Phylogeny and Physiology of Marine Dissimilatory Perchlorate Reducing Bacteria

Perchlorate (ClO4-) is a toxic, water-soluble oxyanion of chlorine that is naturally and anthropogenically produced. Because of its extensive industrial uses and unregulated disposal prior to 1997, perchlorate contamination of water sources is widespread. In humans, perchlorate inhibits iodide uptake by the thyroid gland, which can lead to hypothyroidism. As a result, the Environmental Protection Agency decided to regulate perchlorate under the Safe Water Drinking Act of 2012./nDissimilatory perchlorate reducing bacteria (DPRB) can grow by respiring perchlorate (ClO4-) or chlorate (ClO3-) [collectively denoted (per)chlorate] to innocuous chloride (Cl-). Even though about 80 strains of DPRB have been isolated to date (Chapter 1), most of these isolates have been obtained from freshwater, mesophilic, neutral pH environments. As a result, most of these microorganisms do not tolerate high temperatures, salinities, or extreme pH. With few exceptions, most DPRB belong to the Alpha, Beta, Gamma and Epsilon classes of the phylum Proteobacteria. The current work aims to expand the known environmental range, metabolic strategies, and taxonomic diversity of DPRB./nShallow sediment samples from a marina in Berkeley, CA were used to study marine perchlorate reduction. Enrichments were set up at 1, 3, 5, 7, and 10% NaCl with acetate as an electron donor and perchlorate as an electron acceptor; perchlorate was consumed in salinities of up to 7% NaCl (Chapter 2). Microbial community analysis revealed that the most active members of the community were in families Rhodocyclaceae (1% and 3% NaCl), Pseudomonadaceae (1% NaCl), Campylobacteraceae (1%, 5%, and 7% NaCl), Sedimenticolaceae (3% NaCl), Desulfuromonadaceae (5% & 7% NaCl), Pelobacteraceae (5% NaCl), Helicobacteraceae (5% & 7% NaCl), and V1B07b93 (7% NaCl; phylum Deferribacteres) (Chapter 2). DPRB in the genera Sedimenticola, Azoarcus, Pseudomonas, Denitromonas, and Marinobacter (Chapter 2) were isolated./nTo further study the physiology and metabolic potential of marine DPRB, an Arcobacter sp. was isolated from marine sediment from the Berkeley Bay, and was fully characterized and genome sequenced (Chapter 3). Arcobacter sp. CAB is the only perchlorate reducing bacterium (PRB) in pure culture belonging to the Epsilonproteobacteria. Interestingly, CAB lacks the pcrC gene previously thought to be essential for perchlorate reduction. Additionally, CAB can couple the oxidation of the aromatic compound catechol to perchlorate reduction in anaerobic conditions. However, it utilizes an aerobic pathway that requires oxygen as a co-substrate for an oxygenase. Thus, Arcobacter sp. CAB represents the first example of a PRB that can utilize an aerobic pathway for aromatic degradation with perchlorate as an electron acceptor by utilizing oxygen produced from chlorite dismutation in otherwise anaerobic conditions./nThe PRB Sedimenticola selenatireducens CUZ, also isolated from Berkeley Bay sediment, and the chlorate reducing bacteria (CRB) Dechloromarinus chlorophilus NSS, isolated from San Diego Bay sediment, were fully characterized and represent the first case of two highly related microorganisms (99% 16S rRNA identity), one of which is a perchlorate-reducing bacterium (PRB) and one of which is a chlorate-reducing bacterium (CRB; Chapter 4). Both strains are metabolically versatile, and can oxidize the aromatic compounds benzoate and phenylacetate coupled to the reduction of oxygen, perchlorate, and nitrate (Chapter 5). Both strains encode aerobic-hybrid and anaerobic pathways of phenylacetate and benzoate degradation. While S. selenatireducens acts as a true anaerobe and predominantly utilizes the anaerobic pathways with perchlorate as an electron acceptor, D. chlorophilus NSS may utilize a mixture of aerobic and anaerobic pathways when respiring on chlorate (Chapter 5)./nThe marine, perchlorate-reducing microbial community and novel isolates studied in this work greatly contribute to the current knowledge in the field of microbial perchlorate reduction. With the exception of Marinobacter, these new isolates represent the first known perchlorate-reducers in each genus (Chapter 2), thus expanding the known phylogeny of DPRB which are dominated by the genera Dechloromonas and Azospira (Chapter 1). Further, the characterization of the PRBs Arcobacter sp. CAB and S. selenatireducens CUZ and the CRB Dechloromarinus chlorophilus NSS has revealed several mechanisms by which DPRB degrade aromatic compounds and the choices these microorganisms make in respect to the oxygen they produce from the dismutation of chlorite. Further characterization and genome sequencing of the other novel DPRB obtained in this study is of great importance, and will likely aid in the study of the evolution of perchlorate metabolism and in understanding the role each of these isolates in microbial communities.

Advisor: Coates, John D