Detecting nitrous oxide reductase (NosZ) genes in soil metagenomes: method development and implications for the nitrogen cycle.

UNLABELLED: Microbial activities in soils, such as (incomplete) denitrification, represent major sources of nitrous oxide (N2O), a potent greenhouse gas. The key enzyme for mitigating N2O emissions is NosZ, which catalyzes N2O reduction to N2. We recently described "atypical" functional NosZ proteins encoded by both denitrifiers and nondenitrifiers, which were missed in previous environmental surveys (R. A. Sanford et al., Proc. Natl. Acad. Sci. U. S. A. 109:19709-19714, 2012, doi:10.1073/pnas.1211238109). Here, we analyzed the abundance and diversity of both nosZ types in whole-genome shotgun metagenomes from sandy and silty loam agricultural soils that typify the U.S. Midwest corn belt. First, different search algorithms and parameters for detecting nosZ metagenomic reads were evaluated based on in silico-generated (mock) metagenomes. Using the derived cutoffs, 71 distinct alleles (95% amino acid identity level) encoding typical or atypical NosZ proteins were detected in both soil types. Remarkably, more than 70% of the total nosZ reads in both soils were classified as atypical, emphasizing that prior surveys underestimated nosZ abundance. Approximately 15% of the total nosZ reads were taxonomically related to Anaeromyxobacter, which was the most abundant genus encoding atypical NosZ-type proteins in both soil types. Further analyses revealed that atypical nosZ genes outnumbered typical nosZ genes in most publicly available soil metagenomes, underscoring their potential role in mediating N2O consumption in soils. Therefore, this study provides a bioinformatics strategy to reliably detect target genes in complex short-read metagenomes and suggests that the analysis of both typical and atypical nosZ sequences is required to understand and predict N2O flux in soils. IMPORTANCE: Nitrous oxide (N2O) is a potent greenhouse gas with ozone layer destruction potential. Microbial activities control both the production and the consumption of N2O, i.e., its conversion to innocuous dinitrogen gas (N2). Until recently, consumption of N2O was attributed to bacteria encoding "typical" nitrous oxide reductase (NosZ). However, recent phylogenetic and physiological studies have shown that previously uncharacterized, functional, "atypical" NosZ proteins are encoded in genomes of diverse bacterial groups. The present study revealed that atypical nosZ genes outnumbered their typical counterparts, highlighting their potential role in N2O consumption in soils and possibly other environments. These findings advance our understanding of the diversity of microbes and functional genes involved in the nitrogen cycle and provide the means (e.g., gene sequences) to study N2O fluxes to the atmosphere and associated climate change.

The chimeric genome of Sphaerochaeta: nonspiral spirochetes that break with the prevalent dogma in spirochete biology.

UNLABELLED: Spirochaetes is one of a few bacterial phyla that are characterized by a unifying diagnostic feature, namely, the helical morphology and motility conferred by axial periplasmic flagella. Their unique morphology and mode of propulsion also represent major pathogenicity factors of clinical spirochetes. Here we describe the genome sequences of two coccoid isolates of the recently described genus Sphaerochaeta which are members of the phylum Spirochaetes based on 16S rRNA gene and whole-genome phylogenies. Interestingly, the Sphaerochaeta genomes completely lack the motility and associated signal transduction genes present in all sequenced spirochete genomes. Additional analyses revealed that the lack of flagella is associated with a unique, nonrigid cell wall structure hallmarked by a lack of transpeptidase and transglycosylase genes, which is also unprecedented in spirochetes. The Sphaerochaeta genomes are highly enriched in fermentation and carbohydrate metabolism genes relative to other spirochetes, indicating a fermentative lifestyle. Remarkably, most of the enriched genes appear to have been acquired from nonspirochetes, particularly clostridia, in several massive horizontal gene transfer events (>40% of the total number of genes in each genome). Such a high level of direct interphylum genetic exchange is extremely rare among mesophilic organisms and has important implications for the assembly of the prokaryotic tree of life. IMPORTANCE: Spiral shape and motility historically have been the unifying hallmarks of the phylum Spirochaetes. These features also represent important virulence factors of highly invasive pathogenic spirochetes such as the causative agents of syphilis and Lyme disease. Through the integration of genome sequencing, microscopy, and physiological studies, we conclusively show that the spiral morphology and motility of spirochetes are not universal morphological properties. In particular, we found that the genomes of the members of the recently described genus Sphaerochaeta lack the genes encoding the characteristic flagellar apparatus and, in contrast to most other spirochetes, have acquired many metabolic and fermentation genes from clostridia. These findings have major implications for the isolation and study of spirochetes, the diagnosis of spirochete-caused diseases, and the reconstruction of the evolutionary history of this important bacterial phylum. The Sphaerochaeta sp. genomes offer new avenues to link ecophysiology with the functionality and evolution of the spirochete flagellar apparatus.

Fate and origin of 1,2-dichloropropane in an unconfined shallow aquifer.

