Evidence for Perchlorate-Coupled Molybdenum and Nickel Carbon Monoxide Dehydrogenase CO Oxidation and Characterization of Novel Perchlorate-Reducing Haloarchaea

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Marisa Russell Myers Louisiana State University and Agricultural and Mechanical College

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Recommended Citation Myers, Marisa Russell, "Evidence for Perchlorate-Coupled Molybdenum and Nickel Carbon Monoxide Dehydrogenase CO Oxidation and Characterization of Novel Perchlorate-Reducing Haloarchaea" (2021). LSU Doctoral Dissertations. 5555. https://digitalcommons.lsu.edu/gradschool_dissertations/5555

This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. EVIDENCE FOR PERCHLORATE-COUPLED MOLYBDENUM AND NICKEL CARBON MONOXIDE DEHYDROGENASE CO OXIDATION AND CHARACTERIZATION OF NOVEL PERCHLORATE-REDUCING HALOARCHAEA

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

The Department of Biological Sciences

by Marisa Russell Myers B.S., Louisiana State University, 2014 August 2021 ACKNOWLEDGEMENTS

I want to thank my mentor and chair of my dissertation committee, Dr. Gary King for first welcoming me into the lab as a young undergraduate and guiding me throughout both my

Bachelor and Doctoral programs. His leadership and engagement made these projects possible and successful. I would also like to thank my committee members, Dr. Gregg Pettis, Dr. William

Doerrler, and former committee members Dr. J. Cameron Thrash, Dr. Brent Christner, and Dr.

Yuchen Liu for their thoughtful guidance and feedback. I also thank the current and former members of the King lab, Dr. Caitlin King, Craig Judd, Dr. Shannon McDuff Johnson, Amber

DePoy, and Eric Martinez for your mentoring, feedback, and support.

To my friends, Kristen Campbell, Lindsey Morphis, Reagan Dugan, Scott Grimmell,

Katie Rose DeLeo, Andrew McGuffey, Charles Zimmerman, Grigor Sargsyan, Aheli Mukerji,

Wesley Morgan, Emily Morgan, David Alspaugh, and Frederic Marazzato, thank you for always listening, giving feedback, providing endless encouragement and laughs, and reminding me it is ok to leave the lab and have a solid climbing send session. I can truly say I wouldn’t have been as happy and motivated without ya’ll in my life.

To my family, Marion, Lisa, and Marty, thank you for your love, support, and teaching me hard work, without which no graduate student can be successful. To my in-laws, Douglas,

Florence, Daniel, Katie, and little Addison, thank you for welcoming, supporting, and loving me as one of your own.

Finally, I am eternally grateful for my husband, David Myers. I could not have done this without you, you have shown me strength, commitment, graciousness, and leadership which allowed our friendship and marriage to thrive after nearly seven years of long distance. You remind me every day to not give up, support me in my choices, and encourage me in my goals.

This research was funded by the US National Science Foundation award EAR-15654499, and NASA awards NNX15AE82A, and 15-EXO15_2-0147.

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TABLE OF CONTENTS

Acknowledgements ...... ii

Abstract ...... v

Chapter 1. Introduction ...... 1

Chapter 2. Perchlorate-Coupled Carbon Monoxide (CO) Oxidation: Evidence for a Plausible Microbe-Mediated Reaction in Martian Brines ...... 19

Chapter 3. Addendum: Perchlorate-Coupled Carbon Monoxide (CO) Oxidation: Evidence for a Plausible Microbe-Mediated Reaction in Martian Brines ...... 36

Chapter 4. Halobacterium bonnevillei sp. nov., Halobaculum saliterrae sp. nov., and Halovenus carboxidivorans sp. nov., three novel carbon monoxide-oxidizing Halobacteria from saline crusts and soils ...... 42

Chapter 5. Halanaeroarchaeum oxyrespirans sp. nov., a novel oxygen-respiring, perchlorate-reducing extreme halophile ...... 59

Chapter 6. Perchlorate-Coupled Carbon Monoxide (CO) Oxidation in Moorella glycerini: A Novel Mechanism for Nickel-Dependent Carbon Monoxide Dehydrogenases ...... 83

Chapter 7. Conclusions and Future Directions ...... 92

Appendix A. Supplementary Material for Chapter 2 ...... 96

Appendix B. Supplementary Material for Chapter 4 ...... 99

Appendix C. Supplementary Material for Chapter 5 ...... 103

Appendix D. Permission to Reproduce Chapters 2 and 3 ...... 107

Appendix E. Permission to Reproduce Chapter 4 ...... 109

References ...... 111

Vita ...... 126

ABSTRACT

Carbon monoxide (CO) has been exploited as a microbial energy source for much of life’s evolutionary history. A phylogenetically diverse array of microorganisms can oxidize CO using two distinct CO dehydrogenases, molybdenum-dependent (Mo-CODH) and nickel- dependent (Ni-CODH). Aerobes and facultative organisms contain Mo-CODHs which allow them to utilize oxygen as an electron acceptor in addition to alternatives such as nitrate and sulfate. Obligate anaerobic organisms contain Ni-CODHs, which oxidize CO at elevated concentrations, but cannot utilize oxygen. In systems where organic matter deposits are limited or absent, atmospheric trace gases such as CO are thought to assist in supporting the growth and survival of microbial communities. Extraterrestrially, the Martian atmosphere is dominated by

CO2, however, CO also has also been documented at substantial levels. Martian regolith contains only trace levels of organic carbon, leaving CO as potentially the most abundant and available substrate capable of supporting near-surface microbial activity. However, Martian regolith also contains perchlorate, while potentially toxic it also serves an abundant potential oxidant. At locations such as the recurrent slope lineae, hypersaline perchlorate-based brines are thought to exist. Though previously unexplored for CO oxidation, chlorine oxyanions may act as suitable electron acceptors, expanding the current range of both Mo-CODH and Ni-CODH CO oxidizing microorganisms. This study used a variety of culture-dependent approaches, cultivating four novel haloarchaea from the Bonneville Salt Flats and surrounding saline soils capable of utilizing

- CO, ClO4 , or a metabolic coupling. Halovenus carboxidivorans was capable of CO oxidation, while Halobacterium bonnevillei and Halobaculum saliterrae, represent the first microbes

- capable of perchlorate-coupled CO oxidation at concentrations up to 1 M ClO4 . All three isolates contained Mo-CODHs. The provisional species, Halanaeroarchaeum oxyrespirans, is

v capable of growth via perchlorate reduction, independently of nitrate, a first for haloarchaeal cryptic perchlorate reduction. Additionally, H. oxyrespirans, can respire oxygen, expanding the known capacities of the genus. Perchlorate-coupled CO oxidation was further expanded to include carboxidotrophic Ni-CODH containing microbes by using the thermophilic Firmicute

Moorella glycerini DSM 11254T as a model organism. Collectively, the isolation of these haloarchaeal cultivars contributed to the expansion of haloarchaeal diversity through both physiological and genomic characterization.

CHAPTER 1. INTRODUCTION

Microbes have exploited various anaerobic metabolic processes for energy since the origin of life. Carbon monoxide (CO), the second most common molecule in the universe, has contributed to diverse and significant processes throughout the history of terrestrial life (Aylward and Bofinger, 2001; Miyakawa et al., 2002; King and Weber, 2007). Life on Earth is likely to have exploited CO for much of its evolutionary history, including possible early chemolithotrophs near anoxic hydrothermal vent systems (Svetlichny et al., 1991; Sokolova et al., 2004). In the absence of organic matter deposits, atmospheric trace gases such as CO may aid in supporting the growth and survival of microbial communities (King, 2003; King et al., 2008).

Microbial carbon monoxide oxidation is known to proceed under both aerobic and facultative conditions using a molybdenum-dependent carbon monoxide dehydrogenase (Mo-CODH) as well as anaerobic conditions using a nickel-dependent carbon monoxide dehydrogenase (Ni-

CODH). Thus far, electron acceptors such as oxygen, nitrate, and sulfate have been identified for

CO oxidation in a wide array of bacteria and archaea. Though previously unexplored for CO oxidation, chlorine oxyanions may act as suitable electron acceptors, expanding the current knowledge and diversity of both Mo-CODH and Ni-CODH CO oxidizing microorganisms.

Metabolic processes involving chlorine oxyanions are understudied when compared to other anaerobic metabolisms such as sulfate and nitrate reductions, methanogenesis, and anoxygenic photosynthesis. Found worldwide and extraterrestrially, the most oxidized chlorine

- oxyanion, perchlorate (ClO4 ), is capable of being reduced by a phylogenetically large diversity of microorganisms, though in cultured isolates canonical reduction primarily takes place in

Proteobacteria. Perchlorate reduction canonically results in the production of molecular oxygen and chloride, however alternative pathways exist and commonly result in the production and

1 abiotic detoxification of chlorite. Beyond Proteobacteria, members of Firmicutes, Crenarchaeota, and Euryarchaeota have also been documented to reduce perchlorate. Currently, several haloarchaea within the Euryarchaeota have been documented to reduce perchlorate using non- canonical perchlorate reduction pathways such as cryptic perchlorate reduction, however growth via cryptic perchlorate reduction in all cultured isolates has been nitrate-dependent (Martinez-

Espinosa et al., 2015; Myers and King; 2017; 2019; 2020).

The research performed for this dissertation sought to determine if perchlorate can act as a CO electron acceptor for Mo-COX and Ni-COX isolates, isolate and characterize novel halophilic CO oxidizers, and investigate how perchlorate reduction differs between canonical perchlorate reducers and those that utilize alternative pathways such as cryptic perchlorate reduction. To do this, this study used a variety of culture-dependent approaches on novel haloarchaea cultivated from the Bonneville Salt Flats and surrounding saline soils as well as the known thermophilic Firmicute Moorella glycerini DSM 11254T. Chapters two and three focus on evidence of novel denitrifying and nitrate-respiring euryarchaeal extreme halophiles which can couple Mo-CODH CO oxidation to perchlorate at elevated sodium chloride concentrations, establishing that perchlorate can act as a Mo-CODH CO oxidation electron acceptor. Chapter four centers on the cultivation and characterization of three novel CO oxidizing haloarchaea isolates, promoting a greater understanding of CO oxidation in extremely halophilic environments. Chapter five explores the provisional haloarchaeal isolate, Halanaeroarchaeum oxyrespirans sp. nov., a haloarchaeal species with novel cryptic perchlorate-reduction features as well as the capacity for oxygen respiration which expands the currently known characteristics of the genus. Chapter six focuses on expanding perchlorate-coupled carbon monoxide oxidation to organisms containing Ni-CODH, using Moorella glycerini DSM 11254T as a model species.

Significance and History of Natural and Anthropogenic Sources of Perchlorate

Of the four types of chlorine oxyanions: perchlorate, chlorate, chlorite, and hypochlorite, perchlorate specifically is of environmental concern due to its toxicity to mammals (Crump et al.,

2000; Coates and Achenbach, 2004; Dohan et al., 2007; Tran et al., 2008, Bardiya and Bae,

2011). Due to perchlorate’s toxicity, and the lack of knowledge about environmental concentration and prevalence, surveys of perchlorate distribution have been conducted to evaluate distribution and concentrations as well as used to identify natural and anthropogenic sources. These surveys have led to the discovery of elevated perchlorate levels in both ground and surface waters of several western states in the United States, particularly along the Colorado

River (Urbansky, 1998).

Anthropogenic activities are the primary source of perchlorate introduction into the environment. Perchlorate salts have a broad range of industrial applications such as lubricating oils, fireworks, airbag initiators for vehicles, matches, signal flares, paints, and notably munitions manufacturing and handling. In munitions specifically, ammonium perchlorate (NH4ClO4) is used as an oxidant in solid rocket fuels which account for approximately 90% of perchlorate salts manufactured, and for more than 15.9 million kg of perchlorate discharged into the environment since the 1950s (Urbansky, 2002). Natural sources of perchlorate are geographically widespread, but uncommonly found. As early as the 1880s perchlorate was identified in the Atacama Desert

(Chile) within terrestrial nitrate deposits, which are considered the largest and most concentrated perchlorate source, averaging ~ 0.03 wt % and peaking at ~ 0.6 wt %.

Lower concentrations of natural perchlorate can also be found in the southwestern United

States (subsoils), California and Nevada (salt evaporites), New Mexico (Pleistocene groundwater), New Mexico and Canada (Potash), and Antarctica (Orris et al., 2003; Duncan et

3 al., 2005). Specific processes for natural perchlorate production remain poorly understood but are thought to involve photochemical processes in the atmosphere, ozone oxidation of chloride in both aqueous and dry systems, and electrical discharge. Research has shown that Atacama perchlorate originated in the stratosphere, indicating a likelihood that perchlorate is globally produced, though accumulation only occurs in arid locations due to its high solubility in aqueous solutions (Bao and Gu, 2004; Bohlke et al., 2005; Motzer et al., 2006; Sturchio et al., 2009;

Catling et al., 2010). Widespread stratospheric production is supported by the presence of trace perchlorate levels in rainwater across the North American continent (Rajagopalan et al., 2009).

Perchlorate has also been documented extraterrestrially in Martian regolith, lunar samples, and chondrite meteorite samples (Jackson et al., 2015). In particular, Martian regolith samples contain perchlorate at elevated concentrations. Concentrations of ~0.6 wt % were first detected in Martian regolith by the Wet Chemistry Laboratory onboard the Phoenix lander in

2008, this was later supported by results from the Martian lander Curiosity (Hecht et al., 2009;

Glavin et al., 2013). Martian perchlorate production pathways were initially thought to occur in similar ways to Earth, primarily a photochemical reaction via oxidation of chlorine by ozone

(O3) (Dasgupta et al., 2005; Catling et al., 2010). However, studies have shown that the amount found cannot be solely explained by these interactions, and research has shown that additional mechanisms for Martian oxychlorine production exist (Smith et al., 2014; Carrier and Kounaves,

2015). Carrier and Kounaves (2015) results indicate that under current Martian environmental conditions chloride-bearing mineral surfaces can react with atmospherically or photochemically produced oxidants, globally producing oxychlorines on Mars. If present, Martian water is speculated to exist as brines and pockets of hydrated salts which would likely be dominated by

(per)chlorates which lowers the freezing temperature (Hecht et al., 2009, Kounaves et al., 2014a;

- - Clark and Kounaves, 2016). Additionally, perchlorate, chlorate (ClO3 ), and nitrate (NO3 ) have also been found in Martian meteorite EETA79001, further supporting the hypothesis that perchlorate is ubiquitous on Mars (Kounaves et al., 2014b).

Significant abiological perchlorate decomposition does not occur naturally due to its high activation energy. Industrial processes such as ion exchange, electro-dialysis, and reverse osmosis, among others have been employed but have limited application due to non-selectivity and high cost. However, biologically mediated perchlorate reduction occurs readily under some circumstances, can be exploited for bioremediation of contaminated sites, and consequently is viewed as a cost-effective way to treat waters. Currently, biological reduction is being explored as it would exploit diverse capabilities of ubiquitous dissimilatory perchlorate reducing bacteria.

Biological reactors used to treat perchlorate-contaminated groundwater have been applied at both pilot-scale and full-scale at multiple locations and have been documented to remove perchlorate from more than 30 million liters of groundwater daily (Hatzinger et al., 2005). Notably, two full- scale bioreactors have been constructed to treat high concentrations in military and industrial wastewaters (Hatzinger et al., 2005). Following successful ex situ biological treatment systems, in situ perchlorate treatments are also beginning to be explored. A phylogenetically diverse set of microorganisms including Proteobacteria, Firmicutes, Crenarchaeota, and Euryarchaeota have been shown to reduce perchlorate and be indigenous to many soils and groundwaters. The wide distribution of microorganisms combined with their ability to use common substrates led to the first successful pilot-study of in situ perchlorate bioremediation aimed at treatment of aquifers

(Hatzinger et al., 2006).

Review of Microbial Dissimilatory Perchlorate Reduction

Microorganisms capable of reducing the chlorine oxyanions perchlorate and chlorate have been found at diverse sites including: soils, gold mine drainage sediments, wastewater treatment plants, and underground gas storage facilities as well as extreme environments such as hot springs, deep unsaturated vadose zones, Antarctic sites, and hypersaline salts and soils

(Malmqvist et al. 1994; Rikken et al. 1996; Wallace et al. 1996; Bruce et al. 1999; Achenbach et al. 2001; Logan et al. 2001; Zhang et al. 2002; Bardiya and Bae 2004; Bardiya and Bae 2005;

Bardiya and Bae 2008; Wolterink et al. 2005; Balk et al. 2008; Gal et al. 2008; Weelink et al.

2008; Balk et al. 2010; Thrash et al. 2010; Myers and King, 2020). Whereas anthropogenic perchlorate-contaminated sites characteristically harbor greater numbers of perchlorate-reducing bacteria, natural sites are also well documented to contain perchlorate-reducing bacteria, though their metabolic role is still unclear as perchlorate is not likely to be their primary mode of metabolism. The majority of perchlorate- and chlorate-reducing bacteria grow at neutral pH, mesophilic temperatures, and are typically facultative anaerobes. Some notable exceptions include the thermophilic obligate anaerobes Moorella perchloratireducens, Moorella glycerini, and Carboxydothermus hydrogenoformans, the thermophile crenarchaeal aerobe Aeropyrum pernix, and the extremely halophilic euryarchaeota, Haloferax mediterranei, Halobacterium bonnevillei, and Halobaculum saliterrae (Balk et al., 2008; Liebensteiner et al., 2015; Martínez-

Espinosa et al., 2015; Myers and King, 2020).

Both perchlorate and chlorate, collectively (per)chlorate, can support the metabolism of bacteria and archaea. Dissimilatory perchlorate-reducing microorganisms (DPRM) can use perchlorate as an alternative respiratory electron acceptor in which it is coupled to the oxidation various electron donors. In canonical perchlorate reduction, organisms contain a perchlorate

6 reduction genomic island (PRI) of approximately 10- to 25- kb (Figure 1.1A) (Melnyk et al.,

- 2011). PRIs always contain a perchlorate reductase (pcr) in which ClO4 is anaerobically reduced

- to chlorite (ClO2 ) within the bacterial periplasm. The perchlorate reductase operon always contains the catalytic subunits (pcrAB) and a maturation protein (pcrD). Additional operon subunits frequently found include electron transfer subunits (pcrCO), quinol-oxidizing subunit

(pcrQ), and transcriptional regulation subunits (pcrPSR) though their organizational order may vary (Melnyk et al., 2014; Melnyk et al., 2015; Melnyk and Coates, 2015).

Chlorite is dismutated to end products chloride and molecular oxygen by a chlorite dismutase (cld) (Figure 1.1A). Hypochlorite (ClO-), a reactive transient byproduct chlorine species produced from chlorite dismutation, can be damaging to cells through oxidizing reactions. Hypochlorite has emerged as an important fitness determinant of cells during

(per)chlorate reduction. However, some DPRM possess a suite of key genes to detoxify ClO-, these include a periplasmic methionine sulfoxide reductase (yedYZ), sigma factor/anti-sigma factor system (sigF/nrsF), and a methionine-rich peptide (mrpX) which form a periplasmic methionine-cycling system reducing oxidative damage to proteins by detoxifying hypochlorite

(Melnyk et al., 2015; Melnyk and Coates, 2015). Molecular oxygen generated in this process is not extracellularly released, but instead reduced to water by the same microorganism, commonly by use of a high-affinity cytochrome cbb3 oxidase (Melnyk et al., 2015; Barnum et al., 2018).

Dissimilatory chlorate reducing microorganisms (DCRM) differ from DPRM as they do not possess a pcr and instead contain a chlorate reductase (clr) along with the previously described cld (Figure 1.1B). Fewer DCRM have been cultivated than DPRM and cultivated isolates are currently comprised of various Proteobacteria species such as Ideonella dechloratans, Alicycliphilus denitrificans BC, Shewanella algae ACDC, Dechloromarinus

7 chlorophilus NSS, and various Pseudomonas strains (Malmqvist et al., 1994; Weelink et al.,

2008; Clark et al., 2014; Youngblut et al., 2016). As an alternative to a PRI, the core genes of chlorate reduction reside on a composite transposon termed chlorate reduction composite transposon interior (CRI) (Clark et al., 2014). DCRM organisms cannot reduce perchlorate and

- - - instead reduce ClO3 to ClO2 via the heterotrimer ClrABC, after which the ClO2 is dismutated by the cld in the same manner as seen by DPRM (Thorell et al., 2003; Steinberg et al., 2005).

