Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2014)

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.

Dissimilatory 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.

Shallow 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.

To 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.

The 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).

The 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.

    • Article
    • Peer Reviewed
    LBL Publications (2015)
    Two (per)chlorate-reducing bacteria, strains CUZ and NSS, were isolated from marine sediments in Berkeley and San Diego, CA, respectively. Strain CUZ respired both perchlorate and chlorate [collectively designated (per)chlorate], while strain NSS respired only chlorate. Phylogenetic analysis classified both strains as close relatives of the gammaproteobacterium Sedimenticola selenatireducens. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) preparations showed the presence of rod-shaped, motile cells containing one polar flagellum. Optimum growth for strain CUZ was observed at 25 to 30 °C, pH 7, and 4% NaCl, while strain NSS grew optimally at 37 to 42 °C, pH 7.5 to 8, and 1.5 to 2.5% NaCl. Both strains oxidized hydrogen, sulfide, various organic acids, and aromatics, such as benzoate and phenylacetate, as electron donors coupled to oxygen, nitrate, and (per)chlorate or chlorate as electron acceptors. The draft genome of strain CUZ carried the requisite (per)chlorate reduction island (PRI) for (per)chlorate respiration, while that of strain NSS carried the composite chlorate reduction transposon responsible for chlorate metabolism. The PRI of strain CUZ encoded a perchlorate reductase (Pcr), which reduced both perchlorate and chlorate, while the genome of strain NSS included a gene for a distinct chlorate reductase (Clr) that reduced only chlorate. When both (per)chlorate and nitrate were present, (per)chlorate was preferentially utilized if the inoculum was pregrown on (per)chlorate. Historically, (per)chlorate-reducing bacteria (PRB) and chlorate-reducing bacteria (CRB) have been isolated primarily from freshwater, mesophilic environments. This study describes the isolation and characterization of two highly related marine halophiles, one a PRB and the other a CRB, and thus broadens the known phylogenetic and physiological diversity of these unusual metabolisms.
      Cover page: Phenotypic and genotypic description of Sedimenticola selenatireducens strain CUZ, a marine (per)chlorate-respiring gammaproteobacterium, and its close relative the chlorate-respiring Sedimenticola strain NSS.
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2014)
      We examined the potential for CH4 oxidation to be coupled with oxygen derived from the dissimilatory reduction of perchlorate, chlorate, or via chlorite (ClO(-) 2) dismutation. Although dissimilatory reduction of ClO(-) 4 and ClO(-) 3 could be inferred from the accumulation of chloride ions either in spent media or in soil slurries prepared from exposed freshwater lake sediment, neither of these oxyanions evoked methane oxidation when added to either anaerobic mixed cultures or soil enriched in methanotrophs. In contrast, ClO(-) 2 amendment elicited such activity. Methane (0.2 kPa) was completely removed within several days from the headspace of cell suspensions of Dechloromonas agitata CKB incubated with either Methylococcus capsulatus Bath or Methylomicrobium album BG8 in the presence of 5 mM ClO(-) 2. We also observed complete removal of 0.2 kPa CH4 in bottles containing soil enriched in methanotrophs when co-incubated with D. agitata CKB and 10 mM ClO(-) 2. However, to be effective these experiments required physical separation of soil from D. agitata CKB to allow for the partitioning of O2 liberated from chlorite dismutation into the shared headspace. Although a link between ClO(-) 2 and CH4 consumption was established in soils and cultures, no upstream connection with either ClO(-) 4 or ClO(-) 3 was discerned. This result suggests that the release of O2 during enzymatic perchlorate reduction was negligible, and that the oxygen produced was unavailable to the aerobic methanotrophs.
        Cover page: Methane oxidation linked to chlorite dismutation
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2018)
        Dissimilatory perchlorate reduction is an anaerobic respiratory pathway that in communities might be influenced by metabolic interactions. Because the genes for perchlorate reduction are horizontally transferred, previous studies have been unable to identify uncultivated perchlorate-reducing populations. Here we recovered metagenome-assembled genomes from perchlorate-reducing sediment enrichments and employed a manual scaffolding approach to reconstruct gene clusters for perchlorate reduction found within mobile genetic elements. De novo assembly and binning of four enriched communities yielded 48 total draft genomes. In addition to canonical perchlorate reduction gene clusters and taxa, a new type of gene cluster with an alternative perchlorate reductase was identified. Phylogenetic analysis indicated past exchange between these gene clusters, and the presence of plasmids with either gene cluster shows that the potential for gene transfer via plasmid persisted throughout enrichment. However, a majority of genomes in each community lacked perchlorate reduction genes. Putative chlorate-reducing or sulfur-reducing populations were dominant in most communities, supporting the hypothesis that metabolic interactions might result from perchlorate reduction intermediates and byproducts. Other populations included a novel phylum-level lineage (Ca. Muirbacteria) and epibiotic prokaryotes with no known role in perchlorate reduction. These results reveal unexpected genetic diversity, suggest that perchlorate-reducing communities involve substantial metabolic interactions, and encourage expanded strategies to further understand the evolution and ecology of this metabolism.
          Cover page: Genome-resolved metagenomics identifies genetic mobility, metabolic interactions, and unexpected diversity in perchlorate-reducing communities
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2018)
          Dissimilatory phosphite oxidation (DPO), a microbial metabolism by which phosphite (HPO32-) is oxidized to phosphate (PO43-), is the most energetically favorable chemotrophic electron-donating process known. Only one DPO organism has been described to date, and little is known about the environmental relevance of this metabolism. In this study, we used 16S rRNA gene community analysis and genome-resolved metagenomics to characterize anaerobic wastewater treatment sludge enrichments performing DPO coupled to CO2 reduction. We identified an uncultivated DPO bacterium, Candidatus Phosphitivorax (Ca. P.) anaerolimi strain Phox-21, that belongs to candidate order GW-28 within the Deltaproteobacteria, which has no known cultured isolates. Genes for phosphite oxidation and for CO2 reduction to formate were found in the genome of Ca. P. anaerolimi, but it appears to lack any of the known natural carbon fixation pathways. These observations led us to propose a metabolic model for autotrophic growth by Ca. P. anaerolimi whereby DPO drives CO2 reduction to formate, which is then assimilated into biomass via the reductive glycine pathway.
            Cover page: Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2 fixation pathway
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2015)