A shallow aquifer with different redox zones overlain by intensive agricultural activity was monitored for the occurrence of 1,2-dichloropropane (DCP) to assess the fate and origin of this pollutant. DCP was detected more frequently in groundwater samples collected in aerobic and nitrate-reducing zones than those collected from iron-reducing zones. Simulated DCP concentrations for groundwater entering an iron-reducing zone were calculated from a fate and transport model that included dispersion, sorption, and hydrolysis but not degradation. Simulated concentrations were well in excess of measured values, suggesting that microbial degradation occurred in the iron-reducing zone. Microcosm experiments were conducted using aquifer samples collected from iron-reducing and aerobic zones to evaluate the potential for microbial degradation of DCP and to explain field observations. Hydrogenolysis of DCP and production of monochlorinated propanes in microcosm experiments occurred only with aquifer materials collected from the iron-reducing zone, and no dechlorination was observed in microcosms established with aquifer materials collected from the aerobic zones. Careful analyses of the DCP/1,2,2-trichloropropane ratios in groundwater indicated that older fumigant formulations were responsible for the high levels of DCP present in this aquifer.

Initial Characterization of a Reductive Dehalogenase from Desulfitobacterium chlororespirans Co23.

Desulfitobacterium chlororespirans Co23 is capable of using 3-chloro-4-hydroxybenzoate as terminal electron acceptor for growth. Membrane preparations from cells grown fermentatively on pyruvate in the presence of 3-chloro-4-hydroxybenzoate dechlorinated this compound at a rate of 3.9 nmol min(sup-1) mg of protein(sup-1). Fivefold-greater dechlorination rates were measured with reduced methyl viologen as the artificial electron donor. Reduced benzyl viologen, NADH, NADPH, reduced flavin adenine dinucleotide, and reduced flavin mononucleotide could not substitute for reduced methyl viologen. The maximal initial rate of catalysis was achieved at pH 6.5 and 60(deg)C. The membrane-bound dechlorinating enzyme system was not oxygen sensitive and was stable at 57(deg)C for at least 2 h. Sulfite inhibited dechlorination in cell-free assays, whereas sulfate did not. Several chlorophenols were dehalogenated exclusively in the ortho position by cell extracts.

Dechlorination of chloroethenes is inhibited by 2-bromoethanesulfonate in the absence of methanogens.

2-Bromoethanesulfonate (BES) inhibited the reductive dechlorination of chloroethenes in several sediment-free enrichment cultures in the absence of methanogenic archaea. Archaeon-specific PCR primers confirmed the absence of methanogens in the enrichment cultures. BES should not be used to attribute dechlorination activities to methanogens.

Fraction of electrons consumed in electron acceptor reduction and hydrogen thresholds as indicators of halorespiratory physiology.

Measurements of the hydrogen consumption threshold and the tracking of electrons transferred to the chlorinated electron acceptor (f(e)) reliably detected chlororespiratory physiology in both mixed cultures and pure cultures capable of using tetrachloroethene, cis-1, 2-dichloroethene, vinyl chloride, 2-chlorophenol, 3-chlorobenzoate, 3-chloro-4-hydroxybenzoate, or 1,2-dichloropropane as an electron acceptor. Hydrogen was consumed to significantly lower threshold concentrations of less than 0.4 ppmv compared with the values obtained for the same cultures without a chlorinated compound as an electron acceptor. The f(e) values ranged from 0.63 to 0.7, values which are in good agreement with theoretical calculations based on the thermodynamics of reductive dechlorination as the terminal electron-accepting process. In contrast, a mixed methanogenic culture that cometabolized 3-chlorophenol exhibited a significantly lower f(e) value, 0.012.

Characterization of Desulfitobacterium chlororespirans sp. nov., which grows by coupling the oxidation of lactate to the reductive dechlorination of 3-chloro-4-hydroxybenzoate.

Strain Co23, an anaerobic spore-forming microorganism, was enriched and isolated from a compost soil on the basis of its ability to grow with 2,3-dichlorophenol (DCP) as its electron acceptor, ortho chlorines were removed from polysubstituted phenols but not from monohalophenols. Growth by chlororespiration was indicated by a growth yield of 3.24 g of cells per mol of reducing equivalents (as 2[H]) from lactate oxidation to acetate in the presence of 3-chloro-4-hydroxybenzoate but no growth in the absence of the halogenated electron acceptor. Other indicators of chlororespiration were the fraction of electrons from the electron donor used for dechlorination (0.67) and the H2 threshold concentration of < 1.0 ppm. Additional electron donors utilized for reductive dehalogenation were pyruvate, formate, butyrate, crotonate, and H2. Pyruvate supported homoacetogenic growth in the absence of an electron acceptor. Strain Co23 also used sulfite, thiosulfate, and sulfur as electron acceptors for growth, but it did not use sulfate, nitrate or fumarate. The temperature optimum for growth was 37 degrees C; however, the rates of dechlorination were optimum at 45 degrees C and activity persisted to temperatures as high as 55 degrees C. The 16S rRNA sequence was determined, and strain Co23 was found to be related to Desulfitobacterium dehalogenans JW/IU DC1 and Desulfitobacterium strain PCE1, with sequence similarities of 97.2 and 96.8%, respectively. The phylogenetic and physiological properties exhibited by strain Co23 place it into a new species designated Desulfitobacterium chlororespirans.

