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UNIVERSITY of CALIFORNIA, SAN DIEGO Vanadium-Dependent UNIVERSITY OF CALIFORNIA, SAN DIEGO Vanadium-dependent bromoperoxidase in a marine Synechococcus A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Marine Biology by Todd L. Johnson Committee in charge: Brian Palenik, Chair Bianca Brahamsha, Co-Chair Lihini Aluwihare James Golden Jens Mühle Bradley Moore 2013 Copyright Todd L. Johnson, 2013 All rights reserved. The dissertation of Todd L. Johnson is approved, and it is acceptable in quality and form for publication on microfilm and electronically: ________________________________________________________ ________________________________________________________ ________________________________________________________ ________________________________________________________ ________________________________________________________ Co-Chair ________________________________________________________ Chair University of California, San Diego 2013 iii DEDICATION To Janet, Tim, and Andrew Johnson, for unconditional love and support. iv TABLE OF CONTENTS Signature Page……………………………………………………………………………iii Dedication ………………………………………………………………………………..iv Table of Contents………………………………………………………………………….v List of Abbreviations………………………………………………………………......…vi List of Figures………………………………………………………………………...…viii List of Tables…………………………………………………………..…………………xi Acknowledgements………………………………………….…………………….……xiii Vita……………………………………………………….………………………...……xiv Abstract…………………....……………………………………………………...…...…xv Chapter 1: Introduction………..………………………………………………….….……1 Chapter 2: Characterization of a functional vanadium-dependent bromoperoxidase in the marine cyanobacterium Synechococcus sp. CC9311 ..…………………………..………10 Chapter 3: Expression patterns of vanadium-dependent bromoperoxidase in Synechococcus sp. CC9311 as a response to physical agitation and a bacterial assemblage …………..………………………………………………………………………………..27 Chapter 4: Halomethane production by vanadium-dependent bromoperoxidase in marine Synechococcus………………………………………………………………………….107 Chapter 5: Conclusions………………..………………….………………….…………149 Appendix 1……………………………………………………………………………...154 v LIST OF ABBREVIATIONS VBPO – vanadium-dependent bromoperoxidase bp – base pair kDa – kilodalton °C – degrees Celsius DNA – deoxyribonucleic acid PCR – polymerase chain reaction MES - 2-(N-morpholino)ethanesulfonic acid Na3VO4 – sodium orthovanadate H2O2 – hydrogen peroxide hr - hour min – minute μmol - micromole μM - micromolar nM – nanomolar ppt – parts per trillion SDS-PAGE – sodium dodecyl sulfate - polyacrylamide gel electrophoresis Br- – bromide I- - iodide Cl- - chloride ppt – parts per trillion mTorr – millitorr VHOC – volatile halogenated organic compounds vi CH3Br – methyl bromide CH3I – methyl iodide CH3Cl – methyl chloride CH2Br2 - dibromomethane CH2Cl2 - dichloromethane CHBr3 - bromoform CHCl3 - chloroform vii LIST OF FIGURES Figure 1: SDS-PAGE gels of total proteins from Synechococcus strains WH8102, CC9311, and VBPO from Corallina officinialis (Co VBPO, Sigma) stained with Sypro Ruby (left) and with phenol red (right) for bromoperoxidase activity. The migrations of molecular mass standards are indicated on the left in kDa………………...…………….13 Figure 2: Amino acid alignment of VBPO from Synechococcus sp. strain CC9311 and strain WH8020. Active site is shown by the solid bar while vanadium-coordinating residues are shown in bold, as determined by homology to Ascophyllum nodosom. Arrows indicate residues used for the design of environmental probes ………………...14 Figure 3: Neighbor-joining tree of known bromoperoxidase sequences and related genes. Amino acid sequences obtained from NCBI using Synechococcus sp. strain CC9311 (YP_731869.1) in a BLASTp. Asterisk (*) indicates only node with less than 50% bootstrap support. Accession numbers are shown in Supplementary Table 1……..….…14 Figure 4: Neighbor-Joining tree of environmental VBPO sequences. Nucleotide sequences were trimmed to a conserved region of approximately 180-bp (alignment shown in Supplementary Figure 2). “Pier#” samples were obtained from a degenerate primer (PierF and PierR) clone library from the Scripps Pier in La Jolla, CA………..