Isolation and genomic characterization of a Dehalococcoides strain suggests genomic rearrangement during culture.

We have developed and characterized a bacterial consortium that reductively dechlorinates trichloroethene to ethene. Quantitative PCR analysis for the 16S rRNA and reductive dehalogenase genes showed that the consortium is highly enriched with Dehalococcoides spp. that have two vinyl chloride reductive dehalogenase genes, bvcA and vcrA, and a trichloroethene reductive dehalogenase gene, tceA. The metagenome analysis of the consortium by the next generation sequencer SOLiD 3 Plus suggests that a Dehalococcoides sp. that is highly homologous to D. mccartyi 195 and equipped with vcrA and tceA exists in the consortium. We isolated this Dehalococcoides sp. and designated it as D. mccartyi UCH-ATV1. As the growth of D. mccartyi UCH-ATV1 is too slow under isolated conditions, we constructed a consortium by mixing D. mccartyi UCH-ATV1 with several other bacteria and performed metagenomic sequencing using the single molecule DNA sequencer PacBio RS II. We successfully determined the complete genome sequence of D. mccartyi UCH-ATV1. The strain is equipped with vcrA and tceA, but lacks bvcA. Comparison with tag sequences of SOLiD 3 Plus from the original consortium shows a few differences between the sequences. This suggests that a genome rearrangement of Dehalococcoides sp. occurred during culture.

Genome sequence determination and metagenomic characterization of a Dehalococcoides mixed culture grown on cis-1,2-dichloroethene.

A Dehalococcoides-containing bacterial consortium that performed dechlorination of 0.20 mM cis-1,2-dichloroethene to ethene in 14 days was obtained from the sediment mud of the lotus field. To obtain detailed information of the consortium, the metagenome was analyzed using the short-read next-generation sequencer SOLiD 3. Matching the obtained sequence tags with the reference genome sequences indicated that the Dehalococcoides sp. in the consortium was highly homologous to Dehalococcoides mccartyi CBDB1 and BAV1. Sequence comparison with the reference sequence constructed from 16S rRNA gene sequences in a public database showed the presence of Sedimentibacter, Sulfurospirillum, Clostridium, Desulfovibrio, Parabacteroides, Alistipes, Eubacterium, Peptostreptococcus and Proteocatella in addition to Dehalococcoides sp. After further enrichment, the members of the consortium were narrowed down to almost three species. Finally, the full-length circular genome sequence of the Dehalococcoides sp. in the consortium, D. mccartyi IBARAKI, was determined by analyzing the metagenome with the single-molecule DNA sequencer PacBio RS. The accuracy of the sequence was confirmed by matching it to the tag sequences obtained by SOLiD 3. The genome is 1,451,062 nt and the number of CDS is 1566, which includes 3 rRNA genes and 47 tRNA genes. There exist twenty-eight RDase genes that are accompanied by the genes for anchor proteins. The genome exhibits significant sequence identity with other Dehalococcoides spp. throughout the genome, but there exists significant difference in the distribution RDase genes. The combination of a short-read next-generation DNA sequencer and a long-read single-molecule DNA sequencer gives detailed information of a bacterial consortium.

Degradation of natural estrogen and identification of the metabolites produced by soil isolates of Rhodococcus sp. and Sphingomonas sp.

Five bacterial strains capable of utilizing 17beta-estradiol (E2) and estrone (E1) were isolated from soil samples. Using their morphological and physiological features and 16S rDNA sequences, we classified these isolates into two groups: Group A (Rhodococcus sp. strains ED6, ED7, and ED10) and Group B (Sphingomonas sp. strains ED8 and ED9). All isolates used E2 and E1 as the sole carbon sources and showed high E1 and E2 degradation activities. In all strains, more than 50% of 0.8 mg of E1 or E2 was degraded in 4 mL of inorganic medium over 24 h, and 90% was degraded over 120 h. By incubating the resting ED8 cells with E2 and the meta-cleavage inhibitor 3-chlorocatechol, we identified two metabolites, 4-hydroxyestrone (4-OH-E1) and 4-hydroxyestradiol (4-OH-E2), and confirmed their identity using authentic chemicals. The 4-OH-E1 and 4-OH-E2 compounds were assumed to be intermediate metabolites formed before meta-cleavage, as they were not identified in culture without 3-chlorocatechol. Degradation of E2 by strain ED8 can be initiated by hydroxylation of the C-4 position, followed by meta-cleavage of the benzene ring. When strains ED8 degraded E2, we further identified hydroxy-E2, keto-E1 and -E2, and an additional degradation product via mass spectrometry. The presence of these compounds implied degradation through a second pathway initiated through an attack of the saturated ring.