16S rRNA gene-based detection of tetrachloroethene-dechlorinating Desulfuromonas and Dehalococcoides species.

Members of the genera Desulfuromonas and Dehalococcoides reductively dechlorinate tetrachloroethene (PCE) and trichloroethene. Two primer pairs specific to hypervariable regions of the 16S rRNA genes of the Dehalococcoides group (comprising Dehalococcoides ethenogenes and Dehalococcoides sp. strain FL2) and the acetate-oxidizing, PCE-dechlorinating Desulfuromonas group (comprising Desulfuromonas sp. strain BB1 and Desulfuromonas chloroethenica) were designed. The detection threshold of a nested PCR approach using universal bacterial primers followed by a second PCR with the Desulfuromonas dechlorinator-targeted primer pair was 1 x 10(3) BB1 cells added per gram (wet weight) of sandy aquifer material. Total community DNA isolated from sediments of three Michigan rivers and six different chloroethene-contaminated aquifer samples was used as template in nested PCR. All river sediment samples yielded positive signals with the BB1- and the Dehalococcoides-targeted primers. One chloroethene-contaminated aquifer tested positive with the Dehalococcoides-targeted primers, and another contaminated aquifer tested positive with the Desulfuromonas dechlorinator-targeted primer pair. Restriction fragment analysis of the amplicons could discriminate strain BB1 from other known Desulfuromonas species. Microcosm studies confirmed the presence of PCE-dechlorinating, acetate-oxidizing Desulfuromonas and hydrogenotrophic Dehalococcoides species in samples yielding positive PCR signals with the specific primers.

Pseudomonas sp. strain 273, an aerobic alpha, omega-dichloroalkaneDegrading bacterium.

A gram-negative, aerobic bacterium was isolated from soil; this bacterium grew in 50% (vol/vol) suspensions of 1,10-dichlorodecane (1,10-DCD) as the sole source of carbon and energy. Phenotypic and small-subunit ribosomal RNA characterizations identified the organism, designated strain 273, as a member of the genus Pseudomonas. After induction with 1,10-DCD, Pseudomonas sp. strain 273 released stoichiometric amounts of chloride from C5 to C12 alpha, omega-dichloroalkanes in the presence of oxygen. No dehalogenation occurred under anaerobic conditions. The best substrates for dehalogenation and growth were C9 to C12 chloroalkanes. The isolate also grew with nonhalogenated aliphatic compounds, and decane-grown cells dechlorinated 1,10-DCD without a lag phase. In addition, cells grown on decane dechlorinated 1,10-DCD in the presence of chloramphenicol, indicating that the 1,10-DCD-dechlorinating enzyme system was also induced by decane. Other known alkane-degrading Pseudomonas species did not grow with 1,10-DCD as a carbon source. Dechlorination of 1,10-DCD was demonstrated in cell extracts of Pseudomonas sp. strain 273. Cell-free activity was strictly oxygen dependent, and NADH stimulated dechlorination, whereas EDTA had an inhibitory effect.

Complete reductive dechlorination of 1,2-dichloropropane by anaerobic bacteria.

The transformation of 1,2-dichloropropane (1,2-D) was observed in anaerobic microcosms and enrichment cultures derived from Red Cedar Creek sediment. 1-Chloropropane (1-CP) and 2-CP were detected after an incubation period of 4 weeks. After 4 months the initial amount of 1,2-D was stoichiometrically converted to propene, which was not further transformed. Dechlorination of 1,2-D was not inhibited by 2-bromoethanesulfonate. Sequential 5% (vol/vol) transfers from active microcosms yielded a sediment-free, nonmethanogenic culture, which completely dechlorinated 1,2-D to propene at a rate of 5 nmol min(sup-1) mg of protein(sup-1). No intermediate formation of 1-CP or 2-CP was detected in the sediment-free enrichment culture. A variety of electron donors, including hydrogen, supported reductive dechlorination of 1,2-D. The highest dechlorination rates were observed between 20(deg) and 25(deg)C. In the presence of 1,2-D, the hydrogen threshold concentration was below 1 ppm by volume (ppmv). In addition to 1,2-D, the enrichment culture transformed 1,1-D, 2-bromo-1-CP, tetrachloroethene, 1,1,2,2-tetrachloroethane, and 1,2-dichloroethane to less halogenated compounds. These findings extend our knowledge of the reductive dechlorination process and show that halogenated propanes can be completely dechlorinated by anaerobic bacteria.