Clone-FISH

Clone-FISH was performed as previously described⁵¹. In brief, a purified plasmid containing the ‘Ca. A. ciliaticola’ 16S rRNA gene sequence (as described in ‘Clone library construction and Sanger sequencing’) in the correct orientation (confirmed by PCR using M13F and 1492R primers) was transformed into electrocompetent Escherichia coli JM109(DE3) cells (Promega) by electroporation using the Cell Porator and Voltage Booster System (Gibco) with settings 350 V, 330 μF capacitance, low ohm impedance, fast charge rate and 4 kΩ resistance (Voltage Booster). After electroporation, cells were transferred into SOC medium (Sigma Aldrich), incubated for 1 h at 37 °C and plated onto LB plates containing 100 mg l⁻¹ kanamycin. After incubation overnight at 37 °C, 4 clones were picked and the presence of the insert was checked with PCR (primers M13F and 1492R) as described in ‘Clone library construction and Sanger sequencing’, followed by gel electrophoresis. An insert-positive clone was selected at random and grown in LB medium containing 100 mg l⁻¹ kanamycin at 37 °C until optical density at 600 nm reached 0.37. Transcription of the plasmid insert was induced using isopropyl β-d-1-thiogalactopyranoside (IPTG) (1 mM final concentration). After addition of IPTG, cells were incubated for 1 h at 37 °C followed by addition of 170 mg l⁻¹ chloramphenicol and subsequent incubation for 4 h. Cells were collected by centrifugation, fixed in 2% formaldehyde solution for 1 h at room temperature, washed and stored at 4 °C in phosphate-buffered saline (pH 7.4) containing 50% ethanol until further processing. Formamide melting curves⁵² were carried out using a ‘Ca. Azoamicus’-specific, HRP-labelled probe (eub62A3_813) (Extended Data Fig. ​Fig.5).5). In brief, cells were applied to glass slides. Permeabilization with lysozyme, peroxidase inactivation, hybridization (10%, 30%, 35%, 40%, 45% and 50% formamide) and tyramide signal amplification (Oregon Green 488) was performed as previously described⁵³. For each formamide concentration, images of three fields of view were recorded using the same exposure time for all formamide concentrations, which was optimized at 10% formamide all same settings using an Axio Imager 2 microscope (Zeiss) and analysed using Daime 2.1⁵⁴.