ClrA, the α-subunit, is predicted to contain a bis(molybdopterin guanine dinucleotide)- molybdenum cofactor and an iron cluster (4Fe-4S] (Thorell et al., 2003). ClrB, the β-subunit is predicted to contain four Fe-S clusters which form an electron transfer pathway between the α and γ subunit (ClrC) (Karlsson et al., 2005). An additional δ-subunit is commonly found and likely participates in proper insertion of the molybdenum cofactor, though it is not considered part of the active enzyme (Clark et al., 2013).

Figure 1.1. Canonical dissimilatory microbial (per)chlorate- and chlorate-reducing pathways with associated chlorine oxidation states. (A) Canonical perchlorate reduction. Perchlorate reductase (Pcr) and chlorite dismutase (Cld) contained within a perchlorate reduction genomic island (PRI) performs the biological reduction of perchlorate to molecular oxygen and chloride. (B) Canonical chlorate reduction. Chlorate reductase (Clr) and chlorite dismutase (Cld) contained within a chlorate-reduction composite transposon interior (CRI) performs the biological reduction of chlorate to molecular oxygen and chloride.

While most research has been conducted on canonical (per)chlorate- and chlorate- reducing microbes, alternative options for reduction exist including chlorate reduction by nitrite- oxidizing bacteria, cryptic reduction, and symbiotic per(chlorate) reduction (Figure 1.2). Some nitrite-oxidizing bacteria in the genera Nitrospira and Nitrobacter contain a Cld predicted to have a cytoplasmic localization comparable to nitrite oxidoreductase (Nxr) (Figure 1.2A)

(Maixner et al., 2008; Melnyk et al., 2011). Additionally, Nitrobacter winogradskyi has been shown to couple nitrite oxidation to chlorate reduction, though limited growth was observed,

- possibly due to an elevated ClO3 concentration (Melnyk et al., 2011). Cld-like proteins located in nitrite-oxidizing genera are termed “Lineage II” as opposed to “Lineage I” in canonical

(per)chlorate- and chlorate-reducers (Maxiner et al., 2008; Youngblut et al., 2016). Lineage I

Clds are located in the periplasm; whereas lineage II Clds are approximately 30% shorter and have a largely unknown physiological role (Youngblut et al., 2016).

Some organisms which lack a PRI, CRI, or Cld in isolation are still able to reduce

- (per)chlorate, though they rely on chemical reactions to remove ClO2 , these organisms perform cryptic per(chlorate) reduction. As an alternative to canonical (per)chlorate reductase genes they harbor a closely related periplasmic Nar-like molybdopterin nitrate reductase (pNar, pnarGHIJ), which reduces (per)chlorate to the end-product chlorite (Figure 1.2B) (Liebensteiner et al., 2013;

Liebensteiner et al., 2015; Marangon et al., 2012; Martinez-Espinosa et al., 2015; Oren et al.,

2014; Youngblut et al., 2016). pNar periplasmic location is identified by a twin-arginine transport (TAT) signal sequence. Thus far, no pNar tested lacks perchlorate reductase activity, with examples from both bacteria and archaea. Notably, pNarG contains key amino acid residues

(α, β, γ positions) similar to PcrA which influence its affinity for perchlorate by helping shape the active site. Bacterial examples have been observed in Firmicutes such as Carboxydothermus hydrogenoformans and various Moorella species. There are no documented canonical

(per)chlorate reducers within Archaea, instead all documented species thus far rely on cryptic reduction including the hyperthermophilic Crenarchaeote Aeropyrum pernix, the hyperthermophilic Euryarchaeote Archaeoglobus fulgidus VC-16, and some extremely halophilic members of Halobacteria (Liebensteiner et al., 2015; Martinez-Espinosa et al., 2015). While the presence of pcr or pnar in combination with cld suggests the specialization for perchlorate reduction, the absence of cld in many pnar-containing organisms likely suggests a more inadvertent reduction of perchlorate. Of note however, a pnar-cld gene cluster forms a separate deep-branching monophyletic clade when compared to pNarG proteins from other organisms

(Barnum et al., 2018).

During cryptic per(chlorate) reduction toxic chlorite may accumulate and is currently thought to be removed by abiological chemical means (Figure 1.2B). For some sulfur-reducing bacteria the potential chlorite and hypochlorite produced could be detoxified with various sulfur species such as sulfide or thiosulfate (Liebensteiner et al., 2015; Liebensteiner et al., 2013).

Perchlorate-dependent sulfide oxidation (PSOX) has been suggested to support non-canonical

DPRM bacteria, including Azospira suillum PS (Gregoire et al., 2014; Mehta-Kolte et al., 2017).

PSOX includes an enzymatic oxidation of sulfide by PcrAB and an additional abiotic reaction

0 2- with chlorite, hypochlorite, and oxygen in which elemental sulfur (S ) and polysulfide (Sn ) are produced as byproducts. Additionally, enzymatic oxidation may not be needed by the per(chlorate) reducing species in the natural environment, instead it may sequester it from its surroundings depending on the site and local microbial community.

Symbiotic (per)chlorate reduction is characterized as the reduction of (per)chlorate or chlorate to chlorite by one organism and the removal of generated chlorite by a second organism

(Figure 1.2C). In a laboratory setting, Clark et al., (2016) used a co-culture of Pseudomonas

- - stutzeri PDA mutants with varying Cld expression to demonstrate reduction of ClO3 to ClO2 in

- one strain (lacking Cld) and the subsequent removal of ClO2 by a second strain (containing Cld).

- - - Clark additionally demonstrated reduction of ClO4 to ClO2 and the subsequent removal of ClO2 by a co-culture of A. suillum PS (mutants). Symbiotic reduction has yet to be documented in nature, however it has been speculated to occur as it would provide microbial communities with a simple mechanism to overcome an environmental (per)chlorate influx. Recently, Lynch et al.,

(2019) presented results of microbial perchlorate reduction in the Pilot Valley paleolake basin

(Utah, USA), phylogenetic data of resident species, and qPCR results of cld and pcrA gene copy numbers. While unable to demonstrate if canonical or alternative (per)chlorate reduction was the

11 primary factor, results indicating species and genes for both (per)chlorate and chlorate reduction as well as species representative of both canonical and alternative pathways.

Figure 1.2. Alternative microbial (per)chlorate and chlorate reducing pathways. (A) Chlorate reduction by Nitrite-oxidizing bacteria. Cytoplasmically located nitrite oxidoreductase (Nxr) reduces chlorate to chlorite while a lineage II Cld dismutates chlorite to molecular oxygen and chloride. (B) Cryptic (per)chlorate reduction. Presence of pNar which biologically reduces perchlorate to chlorite. Abiological elimination of chlorite to chloride, thus far observed for z- reduced sulfur compounds. “Ax” indicates a reduced sulfur species while AxOy represents an oxidized form. (C) Symbiotic (per)chlorate and chlorate reduction. Multi-organism reaction in which organism “1” reduces perchlorate to chlorite which is subsequently eliminated via abiological means or a secondary organism containing a functional Cld.

The Significance of Carbon Monoxide

Carbon monoxide, an odorless, tasteless, and notoriously toxic gas, is one of the most versatile molecules on the planet having participated in diverse processes from cellular to global levels (King and Weber, 2007). As an often-overlooked portion of the global carbon cycle, CO is an important atmospheric trace gas. Specifically, CO can remove atmospheric hydroxyl radicals 12

(OH·) by forming carbon dioxide (CO2); consuming ~80% of total annual CO emissions

(Monson and Holland, 2001). Serving as the dominant greenhouse gas sink, hydroxyl radicals are responsible for cleansing the atmosphere of various greenhouse gases such as methane, and carbon dioxide; however, as CO and other trace gases increase, OH· levels decrease, resulting in higher concentrations as well as extended decay times for other greenhouse gases.

Although present at low atmospheric concentrations (50 – 350 ppb) CO has been identified as one of the six primary air pollutants by the Environmental Protection Agency to also include lead, nitrogen oxides, ground-level ozone, particle pollution (often referred to as particulate matter), and sulfur oxides. Approximately 2.6 petagrams (Pg) of CO per year is produced, with anthropogenic activities responsible for approximately 60% of CO emissions, while naturally generated CO formed via processes such as photochemical reactions, forest fires, and volcanic activity attribute to approximately 40% (Khalil and Rasmussen, 1990).

Atmospheric CO has a relatively short lifespan of approximately 10 – 52 d, during which two major processes occur to oxidize the CO, geochemical oxidation via hydroxyl radicals (~85%) and biological oxidation (~10%) (Khalil and Rasmussen, 1990; Cordero et al., 2019).

Biological oxidation of gases such as carbon monoxide contribute to the survival and growth of microbial communities in organic limited systems (King and Weber, 2008). While microbial CO oxidation can occur under aerobic and anaerobic conditions, early in Earth’s history anaerobic CO-oxidation is likely to have occurred due to elevated atmospheric CO concentrations (>100 ppm) in the Archean atmosphere (Miyakawa et al., 2002). The timing of aerobic CO oxidation evolution is more uncertain, though it thought to be linked to aerobic respiration and atmospheric oxygen accumulation (King and Weber, 2007). Beyond Earth, the

Martian atmosphere is dominated by CO2, however, CO also has also been documented at

13 appreciable levels (>800 ppm) (Weiss et al., 2000; Sindoni et al., 2011). Additionally, the lifespan of atmospheric Martian CO is much longer than that on Earth at around 6 years, plays an important role in atmospheric chemical stability, and it is effectively treated as an atmospheric passive tracer (McElroy and Donahue, 1972; Krasnopolsky et al., 2007; Holmes et al., 2019).

While no evidence for Martian life currently exists, estimates for putative Martian microbes use of atmospheric CO suggested if it occurred it would take place at low rates (globally). However, at local sites such as the recurrent slope lineae (RSL) thought to have perchlorate-based brines, substantial activity could occur (Weiss et al., 2000; King, 2015).

Review of Microbial Carbon Monoxide Oxidation

Carbon monoxide oxidizers which couple elevated levels of CO (>1%) to growth are deemed carboxidotrophic, while carboxidovores use relatively low CO concentrations (< 0.1%) and are incapable of CO-dependent growth (King and Weber, 2007). While both aerobic and anaerobic bacteria can utilize CO as an energy source with the same overall reaction (CO + H2O

+ - → CO2 + 2H + 2e ), the reactions are catalyzed by different variations of the enzyme carbon monoxide dehydrogenase (CODH). CODH genes cluster according to metabolic function rather than phylogeny, suggesting likely horizontal gene transfer events (Techtmann et al., 2012).

Under obligately anaerobic conditions microbial CO oxidation occurs in a more limited group of bacteria using Ni-CODH. The physiology and biochemistry of the Ni-CODH have been thoroughly documented and classified into six distinct clades (A to F). Ni-CODH clade A contains Cdh-type Ni-CODHs, nearly all of which are found in archaea, while the remaining clades contain CooS-type Ni-CODHs more frequently found in bacteria (Techtmann et al., 2012;

Inoue et al., 2019). Though culturally limited, Ni-CODH containing organisms are metabolically

14 diverse utilizing CO as a substrate for various fermentative anaerobic metabolisms including hydrogenogenesis, methanogenesis, and acetogenesis (Oelgeschlager and Rother, 2008). The majority of cultivated Ni-CODH containing organisms are thermophiles, specifically thermophilic Firmicutes (Sokolova et al., 2009).

Under aerobic or facultative conditions microbial CO oxidation occurs via a phylogenetically broad group of bacteria and archaea which oxidize CO to CO2 using Mo-

CODH (Mo-COX). Mo-CODH’s three structural protein subunits are arranged in the transcriptional order coxM, coxS, and coxL, encoding for medium, small, and large respectively.

The coxM subunit is a flavin adenine dinucleotide (FAD), coxS is an iron-sulfur protein utilized in electron transport, and coxL contains the active site of the enzyme. CO is oxidized at the molybdenum ion of the active site and electrons are then transferred to the small and medium subunits prior to reaching the final electron acceptor. Typically, the final electron acceptor is oxygen, however alternative acceptors such as nitrate and perchlorate may be substituted under anaerobic conditions (King and Weber, 2007; Myers and King, 2017; 2019; 2020). In both terrestrial and aquatic systems, Mo-dependent CO oxidation has been documented in cultured isolates or inferred by genomic isolates in a wide diversity of organisms including

Proteobacteria, Actinobacteria, Firmicutes, Chloroflexi, Bacteroidetes, Deinococcus-Thermus,

Crenarchaeota, Geoarchaeota, and Euryarchaeota (King, 1999; King and Weber, 2007; King and

King, 2014; McDuff et al., 2016; Islam et al., 2019). Most CO oxidizers isolated thus far are mesophiles and neutrophiles with thermotolerant or thermophiles being the primary extremophile in culture. However, recent work has shown that moderate and extreme halophilic systems have documented CO uptake. Documented moderately halophilic systems and isolates include numerous marine species and the moderate halophilic alkaliphile Alkalilimnicola ehrlichii

MLHE1; while extremely halophilic systems include salt deposits, hypersaline muds and soils from coastal Hawai’i, the Bonneville Salt Flats, and hypersaline pools within the Atacama Desert

(King and Weber, 2007; Hoeft et al., 2007; Weber and King, 2009; King, 2015; McDuff et al.,

2016). Additionally, the first CO-oxidizing extreme halophile to use a form I Mo-CODH has also been cultivated, Haloferax namakaokahaiae Mke2.3T (McDuff et al., 2016). These recent results suggest that hypersaline environments may ubiquitously contain CO oxidizing extreme halophiles.

Haloarchaea and Hypersaline Environments

Halophilic organisms are found in all three branches of life, Bacteria, Archaea, and

Eukarya. Halotolerant species do not require a specific amount of salt for growth but are capable of growing in a variety of elevated concentrations. Halophilic species will not grow in the absence of salt and commonly require elevated concentrations greater than 1 M. Extreme halophiles grow optimally between 2.5 – 5.2 M NaCl, are most commonly members of the phylum Euryarchaeota (class Halobacteria), frequently termed Haloarchaea. For optimal growth, haloarchaea require a salt concentration of between 10-15% (w/v), and when environmental salt concentrations exceed 16%, they persist as the dominant microbial population. Hypersaline environments such as solar salterns, hypersaline soils, salt marshes, sea floor brines, and ancient evaporite deposits serve as the major habitats for halotolerant, halophilic, and extremely halophilic species.

Waters with a higher total salt concentration than seawater are defined as hypersaline.

Sites, such as solar salterns typically derived from seawater, will initially have a proportional and similar composition of ions to prior seawater, as such are termed thalassohaline. In addition to

16 solar salterns, thalassohaline brines can be found in salt mine drainage waters, playas, and some brine springs from underground salt deposits. In contrast, athalassohaline environments are not of marine origin and are dominated by magnesium, calcium, and sulfates. The Dead Sea is an example where halite (NaCl) precipitation has occurred and brines are dominated by Mg2+ and

Cl-, leaving the waters more acidic. Microbial populations in hypersaline environments are largely influenced by pH in addition to ionic composition and total salinity. The amount of calcium (Ca2+) and to a lesser extent magnesium (Mg2+) play a critical role in determining the

2+ 2- final environmental pH, the presence of Ca can remove alkaline CO3 (a key buffering agent

- along with HCO3 and CO2) through precipitation of calcite (CaCO3). Alkaline lakes (soda lakes) typically develop based on surrounding geology (deficient in Ca2+) such as in the East African

Rift Valley’s Lake Magadi. The precipitation of gypsum (CaSO4 ·2H2O) results in the depletion of calcium and sulfate, leaves sodium chloride as the most abundant salt (Grant, 2004).

Haloarchaea, frequently observed as facultative aerobes, heterotrophic, slightly thermophilic, and phototrophic, are considered the most significant group of extremely halophilic organisms. To adapt to high salinity stressors haloarchaea have developed numerous unique molecular adaptations including: a high density of acidic residues of proteins as well as a decrease in hydrophobic residues (Matarredona et al., 2020). Negative surface charges allow hydrated ion binding networks which aid in the prevention of protein precipitation in the presence of a high cytoplasmic KCl concentration as a result of their salt-in strategy.

Haloarchaea, generally adopt a salt-in strategy which makes their cytosolic salinity equal to their environmental surroundings. A salt-in strategy primarily consists of higher cytosolic levels of

KCl to provide osmotic balance, the accumulation of K+ and to an extent Na+ is maintained by

Na+/H+ antiporters and ATPase dependent ion transporters. Most bacterial halophiles adopt a

“low salt-in” strategy and typically accumulate a low concentration of compatible osmotic solutes such as sugar derivates, betaines, amino acids, and glutamine derivates. Few haloarchaea are known to adopt this strategy though some in the genera Natronobacterium and

Natronococcus accumulate 2-sulphotrehalose and glycine solutes (Matarredona et al., 2020).

The class Halobacteria was recently divided into an emended three orders containing a total of five families, these include: Natrialbales (Natrialbaceae), Haloferacales (Haloferacacae and Halorubraceae), and Halobacteriales (Haloarculaceae, Halobacteriaceae, and

Halococcaceae). Halobacteria members typically lead an aerobic heterotrophic lifestyle using the tricarboxylic acid cycle (TCA) for carbon degradation and occasionally the glyoxylate cycle and respiratory electron transport. Oxygen can often become a limiting factor for halophilic Archaea as well as it has a low solubility in salt-saturated brines, as such some halophilic archaea have the capacity to grow as facultative anaerobes. Facultative anaerobes have been documented to use alternative electron acceptors such as nitrate, dimethyl-sulfoxide, fumarate, or fermentation of arginine, additionally some organisms may use photoheterotrophy to drive anaerobic growth.

Until recently, obligately anaerobic haloarchaea were unknown. This changed with the discovery of Halanaeroarchaeum sulfurireducens and Halodesulfurarchaeum formicium (Sorokin et al.,

2016; 2017). Halanaeroarchaeum sulfurireducens HSR2T, the first obligately anaerobic haloarchaea capable of growth using anaerobic dissimilatory sulfur respiration.

CHAPTER 2. PERCHLORATE-COUPLED CARBON MONOXIDE (CO) OXIDATION: EVIDENCE FOR A PLAUSIBLE MICROBE-MEDIATED REACTION IN MARIAN BRINES

Introduction

The prospects for extraterrestrial life depend on two major factors: liquid water availability and the availability of reduced inorganic and organic substrates that can be used to sustain biochemical reactions (Brack et al., 2010). While discoveries of liquid water on Europa and Enceladus have addressed the former (Kargel et al., 2000; Zolotov et al., 2004; Zolotov,

2007; Waite et al., 2009; Ojha et al., 2014, 2015), much speculation remains about the latter. For

Europa and Enceladus, simple organic and inorganic compounds have been identified as substrates for putative anaerobic processes (Chyba, 2000; Chyba and Phillips, 2002; McKay et al., 2008; Waite et al., 2017). Some of these substrates, especially hydrogen and formate, might be produced by serpentinization or other reactions beneath or associated with ice-capped oceans

(Chyba, 2000). Evidence for liquid water has also been reported on Mars (Ojha et al., 2014,

2015), though Dundas et al. (2017) have indicated that volumes are likely low. Regardless, suites of organics at substrate level concentrations have not yet been identified in the regolith (Benner et al., 2000). Although hydrothermal activity and serpentinization might occur in the deep sub- surface of Mars, these processes are unlikely to contribute reactants to surface brines that might host microbes.

This chapter previously appeared as Myers, Marisa R., and Gary M. King. "Perchlorate-coupled carbon monoxide (CO) oxidation: evidence for a plausible microbe-mediated reaction in Martian brines." Frontiers in microbiology 8 (2017): 2571.

