Metagenomics-based analysis of viral communities in dairy lagoon wastewater.

Microbial populations, especially those of viruses, are poorly studied in dairy wastewater treatment operations. Here we report signature nucleic acid metagenomic sequences obtained by pyrosequencing viromes of virus-like particles that were extracted from two dairy waste treatment lagoons. The lagoons are operated in series, with Lagoon I being used as the primary stage and Lagoon II as the secondary stage of wastewater treatment. An average of 2000 sequences was obtained from each lagoon. More than 300 signatures from each lagoon matched sequences in the virus database of the National Center for Biotechnology Information (NCBI). We utilized a bioinformatics approach and transmission electron microscopy (TEM) to characterize the viral diversity and presence of potential viral pathogens within the lagoons. Our results showed differences in viral community compositions between Lagoon I and Lagoon II, suggesting that the viral community changes significantly in the transition of water between the two lagoons. Furthermore, the diverse viral community in the lagoon samples contained signature sequences of a variety of bacterial, plant, and animal viruses. Bacteriophage sequences dominated the viral community metagenomes in both lagoons. Ultimately these results can be used to identify viral bioindicators to rapidly assess wastewater treatment quality and the potential impacts of dairy operations on watersheds. Our viral metagenomic sequences have been submitted to GenBank (GPID 65805) and can provide insight into the composition and structure of viral communities within wastewaters of dairy lagoon systems.

The effects of screw placement on plate strain in 3.5 mm dynamic compression plates and limited-contact dynamic compression plates.

The objective of this study was to evaluate the effect of screw omission on plate strain during axial load to failure and cycling using a Delrin rod gap model. In addition, the differences between the 3.5 mm limited-contact dynamic compression plate (LC-DCP) and the 3.5 mm dynamic compression plate (DCP) were evaluated. Six, 12-hole LC-DCP and DCP plates were applied to Delrin rods with a 1 cm gap located within the central portion of the plate. Six screw configurations were tested with the following variations: three proximal and three distal (six open), four proximal and three distal (five open), four proximal and four distal (four open), five proximal and four distal (three open), five proximal and five distal (two open), six proximal and five distal (one open). Three strain gauges were mounted on each plate within the gap (gauge three) and extended proximally. Additionally, three constructs (six, three, and one open hole) were tested to failure in cyclic loading. The strain measured within the gap (gauge three) was significantly greater than the strain at other gauges for each screw configuration. Strain within the gap did not significantly change with any screw configuration, but did significantly increase at other locations as screws were omitted. Overall, the DCP withstood significantly more cycles than the LC-DCP. Differences were noted within the DCP group with the 6/5 screw configuration lasting for significantly more cycles than the 5/4 and 3/3 constructs. Although overall strain at the gap did not significantly increase with screw omission, the clinical significance remains to be determined.

The nature of plant growth-promoting effects of a pseudoalteromonad associated with the marine algae Laminaria japonica and linked to catalase excretion.

AIMS: The goal of this study was to identify a marine algae-associated bacterium isolated from Laminaria japonica and investigate this microorganism's growth-promoting effects on plants. METHODS AND RESULTS: The bacterium, identified as Pseudoalteromonas porphyrae, was determined to display a biostimulatory activity for seed germination and shoot growth in several agricultural plants and also for growth in ginseng callus cell culture. This biostimulatory activity was linked to a catalase enzyme that was excreted in the maximal amount during the transition from logarithmic growth phase to stationary growth phase. In addition, selected shifts in growth temperature and medium salinity affected the amount of enzyme excreted. The purified catalase was determined to be composed of identical subunits. The catalase of interest displayed significantly higher biostimulatory activity than the catalase from bovine liver. CONCLUSIONS: The catalase investigated in this study is unique in that it promotes growth in and possibly contributes to stress tolerance of plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The catalase of interest has the potential for use in treatments that aim to improve percent seed germination as well as obtaining tall shoots in a shorter time period.

Diversity of microorganisms within rock varnish in the Whipple Mountains, California.

Rock varnish from Arizona's Whipple Mountains harbors a microbial community containing about 10(8) microorganisms g(-1) of varnish. Analyses of varnish phospholipid fatty acids and rRNA gene libraries reveal a community comprised of mostly Proteobacteria but also including Actinobacteria, eukaryota, and a few members of the Archaea. Rock varnish represents a significant niche for microbial colonization.

Antimicrobial properties of pyridine-2,6-dithiocarboxylic acid, a metal chelator produced by Pseudomonas spp.