Identification of putative benzene-degrading bacteria in methanogenic enrichment cultures.

Anaerobic benzene-degrading enrichment cultures performing methanogenesis were obtained from non-contaminated lotus field soil. Stable isotope probing with 13C benzene was used to detect the bacteria that were involved in benzene degradation. Denaturing gradient gel electrophoresis (DGGE) of fractionated samples exhibited an obvious shift of some DGGE bands to a heavier DNA fraction. An almost full-length 16S rRNA gene sequence corresponding to the DGGE band, namely Hasda-A, was obtained by constructing a clone library of the heavier fraction. The Hasda-A sequence showed only 85.1% identity with the closest identified bacterium, Syntrophus gentianae. Hasda-A may be an important bacterium involved in the initial steps of benzene degradation under methanogenic conditions, as it was the most prominent bacterium that assimilated labeled benzene early in the process of benzene degradation. A primer set was designed to quantify the gene copies of Hasda-A by quantitative PCR. Hasda-A was present at a concentration of (3.5+/-0.8) x 10(6) copies/mL and represented 8.4% of gene copies among bacteria in the enrichment culture. The enrichment culture consisted of three dominant bacterial groups: Hasda-A and both aceticlastic and hydrogenotrophic methanogens. Methane is believed to be produced from benzene by the sequential degradation of benzene by fermenting bacteria, hydrogen-producing acetogens, and methanogens.

Development of an oligonucleotide microarray to detect di- and monooxygenase genes for benzene degradation in soil.

Diverse environmental genes have been identified recently. To characterize their functions, it is necessary to understand which genes and what combinations of those genes are responsible for the biodegradation of soil contaminants. In this article, a 60-mer oligonucleotide microarray was constructed to simultaneously detect di- and monooxygenase genes for benzene and related compounds. In total, 148 probes were designed and validated by pure-culture hybridizations using the following criteria to discriminate between highly homologous genes: < or =53-bp identities and < or =25-bp continuous stretch to nontarget sequences. Microarray hybridizations were performed using PCR products amplified from five benzene-amended soils and two oil-contaminated soils. Six of the probes gave a positive signal for more than six soils; thus, they may represent key sequences for benzene degradation in the environment. The microarray developed in this study will be a powerful tool for the screening of key genes involved in benzene degradation and for the rapid profiling of benzene oxygenase gene diversity in contaminated soils.

Analysis of the phylogenetic diversity of estrone-degrading bacteria in activated sewage sludge using microautoradiography-fluorescence in situ hybridization.

In situ uptake of [2,4,6,7-3H(N)]estrone ([3H]E1) by the major phylogenetic groups present in activated sludge samples from two different municipal wastewater treatment plants was investigated using microautoradiography-fluorescence in situ hybridization (MAR-FISH). Approximately 1-2% of the total cells confined in the samples by an EUB probe mix contributed to E1 assimilation. Almost all the detected E1-assimilating cells involved in the early phase of E1 degradation were affiliated with the Beta- and Gammaproteobacteria. In the early phase of E1 degradation, no E1-assimilating cells affiliated with the Alphaproteobacteria, Actinobacteria, the Cytophaga-Flavobacterium cluster of phylum Bacteroidetes, or the phyla Chloroflexi, Nitrospira and Planctomycetes were detected. Bacteria affiliated with the Betaproteobacteria in the shape of long rods or chains of rods were found to contribute most to in situ E1 degradation. They contributed 61% and 82% of total E1-assimilating cells in cultures from two sources of activated sludge spiked with [3H]E1. The E1-degrading bacteria related to the Betaproteobacteria differed phylogenetically from the aerobic E1-degrading bacterial isolates reported in previous studies. In addition, MAR-FISH revealed the significant contribution of E1-degrading bacteria affiliated with the Gammaproteobacteria in the degradation of E1 in activated sludge.

Development of a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity.

