Kelly S. Bender

Anaerobic benzene oxidation coupled to nitrate reduction in pure culture by two strains of Dechloromonas

Benzene contamination is a significant problem. It is used in a wide range of manufacturing processes and is a primary component of petroleum-based fuels. Benzene is a hydrocarbon that is soluble, mobile, toxic and stable, especially in ground and surface waters. It is poorly biodegraded in the absence of oxygen. However, anaerobic benzene biodegradation has been documented under various conditions. Although benzene biomineralization has been demonstrated with nitrate, Fe(III), sulphate or CO2 as alternative electron acceptors, these studies were based on sediments or microbial enrichments. Until now there were no organisms in pure culture that degraded benzene anaerobically. Here we report two Dechloromonas strains, RCB and JJ, that can completely mineralize various mono-aromatic compounds including benzene to CO2 in the absence of O2 with nitrate as the electron acceptor. This is the first example, to our knowledge, of an organism of any type that can oxidize benzene anaerobically, and we demonstrate the potential applicability of these organisms to the treatment of contaminated environments.

Identification, Characterization, and Classification of Genes Encoding Perchlorate Reductase

The reduction of perchlorate to chlorite, the first enzymatic step in the bacterial reduction of perchlorate, is catalyzed by perchlorate reductase. The genes encoding perchlorate reductase (pcrABCD) in two Dechloromonas species were characterized. Sequence analysis of the pcrAB gene products revealed similarity to alpha- and beta-subunits of microbial nitrate reductase, selenate reductase, dimethyl sulfide dehydrogenase, ethylbenzene dehydrogenase, and chlorate reductase, all of which are type II members of the microbial dimethyl sulfoxide (DMSO) reductase family. The pcrC gene product was similar to a c-type cytochrome, while the pcrD gene product exhibited similarity to molybdenum chaperone proteins of the DMSO reductase family members mentioned above. Expression analysis of the pcrA gene from Dechloromonas agitata indicated that transcription occurred only under anaerobic (per)chlorate-reducing conditions. The presence of oxygen completely inhibited pcrA expression regardless of the presence of perchlorate, chlorate, or nitrate. Deletion of the pcrA gene in Dechloromonas aromatica abolished growth in both perchlorate and chlorate but not growth in nitrate, indicating that the pcrABCD genes play a functional role in perchlorate reduction separate from nitrate reduction. Phylogenetic analysis of PcrA and other alpha-subunits of the DMSO reductase family indicated that perchlorate reductase forms a monophyletic group separate from chlorate reductase of Ideonella dechloratans. The separation of perchlorate reductase as an activity distinct from chlorate reductase was further supported by DNA hybridization analysis of (per)chlorate- and chlorate-reducing strains using the pcrA gene as a probe.

Gibberellin biosynthesis in bacteria: Separate ent-copalyl diphosphate and ent-kaurene synthases in Bradyrhizobium japonicum

Gibberellins are ent‐kaurene‐derived diterpenoid phytohormones produced by plants, fungi, and bacteria. The distinct gibberellin biosynthetic pathways in plants and fungi are known, but not that in bacteria. Plants typically use two diterpene synthases to form ent‐kaurene, while fungi use only a single bifunctional diterpene synthase. We demonstrate here that Bradyrhizobium japonicum encodes separate ent‐copalyl diphosphate and ent‐kaurene synthases. These are found in an operon whose enzymatic composition indicates that gibberellin biosynthesis in bacteria represents a third independently assembled pathway relative to plants and fungi. Nevertheless, sequence comparisons also suggest potential homology between diterpene synthases from bacteria, plants, and fungi.

Metabolic Primers for Detection of (Per)chlorate-Reducing Bacteria in the Environment and Phylogenetic Analysis of cld Gene Sequences

ABSTRACT Natural attenuation of the environmental contaminant perchlorate is a cost-effective alternative to current removal methods. The success of natural perchlorate remediation is dependent on the presence and activity of dissimilatory (per)chlorate-reducing bacteria (DPRB) within a target site. To detect DPRB in the environment, two degenerate primer sets targeting the chlorite dismutase (cld) gene were developed and optimized. A nested PCR approach was used in conjunction with these primer sets to increase the sensitivity of the molecular detection method. Screening of environmental samples indicated that all products amplified by this method were cld gene sequences. These sequences were obtained from pristine sites as well as contaminated sites from which DPRB were isolated. More than one cld phylotype was also identified from some samples, indicating the presence of more than one DPRB strain at those sites. The use of these primer sets represents a direct and sensitive molecular method for the qualitative detection of (per)chlorate-reducing bacteria in the environment, thus offering another tool for monitoring natural attenuation. Sequences of cld genes isolated in the course of this project were also generated from various DPRB and provided the first opportunity for a phylogenetic treatment of this metabolic gene. Comparisons of the cld and 16S ribosomal DNA (rDNA) gene trees indicated that the cld gene does not track 16S rDNA phylogeny, further implicating the possible role of horizontal transfer in the evolution of (per)chlorate respiration.