Pyridine-2,6-dithiocarboxylic acid (pdtc) is a metal chelator produced by Pseudomonas spp. It has been shown to be involved in the biodegradation of carbon tetrachloride; however, little is known about its biological function. In this study, we examined the antimicrobial properties of pdtc and the mechanism of its antibiotic activity. The growth of Pseudomonas stutzeri strain KC, a pdtc-producing strain, was significantly enhanced by 32 microM pdtc. All nonpseudomonads and two strains of P. stutzeri were sensitive to 16 to 32 microM pdtc. In general, fluorescent pseudomonads were resistant to all concentrations tested. In competition experiments, strain KC demonstrated antagonism toward Escherichia coli. This effect was partially alleviated by 100 microM FeCl3. Less antagonism was observed in mutant derivatives of strain KC (CTN1 and KC657) which lack the ability to produce pdtc. A competitive advantage was restored to strain CTN1 by cosmid pT31, which restores pdtc production. pT31 also enhanced the pdtc resistance of all pdtc-sensitive strains, indicating that this plasmid contains elements responsible for resistance to pdtc. The antimicrobial effect of pdtc was reduced by the addition of Fe(III), Co(III), and Cu(II) and enhanced by Zn(II). Analyses by mass spectrometry determined that Cu(I):pdtc and Co(III):pdtc2 form immediately under our experimental conditions. Our results suggest that pdtc is an antagonist and that metal sequestration is the primary mechanism of its antimicrobial activity. It is also possible that Zn(II), if present, may play a role in pdtc toxicity.

Evidence for simultaneous abiotic-biotic oxidations in a microbial-Fenton's system.

The conditions that support the simultaneous activity of hydroxyl radicals (OH.) and heterotrophic aerobic bacterial metabolism were investigated using two probe compounds: (1) tetrachloroethene (PCE) for the detection of OH. generated by an iron-nitrilotriacetic acid (Fe-NTA) catalyzed Fenton-like reaction and (2) oxalate (OA) for the detection of heterotrophic metabolism of Xanthobacter flavus. In the absence of the bacterium in the quasi-steady-state Fenton's system, only PCE oxidation was observed; conversely, only OA assimilation was found in non-Fenton's systems containing X. flavus. In combined Fenton's-microbial systems, loss of both probes was observed. PCE oxidation increased and heterotrophic assimilation of OA declined as a function of an increase in the quasi-steady-state H2O2 concentration. Central composite rotatable experimental designs were used to determine the conditions that provide maximum simultaneous abiotic-biotic oxidations, which were achieved with a biomass level of 10(9) CFU/mL, 4.5 mM H2O2, and 2.5 mM Fe-NTA. These results demonstrate that heterotrophic bacterial metabolism can occur in the presence of hydroxyl radicals. Such simultaneous abiotic-biotic oxidations may exist when H2O2 is injected into the subsurface as a microbial oxygen source or as a source of chemical oxidants. In addition, hybrid abiotic-biotic systems could be used for the treatment of waters containing biorefractory organic contaminants present in recycle water, cooling water, or industrial waste streams.

Carbon tetrachloride dechlorination by the bacterial transition metal chelator pyridine-2,6-bis(thiocarboxylic acid).

A reaction pathway is proposed to explain the formation of end products during defined chemical reactions between carbon tetrachloride (CCl4) and either metal complexes of pyridine-2,6-bis(thiocarboxylic acid) (PDTC) or pure cultures of Pseudomonas stutzeri KC. The pathway includes one-electron reduction of CCl4 by the Cu(II):PDTC complex, condensation of trichloromethyl and thiyl radicals, and hydrolysis of a labile thioester intermediate. Products detected were carbon dioxide, chloride, carbonyl sulfide, carbon disulfide, and dipicolinic acid. Spin-trapping and electrospray MS/MS experiments gave evidence of trichloromethyl and thiyl radicals generated by reaction of CCl4 with PDTC and copper. Experiments testing the effects of transition metals showed that dechlorination by PDTC requires copper and is inhibited by cobalt but not by iron or nickel. PDTC was shown to react stoichiometrically rather than catalytically without added reducing equivalents. With added reductants, an increased turnover was seen along with increased chloroform production.

Metal binding by pyridine-2,6-bis(monothiocarboxylic acid), a biochelator produced by Pseudomonas stutzeri and Pseudomonas putida.

