Strain or plasmid Description a Reference or source b. 168 trpc2 Laboratory. 1A780 trpc2 sigb::spc BGSC

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1 TABLE S1 Bacterial strains and plasmids Strain or plasmid Description a Reference or source b B. subtilis 168 trpc2 Laboratory stock 1A780 trpc2 sigb::spc BGSC BM1010 trpc2 htpx::pgs1582; Em r BM1302 trpc2 amye::pgs1829; Cm r BM1482 trpc2 amye::pgs1829 ykrk::pgs1932; Cm r, Em r BM1495 trpc2 amye::pgs1829 htpx::pgs1582; Cm r, Em r BM1501 trpc2 amye::pgs1829; sigb::spc; Cm r, Sp r BM1506 trpc2 amye::pgs1962; Cm r BM1537 trpc2 amye::pgs1962 ykrk::pgs1932; Cm r, Em r BM1545 trpc2 amye::pgs1994; Cm r BM1574 trpc2 ftsh::pgs2010; Em r BM1579 trpc2 amye::pgs2018; Cm r BM1889 trpc2 htpx::kan; Km r BM1892 trpc2 ftsh::pgs2010 htpx::kan; Em r, Km r BM1907 trpc2 amye::pgs2197; Cm r BM2024 trpc2 amye::pgs2415; Cm r BM2025 trpc2 amye::pgs2415; ykrk::pgs1932; Cm r, Em r BM2026 trpc2 amye::pgs2416; Cm r BM2098 trpc2 amye::pgs2018; ykrk::pgs1932; Cm r, Em r E. coli BL21(DE3) F ompt hsds gal dcm λ(de3) Novagen DH5α F ф80dlacz M15 (laczya-argf) reca1 gyra Laboratory 1

2 JM109 enda1 rela1 supe44 hsdr17 reca1 supe44 enda1 hsdr17 gyra96 rela1 thi Δ(lac-proAB) F'[traD36 proab + laci q laczδm15] stock Takara Plasmids pdg780 pdl pet22b Cloning vector carrying a kanamycin resistance 10 cassette; Ap r Km r amye integration vector for generation of 30 transcriptional fusions to bgab; Ap r, Cm r IPTG-inducible expression vector for producing Novagen His-tagged proteins in E. coli; Ap r pmad Vector used for deletion/replacement of genes; Ap r Em r 2 pqe80 IPTG-inducible expression vector for producing His-tagged proteins in E. coli; Ap r Qiagen prn5101 Vector used for gene disruption; Ap r, Em r 9 pgs1582 prn5101 carrying an internal region close to the N terminus of htpx; htpx disruption plasmid pgs1829 pdl carrying the regulatory region of htpx pgs1932 prn5101 carrying an internal region close to the N terminus of ykrk; ykrk disruption plasmid pgs1962 pdl carrying the regulatory region of ykrk pgs1990 pqe80 carrying the ykrk gene pgs1994 pgs2010 pdl carrying a mutation in the 10 box of the ykrk σ A promoter within the htpx-bgab fusion prn5101 carrying an internal region close to the N terminus of ftsh; ftsh disruption plasmid pgs2018 pdl carrying a mutation in the right arm of the 2

3 pgs2197 inverted repeat in the promoter region of the htpx-bgab fusion pdl carrying a mutation in the 10 box of the htpx σ A promoter of the htpx-bgab fusion pgs2264 pet22b carrying the wild-type htpx gene pgs2277 pmad derivative for htpx deletion pgs2305 pet22b carrying the E156A mutant gene pgs2415 pgs2416 pdl carrying a mutation in the left arm of the inverted repeat in the promoter region of the htpx-bgab fusion pdl carrying a mutation in the 10 box of the ykrk σ A promoter of the ykrk-bgab fusion a Ap r, ampicillin resistant; Cm r, chloramphenicol resistant; Em r, erythromycin resistant; Km r, kanamycin resistant; Sp r, spectinomycin resistant; kan, kanamycin resistance gene; spc, spectinomycin resistance gene. b BGSC, Bacillus Genetic Stock Center. 3

4 TABLE S2 Oligonucleotide primers used in this study Name A024 A025 A073 A275 A454 A455 A569 A570 A591 A592 A657 A658 A898 A899 R752 R935 R936 R978 T544 T545 T586 T587 T682 T859 T901 T902 T944 Sequence (5 3 ) a GCGGGATCCAAGCGTCAGCAAATTAC GCCAAAGCTTCCCGATTGTTGTAAGCA GCACCATATCGGTTCGA CGATTGTTGTAAGCACCA TTAAATATACGGGTTCTTTTGAA TTCAAAAGAACCCGTATATTTAATAAATAAAACATCCATT GCCAACACATATGGCGAAAAGAATTTTTCT GCCACTCGAGTTTAGCTTCCAGCCGTC GCCACTGCAGTTCAAAAGAACCCGTAT GCCAGTCGACATGCTGACAGGGGAG AAGGTGTGCTTGCCCACGCGGTCGCGCATATCACAA CGTGGGCAAGCACACCTT TTTTGAACTTAAAATAACGGAGG CCTCCGTTATTTTAAGTTCAAAACTTGCCGTATATTTAAA GCGGGATCCAGTCGCAAACGCG GCCAAAGCTTGTCTGTTTTAAGCTCTG GCGGAATTCCATCTGCTCCAGCAGC GCCAAAGCTTCTCGTCCAATCAGCG GCCAAAGCTTCCTGGATGACGGGAAAG GCGGGATCCTCTGTATACAGCTTC GCGGGATCCAAGCTCTACGATTTCAT GCAGGTACCGCCATCAGCTGTAA GCGGAATTCTTTAGCTTCCAGCCGTCT GCGGGATCCGTATGTGTTCAAAGTC GCGGGATCCGATACCGCCATCAGCT GCAGGTACCAAGCTCTACGATTTCAT GCGGGATCCAACATTTTTAAACTCTCTCG 4