However, in contrast to Europa and Enceladus, Mars’ atmosphere serves as a reservoir for carbon monoxide (CO), a relatively abundant [about 700 parts per million (ppm), 0.4 Pa] photochemically produced reductant (Sindoni et al., 2011; Mahaffy et al., 2013) that can serve as a microbial metabolite under extreme conditions (King, 2015). Indeed, based on the total mass of

Mars’ atmosphere, its average molar mass (43.34 g mol−1) and a concentration of 700 ppm, CO carbon occurs at 2.8 mol m−2 assuming a uniform distribution across the regolith surface. This is equivalent to the amount of carbon in 1 m3 of regolith with a density of 2 g cm−3 and a carbon concentration of 17 ppm. Since total organic carbon ranges from about 10 to 500 ppm for meteorites derived from Mars (Steele et al., 2012) and Mars mudstones (Ming et al., 2014;

Freissinet et al., 2015), CO might be the single most abundant readily available and renewable resource to drive metabolic activity.

Although diverse lineages of terrestrial bacteria can couple CO to multiple electron acceptors [e.g., molecular oxygen, nitrate, and sulfate (Ragsdale, 2004; King, 2006; King and

Weber, 2007)], hydrated salts and brines on Mars are likely dominated by perchlorate and chlorate (Hecht et al., 2009; Kounaves et al., 2014; Clark and Kounaves, 2016), which have not been shown to support CO oxidation. However, both oxyanions support metabolism of various organic substrates by bacteria and archaea (Coates and Achenbach, 2004; Liebensteiner et al.,

2013; Oren et al., 2014; Martinez-Espinosa et al., 2015; Mehta Kolte et al., 2017) that reduce perchlorate via a dissimilatory perchlorate reductase (only bacteria to date) or a dissimilatory nitrate reductase (bacteria and archaea). To date, no CO oxidizing dissimilatory perchlorate- reducing bacteria have been isolated, but numerous denitrifying and nitrate-respiring CO oxidizers have been reported (King, 2006, 2015; Weber and King, 2017). Results presented here

20 provide the first evidence that some denitrifying and nitrate-respiring euryarchaeal extreme halophiles can couple CO oxidation in brines to perchlorate at concentrations up to 1 M.

Materials and Methods

Isolates

Briefly, several denitrifying or nitrate-respiring CO-oxidizing extremely halophilic euryarchaeotes were obtained from salt crusts or soils in or near the Bonneville Salt Flats (BSF;

Utah, United States). One of the denitrifiers, Haloarcula sp. PCN7, was obtained from enrichments initiated with BSF salt crusts (40◦ 45’ 26.4”, −113◦ 53’ 11.1”) while the nitrate- respiring Halobaculum sp. WSA2 was obtained from enrichments of a saline soil collected south of the BSF (40◦ 25’ 43.0”, −114◦ 00’ 55.4”). Enrichments were conducted in 160-ml serum bottles under anoxic (nitrogen headspace with nitrate as an electron acceptor for Haloarcula sp.

PCN7) or oxic conditions (for Halobaculum sp. WSA2) in medium CM1 (McDuff et al., 2016) with 25 mM pyruvate. After addition of approximately 100 ppm CO (final concentration), serum bottles were incubated at 40◦C with shaking. Headspace CO was monitored by gas chromatographic analysis (McDuff et al., 2016). Enrichments positive for CO oxidation were used to inoculate a series of bottles with fresh CM1 media, which were ultimately used to prepare dilutions that were spread onto CM1-pyruvate agar plates. Distinct colonies were selected and purified through repeated sub-culturing as necessary. Isolates were identified to genus by PCR amplification and sequencing of 16S rRNA genes obtained from genomic extracts using a MoBio Microbial DNA Extraction Kit (Folsom, CA, United States). PCR used an archaeal forward primer, Arch21F [5’-TTCCGGTTGATCCYGCCGGA3’ (Delong, 1992)], and the universal reverse primer, 1492R [5’-CGGTTACCTTGTTACGACTT-3’ (Lane, 1991)].

Purified amplicons (MoBio Ultraclean PCR Clean-Up Kit) were sequenced with an ABI 3130XL

Genetic Analyzer at Louisiana State University. Sequences have been deposited in Genbank as accessions MF767880 and MF767881 for Haloarcula sp. PCN7 and Halobaculum sp. WSA2, respectively.

Genomic DNA extracts were also used to amplify the alpha sub-unit (narG) of nitrate reductase (Martinez-Espinosa et al., 2007, 2015) and the large sub-unit (coxL) of the form I CO dehydrogenase (King, 2003). Primers for narG were designed using nitrate reductase sequences found in the genomes of Haloarcula marismortui 43049T, Haloferax mediterranei 33500T, and other haloarchaea represented in the Integrated Microbial Genomes resource. Primers pNar1F

(5’-ACGAYTGGTAYCACAACGAC-3’) and pNar1R (5’-AGTTCSAGRWACCAGTCGTG-

3’) yield products approximately 990 bp in size. Details of coxL PCR have been published previously (King, 2003); PCR primers used were archcoxF (5’-

GGYGGSTTYGGSAASAAGGT-3’) and PSr (5’-YTCGAYGATCATCGGRTTGA-3’).

Amplicons obtained using these primers were sequenced bi-directionally as above. Phylogenetic analyses of inferred amino acid sequences for coxL and narG were conducted using MEGA7

(Kumar et al., 2015) using a neighbor-joining algorithm and a Poisson correction; all gapped positions were deleted. CoxL sequences have been deposited in Genbank as accessions

MF773971 and MF773972 for Haloarcula sp. PCN7 and Halobaculum sp. WSA2, respectively, and as MF773973 and MF773974 for the respective narG genes.

Assays for nitrate respiration and denitrification were conducted using API 20NE test strips (bioMérieux S.A., Marcy l’Etoile, France) according to the manufacturer’s instructions.

Confirmation of the results for Haloarcula sp. PCN7 was obtained by incubating the isolate in sealed 10-cm3 syringes with CM1 medium containing pyruvate and nitrate. Under these

22 conditions, copious production of gas bubbles presumed to be dinitrogen was interpreted as evidence for denitrification.

Although both Haloarcula sp. PCN7 and Halobaculum sp. WSA2 can utilize CO at concentrations in excess of 100 ppm, growth was not observed for either isolate. This is consistent with results obtained for other CO-oxidizing extreme halophiles (McDuff et al., 2016).

Perchlorate-Coupled CO Oxidation

Haloarcula sp. PCN7 and Halobaculum sp. WSA2 were assessed for their ability to couple CO oxidation to perchlorate reduction using stationary phase cells first grown aerobically in 3.8 M CM1-pyruvate medium to provide a suitable level of cell biomass. Since CO uptake is typically induced by substrate limitation during stationary phase, it was not necessary to preincubate cells with CO. To initiate assays, cells were harvested by centrifugation, washed in

3.8 M CM1 without pyruvate, and resuspended into 3.8 M CM1 with or without 2.5 mM pyruvate to support basal metabolism; treatments included the following: aerobic incubation with no perchlorate; anaerobic incubation (nitrogen headspace) with 0.01 M perchlorate with or without 0.25 mM nitrate; anaerobic incubation with no electron acceptor and anaerobic incubation of autoclaved cells with 0.01 M perchlorate. Resuspended cells (10 ml, A600 ∼ 0.4) were incubated in triplicate 160-ml serum bottles sealed with neoprene rubber stoppers. CO was added to a final headspace (150 ml) concentration of ∼10 ppm (about 1 Pa). Headspace CO concentrations were assayed periodically by removing sub-samples for analysis by gas chromatography. Anoxic treatments contained resazurin to monitor oxygen contamination.

To assess tolerance of elevated perchlorate concentrations cells were grown to stationary phase, centrifuged, and washed as described above. Resuspended cells were aliquoted into 160-ml

23 serum bottles to which either 0.01, 0.1, or 1 M perchlorate was added; each perchlorate treatment was incubated with aerobic (21% oxygen) or anoxic (flushed with nitrogen) headspaces.

Experimental treatments without perchlorate included oxic, no electron acceptor, and an autoclaved kill control. All treatments were performed in triplicate.

Perchlorate concentrations were determined periodically during selected treatments using sub-samples obtained by needle and syringe. Perchlorate concentrations were measured with an ion-selective electrode (Thomas Scientific, Swedesboro, NJ, United States), standardized with perchlorate dissolved in growth medium. Total assay volume was 5 m1 including 500 µ1 of culture and 100 µl of 1 M sodium acetate as an ionic strength adjustment buffer. A 10-fold sample dilution was required due to the high NaCl concentrations in the media.

Chlorate concentrations were monitored via a colorimetric O-tolidine assay (Couture,

1998). O-Tolidine assays contained in a 1-ml final reaction mixture: 4 µl sample, 396 µl deionized water, 100 µl O-tolidine, and 500 µl concentrated (12 M) HCl. After 10 min of incubation, absorbance was read at 448 nm.

Water potentials of growth media were assessed using a WP4-T water potential meter

[Decagon Devices, Pullman, WA, United States (Weber and King, 2009)]. Water potential (with units of pressure, e.g., MPa) is a measure of the chemical potential of water molecules in solutions and varies with temperature and solute concentration and composition (King, 2017).

Pure water has a potential of 0 while solutions have potentials <0 with decreasing values representing increasing physiological water stress. For reference, seawater containing 3.5% NaCl has a water potential of about −2.8 MPa and NaCl saturated brines have potentials about −40

MPa (King, 2015). The water potentials of CM1 media with varied perchlorate concentration were measured to account for variations in water stresses.

Results and Discussion

Perchlorate Linked CO Oxidation by a Denitrifying Extreme Halophile

To assess the feasibility of CO-coupled perchlorate reduction in brines, denitrifying and nitrate-respiring extremely halophilic enrichments and isolates were obtained from the BSF and nearby saline soils (UT, United States). Previous reports have shown that a dissimilatory periplasmic nitrate reductase catalyzes perchlorate, chlorate, and nitrate reduction in the denitrifying extreme halophile, Hfx. mediterranei 33500T, with chlorate reduction rates exceeding those for nitrate (Martinez-Espinosa et al., 2015). However, there has been no evidence to date for dissimilatory perchlorate-reducing extreme halophiles.

Haloarcula sp. PCN7 was selected as a model denitrifier for assays with perchlorate. It oxidizes CO in media with up to 5.2 M NaCl (halite saturation) using molecular oxygen as an electron acceptor; it also oxidizes CO using nitrate during denitrification. It possesses a canonical molybdenum-dependent form I CO dehydrogenase (Appendix Figure A.1), and it contains a nitrate reductase gene as established by PCR amplification of the narGH structural gene and genomic analysis. Sequence and phylogenetic analyses have revealed characteristic motifs for dissimilatory nitrate reductases (Martinez-Espinosa et al., 2007) and a nucleotide identity of

80.3% with the Hfx. mediterranei 33500T narGH gene (Appendix Figure A.2).

Since CO uptake was typically assayed with initial headspace concentrations of about 10 ppm with 10 ml of medium and a headspace of 150 cm3, equivalent to only about 60 nmol total,

CO-coupled perchlorate reduction could not be observed directly by analyses of changes in perchlorate concentrations in hypersaline media. Under the assay conditions, complete CO oxidation would have resulted in a maximum perchlorate decrease of only 1.5 µM out of 0.01 M assuming the following stoichiometries with chlorite as the end-product:

- - CO + C1O4 → CO2 + ClO3

- - CO + ClO3 → CO2 + ClO2

Therefore, coupling of CO oxidation to perchlorate was established by comparing CO uptake in anaerobic assays with no electron acceptors to uptake in aerobic assays (air headspaces), and anaerobic assays with perchlorate only (0.01 M), nitrate only (0.25 or 25 mM), and perchlorate

(0.01 M) with a low concentration of nitrate (0.25 mM) as a nar gene inducer.

Under these conditions, CO uptake occurred at rates of 3.0–6.5 nmol [mg protein]−1 d−1, but only in the presence of oxygen, perchlorate with or without 0.25 mM nitrate, or nitrate at 25 mM; no uptake was observed with 0.25 mM nitrate alone, without electron acceptors or in killed controls (Figures 2.1A, B). In the low nitrate treatment, nitrate was likely unavailable for CO oxidation due to rapid consumption during basal metabolism. Initial CO uptake rates in perchlorate and nitrate treatments were similar, and significantly lower than rates in aerobic assays (Figures 2.1A, B). Although treatments with 0.01 M perchlorate plus 0.25 mM nitrate yielded a small increase in uptake relative to perchlorate only (Figure 2.1A), the effect was not significant and varied among trials. These observations differ from those for Hfx. mediterranei

33500T, which reduced perchlorate and chlorate only after growth in anaerobic media with nitrate (Martinez-Espinosa et al., 2015). This indicates that synthesis of dissimilatory nitrate reductase might be induced by anoxia alone in some denitrifying extreme halophiles as has been observed for some bacteria [e.g., Achromobacter cycloclastes (Coyne and Tiedje, 1990)].

Although nitrate is likely present in Mars’ regolith (Kounaves et al., 2014; Stern et al., 2017), results from Haloarcula sp. PCN7 suggest that it might not be required to initiate perchlorate reduction in brines.

Figure 2.1A.

Figure 2.1. Perchlorate-coupled CO uptake by Haloarcula sp. PCN7. (A) CO uptake rates (nmol CO [mg protein]−1 d−1; solid bars) and uptake thresholds (ppm; cross-hatched bars) for isolate Haloarcula sp. PCN7 during varied oxic and anoxic incubations in 3.8 M NaCl. (B) Headspace CO versus time for isolate Haloarcula sp. PCN7 during varied oxic and anoxic incubations in 3.8 M NaCl; oxic, (○); perchlorate plus nitrate, (■); perchlorate, (□); 25 mM nitrate, (∆); 0.25 mM nitrate, (closed triangle); no electron acceptor, ♦. (C) Perchlorate uptake (circles) and chlorate production (squares) for perchlorate only (open symbols) and perchlorate plus nitrate (closed symbols) during assays with isolate Haloarcula sp. PCN7 in media with 2.5 mM pyruvate. All values are means of triplicate cell suspensions ± 1 standard error. Negative threshold values were not statistically different than zero. (figure cont’d.)

Figure 2.1B.

Figure 2.1C.

Threshold levels for CO uptake did not vary consistently among aerobic, perchlorate, and nitrate treatments (Figure 2.1A). Nonetheless, CO uptake with each of the electron acceptors continued at levels comparable to or less than ambient terrestrial atmospheric values (0.2–0.4 ppm, about 0.02–0.04 Pa), which are far below those in Mars’ atmosphere. This suggests that CO uptake need not be confined to Mars’ surface regolith, but that it could occur in sub-surface horizons that might provide more favorable physical conditions (e.g., lower UV/ionizing radiation exposure).

Results from a separate assay were similar, but by including 2.5 mM pyruvate in the incubation medium as catabolic substrate, it was possible to observe perchlorate reduction and chlorate formation (Figure 2.1C). During the period of maximum CO oxidation, perchlorate decreased by approximately 1 mM, while chlorate increased by about 0.5 mM (Figure 2.1C); chlorite formation from chlorate might have accounted for part or all of the difference. The decrease in perchlorate and increase in chlorate concentrations were greatest in the presence of

0.25 mM nitrate (Figure 2.1C), but perchlorate reduction did not require nitrate, which was consistent with results from CO oxidation. The fact that CO uptake did not occur in the absence of an electron acceptor (e.g., perchlorate or 25 mM nitrate), but occurred while perchlorate was reduced, and chlorate was formed confirmed the potential for CO-coupled perchlorate reduction by extreme halophiles.

Chlorate-Coupled CO Oxidation

Carbon monoxide uptake was also observed with chlorate, an intermediate in the perchlorate reduction pathway (Coates and Achenbach, 2004). CO uptake rates with chlorate were comparable to uptake rates with oxygen and exceeded uptake rates with perchlorate (Figure

2.1A), a pattern consistent with observations of chlorate and perchlorate reduction by Hfx. mediterranei 35000T (Martinez-Espinosa et al., 2015). However, CO uptake threshold values with chlorate were significantly higher than with other oxidants (Figure 2.1A), which indicated that the relative abundance of perchlorate and chlorate could affect CO uptake rates when both co-occur. This possibility was confirmed by comparing results from assays with 9:1 and 1:9 mM perchlorate:chlorate ratios, respectively (Table 2.1 and Appendix Figure A.3). In agreement with prior assays, CO uptake rate constants with 9:1 mM chlorate:perchlorate were greater than uptake rate constants with 1:9 mM perchlorate:chlorate. On the other hand, the addition of 1 mM chlorate to 9 mM perchlorate yielded CO uptake thresholds (1.67 ppm) substantially greater than values observed for perchlorate alone or perchlorate plus 0.25 mM nitrate; thresholds were even higher with 9 mM chlorate plus 1 mM perchlorate (Table 2.1). Nonetheless, all uptake threshold values were lower than ambient atmospheric levels on Mars, and thus would permit surface and sub-surface CO uptake.

Table 2.1. CO uptake rate constants (d-1) and uptake thresholds (ppm) for Haloarcula sp. PCN7. PCN7 incubated under aerobic conditions or with anoxic conditions and varied ratios of perchlorate and chlorate concentrations, concentrations in millimolar. All values are means (±1 standard error) of triplicate determinations. Statistically significant differences based on analysis of variance with a Bonferroni post hoc test are indicated by superscripts.

Treatment Rate constant Threshold Aerobic 0.506 (0.034)a 0.08 (0.05)a 9:1 perchlorate:chlorate 0.389 (0.009)b 1.67 (0.11)b 1:9 perchlorate:chlorate 0.568 (0.023)a 3.75 (0.25)c

Response to Elevated Perchlorate Concentrations

Although CO uptake assays were typically conducted with 0.01M perchlorate, additional assays explored activity at concentrations up to 1 M. No differences in CO uptake rate constants or thresholds were observed when Haloarcula sp. PCN7 was incubated with ambient air and 0–1

M perchlorate in a brine medium with a water potential of −19 MPa (Figure 2.2). Oxic incubations facilitated comparisons with some prior studies. For example, Oren et al. (2014) found no effect of 0.2 M perchlorate on aerobic growth by several archaeal extreme halophiles, but noted partial to substantial inhibition at higher concentrations (0.4–0.6 M). Limited tolerance of 1 M perchlorate has been also reported for several bacterial isolates during aerobic growth assays (Al Soudi et al., 2017), but those assays were conducted at much higher water potentials

(−1.4 to −5.6 MPa, e.g., moderate salt concentrations and less physiological water stress) than used in this study (−19 MPa). Thus, the ability to oxidize CO during substantial water stress (i.e., lower water potentials) and simultaneously high concentrations of a potent chaotrope

[perchlorate (Cray et al., 2013)] is unprecedented.

Figure 2.2. Perchlorate tolerance by Haloarcula sp. PCN7 under oxic conditions. CO uptake by Haloarcula sp. PCN7 during aerobic incubations with no perchlorate (○) or 0.01 (closed circle), 0.1 (□), or 1 M (■) perchlorate. All values are means of triplicate cell suspensions for ±1 standard error.

Haloarcula sp. PCN7 was also able to couple CO uptake with elevated perchlorate concentrations (up to 1 M) under anaerobic conditions. Anaerobic perchlorate tolerance at such

31 high levels has not been previously documented, although 0.05 M perchlorate did not affect CO oxidation by Alkalilimnicola ehrlichii MLHE-1 (King, 2015), which was unable to use it as an electron acceptor. For Haloarcula sp. PCN7 CO uptake rates for 0.01, 0.1, and 1 M treatments did not differ statistically, but uptake rates with 1 M perchlorate were more variable (Figure 2.3) and threshold concentrations were significantly higher (4.9 ± 1.0 ppm) than for 0.01 and 0.1 M perchlorate (not statistically different than zero). Differences in thresholds with 1 M perchlorate could reflect lower water potentials in the assay media (−24.5, −25.4, and −32.6 MPa for 0.01,

0.1, and 1 M, respectively). Collectively, these results indicated that perchlorate was not only tolerated at high concentrations under anaerobic conditions, but that it was exploited as an oxidant. This in turn supports the possibility that high perchlorate concentrations in extraterrestrial brines could sustain microbial life, potentially including biotypes specifically adapted for perchlorate dissimilation.