Pyridine-2,6-bis(monothiocarboxylic acid) (pdtc), a natural metal chelator produced by Pseudomonas stutzeri and Pseudomonas putida that promotes the degradation of carbon tetrachloride, was synthesized and studied by potentiometric and spectrophotometric techniques. The first two stepwise protonation constants (pK) for successive proton addition to pdtc were found to be 5.48 and 2.58. The third stepwise protonation constant was estimated to be 1.3. The stability (affinity) constants for iron(III), nickel(II), and cobalt(III) were determined by potentiometric or spectrophotometric titration. The results show that pdtc has strong affinity for Fe(III) and comparable affinities for various other metals. The stability constants (log K) are 33.93 for Co(pdtc)2(1-); 33.36 for Fe(pdtc)2(1-); and 33.28 for Ni(pdtc)2(2-). These protonation constants and high affinity constants show that over a physiological pH range the ferric pdtc complex has one of the highest effective stability constants for iron binding among known bacterial chelators.

A Pseudomonas stutzeri gene cluster encoding the biosynthesis of the CCl4-dechlorination agent pyridine-2,6-bis(thiocarboxylic acid).

A spontaneous mutant of Pseudomonas stutzeri strain KC lacked the carbon tetrachloride (CCl4) transformation ability of wild-type KC. Analysis of restriction digests separated by pulsed-field gel electrophoresis (PFGE) indicated that the mutant strain CTN1 differed from strain KC by deletion of approximately 170 kb of chromosomal DNA. CTN1 did not produce pyridine-2,6-bis(thiocarboxylic acid) (PDTC), the agent determined to be responsible for CCl4 dechlorination in cultures of strain KC. Cosmids from a genomic library of strain KC containing DNA from within the deleted region were identified by hybridization with a 148 kb genomic Spel fragment absent in strain CTN1. Several cosmids identified in this manner were further screened for complementation of the PDTC biosynthesis-negative (Pdt -) phenotype. One cosmid (pT31) complemented the Pdt- phenotype of CTN1 and conferred CCl4 transformation activity and PDTC production upon other pseudomonads. Southern analysis showed that none of three other P. stutzeri strains representing three genomovars contained DNA that would hybridize with the 25,746 bp insert of pT31. Transposon mutagenesis of pT31 identified open reading frames (ORFs) whose disruption affected the ability to make PDTC in the strain CTN1 background. These data describe the pdt locus of strain KC as residing in a non-essential region of the chromosome subject to spontaneous deletion. The pdt locus is necessary for PDTC biosynthesis in strain KC and is sufficient for PDTC biosynthesis by other pseudomonads but is not a common feature of P. stutzeri strains.

Identification of an extracellular agent [correction of catalyst] of carbon tetrachloride dehalogenation from Pseudomonas stutzeri strain KC as pyridine-2, 6-bis(thiocarboxylate)

Pseudomonas stutzeri strain KC was originally characterized as having, under iron-limiting conditions, novel carbon tetrachloride (CCl(4)) dehalogenation activity, specifically, a net conversion of CCl(4) to CO(2). The exact pathway and reaction mechanisms are unknown, but chloroform is not an intermediate and thiophosgene and phosgene have been identified as intermediates in trapping experiments. Previous work by others using cell-free preparations has shown that cell-free culture supernatants that have been passed through a low-molecular-weight cutoff membrane can confer rapid CCl(4) transformation ability upon cultures of bacteria which otherwise show little or no reactivity toward CCl(4). We used a cell-free assay system to monitor the complete purification of compounds showing CCl(4) degradation activity elaborated by iron-limited cultures of strain KC. Electrospray tandem mass spectroscopy, NMR spectroscopy, and comparisons with synthetic material have identified pyridine-2,6-bis(thiocarboxylate) as a metabolite of strain KC which has CCl(4) transformation activity in the presence of chemical reductants, e.g., titanium[III] citrate or dithiothreitiol, or actively growing bacterial cultures.

Optimization of simultaneous chemical and biological mineralization of perchloroethylene.

Optimization of the simultaneous chemical and biological mineralization of perchloroethylene (PCE) by modified Fenton's reagent and Xanthobacter flavus was investigated by using a central composite rotatable experimental design. Concentrations of PCE, hydrogen peroxide, and ferrous iron and the microbial cell number were set as variables. Percent mineralization of PCE to CO2 was investigated as a response. A second-order, quadratic response surface model was generated and fit the data adequately, with a correlation coefficient of 0.72. Analysis of the results showed that the PCE concentration had no significant effect within the tested boundaries of the model, while the other variables, hydrogen peroxide and iron concentrations and cell number, were significant at alpha = 0.05 for the mineralization of PCE. The 14C radiotracer studies showed that the simultaneous chemical and biological reactions increased the extent of mineralization of PCE by more than 10% over stand-alone Fenton reactions.

