Replacement of native spa promoter with P_xyl/tet to distinguish cleavage from alternative transcriptional starting points.

(A) Plasmid construction of pRAB11-spa-gpmCherry. The first three elements depict source DNA fragments and the last element the final vector. The +1 position of the 5’ end of the spa transcript was cloned into position +1 of the ATc inducible pRAB11 plasmid. When induced, the resulting transcript is expected to be identical to that from the native spa promoter. Part of gpmCherry was integrated into the sequence to enable specific primer binding to the chimeric transcript and therefore eliminate the native spa transcript background. Promoters are depicted as angled arrows, promoter elements as white filled boxes, genes as arrows. (B) Close-up DNA sequence of promoter and transcript region of pRAB11-spa-gpmCherry. Promoter region (-35 and -10) shown in blue, spa transcript in black and green. The first UACAU is underlined and marked in green and the first VUUV’ site is marked in blue. (C) Primer extension result of HG003 and ΔmazEF containing pRAB11-spa-gpmCherry. Full length transcripts (fuzzy circle) were strongly visible upon induction with ATc. The signal at UACAU (arrowhead) occurred only in the HG003 WT, but not in the mazEF deletion mutant strain indicating actual cleavage.