INTRODUCING THE ArsR REGULATED ARSENIC STIMULON by Tara Carolyne Saley A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Land Resources and Environmental Science MONTANA STATE UNIVERSITY Bozeman, Montana July 2017 ©COPYRIGHT by Tara Carolyne Saley 2017 All Rights Reserved ii ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Timothy McDermott for sharing his extensive knowledge of microbial-arsenic interactions, his guidance in performing the research, and his help in preparing and reviewing this thesis. Credit is also due to my committee made up of Dr. Brian Bothner, Dr. Ross Carlson, and Dr. Anthony Hartshorn for their input in the research and thesis writing processes. Thank you to National Science Foundation for funding the research. iii TABLE OF CONTENTS 1. LITERATURE REVIEW ................................................................................................1 Introduction to Arsenic ....................................................................................................1 Environmental Chemistry of As ..............................................................................1 Abiotic Environmental Conditions ................................................................2 Microbial Effects ...........................................................................................3 Sources of As ...........................................................................................................5 Minerals and Rocks........................................................................................5 Soils................................................................................................................5 Water ..............................................................................................................7 Atmosphere ..................................................................................................10 Geothermal Areas ........................................................................................11 Microbial Bioremediation of As ............................................................................12 Biostimulation ..............................................................................................13 Bioaugmentation ..........................................................................................13 Risks of As Exposure .............................................................................................13 Risks to Humans ..........................................................................................14 Microbe-As Interactions ................................................................................................15 ars Operon .............................................................................................................16 Regulatory Proteins ......................................................................................16 As (III) Transporters ....................................................................................18 As (V) Reductases........................................................................................19 Additional Proteins ......................................................................................20 aio Operon .............................................................................................................21 Signal Transduction .....................................................................................22 AioAB ..........................................................................................................22 Additional Known Requirements ................................................................23 pho Regulon ...........................................................................................................23 Signaling System .........................................................................................23 pstSCAB–phoU operon. ...............................................................................24 As Metabolism Genes ............................................................................................24 2. INTRODUCING THE ArsR-REGULATED ARSENIC STIMULON ........................26 Introduction ....................................................................................................................26 Materials and Methods ...................................................................................................29 Reporter Constructs and Deletion Mutants ............................................................28 Bacterial Growth Conditions .................................................................................30 arsR Reporter Assays .............................................................................................31 RNA Extraction .....................................................................................................31 RNA Sequencing ...................................................................................................32 iv TABLE OF CONTENTS CONTINUED Sequence Analysis .................................................................................................32 Results ...........................................................................................................................33 arsR Reporter Assays .............................................................................................33 Gene Expression Changes Related to As(III) Exposure ........................................35 Evaluating Changes in Gene Expression Levels of the arsR Mutants...................41 Genes Potentially Co-Regulated by All Four ArsR Proteins .......................46 Genes Regulated by ArsR1 Only .................................................................47 Genes Regulated by ArsR2 Only .................................................................49 Genes Regulated by ArsR3 Only .................................................................54 Genes Regulated by ArsR4 Only .................................................................55 As(III) Influences on ArsR Regulatory Control ....................................................57 Arsenic Resistance .......................................................................................58 Chemotaxis ..................................................................................................58 Sugar Transporters .......................................................................................59 Iron Homeostasis .........................................................................................60 Discussion .....................................................................................................................61 Purpose of arsR Reiteration ...................................................................................62 Activator Function .................................................................................................64 Functions Regulated by the ArsR Proteins ............................................................65 Significance............................................................................................................70 REFERENCES CITED ......................................................................................................71 v LIST OF TABLES Table Page 1. Bacterial strains and constructs used in this study .............................................30 2. Significant