Table S1. Primers and PCR digestion schemes used in this study

Transcription

1 Table S1. Primers and schemes used in this study PS locus promoter region Primer sequence (5-3 ) Product size (bp) Enzyme Fragment sizes (bp) when promoter is on off psa TGTGTAAATGATAGGAGGCTAGGG GTTGACGGAAATGATCGGTATAG CGCAAAGATTACTTTTCCATTTACTTCG TGCGCTTCGCGGTTTGAACG TGTTGGAGATAATTCCCGGTATCTGCGCTC GTCACCTTTGATACGGACAAACTCACCCTC GCCTTTTCCGTTGCTTACTG ACTTAAGTCCGGGTATGCTTCG CGTTTCATGTAAGGCGGATT CCAGTTCAAAGCGGAAGAAG TTTGCTTGTTGTCCGTTTTG TCGAAGTCACCACCTGTAATAC AAAAAGAAGATGACAAGCTCCCTAT TCCTGTCACACGCACACGCTG 1,076 SspI RsaI ,215 EarI SspI ,081 SfcI ,059 DraI ,281 PacI

2 A Step 1: Isolate bacterial DNA from a broth culture, fecal sample, or abscess. Step 2: Set up PCR using primers flanking the invertible promoter regions of each of the 7 polysaccharide biosynthesis loci on off PS biosynthesis genes PS biosynthesis genes Step 3: Digest PCR products with the appropriate restriction enzyme on 1 2 off 3 4 Step 4: Resolve digested PCR products on an agarose gel Step 5: Measure the intensity of each of the 4 DNA fragments in the gel following staining with ethidium bromide B Number of cycles in the PCR FIG S1. method to quantify PS locus promoter orientations within B fragilis populations. A. Schematic of the method. Illustration of a single PS biosynthesis locus with the invertible promoter region marked with a light gray box. Large black arrows represent the promoter. Thin black lines across the invertible regions mark the restriction enzyme site. The small black arrows represent the primers used in the PCRs. Dark gray boxes represent the beginning of the PS biosynthesis operon. B. PCR products resulting from the amplification of the promoter in both the on and the off orientation. PCRs were performed on B fragilis chromosomal DNA isolated from broth cultures of strains of B. fragilis with the PSE promoter locked in the on or off orientation due to a deletion of the gene encoding Mpi (Δmpi mutant 2 and Δmpi mutant 8, ref 5).

3 Pregnant B. fragilis monocolonized mice. LITTER A LITTER B Moved to SPF facility 9 weeks 9 weeks Left in Gnotobiotic Isolator Moved to SPF facility Left in Gnotobiotic Isolator COMPLEX COLIZED 2 weeks 2 weeks MOCOLIZED COMPLEX COLIZED MOCOLIZED FIG. S2. Housing history of mice from which fecal samples were examined for PS locus promoter orientations. Two age-matched unrelated pregnant gnotobiotic mice were housed with a B. fragilis monocolonized mouse in the same gnotobiotic isolator but were received 45 days apart. The pregnant mice gave birth within three days of colonization with B. fragilis. When the male pups reached 9 weeks of age, was performed on B. fragilis DNA from fecal samples and half of the male pups from each litter were moved to a SPF facility to allow for further acquisition of a complex flora. was performed on bacterial DNA from fecal samples of monocolonized and complex colonized mice two weeks after the mice were moved to the SPF facility.

4 FIG. S3. PS locus promoter orientations from B. fragilis in fecal samples from monocolonized mice. Ethidium bromide-stained agarose gels of the fragments resulting from of bacteria from fecal samples of 9-week old mice monocolonized with B. fragilis from birth. Each lane represents the data from one mouse.

5 MOCOLIZED COMPLEX COLIZED MOCOLIZED COMPLEX COLIZED FIG. S4. B. fragilis PS locus promoter orientations from bacteria in fecal samples of mice after two weeks of complex colonization. Ethidium bromide-stained agarose gels of the fragments resulting from PCR digestion of bacteria isolated from fecal samples of B. fragilis monocolonized mice that had been further colonized with a complex microbiota (COMPLEX COLIZED) for 2 weeks and monocolonized littermate controls (MOCOLIZED). Each lane represents the data from one mouse.

6 FIG. S5. PS locus promoter orientations from B. fragilis in intra-peritoneal abscesses. Mice were injected i.p. with B. fragilis mixed with sterile cecal contents. After seven days, the mice were sacrificed and abscesses were removed. One abscess was isolated from each of seven mice. Each lane represents the data from one mouse.

7 TACCTTTGTGTTTATAAACGAACGTTTTTTGAAACA TACCTTTGTTCAATCAAACGAACGTTTTTTGAAACA TACCTTTGTGCTATTAAACGAACGTTTTTTGAAACA TACCTTTGTTCCATTGAACGAACGTTTTTTGAAACA TATCTTTGCGTTCAATTAAACGAACGTCTATTGAAACACT TATCTTTGCGTTCAATTAGACGATCGTCTATTGAAACA TATCTTTGCGTTCAAATAGACGAACGTTTAAAAACA FIG. S6. Sequences of the inverted repeat regions just downstream of each of the seven polysaccharide biosynthesis loci promoters. All regions are shown in the promoter on orientation. The promoter -7 site is shown in pink font. The inverted repeat regions described by Krinos et al. (Nature, 2001,441:555-8) are underlined with the consensus mpi recognition site shown in blue (Proc. Natl. Acad. Sci : ). The extended fin recombination regions described by Patrick et al. (Microbiology, : ) are shown in italic font.