons (ORF100 alga Chlorella ellipsoidea, 17 respectively. Therefore, we and ORF351) on the same strand (Fig. 1). As shown in designated ORF100 as a putative gene for r-protein S14 Fig. 2A, the predicted amino acid sequence of ORF100 (rpsl4)- In E. coli, the rpsl gene coding for r-protein shows 47% identity (77% similarity) with that of E. colir- S14 is found within the spc operon. 15 However, no r- protein S14, 15 and has 52 and 68% identity to chloroplast protein genes are found in the region adjacent to rpsl4 r-protein CS14 from tobacco 16 and the unicellular green in Synechococcus The polypeptide predicted for

3 No. 3] T. Fujishiro et al. 167 A ORFIOO MAKKAMVERDRKRKKLVEKYAAKREAL-KEQFAAATSQSERLELHRKLQQLPRNSAPNRV 59 Ec S14 -..QS.KA.EV..VA.AD..F...AE.KAIISDVNA.DED.WNAVL...T...D.S.S.Q 59 Ce CS14 S.I AR.IT KN.-LVEIKT...LEDKFN V.S 59 Nt CS14..R.SLIQ.EK..Q..EQ..HSI.RS.K-KEISKVP.L.DKW.IYG...S T.L 59 ORFIOO RNRCWVTGRPRGYYRDFGLCRNVLREMAHQGLLPGWKSSW EC S14 RQ H.FL.K...S.IKV..A.MR.QI..-L.K.- Ce CS14 H...TI F S.H Y.L..F A.. Nt CS14 HR..FL AN SGHI V.AC ATR... B ORF351 MTERRSPLPRSWTSWGVGLLLLLYLGALGADCLAPYNPYSSQADAALLPPSQIFWRNRQG 60 * OppC MMLSKKN-SETLENFSEK 17 ORF351 -QWLGPHVYPV-RQGPIDLETGQREVITDWNAPSPLRIWWGDPYRVLSWKGDRHLLGTT 118 * * * * * * OppC LEVEGRSLWQDARRRFMHNRAAVASLIVLF LIALFVTVAPMLSQFTYFDTDWGMM-SS 74 ORF351 GPGKL HLLGTDDQGRDQFSRLLYGSRISLSVGLVGVILTFPLGILVGGIAGYWGGWVD 176 * * *** *** * * **** ** * * * ** ** * OppC APDMASGHYFGTDSSGRDLLVRVAIGGRISLMVGIAAALVAVIVGTLYGSLSGYLGGKID 134 ORF351 ALIMRLVEVLMTIPSLYLLVALAAVLPPGLSSAERFLLI-VAITSLINWAGLARVIRGQV 235 *** * * * * *** *** * ** *** OppC SVMMRLLEILNSFPFMFFVILLVTFF GQNILLIFVAIGMVS-WLDMARIVRGQT 187 ORF351 LSLREREYVQASQVMGASRLYLLIRHILPQTLTYVIIAATLAVPSFIVAESVLSLVGLGI 295 *** * ***** **** * * * * * *** * ** ** *** OppC LSLKRKEFIEAAQVGGVSTASIVIRHIVPNVLGWWYASLLVPSMILFESFLSFLGLGT 247 ORF351 QQPDPSWGNMLSLATNA-SMLLLNPWLIWPPALLIWAVLAFNWGDGIiRDRLDPRR * * *** ** * ** *** ** ** ** ****** *** 351 OppC QEPLSSWGALLS--DGANSM-EVSPWLLLFPAGFLWTLFCFNFIGDGLRDALDPKDR Figure 2. A. Amino acid sequence alignment of the Synechococcus S14-like protein deduced from ORFIOO, Escherichia coli (Ec) S14, Chlorella ellipsoidea (Ce) chloroplast CS14, and tobacco (Nt) chloroplast CS14. B. Amino acid sequence alignment of the polypeptide deduced from Synechococcus ORF315 and S. typhimurium OppC protein. Asterisks indicate identical residues and dashes denote gaps. ORF351 located downstream of rpsl4 shows substantial homology (43.6% identity) to the C-terminal two-thirds portion of the Salmonella typhimurium oligopeptide permease membrane protein OppC (Figs. 1 and 2B). Even the N-terminal part is completely different from that of the Salmonella OppC, ORF351 is predicted to be a gene for OppC homologue (oppc). Northern blot analysis using RNA preparations from rapidly growing cells showed that rpsl4 produces a transcript of approximately 0.48 kb whereas no transcript was detected from oppc (Fig. 3). This observation raises three possibilities. As typical promoter motifs " 35" and " 10" are present in front of rpsl4 but not in the 80-bp spacer, rpsl4 and oppc are cotranscribed, but the oppc

