mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the ␣ and ␤ diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.

Nitrous oxide (N 2 O) is an important greenhouse gas in the troposphere controlling ozone concentration in the stratosphere through nitric oxide production. In order to quantify bacteria capable of N 2 O reduction, we developed a SYBR green quantitative real-time PCR assay targeting the nosZ gene encoding the catalytic subunit of the nitrous oxide reductase. Two independent sets of nosZ primers flanking the nosZ fragment previously used in diversity studies were designed and tested (K. Kloos, A. Mergel, C. Rösch, and H. Bothe, Aust. J. Plant Physiol. 28:991-998, 2001). The utility of these real-time PCR assays was demonstrated by quantifying the nosZ gene present in six different soils. Detection limits were between 10 1 and 10 2 target molecules per reaction for all assays. Sequence analysis of 128 cloned quantitative PCR products confirmed the specificity of the designed primers. The abundance of nosZ genes ranged from 10 5 to 10 7 target copies g ؊1 of dry soil, whereas genes for 16S rRNA were found at 10 8 to 10 9 target copies g ؊1 of dry soil. The abundance of narG and nirK genes was within the upper and lower limits of the 16S rRNA and nosZ gene copy numbers. The two sets of nosZ primers gave similar gene copy numbers for all tested soils. The maximum abundance of nosZ and nirK relative to 16S rRNA was 5 to 6%, confirming the low proportion of denitrifiers to total bacteria in soils.Nitrous oxide (N 2 O), with a global warming potential approximately 300 times higher than that of carbon dioxide, is an important greenhouse gas, contributing up to 6% of global warming. N 2 O also participates in depletion of the stratospheric ozone layer through stratospheric nitric oxide (NO) production. At present, the N 2 O concentration in the atmosphere is increasing at a rate of about 0.3% per year. The soil is the dominant source of atmospheric nitrous oxide, contributing about 57% (9 Tg year Ϫ1 ) of the total annual global emission (12). N 2 O emissions are highly variable in soils and are primarily produced by biological nitrification and denitrification, although the latter is considered to be the main source (31). N 2 O is an intermediate product in the denitrification pathway, which consists of the sequential reduction of NO 3 Ϫ to N 2 via the metalloenzymes nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase (32). Therefore, N 2 O emission by denitrification is the net result of the balance between production and reduction of N 2 O by denitrifying bacteria. The N 2 O reductase (EC 1.7.99.6) is a homodimeric multicopper enzyme, which has been purified from numerous gram-negative denitrifiers but not yet from a gram-positive bacterium (4,5,11,17,27). Production of N 2 O by denitrifying isolates as an end product of denitrification has been reported by several authors (2, 3, 7). Sequencing of the complete genome of Agrobacterium tumefaciens C58 revealed the presence of a denitrification cluster with genes encoding the periplasmic nitrate reductase, the copper nitrite reductase...

Denitrification is a facultative respiratory pathway in which nitrite (NO2(-)), nitric oxide (NO), and nitrous oxide (N2O) are successively reduced to nitrogen gas (N(2)), effectively closing the nitrogen cycle. The ability to denitrify is widely dispersed among prokaryotes, and this polyphyletic distribution has raised the possibility of horizontal gene transfer (HGT) having a substantial role in the evolution of denitrification. Comparisons of 16S rRNA and denitrification gene phylogenies in recent studies support this possibility; however, these results remain speculative as they are based on visual comparisons of phylogenies from partial sequences. We reanalyzed publicly available nirS, nirK, norB, and nosZ partial sequences using Bayesian and maximum likelihood phylogenetic inference. Concomitant analysis of denitrification genes with 16S rRNA sequences from the same organisms showed substantial differences between the trees, which were supported by examining the posterior probability of monophyletic constraints at different taxonomic levels. Although these differences suggest HGT of denitrification genes, the presence of structural variants for nirK, norB, and nosZ makes it difficult to determine HGT from other evolutionary events. Additional analysis using phylogenetic networks and likelihood ratio tests of phylogenies based on full-length sequences retrieved from genomes also revealed significant differences in tree topologies among denitrification and 16S rRNA gene phylogenies, with the exception of the nosZ gene phylogeny within the data set of the nirK-harboring genomes. However, inspection of codon usage and G + C content plots from complete genomes gave no evidence for recent HGT. Instead, the close proximity of denitrification gene copies in the genomes of several denitrifying bacteria suggests duplication. Although HGT cannot be ruled out as a factor in the evolution of denitrification genes, our analysis suggests that other phenomena, such gene duplication/divergence and lineage sorting, may have differently influenced the evolution of each denitrification gene.

We explored the assembly of intestinal microbiota in healthy male participants during the run-in (5 day) and experimental phases [21-day normoxic bed rest (NBR), hypoxic bedrest (HBR)], and hypoxic ambulation (HAmb) in a strictly controlled laboratory environment, balanced fluid, and dietary intakes, controlled circadian rhythm, microbial ambiental burden, and 24/7 medical surveillance. The fraction of inspired O2 (FiO2) and partial pressure of inspired O2 (PiO2) were 0.209 and 133.1 ± 0.3 mmHg for NBR and 0.141 ± 0.004 and 90.0 ± 0.4 mmHg for both hypoxic variants (HBR and HAmb; ~4,000 m simulated altitude), respectively. A number of parameters linked to intestinal transit spanning Bristol Stool Scale, defecation rates, zonulin, α1-antitrypsin, eosinophil derived neurotoxin, bile acids, reducing sugars, short chain fatty acids, total soluble organic carbon, water content, diet composition, and food intake were measured (167 variables). The abundance, structure, and diversity of butyrate producing microbial community were assessed using the two primary bacterial butyrate synthesis pathways, butyryl-CoA: acetate CoA-transferase (but) and butyrate kinase (buk) genes. Inactivity negatively affected fecal consistency and in combination with hypoxia aggravated the state of gut inflammation (p < 0.05). In contrast, gut permeability, various metabolic markers, the structure, diversity, and abundance of butyrate producing microbial community were not significantly affected. Rearrangements in the butyrate producing microbial community structure were explained by experimental setup (13.4%), experimentally structured metabolites (12.8%), and gut metabolite-immunological markers (11.9%), with 61.9% remaining unexplained. Many of the measured parameters were found to be correlated and were hence omitted from further analyses. The observed progressive increase in two immunological intestinal markers suggested that the transition from healthy physiological state toward the developed symptoms of low magnitude obesity-related syndromes was primarily driven by the onset of inactivity (lack of exercise in NBR) that were exacerbated by systemic hypoxia (HBR) and significantly alleviated by exercise, despite hypoxia (HAmb). Butyrate producing community in colon exhibited apparent resilience toward short-term modifications in host exercise or hypoxia. Progressive constipation (decreased intestinal motility) and increased local inflammation marker suggest that changes in microbial colonization and metabolism were taking place at the location of small intestine.