…15 Fig. S1: Alignment of the well-conserved region between cyanobacterial and eukaryotic algal vanadium-dependent bromoperoxidases (VBPOs) used to construct the phylogenetic tree shown in Figure 3. Sequences were obtained from the National Center for Biotechnology Information’s nr protein database……………………………………20 Fig. S2: Nucleotide alignment of the Scripps Pier clone library to metagenomic sequences (see “Materials and Methods”). This alignment was used for the neighbor- joining analysis and phylogenetic tree construction shown in Figure 4…………………23 Figure 3.1: A diagram of the insertional gene inactivation of VBPO (sync_2681) in Synechococcus CC9311 (shown in A) to form the VMUT genotype (shown in B). Steps: 1. PCR amplification of gene fragment, 2. Clone into TOPO vector to form pTOP2681, 3. Excise new fragment with EcoRI and cloned into pMUT100 form pVBR1, 4………....84 Figure 3.2: Mutant genotyping, a 1% agarose gel showing PCR products from boiling DNA extraction of stock cultures using A) RPOC1 primers (303 bp) and B) verifications primers (750 bp). Lanes left to right: 1 Kb Plus Ladder (bp); CC9311; VMUT; VMUT2; no DNA control…………………………………………………………………………..85 Figure 3.3: Mutant growth curves showing the cell counts (lines) comparing the growth of CC9311 and the original mutant, VMUT, under stirred and still conditions over the first three growth curves (A-C) as well as CC9311 and VMUT2 (D-F). VBPO specific activities (bars) are also shown comparing CC9311 to VMUT2 (D-F) over three……...86 viii Figure 3.4: VBPO specific activity of proteins harvested from cultures of A) CC9311 and B) VMUT2 at different phases of growth. VBPO activities of biological triplicates are shown from cultures grown under still conditions (white bars), cultures grown under still conditions and subjected to a short term (4 hr) period of stirring (grey bars), and ……..87 Figure 3.5: Genotype of VMUT after disappearance of stir-sensitive phenotype. PCR products from boiling DNA extraction shown on 1% agarose gels. Panel A: verifications primers (750 bp) lanes left to right: 1) 1 Kb Plus Ladder (bp); 2) CC9311; 3) VMUT stir; 4) VMUT still; 5) no DNA control. Panel B: RPOC1 primers (303 bp) ………………..88 Figure 3.6: Duplicate SDS-PAGE of proteins extracted from CC9311 and VMUT2 in stationary phase; grown still, still with only four hours of stirring, or constant stirring. Each lane was loaded with 55 μg protein and stained with A) sypro ruby or B) phenol-red in the presence of Na3VO4, KBr, and H2O2 for VBPO activity. Lanes left to right….....89 Figure 3.7: Induction and persistence of VBPO with stirring. A) Cell counts from cultures of CC9311 used to test the induction of VBPO with stirring over time. Vertical solid line shows when stirring was either initiated or halted in cultures. Growth rates of the cultures after the vertical line are 0.56 d-1 for the culture that was stirred from inoculation……..90 Figure 3.8: VBPO and cell resistance to hydrogen peroxide. Phycoerythrin autofluorescence of CC9311 and VMUT cultures after the addition of hydrogen peroxide. Solid lines represent cultures that have been preconditioned with stirring while dotted lines represent cultures that have been grown still from inoculation. Top left: Trial 1….91 Figure 3.9: VBPO specific activities of CC9311 cultures exposed to A) 25 μM hydrogen peroxide and b) 0.1 μM methyl viologen (MV)………………………………………....92 Figure 3.10: Cell counts of Acaryochloris marina cultures tested for VBPO activity (trial 2). ……………………………………………………………………………..…………93 Figure 3.11: Native PAGE Gel of Synechococcus sp. CC9311 and Acaryochloris marina proteins, 21 μg protein per lane, from still and stirred cultures. Gel in panel A was stained with Sypro Ruby for total proteins, Gel in panel B was stained with phenol red for VBPO activity. Lanes left to right A) 1. Benchmark Protein ladder; 2. CC9311, still……......…94 Figure 3.12: SDS-PAGE of Synechococcus CC9311 and Acaryochloris marina proteins, 26.5 μg protein per lane. Gel in panel A was stained with Sypro Ruby for total proteins, Gel in panel B was stained with phenol red for VBPO activity. Lanes left to right A) 1. Benchmark Protein ladder; 2. CC9311, still; 3. CC9311, stirred; 4. A. marina. …..……95 ix Figure 3.13: Neighbor-joining tree showing the 16S gene library constructed of the bacterial assemblage that induces VBPO in Synechococcus CC9311, the 19 isolates, as well known sequences. TJ# refers to a clone from the 16S rRNA gene library and PTB# refers to a strain isolated from the Pteridomonas enrichment. ………………….……....96 Figure 3.14: Alignment of the 421 bp fragment of the 16s rRNA genes from the PTB enrichment clone library (TJ#), 19 isolated strains (PTB#) and known sequences……...97 Figure 4.1: Experimental design used to measure halomethane production in Synechococcus cultures……………………………………………………………….. 140 Figure 4.2: Cell concentrations of cultures (1.5 L) used in the purge-and-trap experiment (solid lines days 2-7). Vertical solid line at day seven indicates when CC9311 (Black) and VMUT2 (light grey) cultures were split and sealed in a serum bottle for 24 hrs with (dotted) and without (dotted) stirring. Vertical dotted line at day 8 indicates when...…141 Figure 4.3: Reaction mechanism for the biosynthesis of polyhalomethanes mediated by bromoperoxidase activity as proposed by Beissner et al. (1981)...……………….……142 Figure 4.4: Reaction scheme for vanadium-dependent
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