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While bacterial communities from McMurdo Dry Valley soils have been studied using molecular techniques, data from permafrost are particularly scarce given the logistical difficulties of sampling. This study examined the molecular diversity and culturability of bacteria in permafrost from the Taylor Valley (TV), Antarctica. A 16S rRNA gene clone library was constructed to assess bacterial diversity, while a clone library of the RNA polymerase beta subunit ( rpoB) gene was constructed to examine amino acid composition of an essential protein-coding gene. The 16S rRNA gene clone library was dominated by Acidobacteria from Gp6 and Gemmatimonadetes. The rpoB gene clone library (created with primers designed in this study) was also dominated by Acidobacteria. The ability of sequence analyses to garner additional information about organisms represented by TV sequences was explored. Specifically, optimum growth temperature was estimated from the stem GC content of the 16S rRNA gene, while potential cold adaptations within translated rpoB sequences were assessed. These analyses were benchmarked using known psychrophiles and mesophiles. Bioinformatic analyses suggested that many TV sequences could represent organisms capable of activity at low temperatures. Plate counts confirmed that c. 103 cells per gram permafrost remained viable and were culturable, while laboratory respiration assays demonstrated that microbial activity occurred at −5 °C and peaked at 15 °C. [ABSTRACT FROM AUTHOR]