            Unlabelled

            The pathways involved in aromatic compound oxidation under perchlorate and chlorate [collectively known as (per)chlorate]-reducing conditions are poorly understood. Previous studies suggest that these are oxygenase-dependent pathways involving O2 biogenically produced during (per)chlorate respiration. Recently, we described Sedimenticola selenatireducens CUZ and Dechloromarinus chlorophilus NSS, which oxidized phenylacetate and benzoate, two key intermediates in aromatic compound catabolism, coupled to the reduction of perchlorate or chlorate, respectively, and nitrate. While strain CUZ also oxidized benzoate and phenylacetate with oxygen as an electron acceptor, strain NSS oxidized only the latter, even at a very low oxygen concentration (1%, vol/vol). Strains CUZ and NSS contain similar genes for both the anaerobic and aerobic-hybrid pathways of benzoate and phenylacetate degradation; however, the key genes (paaABCD) encoding the epoxidase of the aerobic-hybrid phenylacetate pathway were not found in either genome. By using transcriptomics and proteomics, as well as by monitoring metabolic intermediates, we investigated the utilization of the anaerobic and aerobic-hybrid pathways on different electron acceptors. For strain CUZ, the results indicated utilization of the anaerobic pathways with perchlorate and nitrate as electron acceptors and of the aerobic-hybrid pathways in the presence of oxygen. In contrast, proteomic results suggest that strain NSS may use a combination of the anaerobic and aerobic-hybrid pathways when growing on phenylacetate with chlorate. Though microbial (per)chlorate reduction produces molecular oxygen through the dismutation of chlorite (ClO2(-)), this study demonstrates that anaerobic pathways for the degradation of aromatics can still be utilized by these novel organisms.

