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Molecular Cloning, Insertional Inactivation And MOLECULAR CLONING, INSERTIONAL INACTIVATION AND CHARACTERIZATION OF THE CYANATE LYASE GENE FROM THE CYANOBACTERIUM SYNECHOCOCCUS PCC 7942 Farid Jalali A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Botany University of Toronto Q Copyright by Farid Jalali (1997) nbqummbtuvI.J ut IU -V~Y.YI.IV. IY Y. Bibliographic Services services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Cana& Your file Votre rtiference Our fi& Notre réldrence The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant a la National Libraty of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la fome de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or othemise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. MOLECULAR CLONING, INSERTIONAL INACTIVATION AND CHARACTERIZATION OF THE CYANATE LYASE GENE FROM THE CYANOBACTERIUM SYNECHOCOCCUS PCC 7942 Farid Jalali, Master of Science 1997. Department of Botany, University of Toronto. A 4.45 kb region of genomic DNA was isolated from the cyanobacterium Synechococcus PCC 7942 by means of screening subgenomic plasmid and phage libraries. Nucleotide sequence analysis of this region identified four open reading frames. ORF146 is 441 bp in length, coding for a protein of 146 amino acids that had 43% sequence identity to cynS from Escherichia coli The deduced amino acid sequence of the three open reading frames upstream of ORFI 46 showed significant sequence identity to proteins that are members of ABC transport systems. lnsertional inactivation of ORF146 using an antibiotic resistance gene cassette produced a strain (CS1) that was unable to metabotize cyanate. In particular, the CS1 strain was unable to support cyanate-dependent O2 evolution or chlorophyll g fluorescence quenching and did not produce CO2when presented with cyanate. lnsertional inactivation of the first gene of the putative ABC transport group (ORF440) yielded a strain (CAI) that exhibited a phenotype similar to that of CS1. Northern hybridization analysis identified two ORF146 transcript, one that was -0.5 kb and a larger transcript of -3.9 kb. These results provide molecular and physiological evidence for the identification of the cyanate lyase gene (cynS) from the cyanobacterium Synechococcus PCC 7942. Furthermore, these results indicate that the four genes identified form an operon. I have had the privilege of being Dr. George Espie's student for 3 years. I am truly thankful for his constant support and encouragement in every aspect of my work. Over the years he has become more than my supervisor. I consider him a good friend and someone whom I regard with the greatest esteem. I would also like to thank my lab mates for their support and comedic interludes. iii Page ABSTRACT.. ..................................................... ii ..- ACKNOWLEDGEMENTS ............................................~ri TABLE OF CONTENTS ............................................. iv LIST OF FIGURES ................................................viii INTRODUCTION ....................................................1 lnorganic Carbon Transport ......................................... 2 Use of Structural Analogs to Study CO, Transport ....................... 5 Photosynthetic Metabolism of Cyanate in Synechococcus UTEX 625 ........ 7 Escherichia coli Cyanase ............................................10 Research Objectives .................................................15 Bacterial Strains and Growth Conditions ..............................-17 E. coli Strains and Growth Conditions ............................17 Growth Conditions for Synechococcus PCC 7942 ..................17 Nucleic Acid Isolation ...............................................18 Isolation of Genomic DNA from Escherichia coli KI2 ................18 Isolation of Genomic DNA from Synechococcus PCC 7942 ........... 19 Isolation of Plasmid DNA from Bacterial Cells ..................... 20 Isolation of RNA from Synechococcus PCC 7942 .................. 26 DNA Manipulations ................................................. 22 PCR Amplification of Genomic DNA .............................22 Restriction Digests and Agarose Gel Electrophoresis of DNA ......... 23 Gel Purification and Radiolabelling of DNA .........................23 Southern Blotting and Hybridization .............................. 24 Construction and Screening of Plasmid Libraries ................... 