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We examined the bacterial communities in a pool of water under a flowing rock fracture at a depth of 1470 m in the Sanford Underground Research Facility in Lead, SD. High-throughput sequencing of 16S rDNA indicated that Acidobacteria, Chlorobi, Nitrospirae, Planctomycetes, and Gammaproteobacteria (especially Acidiferribacter) were abundant in pool sediments; while Nitrospirae, Plactomycetes, and Proteobacteria (especially Thiobacillus and Methylococcus) were abundant in pool water. Samples of diluted pool water were mixed with agar media, placed in diffusion chambers surrounded by 0.2 µm pore membranes, and incubated in situ within the pool for 19 days. 16S rDNA analysis of media from diffusion chambers indicated Alphaproteobacteria (including Hyphomicrobium), Betaproteobacteria (including Inhella and Methyloversatilis), and Gammaproteobacteria (mainly Pseudomonas) were dominant. Low-nutrient media were used to isolate bacteria from both pool water and media inside diffusion chambers. The majority of these isolates were Methyloversatilis, Inhella, and Bacillus. Although the genera isolated were not very abundant in the pool water, the presence of Methylococcus and Methyloversatilis suggests the importance of methane and methanol oxidation in this community. [ABSTRACT FROM AUTHOR]

Comparison of the organization and sequence of the hao (hydroxylamine oxidoreductase) gene clusters from the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB) Nitrosococcus oceani and the betaproteobacterial aAOB Nitrosospira multiformis and Nitrosomonas europaea revealed a highly conserved gene cluster encoding the following proteins: hao, hydroxylamine oxidoreductase; orf2, a putative protein; cycA, cytochrome C554 and cycB, cytochrome Cm552. The deduced protein sequences of HAO, c554, and Cm552 were highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic inference revealed a broad family of multi-c-heme proteins, including HAO, the pentaheme nitrite reductase, and tetrathionate reductase. The c-hemes of this group also have a nearly identical geometry of heme orientation, which has remained conserved during divergent evolution of function. High sequence similarity is also seen within a protein family, including cytochromes Cm552, NrfH/B, and NapC/NirT. It is proposed that the hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration (denitrification) during the radiation of the Proteobacteria. Conservation of the hydroxylamine oxidation module was maintained by functional pressure, and the module expanded into two separate narrow taxa after a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an orf2 gene, whereas a c554 gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c554 gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB. [ABSTRACT FROM AUTHOR]