Figure 2.3. Perchlorate tolerance by Haloarcula sp. PCN7 under anoxic conditions. CO uptake rates (nmol CO [mg protein]−1 d−1; solid bars) and uptake thresholds (ppm; cross-hatched bars) for isolate Haloarcula sp. PCN7 during aerobic and anaerobic incubations in 3.8 M NaCl with varied concentrations of perchlorate; values are means of triplicate cell suspensions ± 1 standard error; negative threshold values were not significantly different than zero.

Perchlorate-CO Coupling by Nitrate-Respiring Extreme Halophiles

Coupling of CO oxidation with perchlorate reduction was confirmed with a second isolate, Halobaculum sp. WSA2. Like Haloarcula sp. PCN7, Halobaculum sp. WSA2 possesses a form I molybdenum-dependent CODH and a dissimilatory nitrate reductase (Appendix Figures

A.1, 2). However, unlike Haloarcula sp. PCN7, Halobaculum sp. WSA2 produces nitrite as a terminal product from nitrate reduction, and does not possess genes for nitrite or nitrous oxide reduction based on initial results of a genome analysis. It oxidized CO under aerobic conditions, or with nitrate, perchlorate, or perchlorate plus nitrate at rates similar to those for Haloarcula sp.

PCN7 and with similar threshold values (Figure 2.4).

Figure 2.4. Perchlorate-coupled CO uptake by Halobaculum sp. WSA2. CO uptake rates (nmol CO [mg protein]−1 d−1) and uptake thresholds (ppm) for isolate Halobaculum sp. WSA2 during aerobic and anaerobic incubations in 3.8 M NaCl with perchlorate, perchlorate + 0.25 mM nitrate, or with 25 mM nitrate; values are means of triplicate cell suspensions ±1 standard error.

The ability of Halobaculum sp. WSA2 to reduce perchlorate is notable, because a nitrate- respiring gammaproteobacterium, A. ehrlichii MLHE-1, could not do so although it readily oxidized CO with nitrate (King, 2015). This might reflect a fundamental difference in the location of the nitrate reductase active site in the narGH enzyme system of haloarchaea versus that of some bacteria (Martinez-Espinosa et al., 2007). In particular, the haloarchaeal narG active site has been described as periplasmic (Martinez-Espinosa et al., 2007), which could result in greater accessibility to perchlorate than a cytoplasmic orientation in bacteria does. Thus, only a sub-set of CO-oxidizing nitrate respirers may reduce perchlorate, and a similar constraint might apply to denitrifiers.

In summary, this study demonstrates for the first time that perchlorate and chlorate can be coupled as electron acceptors to anaerobic CO oxidation in NaCl brines by diverse euryarchaeal extreme halophiles capable of denitrification or nitrate respiration. Under anoxic conditions, perchlorate concentrations from 0.01 to 1 M in 3.8 M NaCl with or without nitrate supported CO oxidation. Oxidation occurred at CO concentrations comparable to those in Mars’ atmosphere and at concentrations comparable to or lower than those in Earth’s atmosphere.

The ubiquity of CO in the cosmos (Dickman, 1978), its presence at relatively high concentrations in the solar system (Elsila et al., 1997; Greaves et al., 2011; Materese et al.,

2015), and the possibility of coupling CO oxidation to diverse electron acceptors (Ragsdale,

2004; King, 2006; King and Weber, 2007) suggest that CO-based metabolism could fuel microbial activity in some of the exoplanetary systems that have been discovered to date.

Evidence for high perchlorate concentrations on Mars and results from this study also indicate that CO could fuel metabolism by either relict or introduced extreme halophiles in hydrated salts or brines (Ojha et al., 2015).

CHAPTER 3.2 ADDENDUM: PERCHLORATE-COUPLED CARBON MONOXIDE (CO) OXIDATION: EVIDENCE FOR A PLAUSIBLE MICROBE-MEDIATED REACTION IN MARTIAN BRINES

Myers and King (2017) described perchlorate-coupled carbon monoxide oxidation by two haloarchaeal isolates that were presumed to be monocultures, Halobaculum sp. WSA2 and

Haloarcula sp. PCN7. Although both isolates had been subjected to a battery of standard microbiological analyses, results from genome sequence analyses indicated that the former isolate was axenic while the latter was mixed. Subsequent purification and characterization efforts yielded an axenic isolate, Halobacterium sp. PCN9 and a purified Haloarcula sp. PCN7.

Halobacterium sp. PCN9 oxidized CO and reduced perchlorate, and was shown to account for the results originally reported by Myers and King (2017).

Halobaculum sp. WSA2 was identified as a nitrate-respiring (denitratating, reducing nitrate to nitrite) CO oxidizer on the basis of a single 16S rRNA gene sequence deposited with

NCBI as MF767889; it was most closely related to Halobaculum roseum D90T (98.19% identity). Haloarcula sp. PCN7, was identified as a denitrifying (respiring nitrate to dinitrogen)

CO oxidizer that yielded two distinct 16S rRNA gene sequences during PCR amplification as described by McDuff et al. (2016). These two sequences (deposited with NCBI as MK490920 and MK490921) were most closely related to the 16S rRNA gene sequence of Haloarcula taiwanensis and Haloarcula japonica (99.37% and 99.45% identity, respectively) based on

BLAST analyses. Since halobacterial genomes often contain multiple 16S rRNA genes (e.g., Cui et al., 2009), the presence of these two sequences was not considered unusual. However,

This chapter previously appeared as Myers, Marisa R., and Gary M. King. "Addendum: Perchlorate-Coupled carbon monoxide (CO) oxidation: evidence for a plausible Microbe- Mediated reaction in Martian brines." Frontiers in Microbiology 10 (2019): 1158. 36 genomic analyses of paired-end DNA sequences generated on an Illumina Miseq using 2 × 250 bp chemistry at Michigan State University subsequent to the work reported by Myers and King

(2017) revealed that the outcome of enriching Haloarcula sp. PCN7 was a mixed culture, while

Halobaculum sp. WSA2 was confirmed to be a pure culture (IMG Metagenome/Genome IDs

3300032142 and 2756170195, respectively). For the mixed culture, two sets of contigs were evident after read assembly and annotation. One set included the two 16S rRNA genes along with numerous other genes that were most closely related to Haloarcula. Notably, these contigs did not contain genes essential for denitrification (e.g., nitric oxide and nitrous oxide reductase genes), or CO oxidation (e.g., form I CO dehydrogenase genes).

A second set of contigs was comprised of genes most closely related to Halobacterium, including a 16S rRNA gene that was 98.66% identical to Halobacterium noricense (available as

Ga0334598_104419 from IMG metagenome ID 3300032142). In addition, the Halobacterium contigs contained genes essential for CO oxidation (e.g., coxL available as Ga0334598_11585 from IMG metagenome ID 3300032142).

Additional transfers of the original mixed culture ultimately yielded two distinct isolates.

Haloarcula sp. PCN7 was obtained as a monoculture that was morphologically indistinguishable from the original mixed culture, but neither oxidized CO nor denitrified, a finding consistent with results from genome sequence annotation. Absence of Halobacterium from this derivative of the original mixed culture was supported by results from a PCR analysis (Figure 3.1) using primers designed for the Halobacterium 16S rRNA gene sequence identified by genome analysis

(HalobacF: 5′ - ATGCTAGTTGTGCGGGTTCA−3 ′ and reverse primer HalobacR:. 5′ -

CGAGCAGATTTCCCAACGGA−3 ′). PCR with these primers was positive for the original mixed culture, positive for a Halobacterium isolate derived from it, and negative for the

Haloarcula sp. PCN7 monoculture (Figure 3.1). Haloarcula sp. PCN7 has been deposited as an axenic culture with the Deutsche Sammlung von Mikroorganismen und Zellculturen (DSM

103645).

Figure 3.1. Results of PCR using Halobacterium-specific primers based on the 16S rRNA gene sequence of Halobacterium sp. PCN9. Lane 1: l00 bp ladder; 2: negative control; 3: Halobacterium sp. PCN9 monoculture; 4: Haloarcula sp. PCN7 monoculture; 5: Haloarcula sp. PCN7 mixed culture as used in original analyses of CO-coupled perchlorate reduction (Myers and King, 2017).

The second isolate in the original mixed culture was identified as a member of the genus

Halobacterium and designated as Halobacterium sp. PCN9. This isolate has been deposited with the American Type Culture Collection as TSD-126 and with the Belgian Coordinated Collection of Microorganisms as LMG 31022. The 16S rRNA sequence for Halobacterium sp. PCN9 was identical to the Halobacterium sequence found during genomic analysis of the original mixed

Haloarcula sp. PCN7 culture. The genome of Halobacterium sp. PCN9 also harbored genes for

CO oxidation as indicated by successful amplification of the coxL gene as described by McDuff

38 et al. (2016); the sequence for this gene (Genbank accession MK262901) was identical to that observed in the mixed culture.

Halobacterium sp. PCN9 oxidized CO under oxic conditions as described by Myers and

King (2017) in contrast to the Haloarcula sp. PCN7 monoculture (Figure 3.2A). CO uptake assays were also conducted with 10 mM perchlorate as an electron acceptor under anoxic conditions as described by Myers and King (2017). Halobacterium sp. PCN9 reduced perchlorate to chlorate (Figure 3.3) while simultaneously oxidizing CO (Figure 3.2B) as described in Myers and King (2017). Although the Haloarcula sp. PCN7 monoculture could not oxidize CO (Figure 3.2A), a 1:1 mixture (based on A600) of Halobacterium sp. PCN9 and the

Haloarcula sp. PCN7 monoculture oxidized CO, reduced perchlorate, and produced chlorate as reported. (Figures 3.2B, 3.3).

Figure 3.2A.

Figure 3.2B.

Figure 3.2. (A) CO uptake under oxic conditions by monocultures of Halobacterium sp. PCN9 and Haloarcula sp. PCN7, and a 1:1 mixed culture of both. (B) CO uptake under anoxic conditions by monocultures of Halobacterium sp. PCN9 and Haloarcula sp. PCN7, and a 1:1 mixed culture of both with and without perchlorate. Assays as described by Myers and King (2017).

Figure 3.3. Perchlorate reduction (open symbols) and chlorate accumulation (closed symbols) for a monoculture of Halobacterium sp. PCN9 and a 1:1 mixed culture of Halobacterium sp. PCN9 and Haloarcula sp. PCN7 incubated anaerobically with conditions and assays as described by Myers and King (2017).

These results confirmed observations by Myers and King (2017) that extreme halophiles can couple CO oxidation to perchlorate reduction, but they revealed that a member of the genus

Halobacterium (Halobacterium sp. PCN9) rather Haloarcula sp. PCN7 was responsible for the reported activity. In addition, analysis of the Halobaculum sp. WSA2 genome sequence confirmed that it was a monoculture, and thus responsible for CO-coupled perchlorate reduction as described by Myers and King (2017).

CHAPTER 4.3 HALOBACTERIUM BONNEVILLEI SP. NOV., HALOBACULUM SALITERRAE SP. NOV. AND HALOVENUS CARBOXIDIVORANS SP. NOV., THREE NOVEL CARBON MONOXIDE- OXIDIZING HALOBACTERIA FROM SALINE CRUSTS AND SOILS

Introduction

Extremely halophilic archaea, phylum Euryarchaeota, class Halobacteria, occur ubiquitously in anthropogenic and natural saline and hypersaline environments (Oren, 2015;

Ventosa et al., 2015; Mora-Ruiz et al., 2018). At the time of this writing at least 65 genera have been validly published, with the overwhelming majority of halobacteria described as aerobic or facultatively anaerobic heterotrophs. Genomic and cultivation-based analyses have shown that many of these halobacteria harbor genes for unexpected traits, e.g., the ability to oxidize carbon monoxide (CO) using the form I molybdenum-dependent CO dehydrogenase (Mo-CODH; King and Weber, 2007). Globally, approximately 10% of annual CO emissions are consumed by microbial species in soils, with the capacity haloarchaeal isolates to oxidize CO only recently discovered (Khalil, et al., 1999; King, 1999; McDuff et al., 2016). Several isolates obtained from the Bonneville Salt Flats (BSF; Utah, USA) that contain these genes have been shown to couple

CO oxidation to perchlorate reduction in addition to molecular oxygen and nitrate reduction

(Myers and King, 2017, 2019).

The Bonneville Salt Flats, located in the western part of the Great Salt Lake Desert, is a large playa known for its salt crust, (Lines, 1979; Jewell et. al 2016; Bowen et. al 2017). A

This chapter previously appeared as Myers, Marisa R., and G. M. King. "Halobacterium bonnevillei sp. nov., Halobaculum saliterrae sp. nov. and Halovenus carboxidivorans sp. nov., three novel carbon monoxide-oxidizing Halobacteria from saline crusts and soils." International journal of systematic and evolutionary microbiology 70.7 (2020): 4261-4268.

42 remnant of Pleistocene Lake Bonneville, it supports a predominantly halite crust, which undergoes partial dissolution during winter and recrystallization during summer. We report here the isolation of three CO-oxidizing haloarchaeal species obtained during an investigation of BSF and neighboring saline soils. Halobacterium PCN9T was isolated from a BSF salt crust, while strains Halobaculum WSA2T and Halovenus WSH3T were isolated from nearby saline soils.

These taxa were recognized as novel extreme halophiles based on their 16S rRNA gene sequences and other distinct traits (Oren et al., 1997; Oren, 2008).

Salt crusts and saline soils were collected from BSF (40.757333, -113.886417) and a site south of Wendover, UT (40.515778, -114.045056) during July 2015. Enrichments were performed by transferring approximately 0.5 g fresh weight (gfw) of crusts or soils into 60-mL serum bottles containing 5 mL of CM1 medium with 3.8 M NaCl and 25 mM pyruvate (Dyall-

Smith, 2008; McDuff et al., 2016); the bottles were then sealed with neoprene rubber stoppers.

Bottle headspaces contained either air (oxic conditions), or oxygen-free nitrogen (anoxic conditions) to which CO was added at a final concentration of 100 ppm. Potassium nitrate was added at a final concentration of 10 mM to anoxic bottles. Bottles were then incubated at 40oC or

60oC. Headspace CO was monitored at intervals as previously described (McDuff et al., 2016).

Sub-samples from enrichments that oxidized CO were used to inoculate fresh 3.8 M NaCl CM1- pyruvate media; this process was repeated three times prior to spreading serial dilutions onto 3.8

M NaCl CM1-pyruvate agar plates. Plates were incubated at 40oC or 55oC corresponding to the initial enrichment incubation temperature. After approximately 7 d, morphologically distinct colonies were selected and used to inoculate 60-mL serum bottles containing 5 mL 3.8 M NaCl

CM1-pyruvate media with air headspaces. Cultures were screened for CO oxidation as before; positive cultures were re-plated onto 3.8 M NaCl CM1-pyruvate plates as needed to obtain pure

43 isolates. Three novel isolates PCN9T, WSA2 T, and WSH3T were selected for detailed characterization.

Two additional strains, Halobaculum roseum D90T and Halobacterium noricense A1T, that were close phylogenetic neighbors of WSA2 T and PCN9T, respectively, were obtained from the Japan Collection of Microorganisms (JCM) and German Collection of Microorganisms and

Cell Cultures (DSMZ) respectively. Both strains were grown in 3.8 M NaCl CM1-pyruvate. CO uptake potential was assessed by adding 10 ppm CO to 5 mL of culture in 60 mL serum bottles containing an aerobic headspace and monitoring as previously described. Neither isolate D90T nor A1T oxidized exogenous CO.

Genomic DNA was extracted from strains PCN9T, WSA2 T, and WSH3T using a MoBio

UltraClean DNA extraction kit following the manufacturer’s instructions (Folsom, CA).

Genomic DNA extracts were used to amplify 16S rRNA genes using archaeal forward primer

Arch21F (5’-TTCCGGTTGATCCYGCCGGA-3’; Miyashita et al., 2009; DeLong et al., 1992) and universal reverse primer 1492R (5’-CGGTTACCTTGTTACGACTT-3’; Lane, 1991).

Amplicons purified with a MoBio UltraClean PCR Clean-up kit were sequenced using an ABI

3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA) at the Louisiana State

University Genomics Facility (Baton Rouge, Louisiana, USA). The resulting bidirectional sequence reads were assembled and edited using Sequencher 4.8 (Gene Codes Corporation, Ann

Arbor, MI, USA). The 16S rRNA gene sequences have been deposited in Genbank as accessions

MK262901, MF767880, and MN128599 for Halobacterium sp. PCN9 T, Halobaculum sp.

WSA2 T, and Halovenus sp. WSH3 T respectively. Based on 16S rRNA gene sequence analysis in EZBiocloud (Yoon et al., 2017a), Halobacterium sp. PCN9T, Halobaculum sp. WSA2 T, and

Halovenus sp. WSH3 T are most closely related to, Halobacterium noricense A1T, Halobaculum

44 roseum D90T, and Halovenus aranensis EB27T with gene sequence identities of 98.71%,

98.19%, and 95.95% respectively (Gruber et al., 2004; Chen et al., 2017; Makhdoumi-Kakhki et al., 2012). All sequence identities are at or below the accepted cutoff for 16S rRNA gene-based species differentiation of 98.7%-99.1% (Yarza et al., 2014). Phylogenetic relationships of the novel isolates and close relatives were determined using a maximum likelihood analysis in

MEGAX (Figure 4.1; Nei and Kumar, 2000; Kumar et al., 2018; Stecher et al., 2020).

Additionally, maximum parsimony and neighbor-joining analyses were performed which support maximum likelihood analysis (Appendix Figures B.2, 3).

Figure 4.1. Evolutionary analysis by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method and General Time Reversible model. The tree with the highest log likelihood (-4477.23) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.2043)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 67.09% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 19 nucleotide sequences. All positions containing gaps and missing data were eliminated (complete deletion option). There were a total of 1108 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.

The large sub-unit gene for CO dehydrogenase (coxL) was amplified using primers

ArchcoxF (5’-GGYGGSTTYGGSAASAAGGT-3’) and PSr (5’-

YTCGAYGATCATCGGRTTGA-3’). The PCR reaction mixture consisted of 0.2 µL Hotmaster

Taq DNA Polymerase (Quantabio, Beverly, MA, USA), 34.8 µL nuclease-free H2O, 5 µL

Hotmaster Taq Buffer, 3 µL DMSO, 2 µL dNTPs, 2 µL ArchcoxF primer, 2 µL PSr primer, and

1 µL template. Thermocycler conditions included: initial denaturation at 94oC for 3 min, followed by 35 cycles of denaturation at 94oC 45 sec, annealing at 58oC 1 min, and elongation at

72oC 2 min, followed by a final elongation at 68oC for 10 min. Amplicons were purified and sequenced as above. CoxL sequences have been deposited in Genbank as accessions MN136860,

MF773972, and MN136859 or Halobacterium sp. PCN9 T, Halobaculum sp. WSA2 T, and

Halovenus sp. WSH3 T respectively.

Genomic analyses for WSA2T and WSH3T were conducted using paired-end DNA sequences generated on an Illumina Miseq with 2 x 250 bp chemistry at Michigan State

University. Genomes for Halobacterium noricense A1T and Halobaculum roseum D90T were sequenced similarly, but with Illumina Miseq and 2 x 150 bp chemistry. Genomic analysis for

PCN9T was performed using an Ion Torrent S5 with a 530 chip at Louisiana State University. A total of 8,132,138; 1,402,189; 1,345,632; 1,707,515; and 1,635,149 reads were generated for

PCN9T, WSA2T, WSH3T, D90T, and A1T, respectively. Reads were assembled using a5_miseq_macOS_20140604 (Coil, Jospin, Darling, 2014) for WSA2T, WSH3T, D90T, and A1T, and SPAdes-3.10.0-Darwin for PCN9T (Bankevich et al., 2012). Assemblies were submitted to the Integrated Microbial Genomes Microbial Annotation Pipeline (IMG) for annotation

(Markowitz et al., 2013). Genomes for PCN9T, WSA2T, and WSH3T are available within the

Genomes OnLine database (GOLD) under study ID Gs0116871 as projects Gp0375023,

Gp0252472, and Gp0252468 respectively (Mukherjee et al., 2018). Additionally, Whole

Genome Shotgun projects have been deposited at DDBJ/ENA/GenBank under the accessions

WUUU00000000, WUUS00000000, WUUT00000000 for PCN9T, WSA2T, and WSH3T

47 respectively. The versions described in this paper are versions WUUU01000000,

WUUS01000000, WUUT01000000.