25 Construction and Screening of Lambda Phage Library ..............27 Insertional Inactivations .............................................29 RNA Gels and Northern Analysis ...................................... 31 Measurement of Photosynthetic O, Evolution ............................ 33 Fluorometry ........................................................34 Mass Spectrometry.................................................. 35 RESULTS ......................................................... 38 Construction of Subgenomic Libraries and Isolation of a Genomic Region ...38 Sequence Analysis of pBH22. pE56 and pK50 .......................... 44 ORF146 ..................................................... 49 ORF289 ...................................................... 51 ORF263 ...................................................... 52 ORF440 ...................................................... 56 Insertional Inactivation of ORF146 and ORF440 ..........................58 Cyanate Dependent O, Evolution and Fluorescence Quenching in WT and CS1 Cells of Synechococcus PCC 7942 .......................67 Mass Spectrometric Analysis of Synechococcus PCC 7942 W and CS1 .....71 Cyanate-Dependent O, Evolution and Fluorescence Quenching in WT and CA1 Cells of Synechococcus PCC 7942 .......................74 Cyanate Transport in WT, CSI, and CA1 Cells of SynechococcusPCC7942 ...........................................78 Northern Analysis of RNA Transcript Size From WT, CS1 and CA1 Cells of Synechococcus PCC 7942 ....................-79 cynS is Responsible for Cyanate Metabolism in SynechococcusPCC7942 ............................................82 cynS is Expressed as Part of an Operon .................................85 Cyanate Uptake in Synechococcus PCC 7942 .......................... -88 Physiological Significance of Cyanate Lyase in Synechococcus PCC 7942 ...93 Future Research Objectives .......................................... 96 Transcript Analysis ............................................96 Cyanate Transport ............................................ -97 Physiological Significance of Cyanate Lyase in SynechococcusPCC7942 ...................................... 99 Concluding Remarks ................................................ 99 REFERENCES .....................................................101 Page Figure 1. Southern hybridization analysis of restriction enzyme digested genomic DNA from Synechococcus PCC 7942 ....................... Figure 2. Organization of the genomic region isolated from Synechococcus PCC7942 ..................................................... Figure 3. Nucleotide and deduced amino acid sequence of the 4453 bp region identified from Synechococcus PCC 7942 .......................... 45 Figure 4. Alignment of the deduced amino acid sequence of ORFI 46 with that of CynS from E. coli, and that of ORF289 with that of NrtD from SynechococcusPCC7942 ......................................... Figure 5. Alignrnent of the deduced amino acid sequence of ORF263 with that of NrtB from Synechococcus PCC 7942 ........................... Figure 6. Hydropathy profile of the deduced amino acid sequence of ORF263 ....................................................... viii that of NrtA f rom Synechococcus PCC 7942 . 57 Figure 8. Cloning strategy to generate plasmid pXH::ORF146 for insertional inactivation of ORF146 from Synechococcus PCC 7942 . 59 Figure 9. Cloning strategy for construction of plasmid pP12::ORF440 to insertionally inactivate ORF440 from Synechococcus PCC 7942 . .61 Figure 10. Southern hybridization analysis of Synechococcus PCC 7942 mutants, CS1 and CA1 . .64 Figure 11. Ci- and cyanate-dependent fluorescence quenching and 0, evolution in wild type and CS1 cells of Synechococcus PCC 7942 . .69 Figure 12. Mass spectrometric analysis of cyanate-dependent CO, eff lux from wild type and CS1 cells of Synechococcus PCC 7942 . .72 Figure 13. Ci and cyanate-dependent fluorescence quenching and 0, evolution from wild type and CA1 cells of Synechococcus PCC 7942 . .75 Y * * I wild type, CS1 and CA1 cells of Synechococcus PCC 7942 . -80 ABC, ATP-binding cassette; BME, bmercaptoethanol; BTP, 1.3- bis[tris(hydroxymethyl)-methylamino] propane; Ci, inorganic carbon; CA, carbonic anhydrase; CA1 , Synechococcus PCC 7942 cynA insertional inactivant; CP, carbamy l phosphate; CPase, carbarnyl phosphate synthetase; CS 1, Synechococcus PCC 7942 cynS insertional inactivant; CCM,carbon concentrating mechanism; COS, carbon oxysulf ide; CCCP, 3-chloro-carbonylcyanidephenylhydrazone;
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