4 168 rpsl4 and oppc Homologues of Synechococcus PCC 6301 [Vol. 3, mrna portion is specifically degraded. The second possibility is that the rpsl4 gene is transcribed monocistronically, and probably terminated at the inverted repeat present in the spacer. In this case, the oppc could be expressed by recognition of an atypical promoter with a non-<7 70 -type RNA polymerase under specific environmental conditions.the third possibility is that oppc is a psudogene and is not transcribed. kb 0 53-gH flbf 1, H p r23s -16S Figure 3. Detection of transcripts from rpsl4 and oppc. Total RNA was prepared from Synechococcus 6301 cells as described. The RNA (30 /jg for rpsl4 or 60 /ig for oppc) was separated on a 1.2% formaldehyde agarose gel and blotted onto a nylon membrane (Hybond-N). Northern hybridization was carried out using 32 P-labeled 433 bp Stu I (rpsl4) and 1,213 bp Stu I-EcoRl (oppc-like gene) DNA fragments as gene-specific probes (see Fig. 1). Size markers are endogeneous rrnas (23S and 16S) and an RNA ladder ( kb, Gibco BRL). A kbp 8.5 -«5.9 - kbp B 275 kbp- (W275) < kbp (CW165) 2. Chromosomal Position of rpsl4 Southern blot analysis of genomic DNA resulted in single bands of 2.0 kbp Pst I, 5.9 kbp EcoRI or 8.5 kbp BamHI fragments that hybridized to a rpsi^-specific probe (Fig. 4A), indicating that rpsl4 is present as a single-copy gene on the Synechococcus genome. To localize rpsl4, pulsed-field electrophoresis and blot hybridization were performed. 19 The Pst I fragment containing rpsl4 and oppc hybridized to 275-kb Swa I (275W) and 165-kbp I Ceul/Swa I (CW165) fragments (Fig. 4B), and to the 600 kbp l-ceu I fragment (C600) (data not shown). The rpsl4 gene therefore maps to a 165-kb region located upstream of rrna and is not located in the spclike operon on the Synechococcus 6301 chromosome (Fig. 4C). Similar ORFs of 100 and 294 codons (slro628 and slro324) have recently been found in Synechocystis PCC Interestingly, the corresponding rpsl4 and oppc rps1 Str operon Figure 4. Genomic Southern analyses of rpsl4- A. Genomic DNAs (l^ig each) were digested with BamHI, EcoRI or Pst I, separated on a 1% agarose gel, and transferred to a nylon membrane (Hybond-N, Amersham). The 433-bp Stu I DNA fragment (rpsl4) was used as a gene-specific probe. B. DNA fragments digested with Swa I or l-ceu I and Swa I in agarose beads were separated on 1% SeaKem GTG agarose (FMC BioProducts, USA) under the following pulsed conditions; 5 s for 15 h, 15 s for 20 h then 20 s for 5 h. Southern blotting and hybridization of DNA fragments were performed as described. 19 Labelling of the 2-kbp Pst I DNA probe and detection of hybridized fragments were carried out according to the ECL gene detection system protocol (Amersham). C. Chromosomal location of rpsl4- Physical map of the genome (2,700 kbp) and the gene location of rrna, rrnb, rpsl and str operon are indicated. 19 Numbers indicate sizes of restriction fragments produced by digestion with Swa I (W), Pme I (P), l-ceu I (C), l-ceu I and Swa I (CW), or l-ceu I and Pme I (CP). 19

5 No. 3] T. Fujishiro et al. 169 homologues are separated by 370 kbp in Synechocystis PCC Thus, the gene organization around rpsl4 in Synechococcus 6301 apparently differs from that in Synechocystis 6803 and E. coli. Acknowledgements: We thank Dr. T. Matsubayashi for valuable discussions and C. Sugita for gene mapping of rpsl4- We also thank Dr. Jon Y. Suzuki for critical reading of this manuscript. This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture (Japan). References 1. Giovannoni, S. J., Turner, S., Olsen, G. J., Barns, S., Lane, D. J., and Pace, N. R. 1988, Evolutionary relationships among cyanobacteria and green chloroplasts, J. Bacterioi, 170, Gray, M. W. 1989, The evolutionary origins of organelles, Trends