The PCN9T genome was composed of 3,006,633 bp with 85.62% coding DNA and 4,003 genes of which 3,952 (98.73%) were protein coding. There were 2,576 (64.35%) protein coding genes 808 of which were associated with KEGG pathways, 1,949 with COGs, and 620 with

MetaCyc pathways. Additionally, one CRISPR, one 16S rRNA gene, and 44 tRNA genes were identified. The WSA2T genome was composed of 3,775,766 bp with 87.20% coding DNA and

3,800 genes of which 3,737 (98.34%) were protein coding, with 855 associated with KEGG pathways, 2,062 with COGs, and 681 with MetaCyc pathways. Two CRISPR, two distinct 16S rRNA genes, and 51 tRNAs were identified. The WSH3T genome was composed of 3,245,916 bp with 90.17% coding DNA and 3,337 genes of which 3,288 (98.53%) are protein coding with 836 associated with KEGG pathways, 1,926 with COGs, and 676 with MetaCyc pathways. One 16S rRNA gene, and 44 tRNA were identified, but no CRISPR were found. Each of the genomes harbored a cox operon comprised of the canonical form I structural genes for the molybdenum- dependent CO dehydrogenase, coxM, coxS, coxL, along with several accessory genes (e.g., King and Weber, 2007).

Genome sequences for D90T and A1T were used with sequences for WSA2T and PCN9T, respectively, to conduct digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses using the genome-to-genome distance calculator for dDDH

(https://ggdc.dsmz.de/; Meier-Koltoff et al., 2013), and the EZBiocloud ANI calculator (Yoon et al., 2017b; https://www.ezbiocloud.net/tools/ani). Genome comparisons of WSA2T with

Halobaculum roseum D90T yielded dDDH and ANI values of 31.1% and 86.34%, respectively; comparisons of PCN9T with Halobacterium noricense A1T yielded dDDH and ANI values of

25.7% and 82.00%, respectively. The ANI for WSH3T and Halovenus aranensis EB27T, 75.2%, was determined using the Pairwise ANI application of Integrated Microbial Genomes and

Microbiomes (img.jgi.doe.gov). The ANI value for WSH3T and dDDH and ANI values for both

WSA2T and PCN9T were consistent with species level differentiation from their closest phylogenetic neighbors.

Cell and colony characteristics were determined after growth for 6 d at 40oC (PCN9T and

WSA2T) or 55oC (WSH3T) on 3.8 M NaCl CM1-pyruvate medium. For Gram-staining, a modified procedure was used that consisted of a rinse of CM1 containing 1 M NaCl instead of deionized water to prevent cell lysis. All isolates stained Gram-negative. Cell morphology and dimensions were determined using a Zeiss Axioscope and an AxioCam MR digital camera. Cells occurred singly with the following dimensions: PCN9T, 2.15 ± 0.31 μM x 0.70 ± μM; WSA2T

2.33 ± 0.71 μM x 0.67 ± 0.15 μM; and WSH3T, 2.28 ± 0.18 μM x 0.80 ± 0.18 μM. Cells were additionally imaged at LSU’s Shared Instrument Facility with a JEOL 6610LV scanning electron microscope (Figure 4.2a, 4.2b, 4.2c). For SEM imaging, cells were collected by filtration and fixed on a 0.4μm polycarbonate filter in 2% glutaraldehyde, 2% formaldehyde, 1% OsO4 for 30 min. Cells were then rinsed with distilled water for 10 min (x3), dehydrated with a graded ethanol series, and dried with a graded HMDS series (hexamethyldisilazane reagent

EMS#16700). The filter paper was mounted on aluminum specimen stubs and coated with platinum in an EMS550X Sputter Coater. Both PCN9T and WSA2T formed round, smooth, convex, shiny, opaque, pink-pigmented colonies, while WSH3T formed small round, smooth, convex, shiny, opaque, red-pigmented colonies.

Figure 4.2A.

Figure 4.2B.

Figure 4.2C.

Figure 4.2. Scanning electron microscopy images of, (a) Halobacterium bonnevillei PCN9T; (b) Halobaculum saliterrae WSA2T; (c) Halovenus carboxidivorans WSH3T.

All physiological and biochemical assays were conducted with media based on 3.8 M

NaCl CM1-pyruvate, and growth was measured by monitoring absorbance at 600 nm in triplicate

50 unless otherwise noted. Temperature ranges and optima were assessed at intervals from 15oC to

65oC for 14 d. PCN9T grew optimally at 40oC, while WSA2T and WSH3T grew optimally at

50oC with ranges of 15oC to 55oC (PCN9T), 25oC to 60oC (WSA2T), and 30oC to 60oC (WSH3T).

The ranges and optimal NaCl concentrations for growth were determined using CM1-pyruvate based media with NaCl concentrations adjusted to 0.5 M, 1 M, 1.5 M, 2 M, 3 M, 4 M, and 5.4 M; growth was measured for 14 d. PCN9T and WSA2T grew optimally at 3 M with a range of 2 M to

5.4 M, WSH3T grew optimally at 4 M with a range of 3 M to 5.4 M (Table 4.1).

The minimum concentration of added magnesium (Mg2+) required for growth was assessed using a modified 3.8 M NaCl CM1 medium in which magnesium sulfate was replaced with sodium sulfate, and magnesium chloride was replaced with sodium chloride. Magnesium chloride was then added to final concentrations of 0 mM, 1 mM, 5 mM, and 10 mM; growth was monitored for 14 d. PCN9T and WSH3T do not require added Mg2+ for growth, while WSA2T required addition of 1 mM (Table 4.1).

The pH range for growth was assessed from pH 5.0 to 9.0 with 0.5-unit intervals. pH was adjusted using the following buffers at 50 mM: MES (5.0, 6.0, 6.5), HEPES (7.0, 7.5, 8.0),

MOPS (7.0), Tricine (7.5, 8.0), TAPS (8.0), CHES (8.5, 9.0). The magnesium concentration in test media was reduced to 10 mM, and media were filter sterilized to avoid precipitation.

Following incubation at 40oC for PCN9T and WSA2T or 50oC for WSH3T the pH of all buffered media shifted so that final pH was closer to the optima (Appendix Table B.1). pH optima and growth ranges were determined based on initial pH of the media. PCN9T and WSH3T grew optimally between 7 and 8, with a range of 6.5 to 8.5. WSA2T grew optimally between 6.5 and

8.0, with a range of 6.0 to 8.5 (Table 4.1). All grew aerobically or weakly under anoxic conditions with nitrate and some cells exhibited motility in early exponential phase under oxic

51 conditions. PCN9T and WSA2T were also capable of reducing perchlorate to chlorite under anoxic conditions as well as oxidizing carbon monoxide as described by Myers and King (2017,

2019). Halovenus sp. WSH3T was also shown capable of carbon monoxide oxidation under aerobic conditions (Appendix Figure B.1).

Substrate utilization was assessed with single carbon sources at a final concentration of

25 mM in 3.8 M NaCl CM1 medium. Cells used for inoculating media were harvested by centrifugation (10 min, 4oC, 10,000 x g), washed twice and re-suspended in substrate-free CM1; growth was monitored spectrophotometrically (OD600) for 14 d. Substrates included malic acid, glycine, sodium glycolate, glucuronic acid, gluconic acid, pyruvic acid, succinic acid, proline, mannitol, alanine, malonic acid, sodium citrate, aspartic acid, valine, serine, sodium acetate, propionic acid, sodium tartrate, glycine betaine, sodium fumarate, trimethylamine, dimethylamine, methylamine, sodium lactate, glycerol, acetone, ethanol, isopropanol, methanol, glucose, galactose, lactose, sucrose, ribose, mannose, arabinose, xylose, fructose, peptone, casamino acids, and tryptone. PCN9T grew with the following sole carbon sources: pyruvic acid, proline, alanine, sodium acetate, glycerol, glucose, mannose, and tryptone. WSA2T grew with gluconic acid, succinic acid, pyruvic acid, proline, alanine, sodium acetate, sodium fumarate, sodium lactate, glucose, tryptone, and peptone. WSH3T grew with pyruvic acid, mannitol, sodium acetate, and sodium lactate (Table 4.1).

Catalase activity was determined by combining a colony and hydrogen peroxide and observing formation of oxygen bubbles. Oxidase activity was determined by adding oxidase reagent onto colonies grown on 3.8 M NaCl CM1-pyruvate agar plates. WSA2T and WSH3T were positive for catalase while PCN9T was negative. PCN9T, WSA2T and WSH3T were positive for oxidase activity. PCN9T produced indole from tryptophan, while WSA2T and WSH3T did not.

Hydrolysis of starch (0.2%), gelatin (1%), casein (0.1%), tween 80 (0.1%) and tween 20 (0.1%) were determined by supplementing plated CM1 media. PCN9T could hydrolyze gelatin. WSA2T could hydrolyze starch and gelatin. WSH3T could hydrolyze Tween 20 and Tween 80. None of the isolates hydrolyzed casein or grew under anoxic conditions with arginine as an electron acceptor (Table 4.1).

Polar lipids for all strains were determined commercially by the Identification Service of

DSMZ (Braunschweig, Germany). All assays followed standard methods (Bligh and Dyer, 1959;

Tindall, 1990a, Tindall et al., 2007). Major polar lipids of PCN9T include: glycolipid (GL), phospholipid (PL), phosphatidylglycerol (PG), and phosphatidylglycerol phosphate methylester

(Me-PGP). Major polar lipids of WSA2T include: glycolipid (GL), phospholipid (PL), phosphatidylglycerol (PG), phosphatidylglycerol sulfate (PGS), phosphatidylglycerol phosphate methylester (Me-PGP). Major polar lipids of WSH3T include: phosphatidylglycerol (PG), phosphatidylglycerol sulfate (PGS), and phosphatidylglycerol phosphate methylester (Me-PGP).

Differences when compared to their closest relatives include: the additional presence of phosphatidylglycerol sulfate (PGS), triglycosyl diether, and sulfated tetraglycosyl diether in

Halobacterium noricense; the absence of phosphatidylglycerol sulfate (PGS) in both

Halobaculum roseum and Halovenus aranensis.

The DNA base composition (mol% G+C) for all strains was calculated from genome sequence data. Values of 66.75 mol%, 67.62 mol%, and 63.97 mol% were obtained for PCN9T,

WSA2T, and WSH3T, respectively (Table 4.1).

Table 4.1. Differential characteristics of strains PCN7T, WSA2T, and WSH3T with closest related species identified by 16S rRNA gene similarity. Taxa: 1, PCN9T; 2, Halobacterium noricense A1T; 3, WSA2T; 4, Halobaculum roseum D90T; 5, WSH3T. +, positive; -, negative; ND, not determined Characteristic 1 2 3 4 5 Cell shape Rods Rods Rods Rods Rods Colony color Pink Red Pink Pink Red Temperature (oC) Optimum 40 45 50 40 50 Range 15-55 28- 50 25 – 60 25- 50 30 - 60 pH Optimum 7 - 8 5.2 – 7.0 6.5 – 8.0 7 7 - 8 Range 6.5 – 8.5 5.1 – 8.8 6.0 – 8.5 6.5 – 8.0 6.5 – 8.5 NaCl (M) Optimum 3 3 3 3.4 4 Range 2 – 5.4 2.1 - ND 2 – 5.4 1.7-5.1 3.0 – 5.4 Catalase - + + + + Oxidase + - + - + Denitrification - - - - - Nitrate + - + + - reduction to nitrite CO oxidation + - + - + DNA G+C 66.75 54.3 – 54.5 67.62 65.9 63.97 (mol%) Hydrolysis of: Tween 20 - - - - + Tween 80 - - - - + Gelatin + - + - - Starch - - + - - Growth Substrates Gluconic acid - + + - - Pyruvic acid + + + + + Succinic acid - + + + - Proline + - + - - Mannitol - - - + + Alanine + - + - - Sodium citrate - + - - - Sodium acetate + + + - + Sodium - + + + - fumarate Sodium lactate - - + - + (table cont’d)

Characteristic 1 2 3 4 5 Glycerol + + - + - Glucose + - + + - Galactose - - - + - Lactose - - - - - Sucrose - - - + - Ribose - - - - - Mannose + - - + - Xylose - - - - - Fructose - + - + - Tryptone - + + + - Peptone - + + + - Casamino - + - - - acids

Description of Halobacterium bonnevillei sp. nov.

Halobacterium bonnevillei (bon.ne.vil’le.i N.L. gen. n. bonnevillei of Bonneville, honors Major

Benjamin Bonneville, for whom Bonneville Salt Flats is named.

Cells are Gram-stain-negative, sometimes motile rods of varying length. When grown on solid media colonies are round, smooth, convex, shiny, with opaque pink pigmentation. Growth occurs between 2 M and 5.4 M NaCl (optimum at 3 M); between 15oC and 55oC (optimum

40oC); and pH between 6.5 and 8.5 (optimum at pH 7-8). Added magnesium is not required.

Cells can reduce nitrate to nitrite and reduce perchlorate to chlorite. Cells are positive for gelatin hydrolysis and oxidase activity. Capable of CO oxidation; possesses a form I large sub-unit CO dehydrogenase gene (coxL) and a canonical form I cox operon. Utilizes the following substrates as sole carbon sources for growth: pyruvic acid, proline, alanine, sodium acetate, glycerol, glucose, and mannose. Substrates not utilized for growth include: malic acid, glycine, sodium glycolate, glucuronic acid, gluconic acid, succinic acid, mannitol, malonic acid, sodium citrate, aspartic acid, valine, serine, propionic acid, sodium tartrate, sodium lactate, glycine betaine, sodium fumarate, trimethylamine, dimethylamine, methylamine, acetone, ethanol, isopropanol,

55 methanol, sucrose, fructose, galactose, lactose, ribose, arabinose, xylose, peptone and casamino acids. The major polar lipids include: glycolipid (GL), phospholipid (PL), phosphatidylglycerol

(PG), and phosphatidylglycerol phosphate methylester (Me-PGP). The DNA G+C content is

66.75 mol%. The type strain is PCN9T (= JCM 323472, = LMG 31022, =ATCC TSD-126) isolated from a salt crust of the Bonneville Salt Flats (UT, USA).

Description of Halobaculum saliterrae sp. nov

Halobaculum saliterrae (sa.li.ter’rae. L. masc. n. sal salt; L. fem. n. terrae land; N.L. gen. n. saliterrae of saline soil).

Cells are Gram-negative, non-motile, pleomorphic rods. When grown on solid media colonies are round, smooth, convex, shiny, with opaque pink pigmentation. Growth occurs between 2 M and 5.4 M NaCl (optimum at 3 M); between 25oC and 60oC (optimum at 50oC); between pH 6.0 to 8.5 (optimally between 6.5 and 8.0). Cells require a minimum addition of

1mM Mg2+, and can reduce nitrate to nitrite. Cells are weakly positive for catalase and oxidase.

Capable of starch and gelatin hydrolysis. Cells are able to reduce nitrate to nitrite via dissimilatory nitrate reductase, as well as reduce perchlorate to chlorite. Capable of carbon- monoxide oxidation and possesses a form I large sub-unit CO dehydrogenase gene (coxL) and a canonical form I cox operon. Can utilize the following substrates as carbon sources for growth: gluconic acid, succinic acid, pyruvic acid, proline, alanine, sodium acetate, sodium fumarate, sodium lactate, glucose, tryptone, and peptone. Substrates not utilized for growth include: malic acid, glycine, sodium glycolate, glucuronic acid, mannitol, malonic acid, sodium citrate, aspartic acid, valine, serine, propionic acid, sodium tartrate, glycine betaine, trimethylamine, dimethylamine, methylamine, glycerol, acetone, ethanol, isopropanol, methanol, galactose, lactose, sucrose, ribose, mannose, arabinose, xylose, fructose, and casamino acids. The major

56 polar lipids include: glycolipid (GL), phospholipid (PL), phosphatidylglycerol (PG), phosphatidylglycerol sulfate (PGS), phosphatidylglycerol phosphate methylester (Me-PGP). The

DNA G+C content is 67.62 mol%. The type strain is WSA2T (=JCM 32473T, =ATCC TSD-

127T), isolated from saline soil south of Wendover, Utah, USA.

Description of Halovenus carboxidivorans sp. nov

Halovenus carboxidivorans (car.bo.xi.di.vo’rans. N.L. neut n. carboxidum carbon monoxide; L. pres. part. adj. vorans devouring; N.L. part. adj. carboxidivorans carbon monoxide-devouring).

Cells are Gram-negative, non-motile, pleomorphic rods. When grown on solid media colonies are round, smooth, convex, shiny, with opaque red pigmentation. Growth occurs between 3 M – 5.4 M NaCl (optimum at 4 M); between 30oC – 60oC (optimum at 50oC); and pH

6.5 to 8.5 (optimally between 7 and 8). Added magnesium is not required for growth. Cells are positive for catalase and oxidase. Nitrate is reduced to nitrite. Hydrolysis of tween 20 and tween

80. Capable of CO oxidation, and possesses a form I large sub-unit CO dehydrogenase gene

(coxL) and a canonical form I cox operon. Can utilize the following substrates as carbon sources for growth: pyruvic acid, mannitol, sodium acetate, and sodium lactate. Substrates not utilized for growth include: gluconic acid, succinic acid, proline, alanine, sodium fumarate, glucose, tryptone, peptone, malic acid, glycine, sodium glycolate, glucuronic acid, malonic acid, sodium citrate, aspartic acid, valine, serine, propionic acid, sodium tartrate, glycine betaine, trimethylamine, dimethylamine, methylamine, glycerol, acetone, ethanol, isopropanol, methanol, galactose, lactose, sucrose, ribose, mannose, arabinose, xylose, fructose, and casamino acids The major polar lipids include: phosphatidylglycerol (PG), phosphatidylglycerol sulfate (PGS), and phosphatidylglycerol phosphate methylester (Me-PGP). The DNA G+C content is 63.97 mol%.

The type strain is WSH3T (= JCM 32474T, = ATCC TSD-128T) isolated from saline soil south of

Wendover, Utah, USA.

CHAPTER 5. HALANAEROARCHAEUM OXYRESPIRANS, SP. NOV., A NOVEL OXYGEN-RESPIRING, PERCHLORATE-REDUCING EXTREME HALOPHILE

Introduction

The extremely halophilic class Haloarchaea is comprised of a unique group of microorganisms that dominant terrestrial, marine, and anthropogenic hypersaline environments.

They have been classified predominantly as aerobic heterotrophs with relatively few examples of facultative anaerobes (Oren and Trüper, 1990; Oren, 1991; Antunes et al., 2008; Bonete et al.,

2008). Until recently, obligately anaerobic haloarchaea were unknown, as were their potential roles in anoxic hypersaline habitats worldwide. This changed with the discovery of obligate anaerobic haloarchaea capable of anaerobic dissimilatory sulfur respiration, Halanaeroarchaeum sulfurireducens and Halodesulfurarchaeum formicium (Sorokin et al., 2016; 2017).

Halanaeroarchaeum sulfurireducens HSR2T, the first obligately anaerobic haloarchaeon, grows via elemental sulfur respiration with either pyruvate or acetate as the electron donor/carbon source. Halodesulfurarchaeum formicium HSR6T, the first example of a haloarchaeon with lithotrophic metabolism, is capable of growth using elemental sulfur, DMSO, or thiosulfate

(some strains) as electron acceptors coupled to oxidation of formate or hydrogen (some strains).

Genomic analyses of these species have revealed the crucial absence of cytochrome c oxidase, further supporting their strictly anaerobic lifestyle. A group of facultatively anaerobic natronoarchaea cultivated from hypersaline sediments comprise the third group capable of anaerobic dissimilatory sulfur respiration, containing the genera Halalkaliarchaeum and

Natrachaeobaculum with more versatile sulfur respiration metabolic properties including use of formate/H2, C4-C9 fatty acids, and peptone (Sorokin et al., 2018, 2019). Recently, a fourth group of sulfur-reducing haloarchaea, the halo(natrono)archaea containing provisional genus 59

Halarchaeoglobus, were discovered to utilize various carbohydrates as alternative electron donors including alpha-glucans, oligo- and monomeric sugars, and glycerol while sulfur, thiosulfate or DMSO were used as electron acceptors (Sorokin et al., 2021). Upon these groups’ recent discoveries, it has been suggested that the physiology and ecological role of haloarchaea role as aerobic organoheterotrophs be revised as their role in anoxic sediments continues to expand (Sorokin et al., 2021).

