Generation and characterization of the spo0A mutant. (A) Confirmation of pure culture of the spo0A mutant by PCR, using different combinations of primers. Amplification of intron-spo0A junction regions using one primer in the genome and the other in the intron (Spo0AF/intronF1 and intronR1/Spo0AR) resulted in bands from the Ccel_1894 mutant (lanes 1 and 2), but not in wild-type (WT) cells (lanes 4 and 5). In PCR reactions using Spo0AF/Spo0AR primers, the mutant showed a single band (lane 3), which was 915 bp larger than the single band (lane 6) in WT cells, confirming the expected intron insertion. (B) Southern blot with biotin-labeled intron-specific probes showing a single intron insertion band for spo0A mutant (lane 3), and no band for WT (lane 2). The intron donor plasmid was used as a positive control (lane 1). (C) Agar plate showing that the colonial morphology of spo0A mutant (right) was flatter and more translucent compared to WT (left). (D) Comparison of cellulose fermentation broth showing that spo0A mutant culture produced a yellow-green pigment (right), which was not observed for WT (left).