UC Santa Cruz UC Santa Cruz Electronic Theses and Dissertations Title Comparative genomics and natural distributions of phenotypically distinct strains of the nitrogen-fixing cyanobacterium Crocosphaera watsonii Permalink https://escholarship.org/uc/item/2sh8r9q6 Author Bench, Shellie Publication Date 2012 Supplemental Material https://escholarship.org/uc/item/2sh8r9q6#supplemental Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA SANTA CRUZ COMPARATIVE GENOMICS AND NATURAL DISTRIBUTIONS OF PHENOTYPICALLY DISTINCT STRAINS OF THE NITROGEN-FIXING CYANOBACTERIUM CROCOSPHAERA WATSONII A dissertation submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in OCEAN SCIENCES by Shellie R. Bench June 2012 The Dissertation of Shellie R. Bench is approved: ______________________________ Professor Jonathan P. Zehr, Chair ______________________________ Professor Fitnat Yildiz ______________________________ Professor Jack Meeks ______________________________ Professor Eric Webb ______________________________ Tyrus Miller Vice Provost and Dean of Graduate Studies Copyright © by Shellie R. Bench 2012 Table of Contents Title ........................................................................................................................ i Copyright .............................................................................................................. ii Table of contents .................................................................................................. iii List of Figures and Tables .................................................................................... vi Abstract ................................................................................................................ ix Dedication ............................................................................................................ xi Acknowledgements ............................................................................................. xii Chapter 1: Introduction ...................................................................................... 1 Nitrogen-fixing cyanobacteria in the global oceans ............................... 1 Physiological studies in cultivated Crocosphaera .................................. 2 Phenotypic variation in Crocosphaera isolates ....................................... 3 Previous studies of genetic variation in Crocosphaera populations and isolates ............................................................................................... 6 Unanswered questions and focus of the dissertation .............................. 9 References .............................................................................................. 11 Chapter 2: Two strains of Crocosphaera watsonii with highly conserved genomes are distinguished by strain-specific features .................................................. 16 Abstract ................................................................................................. 17 Introduction ............................................................................................18 Materials and Methods .......................................................................... 23 WH0003 genomic DNA amplification and 454 sequencing .... 23 Sequence assembly and analysis and ORF identification ......... 24 Transposase annotation ............................................................. 26 Genome comparisons ................................................................. 27 iii Microarray gene expression ...................................................... 28 Results and Discussion ......................................................................... 29 Broad Genome Comparison ...................................................... 29 Shared Genome Features ........................................................... 30 Strain-specific Genome Features .............................................. 32 Transposase Gene Comparisons ............................................... 38 Conclusions ............................................................................... 40 Acknowledgements ............................................................................... 41 References ............................................................................................. 42 Tables .................................................................................................... 49 Figures ................................................................................................... 53 Chapter 3: Investigation of Crocosphaera watsonii phenotypes through whole genome comparison of six strains ..................................................................... 58 Abstract ................................................................................................ 59 Introduction ............................................................................................61 Materials and Methods .......................................................................... 63 Strain growth and genomic DNA isolation and sequencing ..... 63 Genome assembly and annotation ............................................ 65 Genome comparisons ................................................................. 67 Analysis and PCR of specific groups of functional genes ........ 68 Results ................................................................................................... 73 Genome characteristics and sequence replication within genomes .......................................................................... 73 Shared and unique ORFs among the six genomes .................... 74 Phylogenetic analysis and comparison of metabolic capabilities ................................................................................. 78 Discussion ............................................................................................. 82 Conclusions ............................................................................................ 91 References ............................................................................................. 93 iv Figures and Tables ................................................................................ 98 Chapter 4: Natural abundances of two Crocosphaera types in the North and South Pacific .................................................................................. 115 Abstract ............................................................................................... 116 Introduction ..........................................................................................117 Methods ............................................................................................... 121 Design and testing of qPCR assays .......................................... 121 Sample collection, DNA extraction and qPCR of cruise samples .......................................................................... 123 Results and Discussion ....................................................................... 126 Conclusions .......................................................................................... 132 References ............................................................................................ 133 Figures and Tables .............................................................................. 136 Chapter 5: Conclusions and future directions .............................................. 142 Appendix 1: Additional analysis of WH0003 and WH8501 transposase ORFs.............................................................................................. 146 Methods ............................................................................................... 146 Results and Discussion ....................................................................... 147 References ............................................................................................ 151 Figures ................................................................................................. 153 Appendix 2: Chapter 2 supplemental tables ................................................. 157 Appendix 3: Chapter 3 supplemental tables and figures ............................. 160 Appendix 4: Chapter 4 supplemental tables and figures ............................. 165 List of additional supplementary files ................................................................ 168 v List of Figures and Tables Chapter 2: Two strains of Crocosphaera watsonii with highly conserved genomes are distinguished by strain-specific features Table 1. Genome assembly information and annotation summary .................... 49 Table 2. ORFs within WH0003 strain specific genome region ......................... 50 Table 3. Transposase IS family distribution in both genomes ........................... 52 Figure 1. Nucleotide BLAST similarity of open reading frames (ORFs) and intergenic spaces (IGSs) of the two C. watsonii genomes to the opposite strain ................................................................................. 53 Figure 2. Comparison of open reading frames and intergenic spaces between two C. watsonii genomes ..................................................................... 54 Figure 3. Taxonomic distribution of 930 ORFs in the WH0003 genome that show little or no sequence similarity to the WH8501 genome ..... 55 Figure 4. Alignment of WH8501 and WH0003 contigs illustrating