- Often overlooked for its role as an electron acceptor, perchlorate (ClO4 ) can be reduced by a phylogenetically diverse array of microorganisms in an anaerobic process termed microbial dissimilatory perchlorate reduction (DPRM). While natural sources of perchlorate are uncommonly found, sources are geographically widespread with the highest natural levels found within the Atacama Desert (Chile) in terrestrial nitrate deposits averaging ~0.03 wt %.

Extraterrestrial environments, such as Martian regolith, have also been discovered to contain elevated levels of perchlorate first measured by the Phoenix lander in 2008 at ~0.6 wt % (Hecht et al., 2009; Glavin et al., 2013). Notably, perchlorate on Mars also has the potential to lead to transient hypersaline brines, thereby playing a large role in the limited Martian hydrological cycle.

Though DPRM has been documented in both bacteria and archaea, bacteria typically utilize canonical perchlorate reduction by means of a dissimilatory perchlorate reductase (pcr) and a chlorite dismutase (cld), which reduces perchlorate to molecular oxygen and chloride.

Archaea and some bacteria have been shown to reduce perchlorate through alternative mechanisms, such as cryptic perchlorate reduction (Youngblut et al., 2016). Cryptic perchlorate reducers contain a closely related periplasmic Nar-like molybdopterin nitrate reductase (pNar) as an alternative to pcr, however they lack a functional chlorite dismutase thus forcing them to

60 depend on chlorite sinks for abiotic removal. Recently, haloarchaea such as Haloferax mediterranei, Halobacterium bonnevillei, and Halobaculum saliterrae have also been shown to use perchlorate at concentration up to 1 M, in addition to some species being able to couple perchlorate reduction to carbon monoxide oxidation (Oren et al., 2014; Martínez-Espinosa et al.,

2015; Myers and King, 2017, 2019, 2020). Current examples of haloarchaeal DPRM are nitrate dependent, meaning for pNar induction the cells must either be grown in the presence of small amounts of nitrate in conjunction with perchlorate (H. bonnevillei, and H. saliterrae), or culture inoculum must be previously grown on nitrate after which perchlorate-reducing capabilities diminish with subsequent transfers (H. mediterranei) (Martinez-Espinosa et al., 2015; Myers and

King 2017; 2019; 2020).

This study has identified Halanaeroarchaeum sp. oxyrespirans BSP3T as a unique member of Haloarchaea cultivated from salt-saturated sediment of the Bonneville Salt Flats.

Belonging to a genus previously assumed to contain strict anaerobes, BSP3T is a novel species of diverse metabolism, able to use electron acceptors including oxygen, nitrate, and perchlorate.

Oxygen respiration and current identification into the genus Halanaeroarchaeum suggests that the sole published isolate does not encompass the full traits of the genus, which can no longer be classified as strictly anaerobic. While uncommon there are bacterial examples in which a genus description has been emended to include strict and facultative anaerobes. For more than 90 years

Desulfobibrio were thought to not survive oxygen exposure, additionally Geobacter species were considered strict anaerobes until the discovery that Geobacter sulfurreducens could grow utilizing oxygen as a terminal electron acceptor (Fareleira et al., 2003; Lin et al., 2004). The ability to respire oxygen was documented both with gas chromatographic (GC) analysis and confirmed by genomic analysis which revealed a suite of genes for aerobic respiration, including

61 cytochrome c oxidases previously undocumented in the genus. Additionally, perchlorate reduction in BSP3T is unique among haloarchaeal perchlorate reducers as it possesses a pNar which can support growth on elevated levels of perchlorate independent of nitrate.

Materials and Methods

Enrichment, Isolation, and Cultivation

Salt-saturated sediment collected from a ~10 cm deep hand-dug hole beneath Bonneville

Salt Flats salt crust (40.757333, -113.886417) was collected during July 2017. Sediment was stored in zipper storage bags and transported back to Louisiana State University to begin enrichments. Enrichments were performed by transferring approximately 5 g fresh weight (gfw) of sediment into 60-mL serum bottles containing 5-mL of CM1 with 3.8 M NaCl, 25 mM pyruvate, and 20 mM perchlorate (Dyall-Smith, 2008). The bottles were then sealed with neoprene rubber stoppers and flushed with oxygen-free nitrogen to create an anoxic environment. Bottles were then incubated at 30oC, sub-samples were retrieved using a needle and syringe which were then used to quantify perchlorate concentration using a ion-selective electrode. Replicates positive for perchlorate reduction were used to inoculate fresh media in triplicate, three times over the course of one year. Spread plating of serial dilutions was done after the third transfer of cultures positive for perchlorate reduction. Plates were incubated at

30oC under low oxygen concentrations. Morphologically distinct colonies were selected and used to inoculate fresh media which were then screened for perchlorate reduction until an axenic perchlorate-reducing isolate was obtained.

Growth on Perchlorate

To determine if nitrate induction was required for growth on perchlorate, inocula from cultures previously grown on nitrate and thiosulfate were compared to cultures previously grown on perchlorate and thiosulfate. Cells used for inocula were harvested via centrifugation, washed twice in CM1 medium without electron acceptors and carbon sources, and inoculated at 2.5% of total culture volume into 3.8 M CM1 with 20 mM perchlorate, 16 mM pyruvate, and 20 mM thiosulfate. Triplicate cultures were then sealed in serum bottles with neoprene rubber stoppers and headspaces were flushed with nitrogen (N2) to create an anoxic environment. Sub-samples were obtained to quantify perchlorate, chlorate, chlorite, cell density (absorbance 600 nm), and total protein concentrations (µg/mL) as needed, while CO2 was quantified at the start and end of the experiments.

To confirm that perchlorate reduction occurred biologically, cells were first grown in 3.8

M CM1 supplemented with 50 mM perchlorate and 50 mM thiosulfate which were then washed and resuspended in 3.8 M CM1 without a substrate present. Potential abiological perchlorate interactions were tested by autoclaving washed cell resuspensions for 30 min as well as using media controls which lacked live or dead cells. Protein concentrations were quantified at the start and end of experiments while perchlorate was quantified as periodically.

Additional substrates succinate, and sodium fumarate, were further evaluated for their potential to support growth on perchlorate in comparison to pyruvate. Inoculum from cultures grown on 3.8 M CM1 with 50 mM pyruvate, perchlorate, thiosulfate was harvested and resuspended in 3.8 M CM1 with 50 mM perchlorate and the presence of either 50 mM succinate, sodium fumarate, or pyruvate (all with or without the additional presence of thiosulfate). All cultures were prepared in triplicate as described above. Sub-samples were obtained to quantify

63 perchlorate, chlorate, chlorite, cell density (absorbance 600 nm), and total protein concentrations

-1 (µg ml ) as needed, while CO2 was quantified at start and end of the experiment. Pyruvate cultures were further used to determine the end-product of perchlorate reduction by first quantifying the total increase in CO2 and using that to calculate estimated pyruvate equivalents consumed during growth assuming the production of 3 CO2 molecules for every pyruvate oxidized.

Chlorite and Hypochlorite Interactions

Potential chlorite abiotic reactions with the substrates pyruvate, succinate, and sodium fumarate in 3.8 M CM1 were evaluated. In triplicate, 3.8 M CM1 media was incubated with each substrate and 5 mM chlorite. Chlorite concentration was monitored from sub-samples of media for approximately 6 d.

To determine if potential chlorite accumulation was harmful to cell viability, cultures grown on 3.8 M CM1 with 50 mM pyruvate, perchlorate, and thiosulfate were harvested and washed as described above and resuspended in the following CM1 solutions: 0 mM chlorite (25 mM pyruvate, perchlorate, and thiosulfate) acting as our positive control, 1 mM chlorite (w/ and w/o 25 mM pyruvate), 5 mM chlorite (w/ and w/o 25 mM pyruvate), and 10 mM chlorite (25 mM pyruvate). Cultures were sub-sampled at time zero, 1 d, and 2 d, and observed both under

DIC and fluorescent (MitoView) imaging for changes in cell shape and intactness of the cell membrane. After 2 d of chlorite exposure, cells were inoculated into standard 3.8 M CM1 with

50 mM pyruvate, perchlorate, and thiosulfate in which culture absorbance was monitored for ~1 month to test for cell viability.

Fluorescent, differential interference contrast (DIC) and transmission electron microscopy (TEM) cell imaging were performed at Louisiana State University Shared

Instrumentation Facility. A JEOL JEM-1400 transmission electron microscope was used for

TEM images using a negative stain method performed as follows: drop 3 µL culture sample on a glow discharged 300 mesh carbon filmed grid (EMS: CF300-cu), after 2 min use filter paper to remove excess solution. The grid was then stained with 2% uranyl acetate solution for 10 sec and transferred to the microscope for imaging. A Leica DM6B upright microscope was utilized for

DIC and fluorescent images. Fluorescent cells were prepared by sub-sampling cultures, adding

500 nM of MitoViewTM 633 (Biotium), and incubating at 37oC for 30 mins immediately prior to imaging.

Chemical Analyses

Perchlorate concentrations were measured using an ion-selective electrode (Thomas

Scientific, Swedesboro, NJ, United States), and standardized with perchlorate dissolved in growth medium. Total assay volume was 5 mL to include 100 µL of culture, 100 µL of 1 M sodium acetate acting as an ionic strength buffer, and 4.8 mL of deionized water. Chlorate and chlorite concentrations were monitored via a modified colorimetric O-tolidine assay (Couture,

1998). Assays contained a final volume of 1 mL mixed in the listed order: 4 µL sample, 396 µL deionized water, 100 µL O-tolidine, and 500 µL HCl (12 M – chlorate, 4.8 M – chlorite).

Samples were incubated for 10 min at room temperature and absorbance was read at 448 nm

(chlorate) or 442 nm (chlorite). Headspace oxygen and carbon dioxide (CO2) were monitored by gas chromatographic analysis. Total protein concentrations were quantified

65 spectrophotometrically using a colorimetric detection kit based on the bicinchoninic acid method

(PierceTM BCA Protein Assay Kit, Thermo Scientific).

Molecular Characterization of Halanaeroarchaeum sp. BSP3T

Genomic DNA was extracted from strain BSP3T using a Qiagen DNeasy PowerLyzer

Microbial DNA extraction kit following manufacturer’s instructors. Archaeal forward primer

Arch21F ((5’-TTCCGGTTGATCCYGCCGGA-3’; Miyashita et al., 2009; DeLong et al., 1992) and universal reverse primer 1492R (5’-CGGTTACCTTGTTACGACTT-3’; Lane, 1991) were used to amplify 16S rRNA genes from genomic DNA extracts. Amplicons were purified with

QIAquick PCR Purification kit and sequenced using an ABI 3130XL Genetic Analyzer (Applied

Biosystems, Foster City, CA) at the Louisiana State University Genomics Facility (Baton Rouge,

Louisiana, USA). The resulting bidirectional sequence reads were assembled and edited using

Sequencher 4.8 (Gene Codes Corporation, Ann Arbor, MI, USA). The 16S rRNA gene sequence has been deposited in Genbank as accession MW520126. 16S rRNA gene sequence analysis was completed using via EZBiocloud ([(EzBioCloud, https://www.ezbiocloud.net/tools/ani; Yoon et al., 2017) and a pairwise ANI genome comparison from the Integrated Microbial Genomes and

Microbiomes platform (IMG, img.jgi.doe.gov). Phylogenetic relationships of the novel isolates and close relatives were determined using a maximum likelihood analysis in MEGAX (Kumar et al., 2016).

Physiological characterization of Halanaeroarchaeum sp. BSP3T

All physiological and biochemical assays were conducted with media based on 3.8 M

NaCl CM1-PPT (CM1 media listed above with a final concentration of 25 mM pyruvate,

66 perchlorate, and thiosulfate) and growth was measured by monitoring absorbance at 600 nm in triplicate unless otherwise noted. Temperature ranges and optima were assessed at intervals from

15oC to 50oC. The ranges and optimal NaCl concentrations for growth were determined using media with NaCl concentrations adjusted to 0.5 M, 1 M, 1.5 M, 2 M, 3 M, 4 M, and 5.4 M; growth was measured for 21 d. The minimum concentration of added magnesium (Mg2+) required for growth, optimum and range of growth for pH and NaCl concentrations, and substrate utilization were assessed using media listed above for 21 d based on protocol outlined in Myers and King, 2020. Standard procedures were used to analyze catalase and oxidase production while a modified procedure was used for Gram-staining that consisted of a rinse of

CM1 containing 2 M NaCl as an alternative to deionized water to prevent cell lysis. Polar lipids for all strains were determined commercially by the Identification Service of DSMZ

(Braunschweig, Germany). All assays followed standard methods (Bligh and Dyer, 1959;

Tindall, 1990a, Tindall et al., 2007). Cell morphology and dimensions were determined using a

Zeiss Axioscope and an AxioCam MR digital camera.

Results and Discussion

Enrichment, Isolation, and Cultivation

Salt-saturated sediment collected from an ~10 cm deep hand-dug hole beneath

Bonneville Salt Flats salt crust was collected during July 2017. Approximately 5 gfw of sediment was transferred into 60-mL serum bottles containing 5-mL of CM1 with 3.8 M NaCl to enrich for extreme halophiles, 25 mM pyruvate which acted as an electron donor and is a known substrate for other haloarchaea capable of reducing perchlorate, and 20 mM perchlorate to act as an electron acceptor. Over the course of a year samples were periodically monitored for

67 perchlorate reduction using an ion-selective electrode and three transfers were made after at least half of the perchlorate was reduced. During sub-sampling a strong hydrogen sulfide smell was observed, indicating native sediment likely contained sulfur sources. Transfer two was split to contain triplicates either with or without the addition of thiosulfate, as hydrogen sulfide production indicated potential sulfur species usage. After the third transfer was positive for perchlorate reduction samples were serially diluted and spread plated onto media with or without thiosulfate and incubated at 30oC under low oxygen concentrations. Approximately two weeks later, seven morphologically distinct colonies were observed up to 10-6 plated dilution in the presence of thiosulfate, and 10-4 without thiosulfate. Morphologically distinct colonies were selected and used to inoculate fresh media both with and without thiosulfate which were then screened for perchlorate reduction until an axenic perchlorate-reducing isolate was obtained. A single perchlorate reducing isolate, BSP3T, was cultivated from plates containing thiosulfate at

10-6 dilution, colonies were irregularly shaped, non-pigmented, opaque, raised, and dull. The purity of the single representative isolate was confirmed via 16S and whole genome sequencing.

Isolate Phylogeny and Molecular Characterization

A year-long enrichment process using salt-saturated sediment collected from a ~10 cm deep hand-dug hole beneath Bonneville Salt Flats salt crust yielded a single isolate, BSP3T, capable of reducing perchlorate. Initial 16S rRNA sequencing at Louisiana State University identified the isolate as a novel member of the genus Halaneroarchaeum. The isolate further underwent whole genome Illumina MiSeq sequencing at Michigan State University followed by subsequent annotation using IMG annotation pipeline v.5.0.3. The BSP3T genome was comprised of 2,975,536 bp containing 2,606,145 (87.59%) coding DNA base pairs and 3,125

68 genes of which 3,069 (98.21%) were protein coding. Function prediction was associated with

2,142 genes, 804 were connected to KEGG pathways, 634 to MetaCyc pathways, and 2,268 were associated with COGs. One 16S rRNA (1473 bp) and 48 tRNA genes were identified, no

CRISPRs were identified. BSP3T was phylogenetically distinct from its closest relatives,

Halaneroarchaeum sulfurireducens and Halodesulfurarchaeum formicicum, at 16S rRNA similarities of 98.78% and 95.18%, and pairwise average nucleotide identity values (ANI) of

78.87% and 73.14% respectively. All sequence identities are at or below the accepted cutoff for

16S rRNA gene-based species differentiation of 98.7%-99.1%, whereby we propose the provisional name Halaneroarchaeum oxyrespirans BSP3T (Yarza et al., 2014). Phylogenetic relationships of the novel isolate and close relatives were further displayed using a maximum likelihood analysis of 16S data in MEGA X (Figure 5.1). The DNA base composition (G+C) mol% was calculated to be 60.85%.

Similar identified gene sets between BSP3T and H. sulfurireducens HSR2T include those identified in sulfur metabolism with BSP3T containing an additional thiosulfate sulfurtransferase as well as the TCA and glycolysis cycle with BSP3T containing an additional phosphoenolpyruvate carboxykinase. In general, the number of protein coding genes (COG associated) were also much greater in BSP3T (187) when compared to HSR2T (103) supporting greater diversity in utilized substrates and electron acceptors for growth. Notable genomic differences between BSP3T and Halaneroarchaeum sulfurireducens HSR2T include the smaller size of HSR2T genome at 2,209,738 bp which led to a smaller number of genes (2,373) of which

2,254 were protein coding, and function prediction was associated with 1,577 of those. HSR2T did not contain an identified or similar blast hit for potential nitrate reductase (pNar) genes, or cytochrome c oxidase while BSP3T did, allowing for potential use of nitrate, perchlorate, and

69 oxygen. While annotated as complex iron-sulfur molybdoenzyme family reductase subunit alpha

(IMG Gene ID: 2835723008) within the BSP3T genome, the COG database identified the gene as a likely nitrate reductase alpha subunit (COG5013) flanked by likely beta and gamma subunits, and a sequence similarity of 70% to respiratory nitrate reductase alpha subunit (IMG Gene ID:

2816337284) of the known perchlorate reducer Halobacterium bonnevillei. Additionally, key α,

β, γ residues of known pNarG and PcrA genes were identified, and a likely periplasm location was identified using PRED-TAT software which predicted the likely presence of a twin-arginine translocation (TAT) signal peptide. (Appendix Figure C.1) (Bagos et al., 2010; Barnum et al.,

2018).

Figure 5.1. Evolutionary analysis by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model. The tree with the highest log likelihood (-6742.55) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 27 nucleotide sequences. All positions containing gaps and missing data were eliminated (complete deletion option). There were a total of 1343 positions in the final dataset. Evolutionary analyses were conducted in MEGAX.

Growth on Perchlorate

- Dissimilatory reduction of ClO4 coupled to CO oxidation has been observed up to 1 M concentrations in a denitrifying bacterium (Halobacterium bonnevillei), and lower concentrations in a nitrate-respiring bacterium (Halobaculum saliterrae) however both isolates are nitrate- dependent and require the presence of a small amount of nitrate to induce the periplasmic nitrate

- - reductase (pNar) (Myers and King, 2017, 2019). Nitrate-dependent ClO4 and ClO3 reduction

T has also been observed in Haloferax mediterranei 33500 , in which culture inoculum was

- previously grown in the presence of nitrate. Notably, H. mediterranei cells could only use ClO4 for limited generations after nitrate was removed from the culture (Martinez-Espinosa et al.,

2015).

Halanaeroarchaeum BSP3T was first assessed for the capacity to utilize and grow on perchlorate without the need for nitrate induction of pNar genes. Over the course of two months

- cells previously grown on nitrate were able to reduce 251.4 + 11.8 µM ClO4 reaching a maximum protein concentration of 394 + 34.2 µg ml-1. Cells previously grown on perchlorate

- without nitrate were able to reduce a similar concentration of 211.0 + 16.1 µM ClO4 reaching a maximum protein concentration of 354.2 + 37.1 µg ml-1; similar lag times were observed prior to significant culture growth (Table 5.1, Appendix Figure C.2). These results indicate that perchlorate reduction is coupled to cell growth and notably, cell growth occurred without the need for nitrate to induce pNar. Furthermore, biological perchlorate reduction was confirmed when living cells were compared to both autoclaved (killed) cells and a media control lacking cells (Appendix Figure C.2). At the time of writing over 20 generations have been successfully cultured in the absence of nitrate without loss of cell viability or reduction in perchlorate reduction capacities.

Table 5.1. Maximum protein (µg ml-1), maximum absorbance (600 nm), and perchlorate decrease (µM) observed during BSP3T growth on perchlorate using varied inoculum source. All values are means of triplicate cultures with standard error expressed. Suffix of -N or -P indicate culture inoculum source being previously grown on either nitrate or perchlorate.

