RECENT ADVANCEMENTS IN METAGENOMICS AND SINGLE-CELL GENOMICS HAVE ENABLED UNPRECEDENTED ACCESS TO GENOMES FROM CANDIDATE (YET-UNCULTIVATED) PHYLA OF BACTERIA AND ARCHAEA INCLUDING THOSE INHABITING EXTREME AND EARLY EARTH-LIKE SYSTEMS. THESE DATA HAVE ENABLED INSIGHTS INTO THE BIOLOGY OF THESE ORGANISMS INCLUDING THEIR CATABOLIC AND ANABOLIC POTENTIAL MOLECULAR ADAPTATIONS TO LIFE IN EXTREME ENVIRONMENTS AND INSIGHTS INTO THE EARLY DIVERSIFICATION OF MICROBIAL LINEAGES. HOWEVER THE PREDICTED METABOLIC FUNCTIONS OF THESE ORGANISMS AND THEIR GENES REMAIN LARGELY UNTESTED. THE CENTRAL GOAL OF THIS PROPOSAL IS TO LEVERAGE A FOUNDATION OF GENOMIC EXPLORATION IN GREAT BOILING SPRING (GBS) NV TO ASSESS METABOLIC FUNCTIONS OF THERMOPHILES REPRESENTING CANDIDATE PHYLA OF BACTERIA AND ARCHAEA AND THEIR ROLES IN MAJOR BIOGEOCHEMICAL CYCLES. WE HAVE RECENTLY DESCRIBED THE FIRST SUBSTANTIAL GENOMIC DATA FOR THE CANDIDATE BACTERIAL PHYLA ATRIBACTERIA (OP9) CALESCAMANTES (EM19) AND FERVIDIBACTERIA (OCTSPA1-106) AND KRYPTONIA. TO ADDRESS THE METABOLIC FUNCTIONS OF THESE AND OTHER YET-UNCULTIVATED MAJOR LINEAGES WE WILL USE A COMBINATION OF TARGETED AND BROADBASED APPROACHES. OBJECTIVE 1: ASSESS C AND N ASSIMILATORY METABOLISMS IN SPECIFIC TARGETED TAXA IN MICROCOSMS AND ENRICHMENT CULTURES USING (A) STABLE-ISOTOPE PROBING (SIP) FLUORESCENCE IN SITU HYBRIDIZATION AND NANOSIMS IMAGING (FISH-SIMS) AND (B) IN SITU TRANSCRIPTION OF GENES PREDICTED TO BE INVOLVED IN H C N AND S CYCLING BY QUANTITATIVE REVERSE-TRANSCRIPTASE PCR. OBJECTIVE 2: DETERMINE METABOLIC FUNCTION OF ABUNDANT COMMUNITY MEMBERS USING BROAD-BASED APPROACHES SUCH AS (A) 16S RRNA SIP WITH A CUSTOM PHYLOGENETIC MICROARRAY (CHIP-SIP) TO ASSESS C AND N ASSIMILATION IN MICROCOSMS AND ENRICHMENT CULTURES (B) WHOLECOMMUNITY TRANSCRIPTOMICS AND (C) WHOLE-COMMUNITY PROTEOMICS. ULTIMATELY THE COMBINATION OF BOTH TARGETED AND UNTARGETED APPROACHES WILL PROVIDE ROBUST EVIDENCE OF THE ECOLOGICAL ROLES OF SEVERAL YET-UNCULTIVATED MICROBIAL PHYLA IN GBS AND BEYOND.