UC Berkeley

The anion perchlorate (ClO4-) is a soluble compound that accumulates very slowly in the natural environment as the result of an unknown mechanism. Owing to its excellent oxidizing capability, large quantities of various perchlorate salts are also generated anthropogenically as components of various types of explosives and rocket fuel. Unregulated disposal of perchlorate as well as accidental release has led to elevated amounts of perchlorate in drinking water sources, where it can interfere with thyroid function. Due to its adverse health effects, the United States Environmental Protection Agency has elected to regulate perchlorate under the Safe Water Drinking Act as of 2012.

The high solubility of perchlorate and its strong oxidizing potential make it an attractive terminal electron acceptor for bacteria, and unsurprisingly, there are bacteria that utilize it in such a manner. This metabolism depends on two key enzymes that have been studied extensively: perchlorate reductase (PcrA) and chlorite dismutase (Cld). Cloning and sequencing of these genes revealed that they are part of the same operon, and preliminary phylogenetic analysis indicated that the histories of these genes were not congruent with phylogeny based on the 16S genes of these organisms. These observations, coupled with the fact that perchlorate reducers are found in disparate taxonomic groups, led to the hypothesis that the genetic constituents of perchlorate reduction are horizontally transferred.

The acquisition of several complete (Azospira suillum PS and Dechloromonas aromatica RCB) and draft genomes (Magnetospirillum bellicus VDY and Dechloromonas agitata CKB) of dissimilatory perchlorate reducing bacteria (DPRB) were sequenced in order to understand more about the genomic basis of this metabolism. Another strain that does not reduce perchlorate, Dechloromonas sp. JJ, was also sequenced and used a baseline for comparisons. This led to the identification of a genomic island centered around the pcr and cld genes, but in addition contained many conserved genes of unknown function, with possible roles in electron transport and regulation (Chapter 1). This genomic island was designated the perchlorate reduction genomic island (PRI).

To understand the role, if any, these genes in the PRI play in perchlorate reduction metabolism, a tractable genetic system with methods for mutagenesis and complementation was needed. To fill this role, the organism Azospira suillum PS was developed into a model system for genetic manipulations (Chapter 2). Physiological characterizations were also performed to establish a baseline phenotype with respect to growth on nitrate, chlorate, and perchlorate, as well as combinations thereof. A rich media for rapid and routine aerobic culturing of PS was also developed.

Using the genetic and physiological tools developed, a library of PS transposon mutants were generated and screened for defects specifically during growth on perchlorate. Many of these transposon insertions were found to be in the genes in the PRI, so individual deletions of all 17 genes were made. Eight of these genes were essential for perchlorate reduction, and complementation of each of the eight deletions restored the phenotype. These genetic methods were instrumental in defining the set of essential genes for perchlorate reduction in PS. (Chapter 2).

Several of the genes in the PRI were not essential for perchlorate reduction including homologs of the sigF-nrsF sigma factor/anti-sigma factor system. In PS, this system is responsive to chlorite stress, and governs transcription of an adjacent operon in the PRI (Chapter 3). The genes in this operon consist of a small periplasmic methionine-rich protein (mrpX) and a putative methionine sulfoxide reductase (yedYZ). This system responds to the toxic molecule hypochlorite produced as a side product during perchlorate reduction, which would be specifically scavenged in the periplasm by the MrpX protein and regenerated by YedYZ. A comparative genomic analysis indicates that this mechanism is broadly conserved in the Proteobacteria, and tends to be enriched in organisms with known associations with eukaryotic hosts, both plant and animal.

Further sequencing of DPRB isolates has led to the accumulation of a large library of complete and draft genomes, many of which are from the same genus or species. A comprehensive phylogenomic analysis of these organisms revealed the full extent to which horizontal transfer of the PRI has shaped this metabolism (Chapter 4). In two separate instances, the PRI of divergent members of the same species exhibits almost complete nucleotide identity, indicating that the PRI is under both strong positive selection and very frequent horizontal gene transfer. Additionally, several of the accessory genes in the PRI are polyphyletic and individual lineages can be traced to genes in the chromosomes of phylogenetically similar organisms.

The work presented in this dissertation represents an important transition in the study of the molecular biology of perchlorate reduction. The identification of the PRI provided a plausible mechanism for how this metabolism is horizontally inherited. The genetic tools developed for Azospira suillum PS led to the discovery of many novel genes and also confirmed the important role that reactive chlorine species play in this metabolism. Finally, the large library of DPRB genomes and comparative analysis decribed here provide a solid base as perchlorate reduction research increasingly moves towards community analysis and in situ function.