Phylogeny of Sphingomonas species that degrade pentachlorophenol.

Four pentachlorophenol (PCP)-degrading bacteria isolated from geographically diverse areas have been examined in detail as regards their physiology and phylogeny. According to traditional biochemical methods, these strains had been classified as members of the genera Arthrobacter, Flavobacterium, Pseudomonas, and Sphingomonas. The PCP degradation pathway has been studied extensively in Sphingomonas (Flavobacterium) sp strain ATCC 39723 and the first three degradation steps catalyzed by a PCP-4-monooxygenase (PcpB) and a reductive dehalogenase (PcpC) that functions twice are well established. A fourth step appears to involve ring-fission of the aromatic nucleus (PcpA). Molecular analyses revealed that the PCP degradation pathway in these four strains was rather conserved, leading to a phylogenetic analysis using 16S rDNA. The results revealed a much closer phylogenetic relationship between these organisms than traditional classification indicated, placing them into the more recently established genus Sphingomonas where they may even represent a single species. With 16S rDNA analysis, many bacterial isolates involved in degradation of xenobiotic compounds that were previously classified into diverse genera have been reclassified into the genus Sphingomonas.

Mutational analysis of pcpA and its role in pentachlorophenol degradation by Sphingomonas (Flavobacterium) chlorophenolica ATCC 39723.

Sphingomonas (Flavobacterium) chlorophenolica ATCC 39723 degrades pentachlorophenol (PCP) through a catabolic pathway encoded by multiple genes. One gene required for PCP degradation is pcpA, which encodes information for a 30-kDa polypeptide, PcpA, found in the periplasm of the bacterium. The biological role of PcpA has remained unknown. We disrupted pcpA by replacing it with a defective copy through homologous recombination. The pcpA recombinant, mutant strains accumulated 2,6-dichlorohydroquinone (2,6-DiCH) as a metabolite of PCP. This work confirms that pcpA is essential for degradation of PCP by S. chlorophenolica ATCC 39723 and suggests that it encodes a protein involved in hydrolytic dehalogenation of 2,6-DiCH, an already established primary metabolite of the PCP catabolic pathway.

Production of Clostridium bifermentans Spores as Inoculum for Bioremediation of Nitroaromatic Contaminants.

Spores of Clostridium bifermentans KMR-1 were produced for use as a microbial inoculum for bioremediation and were preserved in both liquid and dry forms. All spore formulations showed good viability and ability to biodegrade the target compound, 2,4,6-trinitrotoluene (TNT), after 4 months of storage. For low-cost bulk spore production, several medium compositions, based on soy peptone, corn steep liquor, and meat peptone, were tested and yielded 10(sup7) spores per ml. A medium pH above 7.0, a low glucose concentration, and a sufficient concentration of protein favored the sporulation of C. bifermentans KMR-1.

2,4,6-Trinitrotoluene (TNT) transformation by clostridia isolated from a munition-fed bioreactor: comparison with non-adapted bacteria.

Several bacterial strains were examined for their ability to degrade the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). The strains examined included various clostridial strains isolated from a 4-year-old munition enrichment, related clostridial strains obtained from a culture collection, two enteric bacteria, and three lactobacilli. All Clostridium species tested were able to reduce TNT rapidly in a complex medium. In cell suspension experiments, these strains were also able to reduce 2,4-diamino-6-nitrotoluene (DANT) to 2,4,6-triaminotoluene (TAT) and to produce a compound that is not yet identified; thus, they could not be distinguished from one another with regard to the pathway of transformation. The enteric strains and the lactobacilli were able to perform the initial reduction of TNT, but none was capable of reducing DANT in cell suspensions.

PCP degradation is mediated by closely related strains of the genus Sphingomonas.

