Extended Data Fig. 7: Determination of phase-variable promoter switching for six loci encoding putative S-layer proteins.

Determination of phase-variable promoter switching for six loci encoding putative S-layer proteins. The hypothesis that the promoters associated with seven additional B. thetaiotaomicron S-layer like lipoproteins are phase variable was validated using a PCR amplicon sequencing strategy. Because of high nucleotide identity in both the regions flanking the 7 additional loci, a nested PCR approach was required to specifically amplify and sequence each site. In the first step, a primer lying in each S-layer gene (Supplementary Table 5 “S-layer gene” primers) was oriented towards the promoter and used in a PCR extension to a primer in the upstream recombinase gene (Supplementary Table 5 “recombinase gene 3” primer). The products of this PCR were purified without gel extraction and used in a second reaction with a nested primer that lies internal to the previous recombinase gene primer (Supplementary Table 5 “recombinase 2” primer). The expected PCR products from this reaction, which are ~1 kb and span promoter sequences in both the ON and OFF orientations, were excised and used for an orientation-specific PCR using the original S-layer gene primer for each site and a universal primer (green schematic) that was designed for each promoter and is oriented to extend upstream of the S-layer gene (e.g., OFF orientation). Resulting products from this third reaction, which should correspond to the ON orientation if a promoter inversion has occurred in some cells, were obtained for 5/7 of the additional identified loci and the BT1927 S-layer locus as a control. In all cases in which an amplicon and sequence were obtained, the expected recombination occurred between the inverted repeat site proximal to the S-layer gene start (new DNA junction), which would orient the promoter to enable expression of the downstream S-layer gene. The sequences shown are the consensus between forward and reverse reads for each amplicon. The putative core promoter -7 sequence is shown in bold/red text, the coding region of each S-layer gene is shown in bold/blue text and the S-layer gene proximal recombination site is noted and highlighted in bold/gold text. Note that the 5′-end of the sequenced amplicon was not resolved for the BT2486 locus.