gene expression changes in wild type A. tumefaciens as a function of As(III) exposure ..............................................................................37 3. Summary of functions influenced by ArsR proteins..........................................45 4. Genes exhibiting expression patterns suggesting they are influenced directly or indirectly by all of the ArsR proteins ...............................................47 5. Gene expression significantly influenced by ArsR1 only .................................48 6. Gene expression significantly influenced by ArsR2 only .................................51 7. Gene expression significantly influenced by ArsR3 only .................................54 8. Gene expression significantly influenced by ArsR4 only .................................56 9. Influence of As(III) on ArsR control of the arsenic resistance response ...........58 10. Influence of As(III) on ArsR control of chemotaxis ........................................59 11. Influence of As(III) on ArsR control of sugar transporters .............................60 12. Influence of As(III) on ArsR control of iron homeostasis ...............................61 vi LIST OF FIGURES Figure Page 1. As speciation based on environmental conditions ...............................................3 2. As in the top 0-5 cm of soil in the United States .................................................6 3. As levels in drinking water around the United States ..........................................9 4. As Levels in Gallatin County, MT .....................................................................10 5. Representations of the arsR1 and arsR2 loci in A. tumefaciens ........................17 6. Organization of the arsR1 and arsR2 loci ..........................................................34 7. Reporter gene assays monitoring expression of arsR1, arsR2, and arsR4 as a function of As(III) exposure and time ..............................................35 8. Volcano plots illustrating the influence of each ArsR on genome-wide transcription in the absence of As(III) ...............................................................43 9. Genes uniquely repressed by ArsR1 ..................................................................49 10. ArsR regulatory hierarchy................................................................................63 11. Functions regulated by the ArsR proteins with no As(III) ..............................66 vii ABSTRACT The United States EPA ranks arsenic as the number one environmental toxin. Since microorganisms are significant drivers of arsenic toxicity and mobility in nature, it is important to understand how microbes detect and react to arsenic. The microbial arsenic resistance operon (ars) is critical for sensing arsenic in the environment and controlling the cellular response to this toxin. The ars operon is minimally comprised of arsRBC, which codes for an ArsR transcriptional repressor, arsenite effluxer, and an arsenate reductase, respectively, with the operon negatively regulated by the transcriptional repressor, ArsR. Our model organism Agrobacterium tumefaciens 5A carries two ars operons, with each containing two arsR genes. We conducted an RNASeq study to examine the regulatory roles of the encoded four ArsR regulatory proteins as a function of +/- arsenite. We report that the regulatory influence of the ArsR proteins extends well beyond the ars operon, with both activation and repression effects. In addition to the expected arsenic resistance response, many cellular functions were impacted, including: phosphate acquisition/metabolism, sugar transport, chemotaxis, copper tolerance, and iron homeostasis. Each of the ArsR proteins uniquely influenced different sets of genes and an arsR regulatory hierarchy was observed, wherein ArsR1 is auto regulatory and negatively regulates arsR4, ArsR4 activates arsR2, and ArsR2 negatively regulates arsR3. ArsR3 is the least active with respect to number of genes regulated. To summarize, this study provides a more complete understanding of how microbial gene expression and biogeochemical cycling may be influenced by arsenic in the environment. 1 LITERATURE REVIEW Introduction to Arsenic Arsenic (As) is at the top of the toxic substance priority list prepared by the ATSDR and the EPA because of the frequency, toxicity, and potential for human exposure at national priority list sites (ATSDR, 2016). The World Health Organization has ranked As among the top ten chemicals that threaten human health (WHO, 2016). While often grouped with heavy metals, As is actually a metalloid meaning it has both metallic and nonmetallic properties. Organic As compounds are molecules wherein As is directly bonded with carbon, whereas inorganic As is bonded with elements such as oxygen or sulfur and are usually more harmful to various forms of life (Kaur, Kamli, & Ali, 2011). As hotspots exist around the world. Some areas may have naturally high levels of As, but anthropogenic sources can contribute substantially to As contamination in soils and waters. Once As has entered an environment, it cannot be destroyed but may change forms or solubility through the actions of bacteria or prevailing environmental chemistry. This literature review will cover sources of As, environmental chemistry of As, risks of As exposure, and microbe-As interactions. Environmental Chemistry of As As can be found in oxidation states of +5, +3, 0, and -3 (Kaur et al., 2011). It is typically found in the environment as arsenite [As(III)] and arsenate [As(V)], whereas elemental As [As(0)] and arsine [As(-III)] are not as common. Speciation of As determines its mobility and toxicity. As(V) tends to be less mobile and less toxic, 2 whereas As(III) is more mobile and more toxic. Speciation is determined by redox potential, pH, and microorganism activity (Kang, Heinemann, Bothner, Rensing, & McDermott, 2012; Kruger, Bertin, Heipieper, & Arsene-Ploetze, 2013). Abiotic Environmental Conditions. Both redox potential and pH of the environment are abiotic factors that influence As speciation (Figure 1). A redox reaction occurs when an electron is passed from one atom to another. The atom that accepts the electron is reduced and the atom that donates the electron is oxidized. Some environmental conditions are conducive to reducing and some are conducive to oxidizing. Redox conditions play a major role in As speciation, with As(V) usually being the dominant species in aerobic conditions and As(III) being dominant under anaerobic conditions. For example, in oxidizing aquatic environments, As(III) is rare, while the majority of As exists as As(V) (Kruger et al., 2013). Redox conditions can also change As solubility by affecting binding sites in soil, which are often oxidized forms of iron, manganese, or aluminum. Both As (V) and As(III) can form complexes with metal oxides, but As(V) is more strongly sorbed at near-neutral pH (such as in most groundwater). As(V) is often associated with iron-(oxy)hydroxides. In oxidizing conditions, the hydroxide is stable and remains in solid state, along with the sorbed As(V). In reducing conditions, the hydroxide becomes less thermodynamically stable and may release to the solution along with the As (Smedley & Kinniburgh, 2002). A major example of this phenomenon is in the Bengal Basin. Reducing conditions were likely the reason for the As contamination in groundwater of this region. It is likely that this As