Sequencing and Transcriptional Analysis of the Chlorite Dismutase Gene of Dechloromonas agitata and Its Use as a Metabolic Probe

ABSTRACT The dismutation of chlorite into chloride and O2 represents a central step in the reductive pathway of perchlorate that is common to all dissimilatory perchlorate-reducing bacteria and is mediated by a single enzyme, chlorite dismutase. The chlorite dismutase gene cld was isolated and sequenced from the perchlorate-reducing bacterium Dechloromonas agitata strain CKB. Sequence analysis identified an open reading frame of 834 bp that would encode a mature protein with an N-terminal sequence identical to that of the previously purified D. agitata chlorite dismutase enzyme. The predicted translation product of the D. agitata cld gene is a protein of 277 amino acids (aa), including a leader peptide of 26 aa. Primer extension analysis identified a single transcription start site directly downstream of an AT-rich region that could represent the −10 promoter region of the D. agitata cld gene. Northern blot analysis indicated that the cld gene was transcriptionally up-regulated when D. agitata cells were grown in perchlorate-reducing versus aerobic conditions. Slot blot hybridizations with a D. agitata cld probe demonstrated the conservation of the cld gene among perchlorate-reducing bacteria. This study represents the first description of a functional gene associated with microbial perchlorate reduction.

Development of a Markerless Genetic Exchange System for Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency

ABSTRACT In recent years, the genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress. In spite of this progress, the current marker exchange deletion method does not allow for easy selection of multiple sequential gene deletions in a single strain because of the limited number of selectable markers available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless deletion system has been developed. The counterselectable marker that makes this deletion system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by upp. In wild-type D. vulgaris, growth was shown to be inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU), whereas a mutant bearing a deletion of the upp gene was resistant to 5-FU. When a plasmid containing the wild-type upp gene expressed constitutively from the aph(3′)-II promoter (promoter for the kanamycin resistance gene in Tn5) was introduced into the upp deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a two-step integration and excision strategy for the deletion of genes of interest. Since this in-frame deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated in a single strain without the accumulation of genes conferring antibiotic resistances. We used this strategy to generate a deletion strain lacking the endonuclease (hsdR, DVU1703) of a type I restriction-modification system that we designated JW7035. The transformation efficiency of the JW7035 strain was found to be 100 to 1,000 times greater than that of the wild-type strain when stable plasmids were introduced via electroporation.

The RNA chaperone Hfq is important for growth and stress tolerance in Francisella novicida.

The RNA-binding protein Hfq is recognized as an important regulatory factor in a variety of cellular processes, including stress resistance and pathogenesis. Hfq has been shown in several bacteria to interact with small regulatory RNAs and act as a post-transcriptional regulator of mRNA stability and translation. Here we examined the impact of Hfq on growth, stress tolerance, and gene expression in the intracellular pathogen Francisella novicida. We present evidence of Hfq involvement in the ability of F. novicida to tolerate several cellular stresses, including heat-shock and oxidative stresses, and alterations in hfq gene expression under these conditions. Furthermore, expression of numerous genes, including several associated with virulence, is altered in a hfq mutant strain suggesting they are regulated directly or indirectly by Hfq. Strikingly, we observed a delayed entry into stationary phase and increased biofilm formation in the hfq mutant. Together, these data demonstrate a critical role for Hfq in F. novicida growth and survival.

The Biochemistry and Genetics of Microbial Perchlorate Reduction

The identification and analysis of the genes encoding perchlorate reductase and chlorite dismutase has provided not only a building block for pathway understanding, but has also provided a tool for bioremediative and phylogenetic studies. On-going genome sequencing will further facilitate transcriptional profiling under perchlorate-reducing conditions via microarray analyses. This analysis will give a more inclusive look into transcriptional expression patterns associated with the perchlorate metabolism. While further advancements in the genetic analysis of perchlorate-reducing bacteria continue, the recent development of a genetic system in D. aromatica will provide an invaluable tool for corroborating microarray results and solidifying hypotheses regarding microbial perchlorate metabolism.

