Novel high-rank phylogenetic lineages within a sulfur spring (Zodletone Spring, Oklahoma), revealed using a combined pyrosequencing-Sanger approach

The utilization of high-throughput sequencing technologies in 16S rRNA gene-based diversity surveys has indicated that within most ecosystems, a significant fraction of the community could not be assigned to known microbial phyla. Accurate determination of the phylogenetic affiliation of such sequences is difficult due to the short-read-length output of currently available high-throughput technologies. This fraction could harbor multiple novel phylogenetic lineages that have so far escaped detection. Here we describe our efforts in accurate assessment of the novelty and phylogenetic affiliation of selected unclassified lineages within a pyrosequencing data set generated from source sediments of Zodletone Spring, a sulfide- and sulfur-rich spring in southwestern Oklahoma. Lineage-specific forward primers were designed for 78 putatively novel lineages identified within the pyrosequencing data set, and representative nearly full-length small-subunit (SSU) rRNA gene sequences were obtained by pairing those primers with reverse universal bacterial primers. Of the 78 lineages tested, amplifiable products were obtained for 52, 32 of which had at least one nearly full-length sequence that was representative of the lineage targeted. Analysis of phylogenetic affiliation of the obtained Sanger sequences identified 5 novel candidate phyla and 10 novel candidate classes (within Fibrobacteres, Planctomycetes, and candidate phyla BRC1, GN12, TM6, TM7, LD1, WS2, and GN06) in the data set, in addition to multiple novel orders and families. The discovery of multiple novel phyla within a pilot study of a single ecosystem clearly shows the potential of the approach in identifying novel diversities within the rare biosphere.Peer reviewedMicrobiology and Molecular Genetic

Comparison of sulfide-oxidizing Sulfurimonas strains reveals a new mode of thiosulfate formation in subsurface environments

Sulfur-oxidizing Sulfurimonas spp. are widespread in sediments, hydrothermal vent fields, aquifers and subsurface environments such as oil reservoirs where they play an important role in the sulfur cycle. We determined the genome sequence of the oil field isolate Sulfurimonas sp. strain CVO and compared its gene expression during nitrate-dependent sulfide oxidation to the coastal sediment isolate Sulfurimonas denitrificans. Formation of elemental sulfur (S-0) and high expression of sulfide quinone oxidoreductase (SQR) genes indicates that sulfide oxidation in both strains is mediated by SQR. Subsequent oxidation of S-0 was achieved by the sulfur oxidation enzyme complex (SOX). In the coastal S. denitrificans, the genes are arranged and expressed as two clusters: soxXY(1)Z(1)AB and soxCDY(2)Z(2)H, and sulfate was the sole metabolic end product. By contrast, the oil field strain CVO has only the soxCDY(2)Z(2)H cluster and not soxXY(1)Z(1)AB. Despite the absence of the soxXY(1)Z(1)AB cluster, strain CVO oxidized S-0 to thiosulfate and sulfate, demonstrating that soxCDY(2)Z(2)H genes alone are sufficient for S-0 oxidation in Sulfurimonas spp. and that thiosulfate is an additional metabolic end product. Screening of publicly available metagenomes revealed that Sulfurimonas spp. with only the soxCDY(2)Z(2)H cluster are widespread suggesting this mechanism of thiosulfate formation is environmentally significant