            Importance

            S. selenatireducens CUZ and D. chlorophilus NSS are (per)chlorate- and chlorate-reducing bacteria, respectively, whose genomes encode both anaerobic and aerobic-hybrid pathways for the degradation of phenylacetate and benzoate. Previous studies have shown that (per)chlorate-reducing bacteria and chlorate-reducing bacteria (CRB) can use aerobic pathways to oxidize aromatic compounds in otherwise anoxic environments by capturing the oxygen produced from chlorite dismutation. In contrast, we demonstrate that S. selenatireducens CUZ is the first perchlorate reducer known to utilize anaerobic aromatic degradation pathways with perchlorate as an electron acceptor and that it does so in preference over the aerobic-hybrid pathways, regardless of any oxygen produced from chlorite dismutation. D. chlorophilus NSS, on the other hand, may be carrying out anaerobic and aerobic-hybrid processes simultaneously. Concurrent use of anaerobic and aerobic pathways has not been previously reported for other CRB or any microorganisms that encode similar pathways of phenylacetate or benzoate degradation and may be advantageous in low-oxygen environments.
              Cover page: (Per)Chlorate-Reducing Bacteria Can Utilize Aerobic and Anaerobic Pathways of Aromatic Degradation with (Per)Chlorate as an Electron Acceptor
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2013)
              A novel dissimilatory perchlorate-reducing bacterium (DPRB), Arcobacter sp. strain CAB, was isolated from a marina in Berkeley, CA. Phylogenetically, this halophile was most closely related to Arcobacter defluvii strain SW30-2 and Arcobacter ellisii. With acetate as the electron donor, strain CAB completely reduced perchlorate (ClO4(-)) or chlorate (ClO3(-)) [collectively designated (per)chlorate] to innocuous chloride (Cl(-)), likely using the perchlorate reductase (Pcr) and chlorite dismutase (Cld) enzymes. When grown with perchlorate, optimum growth was observed at 25 to 30°C, pH 7, and 3% NaCl. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) preparations were dominated by free-swimming straight rods with 1 to 2 polar flagella per cell. Strain CAB utilized a variety of organic acids, fructose, and hydrogen as electron donors coupled to (per)chlorate reduction. Further, under anoxic growth conditions strain CAB utilized the biogenic oxygen produced as a result of chlorite dismutation to oxidize catechol via the meta-cleavage pathway of aerobic catechol degradation and the catechol 2,3-dioxygenase enzyme. In addition to (per)chlorate, oxygen and nitrate were alternatively used as electron acceptors. The 3.48-Mb draft genome encoded a distinct perchlorate reduction island (PRI) containing several transposases. The genome lacks the pcrC gene, which was previously thought to be essential for (per)chlorate reduction, and appears to use an unrelated Arcobacter c-type cytochrome to perform the same function. IMPORTANCE The study of dissimilatory perchlorate-reducing bacteria (DPRB) has largely focused on freshwater, mesophilic, neutral-pH environments. This study identifies a novel marine DPRB in the genus Arcobacter that represents the first description of a DPRB associated with the Campylobacteraceae. Strain CAB is currently the only epsilonproteobacterial DPRB in pure culture. The genome of strain CAB lacks the pcrC gene found in all other DPRB tested, demonstrating a new variation on the (per)chlorate reduction pathway. The ability of strain CAB to oxidize catechol via the oxygenase-dependent meta-cleavage pathway in the absence of external oxygen by using the biogenic oxygen produced from the dismutation of chlorite provides a valuable model for understanding the anaerobic degradation of a broad diversity of xenobiotics which are recalcitrant to anaerobic metabolism but labile to oxygenase-dependent mechanisms.
                Cover page: Physiological and Genetic Description of Dissimilatory Perchlorate Reduction by the Novel Marine Bacterium Arcobacter sp. Strain CAB
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2015)
                Two (per)chlorate-reducing bacteria, strains CUZ and NSS, were isolated from marine sediments in Berkeley and San Diego, CA, respectively. Strain CUZ respired both perchlorate and chlorate [collectively designated (per)chlorate], while strain NSS respired only chlorate. Phylogenetic analysis classified both strains as close relatives of the gammaproteobacterium Sedimenticola selenatireducens. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) preparations showed the presence of rod-shaped, motile cells containing one polar flagellum. Optimum growth for strain CUZ was observed at 25 to 30 °C, pH 7, and 4% NaCl, while strain NSS grew optimally at 37 to 42 °C, pH 7.5 to 8, and 1.5 to 2.5% NaCl. Both strains oxidized hydrogen, sulfide, various organic acids, and aromatics, such as benzoate and phenylacetate, as electron donors coupled to oxygen, nitrate, and (per)chlorate or chlorate as electron acceptors. The draft genome of strain CUZ carried the requisite (per)chlorate reduction island (PRI) for (per)chlorate respiration, while that of strain NSS carried the composite chlorate reduction transposon responsible for chlorate metabolism. The PRI of strain CUZ encoded a perchlorate reductase (Pcr), which reduced both perchlorate and chlorate, while the genome of strain NSS included a gene for a distinct chlorate reductase (Clr) that reduced only chlorate. When both (per)chlorate and nitrate were present, (per)chlorate was preferentially utilized if the inoculum was pregrown on (per)chlorate. Historically, (per)chlorate-reducing bacteria (PRB) and chlorate-reducing bacteria (CRB) have been isolated primarily from freshwater, mesophilic environments. This study describes the isolation and characterization of two highly related marine halophiles, one a PRB and the other a CRB, and thus broadens the known phylogenetic and physiological diversity of these unusual metabolisms.
                  Cover page: Phenotypic and Genotypic Description of Sedimenticola selenatireducens Strain CUZ, a Marine (Per)Chlorate-Respiring Gammaproteobacterium, and Its Close Relative the Chlorate-Respiring Sedimenticola Strain NSS
                  Top