There have been numerous reports in the literature of diverse bacteria capable of degrading pentachlorophenol (PCP). In order to gain further insight into the phylogenetic relationships of PCP-degrading bacteria, we examined four strains: Arthrobacter sp. strain ATCC 33790, Flavobacterium sp. strain ATCC 39723, Pseudomonas sp. strain SR3, and Sphingomonas sp. strain RA2. These organisms were isolated from different geographical locations and all of them degrade high concentrations (100-200 mg/L) of PCP. Southern blot analyses determined that these bacteria all harbour DNA that encodes similar, if not identical, genes involved in PCP degradation. Comparison of the 16S rRNA nucleotide sequences revealed that these organisms were very closely related and, in fact, represent a monophyletic group. The 16S rRNA analyses together with fatty acid and sphingolipid analyses strongly suggest that the four strains are members of the genus Sphingomonas. The close relationship of the four organisms is supported by nucleotide sequence analysis data of the pcpB locus encoding PCP-4-monooxygenase, the first enzyme in the PCP degradative pathway.

Effects of light on the accumulation of abscisic acid and expression of an early cysteine-labeled metallothionein gene in microspores ofTriticum aestivum during induced embryogenic development.

A cloned cDNA to the wheat (Triticum aestivum) early cysteine-labeled metallothionein has many characteristics of a molecular marker for pollen embryogenesis in this plant. This transcript was not detected in uninucleate microspores at the time of culture or in pollen at any stage during normal ontogeny; its mRNA did begin to increase in embryogenic microspores within 6 h of culture, peaked at around 24 h, declined, then leveled off through the 21-day-old embryoid stage. Additionally, the accumulation of the embryoid-abundant EcMt gene transcript showed a direct and positive correlation with an increase of ABA in embryogenic microspores and developing pollen embryoids. Irradiating cultures with high intensity white light or with far-red, or blue light, suppressed EcMt transcript accumulation and the ability of microspores to form embryoids; however, light did not affect ABA concentrations during the early stages of culture. These results suggest that although a promoter of pollen embryogenesis in bread wheat, ABA alone cannot maintain the sporophytic differentiation of microspores subjected to inhibitory regimes of light in vitro. Whether or not light acts directly or indirectly in suppressing EcMt gene expression and pollen embryogenesis remains unknown.

Products of Anaerobic 2,4,6-Trinitrotoluene (TNT) Transformation by Clostridium bifermentans.

Experiments to elucidate the 2,4,6-trinitrotoluene (TNT)-transforming activity of Clostridium bifermentans LJP-1 identified reductive TNT transformations that ultimately produced as end products triaminotoluene (TAT) and phenolic products of TAT hydrolysis. An adduct of TAT, apparently formed by condensation of TAT and pyruvic aldehyde (methyl glyoxal), was also detected.

Changes in abundance of an abscisic acid-responsive, early cysteine-labeled metallothionein transcript during pollen embryogenesis in bread wheat (Triticum aestivum).

A clone for an embryoid-abundant, early cysteine-labeled metallothionein (EcMt) gene has been isolated from a wheat pollen embryoid cDNA library. The transcript of this gene was only expressed in embryogenic microspores, pollen embryoids, and developing zygotic embryos of wheat. Accumulation of the EcMt mRNA showed a direct and positive correlation with an increase of the plant hormone, abscisic acid (ABA) in developing pollen embryoids. Treating cultures with an inhibitor of ABA biosynthesis, fluridone, suppressed not only ABA accumulation but also the appearance of the EcMt gene transcript and the ability of microspores to form embryoids. These results suggest that the EcMt gene may act as a molecular marker for pollen embryogenesis because ABA biosynthesis is accompanied by the increased expression of the EcMt transcript that coincides with the differentiation of pollen embryoids in wheat anther cultures.

Degradation of 2-sec-butyl-4,6-dinitrophenol (dinoseb) by Clostridium bifermentans KMR-1.

A strain of Clostridium bifermentans, KMR-1, degraded 2-sec-butyl-4,6-dinitrophenol (dinoseb) to a level below the limit of detection by high-performance liquid chromatography (0.5 mg/liter) within 96 h, with no accumulation of aromatic intermediates. KMR-1 could not utilize dinoseb as a sole carbon or energy source, and degradation occurred via cometabolism in the presence of a fermentable carbon source. KMR-1 mineralized some dinoseb in anaerobic cultures, evolving 7.2% of the radioactive label in U-ring 14C-labeled dinoseb as 14CO2. The remaining anaerobic degradation products were incubated with aerobic soil bacteria, and 35.4% of this residual radioactive label was evolved as 14CO2. During this mineralization experiment, 38.9% of the initial label was evolved as 14CO2 after both anaerobic and aerobic phases. This is the first demonstration of dinoseb degradation by a pure microbial culture.