We constructed a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity to develop a new technology for simultaneous detection of the functional gene diversity in environmental samples. The diversity of the monooxygenase genes associated with benzene degradation was characterized. A new polymerase chain reaction (PCR) primer set was designed using conserved regions of benzene monooxygenase gene (BO12 primer) and used for PCR-clone library analysis along with a previously designed RDEG primer which targeted the different types of benzene monooxygenase gene. We obtained 20 types of amino acid sequences with the BO12 primer and 40 with the RDEG primer. Phylogenetic analysis of the sequences obtained suggested the large diversity of the benzene monooxygenase genes. A total of 87 60-mer probes specific for each operational taxonomical unit were designed and spotted on a microarray. When genomic DNAs of single strains were used in microarray hybridization assays, corresponding sequences were successfully detected by the microarray without any false-negative signals. Hybridization with soil DNA samples showed that the microarray was able to detect sequences that were not detected in clone libraries. Constructed microarray can be a useful tool for characterizing monooxygenase gene diversity in benzene degradation.

Effects of ammonium and nitrite on communities and populations of ammonia-oxidizing bacteria in laboratory-scale continuous-flow reactors.

This study investigated the effects of ammonium and nitrite on ammonia-oxidizing bacteria (AOB) from an activated sludge process in laboratory-scale continuous-flow reactors. AOB communities were analyzed using specific PCR followed by denaturing gel gradient electrophoresis, cloning and sequencing of the 16S rRNA gene, and AOB populations were quantified using real-time PCR. To study the effect of ammonium, activated sludge from a sewage treatment system was enriched in four reactors receiving inorganic medium containing four different ammonium concentrations (2, 5, 10 and 30 mM NH(4) (+)-N). One of several sequence types of the Nitrosomonas oligotropha cluster predominated in the reactors with lower ammonium loads (2, 5 and 10 mM NH(4) (+)-N), whereas Nitrosomonas europaea was the dominant AOB in the reactor with the highest ammonium load (30 mM NH(4) (+)-N). The effect of nitrite was studied by enriching the enriched culture possessing both N. oligotropha and N. europaea in four reactors receiving 10-mM-ammonium inorganic medium containing four different nitrite concentrations (0, 2, 12 and 22 mM NO(2) (-)-N). Nitrosomonas oligotropha comprised the majority of AOB populations in the reactors without nitrite accumulation (0 and 2 mM NO(2) (-)-N), whereas N. europaea was in the majority in the 12- and 22-mM NO(2) (-)-N reactors, in which nitrite concentrations were 2.1-5.7 mM (30-80 mg N L(-1)).

Development and application of real-time PCR for quantification of specific ammonia-oxidizing bacteria in activated sludge of sewage treatment systems.

In this study, four real-time polymerase chain reaction (PCR) primer sets were developed for the 16S rRNA genes of specific ammonia-oxidizing bacteria (AOB) found in activated sludge of sewage treatment systems. The primer sets target two of several sequence types of the Nitrosomonas oligotropha cluster, members within the Nitrosomonas communis cluster, and all members of the Nitrosomonas europaea-Nitrosococcus mobilis cluster. The detection limit of each primer set was in the range of 3x10(1)-6x10(2) genes reaction(-1). Reliable quantification of the target AOB DNA was obtained when the target AOB DNA comprised more than 0.1% of total AOB DNA in the sample. The application of the primer sets to samples taken from five sewage treatment systems showed that, in all systems, the majority of the AOB population was comprised of one sequence type of the N. oligotropha cluster (3.9+/-1.5x10(9)-1.7+/-0.5x10(10) cell l(-1)) and, in most systems, followed by members within the N. communis cluster (2.8+/-0.3x10(9)-1.0+/-0.1x10(10) cell l(-1)) or/and another sequence type of the N. oligotropha cluster (1.5+/-0.6x10(8)-5.5+/-0.5x10(8) cell l(-1)). N. europaea-N. mobilis cluster arose solely in small numbers (4.9+/-0.8x10(8) cell l(-1)) in one system. Real-time PCR-amplified products obtained from genomic DNA extracted from samples were verified using clone library, and it revealed that only the target AOB DNA were PCR amplified, without amplification of the nontarget sequences.

Communities of ammonia-oxidizing bacteria in activated sludge of various sewage treatment plants in Tokyo.