Impacts of detrital nano- and micro-scale particles (dNP) on contaminant dynamics in a coal mine AMD treatment system

Pollutants in acid mine drainage (AMD) are usually sequestered in neoformed nano- and micro-scale particles (nNP) through precipitation, co-precipitation, and sorption. Subsequent biogeochemical processes may control nNP stability and thus long-term contaminant immobilization. Mineralogical, chemical, and microbiological data collected from sediments accumulated over a six-year period in a coal-mine AMD treatment system were used to identify the pathways of contaminant dynamics. We present evidence that detrital nano- and micron-scale particles (dNP), composed mostly of clay minerals originating from the partial weathering of coal-mine waste, mediated biogeochemical processes that catalyzed AMD contaminant (1) immobilization by facilitating heterogeneous nucleation and growth of nNP in oxic zones, and (2) remobilization by promoting phase transformation and reductive dissolution of nNP in anoxic zones. We found that dNP were relatively stable under acidic conditions and estimated a dNP content of ~0.1g/L in the influent AMD. In the AMD sediments, the initial nNP precipitates were schwertmannite and poorly crystalline goethite, which transformed to well-crystallized goethite, the primary nNP repository. Subsequent reductive dissolution of nNP resulted in the remobilization of up to 98% of S and 95% of Fe accompanied by the formation of a compact dNP layer. Effective treatment of pollutants could be enhanced by better understanding the complex, dynamic role dNP play in mediating biogeochemical processes and contaminant dynamics at coal-mine impacted sites.

Environmental (Per)chlorate Reduction: a collaborative effort in syntrophy?

Biogeochemical cycling of nitrogen, iron, sulfur and even arsenic are common themes of microbial metabolisms. While microbiologists do not readily think about the chlorine cycle, the chloroxyanions perchlorate (ClO24 ) and chlorate (ClO23 ) possess ideal redox potentials for accepting electrons (E0 5 1.28 V and 1.03 V respectively). Perchlorate and chlorate (collectively termed (per)chlorate) are naturally occurring and manufactured anions. Concern over environmental levels of perchlorate stems from its ability to interfere with the uptake of iodine by the thyroid gland. Because of this toxicity, the US has implemented an advisory level of 15 mg L for (per)chlorate (Bardiya and Bae, 2011). While chemical treatment methods are possible, microbial reduction of (per)chlorate is a safe and cost-effective mitigation strategy. Over the past 25 years, numerous (per)chlorateand chlorate-reducing Bacteria and Archaea have been isolated and characterized (as reviewed by Mart ınezEspinosa et al., 2015; Nilsson et al., 2013; Oren et al., 2014). While leveraging the use of environmental contaminants for growth is not a new aspect of microbiology, the phylogenetic, metabolic and genomic diversity associated with microbial respiration of chloroxyanions suggests a fascinating evolutionary history. Central to this diversity are the enzymes involved in the respiration of chloroxyanions. Microorganisms that can couple growth to the reduction of both perchlorate and chlorate possess a perchlorate reductase enzyme (PcrAB), which facilitates sequential reduction of perchlorate to chlorate and chlorate to chlorite. In contrast, chlorate reducers possess the chlorate reductase (ClrABC), which can only reduce chlorate to chlorite. While both reductases belong to the molybdenum-containing type II DMSO reductase superfamily (McDevitt et al., 2002), sequence analysis suggests separate evolutionary tracts for each reductase (Bender et al., 2005; Clark et al., 2013; Nilsson et al., 2013). Once chlorite has been generated, the enzyme chlorite dismutase (Cld) is essential for converting toxic chlorite to chloride and O2. This unique bioproduction of O2 provides an additional electron acceptor for respiration and energy generation. While the origin of chloroxyanion respiration is currently unknown, extensive genomic comparisons by the Coates laboratory have determined that horizontal transfer, specifically transposition of genomic islands, is responsible for disseminating dissimilatory (per)chlorate reduction (Clark et al., 2013, 2014; Melnyk et al., 2011). This is exemplified by the inactivation of the nitrate reductase (nar) operon by a genomic island encoding chlorate reduction in some Pseudomonas strains (Clark et al., 2016). Overall, these analyses suggest that the pathway for (per)chlorate reduction has evolved multiple times from type II DMSO enzymes and the acquisition of a chlorite dismutase (Clark et al., 2013). In this issue of Environmental Microbiology, Clark and colleagues leverage high-throughput genetic tools for the first in-depth genetic analysis of the chlorate reduction pathway in Pseudomonas stutzeri strain PDA. Using elegant barcoded transposon mutant competition assays, they were able to determine that individual chlorite dismutase and chlorate reductase mutants could not grow using chlorate as an electron acceptor. However, the two separate mutants could cooperatively respire chlorate through complementation. This finding was further supported by the recovery of dissimilatory chlorate reduction in Dcld mutants supplemented with exogenous chlorite dismutase. To determine if coordinated intraspecies reactions could lead to perchlorate reduction, Clark and coworkers mixed the wild type chlorate-reducing strain PDA with a Dcld mutant of the perchlorate respirer Azospira suillum strain PS. This experiment illustrated that cooperative *For correspondence. E-mail bender@micro.siu.edu; Tel. 618-4532868; Fax: 618-453-8036