Diversity and Distribution of Thermophilic Bacteria in Hot Springs of Pakistan

Despite recent advances in commercially optimized identification systems, bacterial identification remains a challenging task in many routine microbiological laboratories, especially in situations where taxonomically novel isolates are involved. The 16S rRNA gene has been used extensively for this task when coupled with a well-curated database, such as EzTaxon, containing sequences of type strains of prokaryotic species with validly published names. Although the EzTaxon database has been widely used for routine identification of prokaryotic isolates, sequences from uncultured prokaryotes have not been considered. Here, the next generation database, named EzTaxon-e, is formally introduced. This new database covers not only species within the formal nomenclatural system but also phylotypes that may represent species in nature. In addition to an identification function based on Basic Local Alignment Search Tool (blast) searches and pairwise global sequence alignments, a new objective method of assessing the degree of completeness in sequencing is proposed. All sequences that are held in the EzTaxon-e database have been subjected to phylogenetic analysis and this has resulted in a complete hierarchical classification system. It is concluded that the EzTaxon-e database provides a useful taxonomic backbone for the identification of cultured and uncultured prokaryotes and offers a valuable means of communication among microbiologists who routinely encounter taxonomically novel isolates. The database and its analytical functions can be found at http://eztaxon-e.ezbiocloud.net/.

Next-generation sequencing techniques, and PhyloChip, have made simultaneous phylogenetic analyses of hundreds of microbial communities possible. Insight into community structure has been limited by the inability to integrate and visualize such vast datasets. Fast UniFrac overcomes these issues, allowing integration of larger numbers of sequences and samples into a single analysis. Its new array-based implementation offers orders of magnitude improvements over the original version. New 3D visualization of principal coordinates analysis (PCoA) results, with the option to view multiple coordinate axes simultaneously, provides a powerful way to quickly identify patterns that relate vast numbers of microbial communities. We demonstrate the potential of Fast UniFrac using examples from three data types: Sanger-sequencing studies of diverse free-living and animal-associated bacterial assemblages and from the gut of obese humans as they diet, pyrosequencing data integrated from studies of the human hand and gut, and PhyloChip data from a study of citrus pathogens. We show that a Fast UniFrac analysis using a reference tree recaptures patterns that could not be detected without considering phylogenetic relationships and that Fast UniFrac, coupled with BLAST-based sequence assignment, can be used to quickly analyze pyrosequencing runs containing hundreds of thousands of sequences, revealing patterns relating human and gut samples. Finally, we show that the application of Fast UniFrac to PhyloChip data could identify well-defined subcategories associated with infection. Together, these case studies point the way towards a broad range of applications and demonstrate some of the new features of Fast UniFrac.

The Rehai and Ruidian geothermal fields, located in Tengchong County, Yunnan Province, China, host a variety of geochemically distinct hot springs. In this study, we report a comprehensive, cultivation-independent census of microbial communities in 37 samples collected from these geothermal fields, encompassing sites ranging in temperature from 55.1 to 93.6°C, in pH from 2.5 to 9.4, and in mineralogy from silicates in Rehai to carbonates in Ruidian. Richness was low in all samples, with 21–123 species-level OTUs detected. The bacterial phylum Aquificae or archaeal phylum Crenarchaeota were dominant in Rehai samples, yet the dominant taxa within those phyla depended on temperature, pH, and geochemistry. Rehai springs with low pH (2.5–2.6), high temperature (85.1–89.1°C), and high sulfur contents favored the crenarchaeal order Sulfolobales, whereas those with low pH (2.6–4.8) and cooler temperature (55.1–64.5°C) favored the Aquificae genus Hydrogenobaculum. Rehai springs with neutral-alkaline pH (7.2–9.4) and high temperature (>80°C) with high concentrations of silica and salt ions (Na, K, and Cl) favored the Aquificae genus Hydrogenobacter and crenarchaeal orders Desulfurococcales and Thermoproteales. Desulfurococcales and Thermoproteales became predominant in springs with pH much higher than the optimum and even the maximum pH known for these orders. Ruidian water samples harbored a single Aquificae genus Hydrogenobacter, whereas microbial communities in Ruidian sediment samples were more diverse at the phylum level and distinctly different from those in Rehai and Ruidian water samples, with a higher abundance of uncultivated lineages, close relatives of the ammonia-oxidizing archaeon “Candidatus Nitrosocaldus yellowstonii”, and candidate division O1aA90 and OP1. These differences between Ruidian sediments and Rehai samples were likely caused by temperature, pH, and sediment mineralogy. The results of this study significantly expand the current understanding of the microbiology in Tengchong hot springs and provide a basis for comparison with other geothermal systems around the world.