Optimization of ‘Aigarchaeota’ (G1)-Specific Probes Utilizing Clone-FISH

Event Center BC

‘Aigarchaeota’, a currently uncultivated candidate archaeal phylum, includes both thermophilic and hyperthermophilic microorganisms that reside in terrestrial and marine geothermal environments. This archaeal phylum contains nine proposed genuslevel groups that have been confirmed via 16S rRNA sequencing, with the first group (Aig G1) being the focus of this study. To better detect and quantify Aig G1 in natural samples and enrichment cultures, CloneFISH (Fluorescence in situ hybridization) techniques were used to test possible oligonucleotide probes and to optimize hybridization conditions for FISH. A nearfull length 16S rRNA gene obtained from Aig G1 was cloned into the plasmid pGEM-T. This construct was transformed into E.coli JM109 (DE3) and the Aig G1 16S rRNA was subsequently induced by treatment with isopropyl beta-D-1-thiogalactopyranoside (IPTG) and chloramphenicol. FISH was then conducted with probes targeting the Aig G1 16S rRNA. Fluorescence signal intensities were observed using an epifluorescence microscope for induced and uninduced samples, as well as for a number of negative controls, at various formamide concentrations with fluorescently-labeled bacterial-specific (positive control for hybridization), archaeal-specific, and ‘Aigarchaeaota’-specific oligonucleotide probes. Induced, but not uninduced, cells were positive for the Archaea-specific probe, indicating successful induction of the Aig G1 16S rRNA gene transcript. A previously published Aig G1-specific probe showed specificity but had a relatively low signal intensity in comparison to the archaeal probe at all formamide concentrations, suggesting poor binding of this probe. Two other newly developed ‘Aigarchaeaota’ (G1)-specific probes were observed and compared at five different formamide concentrations, one of which (Aig G1 180 Cy3) was found to have low intensity and the other (Aig G1 1012 Cy3) that was at least 4-fold brighter than the published probe, with an apparent optimal hybridization conditions at 30% formamide. This work will allow for better observation of Aig G1 in natural samples and enrichment cultures, and will facilitate use of FISH coupled with nano-scale stable isotope mass spectrometry to track the uptake of 13C labeled compounds by Aig G1 in future studies of their metabolic capabilities.