5 T945 T955 T956 T998 T999 GCCAAAGCTTGGTGGCGCAGACTATAG GTCAGCAAATTACATTAACATCTATCGTCGAATTCCTTGCAT GATGTTAATGTAATTTGCTGAC TTAAAATAACGGAGGTTGTTATG CATAACAACCTCCGTTATTTTAACAACAAAAGAACCCGTAT a Underlined sequences represent restriction sites inserted. 5

6 Bsu Bpu Bam Bli Bsu Bpu Bam Bli Bsu Bpu Bam Bli ykrk (SD) AATGTTCATT-GTTCACCTCTTTAAACAATTTGACGCGGATATAGGTCT ATGTATAAG TCCACAAAGTGACGTCACAATTTATTCACTTTTTAGCTGTGGTATATGTCAT--AAATATTGAAG CGTGTTCATGG-TTCACCTCT GATTGATTACGTGT AGGTTTCATCATTCCACCTC ATTATGTAT * ** -35 TTCAAAATTCAAGCGTCAGCAAATTACATTAACATCATACGTCGAATTCCTTGCATTTTCAAATG TGACGGGGCTTAAGCCGTTATGATAAATAGAGGAAATGATTCCTTTTTCCTTGCAATTTGAGACG TTACTTTTTAATATCACTGAGAATTACATTAACATCATACGTCGAAATTGTTGCATTTTCAAACA TAAAATG---AAATAGCAAACAATATAAACATTATCTTACGTCTTATCACTTGCATTTTCAATTC * ** * * -10 * * ***** *** * GATGTTT-TATAATTTAAATATACGGGTTCTTTTGAACTTAAAATAA---CGGAGGTTGT-TATG TTTATTTTTATAATTTTATTAATAA-GTTCAAATGAACTTAATATGACAGCGGAGGTTATGTATG GATCATT-TATAATTTAAGTAAATAAGTTCTTTTGATCTTAAAAAAG-A-CGGAGGTAAT-TATG TATGCCG-TATAATTTTATTATACGAGTTCATTTGATCTTATTATAACA-TGGAGGTAAT-CATG * ******** * ** **** *** **** * ****** * *** (SD) htpx FIG S1 Multiple alignments of nucleotide sequences of the ykrk htpx intergenic regions of B. subtilis and some close relatives. Shine Dalgarno sequences (SD) and translational start sequences are underlined and denoted in boldface. Identical nucleotides are indicated by an arterisk. The inverted repeat that serves as the YkrKbinding site is indicated by an inverted pair of solid arrows above the sequence. All nucleotide sequences are taken from GenBank databases. Bsu, B. subtilis 168; Bpu, B. pumilus SAFR-032 ; Bam, B. amyloliquefaciens FZB42; Bli, B. licheniformis ATCC

7 htpx mrna 1 2 rrna FIG S2 Northern blot analysis of heat induction of htpx expression. Wild-type B. subtilis cells were grown at 37 C in 250 ml of LB medium to an OD600 of 0.3, and then the culture was split into two parts. One part was maintained at 37 C for 20 min (lane 1), and the other was shifted to 51 C for 20 min (lane 2). Total RNAs were then individually isolated. A 32 P-labeled htpx-specific probe was used in the hybridization reaction. The upper panel shows autoradiography of a hybridization membrane. The lower panel shows ethidium bromide-stained 16S and 23S rrnas in a denaturing agarose gel.

8 BgaB activity (Miller units) Time (min) FIG S3 Effect of mutation in 10 box of the σ A promoter of ykrk (TAT to ATA) within the regulatory region of the htpx-bgab fusion on BgaB activity. The transcriptional fusion of the regulatory region (containing the wild-type or mutated ykrk promoter) of htpx to bgab was integrated at the amye locus of B. subtilis. Cells were grown at 37 C in LB medium to an OD 600 of 0.3, and then the culture was split into two portions. One portion was maintained at 37 C (circles), and the other was shifted to 51 C (squares) at time zero. The cells were then grown for various times as indicated. Solid symbols, no mutation (BM1302); open symbols, with mutation (BM1545). The values shown are means from two independent experiments. Individual values did not differ by more than 15% from the means.

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tgg agg Supplementary Figure S1.

tgg agg Supplementary Figure S1. ttaggatattcggtgaggtgatatgtctctgtttggaaatgtctccgccattaactcaag tggaaagtgtatagtaatgaatctttcaagcacacagatcacttcaaaagactgtttcaa catcacctcaggacaaaaagatgtactctcatttggatgctgtgatgccatgggtcacag attgcaattcccaagtgcccgttcttttacaccaaaatcaaagaagaatatctccccttt