Reverse transcription-quantitative PCR (RT-qPCR) analysis

cDNA was synthesized from the isolated RNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was performed using Fast Start SYBR Green Master (Roche, Mannheim, Germany) or Power SYBR Green PCR Master Mix (Thermo Fisher Scientific), and the measurement was performed using a C1000 Touch Thermal Cycler (Bio-Rad, CA, USA). The sequences of the primers used for qPCR are listed in Table ^S6. The expression level of gyrA was used as an internal control⁶⁰ to normalize that of nifH and cydA (Figs. ^S1 and ^S2), and the expression level of rho was used for the other experiments (Figs. ^S3, ^S4, ^S5a, ^S6, and ^S10) because the transcriptome analysis results (Fig. 1a) showed that the expression level of rho had less variation among the culture conditions. The relative gene expression was quantified using the standard curve method.