Perchlorate + Thiosulfate – N Perchlorate + Thiosulfate - P

Maximum Protein (µg ml-1) 394.09 + 34.21 354.24 + 37.11

Maximum Absorbance (600 nm) 0.218 + 0.01 0.206 + 0.00

Perchlorate Decrease (µM) 251.39 + 11.77 211.01 + 16.06

Growth on perchlorate was further evaluated with additional substrates succinate and sodium fumarate in comparison to pyruvate, both with and without the addition of thiosulfate.

Over approximately three weeks, quantifiable growth only occurred in the presence of thiosulfate, though growth occurred to a similar degree across substrates tested (Figure 5.2).

While growth was not quantifiable in the absence of thiosulfate, small amounts of perchlorate were reduced in all cultures. In cultures without thiosulfate small amounts of chlorate accumulated, indicating that perchlorate reduction did occur to a degree but could not support quantifiable cell growth (Appendix Figure C.3). As BSP3T grows in hypersaline media, small potential increases in chloride concentrations from perchlorate reduction were not measurable.

As an alternative, the end-product of BSP3T perchlorate reduction was inferred by quantifying total increase in CO2 followed by the amount of needed pyruvate equivalents oxidized to produce the final concentration of CO2 in culture headspaces. Chlorite was determined to be the primary biological end-product of BSP3T perchlorate reduction; this is supported by the absence of cld in the genome, and the requirement of abiological chlorite sinks (thiosulfate) for cell growth to occur (Table 5.2).

Figure 5.2. BSP3T growth on perchlorate in the presence of three different electron donors, Pyruvate (○); pyruvate + thiosulfate (●); succinate (□); succinate + thiosulfate (■); sodium fumarate (Δ); sodium fumarate (▲). All values are means of triplicate cultures with standard error expressed.

Table 5.2. Pyruvate equivalents estimated to have been produced based on total increase in CO2 during BSP3T perchlorate-based cell growth. Possible end-products calculated include chlorite and oxygen and chlorite. All values are means of triplicates with standard error.

Pyruvate equivalents Pyruvate equivalents (Cl– Pyruvate equivalents - (via CO2 production) and O2 end-products) (ClO2 end-product) BSP3T Pyruvate + 276.95 + 13.22 25,379.53 + 58.17 258.75 + 18.33 Thiosulfate

Chlorite and Hypochlorite Interactions

Perchlorate is chemically stable in solution, but chlorate, chlorite, and hypochlorite are considered highly reactive oxidants (Barnum, 2020). Chlorite, the primary biological end- product of perchlorate reduction in BSP3T, may act as a significant source of oxidative stress leading to potential cellular damage affecting cell structure and viability. Abiological chlorite interactions with the substrates pyruvate, succinate, and sodium fumarate in cell-free 3.8 M CM1

74 media were investigated. Over approximately 6 d when exposed to either succinate or sodium fumarate, chlorite concentrations did not decrease relative to the control without added substrate, however, when exposed to pyruvate, chlorite concentrations decreased dramatically (Figure 5.3).

The likely product of a chlorite and pyruvate abiotic interaction was hypochlorite (HClO-), the most oxidizing chlorine oxyanion, known to be lethal to bacteria at micromolar concentrations

(Chesney et al., 1996).

Figure 5.3: Abiological chlorite interactions in 3.8 M CM1 with the substrates pyruvate, succinate, and sodium fumarate. 5 mM chlorite – no substrate (■); 5 mM chlorite – pyruvate (●); 5 mM chlorite – succinate (□); 5 mM chlorite – sodium fumarate (○). All values are means of triplicate cultures with standard error expressed.

To assess if potential chlorite accumulation could cause cellular damage to BSP3T, thus affecting structure and viability, stationary phase cells were exposed to varying levels of chlorite at 0 mM (with pyruvate), 1 mM (with and without pyruvate), 5 mM (with and without pyruvate), and 10 mM (with pyruvate). After 2 d of exposure, cells were observed by DIC and fluorescent

75 imaging (Figure 5.4). Imaging indicated intact cells under all conditions, however, cell shape was visibly abnormal at elevated chlorite concentrations 5 mM and 10 mM, primarily with pyruvate exposure (Figure 5.4). It is suspected that either elevated chlorite concentrations, and/or the spurious byproduct of chlorite dismutation, hypochlorite, likely deformed cells as both chlorine species are known for their potential cell damaging oxidizing reactions.

Figure 5.4: DIC (top) and fluorescent (bottom) images of BSP3T after 2 d of chlorite exposure. (A) 0 mM chlorite (25 mM pyruvate, perchlorate, and thiosulfate), (B) 1 mM chlorite (w/ 25 mM pyruvate), (C) 5 mM chlorite (w/ 25 mM pyruvate), (D) 10 mM chlorite (25 mM pyruvate).

To assess whether cells were viable after chlorite exposure, cultures were sub-sampled, inoculated into fresh 3.8 M CM1 media, and monitored for approximately 1 month. When not exposed to chlorite cells exhibited normal growth, while at 5 mM (with and without pyruvate) and 10 mM chlorite, no significant cell growth occurred (Figure 5.5A). However, although all replicates at 1 mM (with and without pyruvate) eventually grew to standard concentrations, significant variation occurred between replicates (Figure 5.5B). All replicates exposed to 1 mM chlorite experienced an extended lag phase (between 10 and 25 d), implying a likely decrease in

76 total cell viability. Additionally, when exposed to both 1 mM chlorite and pyruvate a minimum lag phase of 20 d was observed in all replicates. These results suggest chlorite alone may not account for loss in cell viability, and that the chlorite dismutation byproduct hypochlorite, may play a larger role.

Figure 5.5A.

Figure 5.5. BSP3T growth after 2 d exposure to varying levels of chlorite. (A) 0 mM chlorite (25 mM pyruvate, perchlorate, and thiosulfate) acting as our positive control (■), 10 mM chlorite (25 mM pyruvate) (●), 5 mM chlorite – pyruvate (□), 5 mM chlorite (▲). (B) Individual replicates of 1 mM chlorite cultures, with pyruvate (closed symbol), without pyruvate (open symbol); replicate 1 (square), replicate 2 (circle), replicate 3 (triangle). All values are means of triplicate cultures with standard error expressed. (figure cont’d.)

Figure 5.5B.

To assess if presumed produced hypochlorite could cause cellular damage and loss in viability stationary phase cells were exposed to 10 mM chlorite with pyruvate, 1 mM hypochlorite, and 5 mM hypochlorite. After 2 d of exposure cells were imaged using TEM and compared to a control without either chlorite or hypochlorite. Imaging of cells exposed to 10 mM chlorite indicated intact though abnormally shaped cells in support of earlier DIC and fluorescent imaging results. However, when exposed to both 1 mM and 5 mM hypochlorite cell lysis occurred. Cells exposed to neither chlorite or hypochlorite retained intact standard rod-shape

(Figure 5.6). Cells were then sub-sampled and inoculated into fresh 3.8 M CM1 media to assess cell viability; after 1 month of incubation no quantifiable growth was detected for chlorite- or hypochlorite-exposed cells further supporting hypochlorite toxicity as the primary cause in loss of cell viability.

Figure 5.6. TEM images of BSP3T after 2 d of varied chlorine oxyanion exposure. (A) 0 mM chlorite (25 mM pyruvate, perchlorate, and thiosulfate), (B) 10 mM chlorite (25 mM pyruvate), (C) 1 mM hypochlorite, (D) 5 mM hypochlorite.

Physiological characterization of Halanaeroarchaeum sp. BSP3T

BSP3T grew optimally at 35oC, with a range of 15oC to 45oC; at 3 M NaCl with a range of 2 M to

5.4 M; and at pH 7.0, with a range of 6.5 to 7.5. Cells occurred singly. BSP3T required addition of 5 mM Mg2+ for growth and cells were weakly positive for catalase and oxidase and stained

Gram-negative (Table 1). Major polar lipids of BSP3T included: phosphatidylglycerol (PG), aminolipid (AL), glycolipid (GL), phosphoglycolipid (PGL), and phospholipid (PL)

Growth occurred on the following carbon sources pyruvic acid, succinate, sodium fumarate, glucose, galactose, lactose, sucrose, ribose, mannose, arabinose, xylose, peptone, casamino acids, yeast extract, and tryptone. Cells were also capable of growth on nitrate. Cells were non-motile under all observed conditions, though potential flagella were observed on TEM images. Cells

79 were able to hydrolyze gelatin but not casein or starch (Table 5.3). Additionally, BSP3T represents a unique member of Halaneroarchaeum due to its ability to respire oxygen (Figure

5.7) and grow anaerobically via nitrate-independent perchlorate reduction. While 16S analysis firmly places BSP3T among the Halaneroarchaeum, the range of growth properties differed greatly from the most closely phylogenetically described species, H. sulfurireducens and H. formicium. BSP3T instead appeared to be physiologically more similar to the facultative anaerobic groups capable of anaerobic dissimilatory sulfur respiration, the natronoarchaea genera

Halalkaliarchaeum and Natrachaeobaculum, and the recently discovered halo(natrono)archaea provisional genus Halarchaeoglobus; all of which contain more versatile metabolic properties.

Figure 5.7. Oxygen respiration by pre-grown BSP3T cells under various oxygen concentrations. 1% O2 (■), 5% O2 (●), 10% O2 (▲), 15% O2 (◊), Atmospheric O2 (□). All values are means of triplicate cell suspensions with standard error expressed.

Table 5.3. Differential characteristics of strain BSP3T with closest related species identified by 16S rRNA gene similarity. Taxa: 1, BSP3T; 2, Halanaeroarchaeum sulfurireducens HSR2T. +, positive; -, negative; ND, not determined. H. sulfurireducens data was assembled from Sorokin et al., 2016. Characteristic 1 2 Source Anoxic saline mud Hypersaline lake Cell shape Rods Coccoid/Rods Temperature (oC) Optimum 35 37-40 Range 15 – 45 ND – 46 pH Optimum 7.0 7.0 – 7.5 Range 6.5 – 7.5 6.7 – 8.0 NaCl (M) Optimum 3 4 Range 2 – 5.2 3 – 5 Catalase + ND Oxidase + ND DNA G+C (mol%) 60.85 62.8 Oxygen Use + -

In summary, we have discovered a novel haloarchaeal species of diverse metabolism,

Halanaeroarchaeum sp. oxyrespirans BSP3T cultivated from salt-saturated sediment of the

Bonneville Salt Flats. This genus was previously assumed to contain only strict anaerobes, however BSP3T changes that assumption and diversifies the genera’s potential metabolisms to include the electron acceptors oxygen, nitrate, and perchlorate. The ability to respire oxygen, suggested by the presence of cytochrome c oxidase and documented by GC analysis indicates the identification of this genus as strictly anaerobic is a misrepresentation. Furthermore, growth on perchlorate is unique among known haloarchaeal cryptic perchlorate reducers as its pNar can support growth on elevated levels of perchlorate independent of nitrate, a first example for haloarchaea.

Emended description of the genus Halanaeroarchaeum (Sorokin et al. 2016)

The description of the genus Halanaeroarchaeum is as given by Sorokin et al., 2016 with the following modifications: contains both obligate and facultative anaerobic haloarchaea; electron acceptors expanded to include oxygen, nitrate, and perchlorate; carbon sources expanded to include pyruvic acid, succinic acid, sodium fumarate, glucose, galactose, lactose, sucrose, ribose, mannose, arabinose, xylose, peptone, casamino acids, yeast extract, and tryptone.

Description of Halanaeroarchaeum oxyrespirans sp. nov.

Halanaeroarchaeum oxyrespirans (ox.y.resp.i’.rans N.L. neut n. oxy oxygen; pres. adj. respirans breathe; N.L. adj. oxyrespirans oxygen breathing).

Cells are Gram-stain-negative, rods of varying length. Growth occurs between 15oC and 45oC

(optimum 35oC); between 2 M and 5.4 M (optimum 3 M); and between pH 6.5 to 7.5 (optimum

7). The addition of 5 mM Mg2+ is required for growth. Cells were largely non-motile, though some potential flagella were observed on TEM images. Cells were able to hydrolyze gelatin but not casein or starch and are weakly positive for catalase and oxidase. Utilizes the following substrates as sole carbon sources for growth: pyruvic acid, succinic acid, sodium fumarate, glucose, galactose, lactose, sucrose, ribose, mannose, arabinose, xylose, peptone, casamino acids, yeast extract, and tryptone. Cells were also capable of growth on perchlorate and nitrate. The major polar lipids include: phosphatidylglycerol (PG), aminolipid (AL), glycolipid (GL), phosphoglycolipid (PGL), and phospholipid (PL). The DNA G+C content is 60.85 mol%. The type strain is BSP3T (=DSM 112023, = JCM 34264) isolated from saline mud beneath the

Bonneville Salt Flats (UT, USA).

CHAPTER 6. PERCHLORATE-COUPLED CARBON MONOXIDE (CO) OXIDATION IN MOORELLA GLYCERINI: A NOVEL MECHANISM FOR NICKEL- DEPENDENT CARBON MONOXIDE DEHYDROGENASES

Introduction

Carbon monoxide (CO) is one of the most versatile molecules on the planet having participated in diverse processes from cellular to global levels (King and Weber, 2007). Early in

Earth’s history, anaerobic CO-oxidation is likely to have occurred due to elevated atmospheric

CO concentrations (>100 ppm) in the Archean atmosphere, however CO is present at lower atmospheric concentrations (50 – 350 ppb) in the modern-day atmosphere (Miyakawa et al.,

2002, Cordero et al., 2019). Extraterrestrially, the Martian atmosphere is dominated by CO2, however, CO also has also been documented at levels (> 800ppm) suitable for microbial metabolism. Martian atmospheric CO serves as potentially the most abundant and available substrate on the planet capable of supporting near-surface microbial activity as Martian regolith contains only trace levels of organic carbon (Steele et al., 2012; King, 2015). Martian regolith also contains perchlorate, and while potentially toxic, it also serves an abundant potential oxidant. Critically, Martian water is speculated to exist as pockets of hydrated salts and hypersaline brines dominated by perchlorate at sites such as the recurrent slope lineae.

In organic limited systems, biological oxidation of gases such as carbon monoxide contribute to the survival and growth of microbial communities (King et al., 2008). Carbon monoxide oxidizers which couple elevated levels of CO (>1%) to growth are deemed carboxidotrophic, while carboxidovores use relatively low CO concentrations (< 0.1%) and are incapable of CO-dependent growth (King and Weber, 2007). Recently, the first evidence of perchlorate-reduction coupled to CO oxidation was observed within carboxidovoric haloarchaea

83 containing molybdenum-dependent carbon monoxide dehydrogenases (Mo-CODHs) at concentrations up to 1 M (Myers and King, 2017, 2019, 2020). Studies have shown that haloarchaea, are capable of cryptic perchlorate reduction using a periplasmic nitrate reductase

(pNar), and most cryptic perchlorate reducers are nitrate-dependent, however recent evidence has indicated that it is not a strict requirement as documented in the provisional isolate

Halanaeroarchaeum oxyrespirans (Oren, 2014; Martinez-Espinosa et al., 2015; Myers and King,

2021 unpublished)

In an effort to expand perchlorate-reduction coupled CO oxidation to carboxidotrophs as well as nickel-dependent carbon monoxide dehydrogenases (Ni-CODHs), Moorella glycerini

DSM 11254T was selected as a model organism. Results presented here provide the first evidence of nitrate-independent, perchlorate-reduction coupled to CO oxidation in a carboxidotrophic organism.

Materials and Methods

Cultivation conditions

The medium used for routine cultivation of Moorella glycerini, termed Moor1-S, is an

-1 altered version of DSMZ medium 793 containing the following (g L ): KH2PO4 0.33, H8N2O4S

0.816, K2SO4 0.772, MgSO4 x 7 H2O 0.394, CaCl2 x 2 H2O 0.33, yeast extract 0.5, NaHCO3 10, ,

L-cysteine-HCl x H2O 0.3, Na-resazurin solution (0.1% w/v) 0.5 mL, FeSO4 x 7 H2O (0.1% w/v in 0.1N H2SO4) 2 mL, glycerol (87%) 3 mL, trace element solution SL-10 (DSMZ medium 320)

1 mL, selenite tungstate solution (DSMZ medium 385) 1 mL, vitamin solution (DSMZ medium

141) 10 mL. The pH was adjusted to 6.5 with 1 M sulfuric acid before filter sterilization. After sterilization media was supplemented with filter sterilized Na2S x 7 H2O (0.5g/L). DSMZ

84 medium 793 was altered to allow for the lowest chloride concentration in base media (~14 mM) which allowed for a more accurate chloride quantification during experimentation. Routine growth conditions included serum-bottles sealed with butyl rubber stoppers, an anoxic carbon

o dioxide (CO2) headspace created by flushing with 100% CO2, and a growth temperature of 58 C.

Perchlorate-Coupled CO Oxidation

Moorella glycerini was assessed for the ability to couple perchlorate reduction to CO oxidation for growth using inoculum from freshly grown stationary phase cells grown in Moor1-

S under routine conditions. Cells were first harvested by centrifugation, washed in Moor1-S without glycerol, and resuspended in Moor1-S with or without glycerol. Washed cells were inoculated at 2.5% of total experimental culture volume. All sample headspaces were then flushed with 100% CO2 to create an anoxic headspace. Experimental treatments included triplicates of the following: glycerol (Moor1-S), glycerol + 20 mM perchlorate (Moor1-S), 25%

CO (Moor1-S without glycerol), and 25% CO + 20 mM perchlorate (Moor1-S without glycerol).

Headspace CO and potential O2 concentrations were assayed periodically by removing headspace sub-samples for gas chromatographic analysis. Perchlorate, chlorate, and chlorite concentrations were determined periodically in relevant treatments using sub-samples obtained by needle and syringe. Perchlorate concentrations were measured with an ion-selective electrode as described by Myers and King, 2017 with the alteration of 100 µL of culture was used.

Chlorate and chlorite concentrations were quantified via a colorimetric O-tolidine assay

(Couture, 1998). Chlorate assay was performed as described by Myers and King, 2017; chlorite assay was performed identically to chlorate with the alteration of 4.8 M HCl used and an absorbance used of 442 nm. Chloride was determined using an altered version of the mercuric

85 thiocyanate spectrophotometric method (Iwasaki et al., 1956). Mercuric thiocyanate assays contained a 1 mL final reaction mixed in the following order in a cuvette: 38 µL sample, 346 µL water, 154 µL ferric ammonium sulfate solution, 77 µL mercuric thiocyanate solution, and 385

µL dioxane which was then mixed and allowed to incubate at room temperature for 10 mins before reading at 460 nm. Ferric ammonium sulfate solution was made fresh at the time of use to avoid precipitation by mixing 6 g ferric ammonium sulfate with 100 mL of 6 N nitric acid.

Mercuric thiocyanate solution was made by mixing 300 mg of mercuric thiocyanate in 100 mL concentrated ethyl alcohol.

Results and Discussion

Selection of Moorella glycerini DSM 11254T as model organism

Perchlorate-coupled CO oxidation has recently been established in carboxidovoric, Mo-

CODH containing extreme halophiles, however it is yet to be shown in carboxidotrophic, Ni-

CODH organisms. Currently, there are two strains of Moorella glycerini in culture, NMP and

DSM 11254T. Moorella glycerini strains are often used interchangeably in the literature and are

99.7% similar when comparing 16S rRNA gene sequences. However, a recent genome-based comparison of all Moorella species suggests that NMP only shows an ANI value of 94% similarity to the type strain (Redl et al., 2020). Redl et al., (2020) further suggests that M. glycerini NMP, M. stamsii DSM 26217T, and M. perchloratireducens may be members of the same species. While previous reports have shown, M. glycerini DSM 11254T with documented growth using CO in concentrations up to 50%, while both strains have been documented as perchlorate reducers (Balk et al., 2008; Alves et al., 2013; Liebensteiner et al., 2015). Neither strain has been documented to couple perchlorate reduction to CO oxidation. To assess the

86 feasibility of perchlorate-coupled CO oxidation in Ni-CODH organisms, the thermophilic

Firmicute, Moorella glycerini DSM 11254T, was selected as a model organism (Slobodkin et al.,

1997).