Genet., 5, Subramanian, A. R. 1993, Molecular genetics of chloroplast ribosomal proteins, Trends Genet., 18, Sugiura, M. 1992, The chloroplast genome, Plant Mol. Biol, 19, Harris, E. H., Boyton, J. E., and Gillham, N. W. 1994, Chloroplast ribosomes and protein synthesis, Microbiol. Rev., 58, Sugita, M., Sugita, C., and Sugiura, M. 1995, Structure and expression of the gene encoding ribosomal protein SI from the cyanobacteium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1, Mol. Gen. Genet., 246, Sanangelantoni, A. M., Calogero, R. C, Buttarelli, R. R., Gualerzi, C. O., and Tiboni, O. 1990, Organization and nucleotide sequence of the genes for ribosomal protein S2 and elongation factor Ts in Spirulina platensis, FEMS Microbiol Lett., 66, Meng, B. Y., Shinozaki, K., and Sugiura, M. 1989, Genes for the ribosomal proteins S12 and S7 and elongation factors EF-G and EF-Tu of the cyanobacterium, Anacystis nidulans: structural homology between 16S rrna and S7 mrna, Mol. Gen. Genet, 216, Buttarelli, R. R., Calogero, R. A., Tiboni, O., Gualerzi, C. O., and Pon, C. L. 1989, Characterization of the str operon genes from Spirulina platensis and their evolutionary relationship to those of other prokaryotes, Mol. Gen. Genet, 217, Sato, N. 1994, A cold-regulated cyanobacterial gene cluster encodes RNA-binding protein and ribosomal protein S21, Plant Mol. Biol., 24, Schmidt, J., Bubunenko, M., and Subramanian, A. R. 1994, A novel operon organization involving the genes for chorismate synthase (aromatic biosynthesis pathway) and ribosomal GTPase center proteins (Lll, LI, L10, L12: rplkajl) in cyanobacterium Synechocystis PCC 6803, J. Biol. Chem., 268, Malakhov, M. P., Wada, H., Los, D. A., Sakamoto, T., and Murata, N. 1993, Structure of a cyanobacterial gene encoding the 50S ribosomal protein L9, Plant Mol. Biol., 21, Sibold, C. and Subramanian, A. R. 1990, Cloning and characterization of the genes for ribosomal proteins L10 and L12 from Synechocystis sp. PCC 6803: comparison of gene clustering pattern and protein sequence homology between cyanobacteria and chloroplasts, Biochim. Biophys. Ada, 1050, Kaneko, T., Tanaka, A., Sato, S., Kotani, H., Sazuka, T., Miyajima, N., Sugiura, M., and Tabata, S. 1995, Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. I. Sequence features in the 1 Mb region from map positions 64% to 92% of the genome, DNA Res., 2, Cerretti, D. P., Dean, D., Davis, G. R., Bedwell, D. M., and Nomura, M. 1983, The spc ribosomal protein operon of Escherichia coli: sequence and cotranscription of the ribosomal protein genes and a protein export gene, Nucl. Acids Res., 11, Ohto, C, Torazawa, K., Tanaka, M., Shinozaki, K., and Sugiura, M. 1988, Transcription of ten ribosomal protein genes from tobacco chloroplasts: a compilation of ribosomal protein genes found in the tobacco chloroplast genome, Plant Mol. Biol., 11, Yoshinaga, K., Iwasaki, H., and Sasaki, T. 1992, Gene organization on Chlorella chloroplast genome is peculiar but suggests common lineage with land plants, Genet. (Life Sci. Adv.), 11, Hiles, I. D., Gallagher, M. P., Jamieson, D. J., and Higgins, C. F. 1987, Molecular characterization of the oligopeptide permease of Salmonella typhimurium, J. Mol. Biol, 195, Kaneko, T., Matsubayashi, T., Sugita, M., and Sugiura, M. 1996, Physical and gene maps of the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 genome, Plant Mol. Biol, 31,

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