Defense Date

1-28-2020

Graduation Date

Spring 5-8-2020

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

Committee Chair

John F. Stolz

Committee Member

Partha Basu

Committee Member

Jan E. Janecka

Committee Member

Nancy Trun

Abstract

The selenium oxyanions selenate (Se(VI)) and selenite (Se(IV)) can be utilized by some bacteria and archaea as terminal electron acceptors in anaerobic respiration. Se(VI) and Se(IV) respiration is mediated by a phylogenetically and ecologically diverse array of organisms, suggesting that selenium respiration is ubiquitous in natural environments. Several respiratory Se(VI) reductases have been characterized in bacteria, revealing that Se(VI) respiration has evolved independently several times in this domain. Se(IV) respiration, in contrast, has yet to be characterized. I have purified and characterized the first respiratory Se(IV) reductase from Bacillus selenitireducens MLS10. The Se(IV) reductase appeared to purify as a single 80 kDa enzyme and contained both iron and molybdenum. The enzyme was highly specific for Se(IV). The genome of MLS10 demonstrated that this enzyme was part of an operon encoding a putative respiratory Se(IV) reductase (Srr) complex. Srr was electrophoretically purified from MLS10 periplasm using non-denaturing gels, and identified using in-gel enzyme assays for Se(IV) reducing activity. Multiple subunits from Srr were identified using liquid chromatography tandem mass spectrometry, confirming the operon codes for a Srr complex. The enzyme was designated SrrA. Phylogenetic analysis determined that SrrA was a member of the polysulfide reductase catalytic subunit (PsrA) and thiosulfate reductase catalytic subunit (PhsA) lineage of molybdopterin or tungstopterin bis(pyranopterin guanine dinucleotide) (Mo/W-bisPGD)-containing molybdoenzymes. Analysis of the operons associated with each catalytic subunit in the phylogeny revealed that putative PsrA, PhsA, and SrrA homologs did not form monophyletic clades with respect to one another. Furthermore, while putative Srr operons were not observed in archaea, Srr was present throughout the PsrA/PhsA lineage in bacteria. To determine if Srr is a reliable marker for Se(IV) respiration, I attempted to ascertain if two organisms, Bacillus beveridgei MLTeJB and Desulfitobacterium hafniense PCP-1, expressed Srr when cultivated in the presence of Se(IV). MLTeJB was shown to express SrrA when grown on both Se(VI) and Se(IV). Definitive identification of the Se(IV) reductase expressed by PCP-1 when grown on Se(VI) was not possible due to the low expression levels of the enzyme. This work represents the first physiological and evolutionary studies of Se(IV) respiration.

Language

English

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