Bacteriophage diversity in haloalkaline environments Shonisani Nemavhulani A thesis submitted in partial fulfillment of the requirements for the degree of Magister Scientiae (M.Sc.) in the Department of Biotechnology, University of the Western Cape Prof M. Tuffin Prof D.A. Cowan Dr S. Easton July 2013 Keywords: Bacteriophages, soda lakes, phage purification, plaque assays, agar spot assay, plaque forming unit (pfu), Transmission electron microscopy (TEM), one step growth curve, multiplicity of infection (MOI), DNA isolation and sequencing. ii Abstract Bacteriophage diversity in haloalkaline environments S. Nemavhulani MSc Thesis, Department of Biotechnology, University of the Western Cape There are limited reports on virus population in haloalkaline environments; therefore the aim of this study was to investigate the genetic diversity and biology of bacteriophage communities in these environments. Bacteria were isolated to be used as phage hosts. One bacterium from Lake Magadi and four bacteria from Lake Shala were successfully isolated from sediment samples. A further two Lake Shala bacterial hosts from the IMBM culture collection were also used to isolate bacteriophages. Bacterial isolates were identified to be most closely related to Bacillius halodurans, Halomonas axialensis, Virgibacillus salarius, Bacillus licheniformis, Halomonas venusta, Bacillus pseudofirmus and Paracoccus aminovorans. Bacteriophages were screened using all bacteria against sediment samples from both Lake Shala and Lake Magadi. One phage was identified from Lake Magadi sediments (MGBH1) and two phages from Lake Shala sediments (SHBH1 and SHPA). TEM analysis showed that these phages belong to three different dsDNA phage families; Siphoviridae (MGBH1), Myoviridae (SHBH1) and Podoviridae (SHPA). All phages showed different genome sizes on agarose gel. Due to the small genome size, phage SHPA was chosen for further investigation. Partial, genome sequence analysis showed homology to both bacterial and phage proteins. A further investigation of phage diversity in this environment is essential using metagenomic approaches to understand these unique communities. July 2013 iii Declaration I declare that Bacteriophage diversity in haloalkaline environments is my own work, that it has not been submitted for any degree or examination in any other university, and that all the sources I have used or quoted have been indicated and acknowledged by complete references. Shonisani Nemavhulani July 2013 iv Acknowledgements I would like to extend my gratitude to: Professor Don Cowan and Professor Marla Tuffin for granting me the opportunity to work at IMBM. I am grateful to have been in your laboratory. NRF for funding which enabled me to pursue this project. My co-supervisors Dr James Cass and Dr Samantha Easton. I thank you for all the support, assistance, patience, encouragement, reading of my research and all the advices you have given me. Your help was really appreciated. Dr William Mavengere, Dr Bronwyn Kirby and Lonnie Van Zyl for their teachings. Dr Heidi Goodman for reading the draft of my thesis and her caring attitude. Everyone who has helped teach me some of the techniques that I have learned over the course of my MSc. My family, thank you for your support and everlasting love and for always believing in me when at times I didn’t believe in my self, you truly are a gift from God. v This thesis is dedicated to my late grandmother Vho-Vele Mamphwe "If you can't fly then run, if you can't run then walk, if you can't walk then crawl, but whatever you do you have to keep moving forward." - Martin Luther King, Jr. vi Table of Contents Keywords .................................................................................................................. ii Abstract .................................................................................................................... iii Declaration ............................................................................................................... iv Acknowledgements ................................................................ .................................. v List of figures ........................................................................................................... x List of tables ........................................................................................................... xii List of symbols and abbreviations ...................................................................... xiii Chapter 1: Literature review .................................................................................... 1 1.1. Bacteriophage introduction and background ................................................. 1 1.1.1. Phage history ............................................................................................... 1 1.1.2. Phage taxonomy and classification .............................................................. 2 1.1.3. Definitions .................................................................................................... 4 1.1.4. Phage structure and sizes ............................................................................ 5 1.1.5. Phage life cycles .......................................................................................... 7 1.1.6. Phage genomics .......................................................................................... 9 1.1.7. Phage impact on the biosphere.................................................................. 25 1.1.8. Current applications in biotechnology ........................................................ 28 1.2. Haloalkaline environments ............................................................................. 34 1.2.1. Introduction to soda lakes .......................................................................... 34 1.2.2. Soda lake formation ................................................................................... 37 1.2.3. Lake Magadi .............................................................................................. 38 1.2.4. Lake Shala ................................................................................................. 40 1.2.5. Microbial diversity of soda lakes................................................................. 42 1.2.6. Survival strategy of microbes in soda lakes ............................................... 45 1.3. Phage diversity in soda lakes ......................................................................... 47 1.4. Aims of the study ........................................................................................... 49 vii Chapter 2: Materials and methods ........................................................................ 50 2.1. Chemicals and reagents ................................................................................ 50 2.2. Growth media, buffers and primers ................................................................ 50 2.2.1. Media ......................................................................................................... 50 2.2.2. Buffers ........................................................................................................ 51 2.2.3. Primers ....................................................................................................... 52 2.3. Sampling sites and collection ......................................................................... 52 2.4. Bacterial hosts, phage isolation and phage growth curve determination........ 52 2.4.1. Bacterial host isolation ............................................................................... 52 2.4.2. Bacterial host growth curve ........................................................................ 53 2.4.3. Phage isolation........................................................................................... 53 2.4.4. Plaque assay.............................................................................................. 54 2.4.5. Phage lysate preparation and purification .................................................. 54 2.4.6. Agar spot assay ......................................................................................... 55 2.4.7. One-step growth curve ............................................................................... 55 2.5. Phage preparation and visualisation by transmission electron microscopy (TEM) .................................................................................................................... 56 2.5.1. Phage lysate purification ............................................................................ 56 2.5.2. Transmission electron microscopy ............................................................. 56 2.6. DNA isolation, quantification and visualization ............................................... 57 2.6.1. Phage DNA isolation .................................................................................. 57 2.6.2. Bacterial host DNA extraction .................................................................... 58 2.6.3. Determination of DNA concentration .......................................................... 59 2.6.4. Agarose gel electrophoresis ....................................................................... 59 2.7. DNA amplification using 16S rRNA primers ................................................... 59 2.7.1. Bacterial 16S rRNA gene amplification ...................................................... 59 2.7.2. PCR product purification ...........................................................................