Perchlorate-Coupled CO Oxidation by Moorella glycerini

Unlike the haloarchaea capable of perchlorate-coupled CO oxidation, M. glycerini is a carboxydotroph, allowing for larger quantities of perchlorate to be reduced which could be readily and directly measured, additionally routine growth media could be altered to contain lower levels of chloride allowing for detection of potential chloride increases.

Moorella glycerini was assessed for the ability to couple perchlorate reduction to CO oxidation for growth using the experimental treatments, glycerol (as control), 25% carbon monoxide, glycerol with 20 mM perchlorate, and 25% carbon monoxide with 20 mM perchlorate. In all replicates with carbon monoxide present, glycerol was removed from the media. Over approximately one month all treatments had documented cell growth, quantifiable cell growth was not observed prior to day 5 (Figure 6.1). Results shown indicate that our altered version of DSM medium 793 could support growth of M. glycerini DSM 11254T, as well as confirmed literature reports of carboxidotrophic CO growth and indicated that perchlorate (20 mM) did not inhibit cell growth (Figure 6.1).

Figure 6.1. Growth of M. glycerini with various substrates glycerol (□), carbon monoxide (■); glycerol + perchlorate (○), carbon monoxide + perchlorate (●). All values are means of triplicate cultures with standard error expressed.

Over approximately one month when grown using CO and perchlorate, M. glycerini oxidized CO to levels < 5 µMol (Figure 6.2), for an average total decrease of 1,451.05 + 9.26

µMol (Table 6.1). Carbon monoxide oxidation was observed to occur in concurrence to perchlorate reduction (Figure 6.2). Perchlorate concentrations reached 357.61 + 6.11 µMol, for an average total decrease of 604.02 + 28.15 µMol (Table 6.1). These results suggest that M. glycerini is able to couple carbon monoxide oxidation to perchlorate reduction for cell growth.

Figure 6.2. Carbon monoxide and perchlorate concentrations observed during growth on perchlorate and carbon monoxide for Moorella glycerini. Carbon monoxide (µMol) (□), perchlorate (µMol) (■). All values are means of triplicate cultures with standard error expressed.

The genome of M. glycerini DSM 11254T, revealed no genes for canonical perchlorate reduction, pcr or cld, and is instead suspected to utilize an alternative pathway, cryptic perchlorate reduction, using a nitrate reductase to reduce (per)chlorate and sulfur species to act as chlorite sinks for abiotic removal. Uniquely, the M. glycerini genome does contain a nitrate reductase, however nitrate reduction has not been documented for the species. Additionally, a periplasmic location was not identified for the nitrate reductase using PRED-TAT software

(Bagos et al., 2010).

To determine the biological end-product of perchlorate reduction, chlorate, chlorite, chloride, and oxygen were quantified. In all replicates no quantifiable increase of chlorate, chlorite, or oxygen was observed. However, chloride concentrations were observed to increase in

89 concurrence with perchlorate decrease. Chloride concentrations increased an average of 625.11 +

26.45 µMol, which accounts for measured perchlorate reduction (Figure 6.3; Table 6.1).

Table 6.1. µMol of CO oxidized, perchlorate reduced, and chloride produced during M. glycerini growth on carbon monoxide and perchlorate. All values are means of triplicate cultures with standard error expressed. CO Oxidized Perchlorate Reduced Chloride Produced

(µMol) (µMol) (µMol)

M. glycerini 1,451.05 + 9.26 604.02 + 28.15 625.11 + 26.45 - ClO4 + CO

Figure 6.3. Chloride increase observed under perchlorate reduction during growth of M. glycerini using carbon monoxide as an electron donor and perchlorate as an electron acceptor. Perchlorate (µMol) (□), chloride (µMol) (■) All values are means of triplicate cultures with standard error expressed.

While chloride was the quantifiable end-product of perchlorate reduction in M. glycerini, it is not suspected to be the biological end-product. The biological end-product of M. glycerini perchlorate reduction was inferred by using the following stoichiometric reactions:

+ - CO + H2O → CO2 + 2H + 2e

- - - - - ClO4 + 2e → ClO3 + 2e → ClO2

With one perchlorate reduced per two CO molecules oxidized both inferred

- stoichiometrically and observed quantifiably, we can infer that chlorite (ClO2 ) is the primary biological end-product of perchlorate reduction in M. glycerini (Table 6.1). This is further supported by the absence of cld in the genome, and the presence of sulfur species sulfide and cysteine in cultivation media which act as abiological chlorite sinks (Myers and King, 2017).

In summary, this study provides evidence of perchlorate-coupled carbon monoxide oxidation in the thermophilic Firmicute, Moorella glycerini DSM 11254T. Utilizing cryptic perchlorate reduction, the biological end-product is inferred to be chlorite which abiological chlorite sinks remove. These results expand the metabolic process of perchlorate-coupled carbon monoxide oxidation for microbial growth to include members of the domain Bacteria (phylum

Firmicutes), carboxidotrphic organisms, and Ni-CODH CO oxidizers.

CHAPTER 7. CONCLUSIONS AND FUTURE DIRECTIONS

Life on Earth has likely exploited carbon monoxide for much of its evolutionary history

(Svetlichny et al., 1991; Sokolova et al., 2004). In the absence of organic matter deposits atmospheric trace gases, such as CO, can be utilized by present day microbes which oxidize CO for energy under aerobic, facultative and obligate anaerobic conditions. Thus far, the phylogenetically wide array of bacteria and archaea which can oxidize CO have used electrons acceptors such as oxygen, nitrate, and sulfate. Though previously unexplored for CO oxidation, chlorine oxyanions could also act as suitable electron acceptors, expanding the current knowledge and diversity of both Mo-CODH and Ni-CODH CO oxidizing microorganisms.

- Found worldwide and extraterrestrially, the chlorine oxyanion perchlorate (ClO4 ), can be reduced by a large diversity of microorganisms. Canonical reduction via perchlorate reductase

(pcr) and chlorite dismutase (cld) results in the end-products of chloride and oxygen and is primarily observed in Proteobacteria. Alternative perchlorate reduction pathways such as cryptic perchlorate reduction commonly use alternative genes such as periplasmic nitrate reductase

(pNar), and result in the biological end-product of chlorite which is then frequently removed by abiological chlorite sinks.

The research conducted for this dissertation aimed to determine if perchlorate can act as a

CO electron acceptor for Mo-COX and Ni-COX isolates, isolate and characterize novel halophilic CO oxidizers, and investigate how perchlorate reduction differs between canonical perchlorate reducers and those that utilize alternative pathways such as cryptic perchlorate reduction. To do this, the studies used a variety of culture-dependent approaches on four novel haloarchaea cultivated from the Bonneville Salt Flats (crusts and salt-saturated sediment) and

92 surrounding saline soils as well as the known thermophilic Firmicute Moorella glycerini.

Collectively, these novel haloarchaeal cultivars along with Moorella glycerini contributed to the expansion of carbon monoxide oxidizer and perchlorate reducer diversity through both physiological and genomic characterization.

Two novel denitrifying and nitrate-respiring euryarchaeal extreme halophiles,

Halobacterium bonnevillei PCN9T and Halobaculum saliterrae WSA2T, provide the first evidence for perchlorate-coupled Mo-CODH CO oxidation (Chapters 2 and 3). Both isolates were able to couple CO with perchlorate under anaerobic conditions using cryptic perchlorate reduction, driven by a periplasmic nitrate reductase and supported by abiological chlorite sinks.

CO oxidation activity was further documented to take place at low water potential (-19 MPa) and perchlorate concentrations ranging from 0.01 to 1 M for H. bonnevillei PCN9T. The ability to oxidize CO under both elevated concentrations of a potent chaotrope in addition to water stress is currently unrivaled. While current isolates limit perchlorate-coupled Mo-CODH based CO oxidation to cryptic perchlorate reducing haloarchaea, further exploration into both bacterial and archaeal phyla may expand this novel metabolic process to canonical perchlorate reducers.

Additionally, perchlorate accumulation, while uncommon, has been documented worldwide; CO oxidizers have also been cultivated worldwide from a wide diversity of environmental conditions, suggesting that perchlorate-coupled Mo-CODH based CO oxidation has the potential to also exist in diverse worldwide locales among diverse phyla.

Halobacterium bonnevillei PCN9T, Halobaculum saliterrae WSA2T, along with additional novel carbon monoxide-oxidizing isolate Halovenus carboxidivorans WSH3T were isolated from the Bonneville Salt Flats (Utah, USA) and surrounding saline soils and selected for further characterization (Chapter 4). All three isolates represented novel haloarchaeal species,

93 and were documented carboxidovores which contained functional Mo-CODHs. These cultivars aid in promoting a greater understanding of CO oxidation in extremely halophilic environments.

The provisional species, Halanaeroarchaeum oxyrespirans BSP3T, cultivated from salt-saturated sediment beneath the Bonneville Salt Flats, greatly expands the metabolic capabilities of its genus (Chapter 5). BSP3T is capable of growth via oxygen respiration as well as nitrate- independent perchlorate reduction, a first for haloarchaeal cryptic perchlorate reduction.

Perchlorate-coupled CO oxidation was further expanded to include carboxidotrophic Ni-CODH containing microbes using the thermophilic Firmicute Moorella glycerini DSM 11254T as a model organism (Chapter 6). The thermophilic Firmicute, Moorella glycerini DSM 11254T was able to utilize cryptic perchlorate reduction coupled to CO oxidation for growth, the biological end-product was inferred to be chlorite which abiological chlorite sinks then removed.

Additional work on Moorella glycerini should be done to further expand our understanding of perchlorate reduction in M. glycerini as well as Ni-CODH containing organisms in general. For instance, while chlorite has been inferred as the biological end-product of perchlorate reduction, the effects on cell viability have yet to be investigated. Additionally, while M. glycerini possesses a nitrate reductase it is incapable of reducing nitrate, though assumed to be responsible for perchlorate reduction as it does not possess a perchlorate reductase. However using sequence analysis the nitrate reductase is not thought to be in a periplasmic location nor contain key residues associated with known perchlorate reductases. Further phylogenetic analysis of this unique nitrate reductase along with comparisons of as yet unknown organisms capable of perchlorate-coupled Ni-CODH based CO oxidation will help elucidate the placement of M. glycerini’s nitrate reductase among known genes capable of perchlorate reduction.

Results presented here expand the known capabilities of both Mo- and Ni-CODH dependent carbon monoxide oxidizers, notably within the haloarchaea. Collectively, results indicate that perchlorate can be tolerated and exploited at elevated concentrations, along with CO oxidation, supporting the possibility that perchlorate and/or CO could sustain microbial life in terrestrial and extraterrestrial brines.

APPENDIX A. SUPPLEMENTARY MATERIAL FOR CHAPTER 2

Figure A.1. Evolutionary relationships of coxL obtained using a neighbor-joining method with a Poisson correction. Gapped and missing data were eliminated; a total of 718 positions were included in the final dataset; distances are in units of amino acid substitutions per site. Numbers at branches indicate bootstrap support > 70%. Arrows indicate isolates used in this study.

Figure A.2. Evolutionary relationships of narG obtained using a neighbor-joining method based on a Poisson correction. Gapped and missing data were eliminated; a total of 717 positions were included in the final dataset; distances are in units of amino acid substitutions per site. Numbers at branches indicate bootstrap support > 70%. Arrows indicate isolates used in this study.

Figure A.3. CO uptake by Haloarcula sp. PCN7 during aerobic incubations with no perchlorate ( ) or anaerobic incubations with 9 mM perchlorate + 1 mM chlorate ( ), 1 mM perchlorate + 9 mM chlorate ( ) or with no electron acceptor ( ) perchlorate. All values are means of triplicate determinations ± 1 standard error

APPENDIX B. SUPPLEMENTARY MATERIAL FOR CHAPTER 4

Table B.1. Buffered CM1 medium starting and post pH following incubation at 40oC for PCN9T and WSA2T or 50oC for WSH3T.

Buffer Initial pH Post 40oC Post 50oC Post Post Post (50mM) PCN9T WSA2T WSH3T MES 5.510 5.385 5.524 5.548 5.820 5.631 MES 6.001 5.925 6.021 6.778 5.906 6.085 MES 6.499 6.461 6.529 7.227 6.933 7.111 HEPES 6.995 6.931 7.018 7.804 7.774 7.600 MOPS 7.001 7.025 7.094 7.525 7.480 7.431 HEPES 7.499 7.403 7.545 8.233 7.820 7.950 Tricine 7.504 7.435 n/a 8.151 7.499 7.999 HEPES 8.004 7.920 n/a 8.531 7.884 8.150 Tricine 8.003 7.959 8.060 8.567 7.824 8.348 TAPS 8.007 7.979 8.040 8.497 8.247 8.099 CHES 8.502 8.167 8.308 8.377 8.606 8.268 CHES 9.007 8.828 8.722 8.557 8.584 8.650

Figure B.1. Maximum Parsimony analysis of taxa. The evolutionary history was inferred using the Maximum Parsimony method. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches[1]. The MP tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm (pg. 126 in ref. (Nei and Kumar, 2000) with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates). This analysis involved 19 nucleotide sequences. All positions containing gaps and missing data were eliminated (complete deletion option). There were a total of 1108 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018; Stecher et al., 2020).

100

Figure B.2. Evolutionary relationships of taxa. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 0.64022851 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and are in the units of the number of base substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 1). This analysis involved 19 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1487 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018; Stecher et al., 2020).

101

) 10

(

O 8

c a

p 6

a e

H 4

0 0 5 10 15 20 25 Time (h) Figure B.3. Carbon monoxide uptake by Halovenus sp. WSH3T. Headspace CO concentrations (ppm) versus time for WSH3T during oxic incubations at 50oC. Data are means of triplicates ±1 standard error. Curve fit represents an exponential decay to a threshold value of 0.03 ppm.

102

APPENDIX C. SUPPLEMENTARY MATERIAL FOR CHAPTER 5

>2835723008 complex iron-sulfur molybdoenzyme family reductase subunit alpha [Ga0397637_01: Halanaeroarchaeum sp. BSP3] MSEQQQEPDVDNRRRDFLKGLAATAAIGAAGVGAASQASQMNRLQVVDDP IGEYPYREWEDLYREQWDWDSISRSTHSINCTGSCSWNVYVKDGQVWREE QAADYPRINDDVPDVNPRGCNKGACYKNYVHGAQRVKYPLKRTGERGEGK WQRISWDDALDDIAQKIIDEVRKGNYDAISGFTPIPAMSPVSFASGSRMI NLLGGVSHSFYDWYSDLPPGQPITWGHQTDNAESADWYNADYIIAWGSNI NVTRIPDAKYFLEARYEGAKTVGVFTDYSQTAIHTDEWVSPDPGTDAALA LGMARTIVNEGLYDEAHLKEQSDMPMLVREDTGKFLRASEVPGMSGSDKV LVMQDGDGNLQAAPGSLGDRAGKYDPDSSIELDFDPSLDVDEIVSTTGGS VRVRSVWNRLREELERYTPDWVYNETGVGEETYQRIAREFARVDKGKIIH GKGVNDWYHNDLGNRAIQLLVTLTGHIGRRGTGVDHYVGQEKIWTFNGWK NLSFPTGSVRGMATTLWTYYHAGIMDEQPELGELIDESVSKGWMPLYPQE RDDGTRPDPSVFFAWRGNYFNQSKAGTAVQETLWPKLDLVVDINYRMDST AMYSDYVLPAASNYEKHDLSMTDMHTYVHPFTKAIEPLGESKSDWEIFRL IAERIQEKAQEANIAPTQDRSFDREIDLQSIHDDYVRDWLGDEDDALEDG LDAAEFILEHSTESNPDGEDPITFDDTVEQPQRFKAAGDHWTSDLEEDET YAPWMDFVQDKEPWPTFTGRQQYYIDHDWFLDLGEELPTHKPPIDEQDPE EYPLRYNTPHGRYSIHSTWRDDKYMLRLNRGQPVVYLHPNDARRRDIEDG DMVRMYNDKGEISAMAKIYPSGEEGVAKMPFAWEYYQFPEEGNFNNLTSM TMKPTQLVQYPEDTGEHLHFFPNFWGPTGVNSDARIEVEKKGGDDQ

Figure C.1. Key perchlorate reductase residue positions in potential BSP3T pNar. α = F; β = Y; γ = E

103

Figure C.2A.

Figure C.2B.

Figure C.2. Growth (protein µg/mL) and perchlorate reduced (µM) observed during BSP3T growth on perchlorate using varied inoculum source. Perchlorate + Thiosulfate (nitrate grown) (■). Perchlorate + Thiosulfate (perchlorate grown) (○) (A) Protein (µg/mL) time course of BSP3T growth via perchlorate reduction. (B) Perchlorate (µM) reduction time course of BSP3T during growth. All values are means of triplicate cultures with standard error expressed.

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Figure C.3A.

Figure C.3B.

Figure C.3. BSP3T perchlorate reduction and cell viability in biotic and abiotic conditions. (A) Perchlorate reduction in live cells (■), autoclaved killed cells (●), and no cell – media blanks (▲). (B) Cell growth expressed as total increase in protein concentration (µg/mL) under abiotic and biotic conditions. All values are means of triplicate cultures with standard error expressed.

105

Figure C.4. Chlorate accumulation after BSP3T growth via perchlorate reduction in the presence of three different electron donors, pyruvate (square), succinate (circle), and sodium fumarate (triangle), with (open) or without (closed) the presence of thiosulfate. All values are means of triplicate cultures with standard error expressed.

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APPENDIX D. PERMISSION TO REPRODUCE CHAPTERS 2 AND 3

Marisa R Myers Fri 2/19/2021 2:51 PM To: [email protected]

To whom it may concern,

I intend to include the following work (cited below) as a chapter in my upcoming doctoral dissertation and would like to clarify your publication policy. Your policies and publication ethics notes "Frontiers allows the inclusion of content which first appeared in an author’s thesis so long as this is the only form in which it has appeared, is in line with the author’s university policy, and can be accessed online." I'm trying to locate and include a publication policy in which the publication was completed prior to appearing a thesis/dissertation to note in my dissertation appendix. If you could please state or link the policy it would be appreciated.

Work: Myers, Marisa R., and Gary M. King. "Perchlorate-coupled carbon monoxide (CO) oxidation: evidence for a plausible microbe-mediated reaction in Martian brines." Frontiers in microbiology 8 (2017): 2571. Myers, Marisa R., and Gary M. King. "Addendum: Perchlorate-Coupled carbon monoxide (CO) oxidation: evidence for a plausible Microbe-Mediated reaction in Martian brines." Frontiers in Microbiology 10 (2019): 1158.

Many thanks, Marisa Myers [email protected]

107

Dear Marisa,

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108

APPENDIX E. PERMISSION TO REPRODUCE CHAPTER 4

Marisa R Myers Fri 2/19/2021 3:04 PM To: [email protected]

To whom it may concern,

I intend to include the following work (cited below) as a chapter in my upcoming doctoral dissertation and would like to clarify your publication policy. If you could please clarify and share the process/paperwork I need to complete for this it would be appreciated.

Work: Myers, Marisa R., and G. M. King. "Halobacterium bonnevillei sp. nov., Halobaculum saliterrae sp. nov. and Halovenus carboxidivorans sp. nov., three novel carbon monoxide-oxidizing Halobacteria from saline crusts and soils." International journal of systematic and evolutionary microbiology 70.7 (2020): 4261-4268.

Many thanks, Marisa Myers [email protected]

109

Hi Marisa,

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VITA

Marisa L.R. Myers was born in Houma, Louisiana in August 1992 to Marion and Lisa Russell

(née Siemssen). Marisa graduated high school summa cum laude in 2010 from Silliman Institute in Clinton, Louisiana. In May 2014 she graduated college with a Bachelor of Science degree in

Microbiology with a minor in Chemistry from Louisiana State University (Baton Rouge,

Louisiana). In June 2014 she married David F. Myers. In Fall 2014 she began her doctoral studies in Biological Sciences at Louisiana State University in the laboratory of Dr. Gary M.

King and will defend her dissertation in April 2021.

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