We investigated ammonia-oxidizing bacteria in activated sludge collected from 12 sewage treatment systems, whose ammonia removal and treatment processes differed, during three different seasons. We used real-time PCR quantification to reveal total bacterial numbers and total ammonia oxidizer numbers, and used specific PCR followed by denaturing gel gradient electrophoresis, cloning, and sequencing of 16S rRNA genes to analyze ammonia-oxidizing bacterial communities. Total bacterial numbers and total ammonia oxidizer numbers were in the range of 1.6 x 10(12) - 2.4 x 10(13) and 1.0 x 10(9) - 9.2 x 10(10)cellsl(-1), respectively. Seasonal variation was observed in the total ammonia oxidizer numbers, but not in the ammonia-oxidizing bacterial communities. Members of the Nitrosomonas oligotropha cluster were found in all samples, and most sequences within this cluster grouped within two of the four sequence types identified. Members of the clusters of Nitrosomonas europaea-Nitrosococcus mobilis, Nitrosomonas cryotolerans, and unknown Nitrosomonas, occurred solely in one anaerobic/anoxic/aerobic (A2O) system. Members of the Nitrosomonas communis cluster occurred almost exclusively in association with A2O and anaerobic/aerobic systems. Solid residence time mainly influenced the total numbers of ammonia-oxidizing bacteria, whereas dissolved oxygen concentration primarily affected the ammonia-oxidizing activity per ammonia oxidizer cell.

Degradation of mono-chlorinated dibenzo-p-dioxins by Janibacter sp. strain YA isolated from river sediment.

Strain YA was newly isolated from an enrichment culture of river sediment and was identified as Janibacter sp. It was able to utilize dibenzofuran as the sole source of carbon and energy. Strain YA degraded > 90% of 1-chloro-dibenzo-p-dioxin (1-CDD) and > 80% of 2-chloro-dibenzo-p-dioxin in 18 hours with each initial concentration at 40 mg/L. A novel metabolite, 2-chloro-2',6-dihydroxydiphenylether, was observed in 1-CDD degradation. From the metabolites detected by gas chromatography-mass spectrometry, strain YA was supposed to have at least two types of oxidation pathways in 1-CDD degradation.

Biotransformation of fluorene, diphenyl ether, dibenzo-p-dioxin and carbazole by Janibacter sp.

Fluorene, diphenyl ether, dibenzo-p-dioxin, and carbazole were used by a dibenzofuran-utilizing Janibacter sp. strain YY-1. Metabolites were identified by GC-MS. Angular dioxygenation was the major pathway for degradation of fluorene, diphenyl ether, and dibenzo-p-dioxin but not for carbazole. Lateral dioxygenation of all tested compounds was indicated by the detection of mono- or di-hydroxylated compounds. The bacterium also catalyzed the monooxygenation of fluorene at the C9 position.

Degradation pathways of trichloroethylene and 1,1,1-trichloroethane by Mycobacterium sp. TA27.

We analyzed the kinetics and metabolic pathways of trichloroethylene and 1,1,1-trichloroethane degradation by the ethane-utilizing Mycobacterium sp. TA27. The apparent Vmax and Km of trichloroethylene were 9.8 nmol min(-1) mg of cells(-1) and 61.9 microM, respectively. The apparent Vmax and Km of 1,1,1-trichloroethane were 0.11 nmol min(-1) mg of cells(-1) and 3.1 microM, respectively. 2,2,2-trichloroethanol, trichloroacetic acid, chloral, and dichloroacetic acid were detected as metabolites of trichloroethylene. 2,2,2-trichloroethanol, trichloroacetic acid, and dichloroacetic acid were also detected as metabolites of 1,1,1-trichloroethane. The amounts of 2,2,2-trichloroethanol, trichloroacetic acid, chloral, and dichloroacetic acid derived from the degradation of 3.60 micromol trichloroethylene were 0.16 micromol (4.4%), 0.11 micromol (3.1%), 0.02 micromol (0.6%), and 0.02 micromol (0.6%), respectively. The amounts of 2,2,2-trichloroethanol, trichloroacetic acid and dichloroacetic acid derived from the degradation of 1.73 micromol 1,1,1-trichloroethane were 1.48 micromol (85.5%), 0.22 micromol (12.7%), and 0.02 micromol (1.2%), respectively. More than 90% of theoretical total chloride was released in trichloroethylene degradation. Chloral and 2,2,2-trichloroethanol were transformed into each other, and were finally converted to trichloroacetic acid, and dichloroacetic acid. Trichloroacetic acid and dichloroacetic acid were not degraded by strain TA27.