Supplementary experimental procedures
Construction of B. subtilis strains
Mutant alleles were first introduced into B. subtilis strain 168 and then transferred into
strain NCIB3610 (Kobayashi, 2007a). Deletion of the bacABCDEF operon was
performed using an overlap-extension PCR technique. A cat cassette was PCR amplified
from pCBB31 (Kobayashi, 2007a) using primers pUC-R and pUC-F; primer sequences
are presented in Table S1. Upstream and downstream regions of the bacA operon were
PCR amplified from NCIB3610 chromosomal DNA using primer pairs ywfBGF1/ywfBG-R1 and ywfBG-F2/ywfBG-R2, respectively. The 5 ywfBG-R1 and ywfBG-F2
sequences were complementary to pUC-R and pUC-F sequences, respectively. To extend
and connect the three PCR fragments, all three were mixed and used as template for a
second PCR amplification using primers ywfBG-F1 and ywfBG-R2. The resultant PCR
products were used for strain 168 transformation.
Deletion of sfp or ppsA was performed using the same procedure and different primer
sets. Upstream and downstream sfp regions were amplified using primer pairs sfp-DF1/sfp-D-R1 and sfp-D-F2/sfp-D-R2, respectively. Upstream and downstream ppsA
regions were amplified using primer pairs ppsA-F1/ppsA-R1 and ppsA-F2/ ppsA-R2,
respectively.
The PpgsB-gfp transcriptional fusion was constructed as follows. The pgsB promoter
region was PCR amplified from NCIB3610 chromosomal DNA using primers pgsB-P-F2
and pgsB-P-R1. The PCR products were digested with MunI and BamHI and inserted into
the corresponding sites of pDCG-1, which harbors the gfp reporter and the amyE
integration sequence (Kobayashi, 2007a). The resultant plasmid, pDCGpgsB, was used
to transform strain 168. The final product was a strain harboring PpgsB-gfp at the genomic
amyE site.
The construction of PsrfA-gfp and PppsA-gfp was performed using the same
procedure and different primer sets. The srfA and ppsA promoter regions were amplified
using primer sets srfAA-P-F/srfAA-P-R and ppsA-P-F/ppsA-P-R, respectively.
Probes for Northern blot analysis
Northern blot analysis and probe preparation were performed as described previously
(Kobayashi, 2007b). To prepare digoxigenin (DIG)-labeled RNA probes, DNA fragments
were PCR amplified using primers listed in Table S1.
Purification of mCherry
The mCherry coding region was PCR amplified using primers mCherry-F1 and
mCherry-R2 (Table S1). The PCR products were digested with NdeI and XhoI and
inserted into the corresponding sites of pET22b, an expression vector for 6  His fusion
proteins (Novagen). The resultant plasmid was designated pETmCherry. E. coli
BL21(DE3)pLysS harboring pETmCherry was grown in 1 l of LB (Difco) supplemented
with 50 mg ampicillin at 30C. When the culture reached OD600 = 0.6, mCherry-6  His
expression was induced with 1 mM isopropy--D-1-thiogalactopyranoside (IPTG), cells
were grown for 2 h, harvested, suspended in 15 ml binding buffer (20 mM Tris-HCl, pH
7.6, 500 mM NaCl, and 10 mM imidazole), and disrupted by sonication. Cell lysate was
centrifuged three times at 8,000 rpm for 10 min, and the supernatant was mixed with 2
ml of Ni-NTA resin (Qiagen) for 1 h. The mixture was loaded onto a column, washed 20
times with 10 ml binding buffer, and mCherry-6  His was eluted with 1.4 ml of elution
buffer (20 mM Tris-HCl, pH 7.6, 500 mM NaCl, and 300 mM imidazole). The elution
fraction was fractionated using a Sephacryl S-200 column (50 cm  2 cm; GE
Healthcare). Fractions containing mCherry-6  His were combined and dialyzed against
buffer (20 mM Tris-HCl, pH 7.6, and 100 mM NaCl). After dialysis, the sample was
stored at 4C.
References
Kobayashi, K. (2007a) Bacillus subtilis pellicle formation proceeds through genetically
defined morphological changes. J Bacteriol 189: 4920-4931.
Kobayashi, K. (2007b) Gradual activation of the response regulator DegU controls serial
expression of genes for flagellum formation and biofilm formation in Bacillus subtilis.
Mol Microbiol 66: 395-409.
Table S1. Primers used in this study
Primers
Sequences (5' to 3')
16S-seqF1
TTAGCGGCGGACGGGTGAGT
16S-seqR1
TGACGGGCGGTGTGTACAAG
pUC-F
GTTTTCCCAGTCACGACG
pUC-R
GAATTGTGAGCGGATAAC
ywfBGF1
TCCATGAGTGTGATTACAGG
ywfBGR1
GTTATCCGCTCACAATTCCTTTGCTGCGTACTCACTGC
ywfBGF2
CGTCGTGACTGGGAAAACGTCGAAGCAGGAGGATCTGC
ywfBGR2
TCAATGCGGACGGCTTGATG
sfp-D-F1
GAAGAATTCTCGCTGGATTGATTGCGTATTATGC
CTAATGGGTGCTTTAGTTGAAGACGATCTCTTCGCTGCGGTGC
sfp-D-R1
AACC
sfp-D-F2
TATGAGATAATGCCGACTGTACTATCGCCATCTATCTCATATGTA
ACG
sfp-D-R2
GGAGGATCCCCAGTAACAGCGCATAGTCATTGCC
ppsA-F1
GAGTGTGGTTTACCGAACTG
ppsA-R1
GTTATCCGCTCACAATTCATGATGGTGTCTCTGGATGC
ppsA-F2
CGTCGTGACTGGGAAAACAAGCCTATGGTACTGATGCC
ppsA-R2
TCTTTCACGAGATCTTCGATG
pgsB-PF2
CCAACAATTGCATCTCGAACCTCCTTAGTC
pgsB-P-R
GGAGGATCCTGACCAGTATGACAGCACAG
srfAA-PF
GAAGAATTCGCGCGGTACACATAGTCATG
srfAA-PR
GGAGGATCCAATTCGTTTTTGTGCATCCG
ppsA-P-F
GAAGAATTCCTAAATTCTTTACCTGTTGC
ppsA-P-R
GGAGGATCCCACACAAGCTGTAAGATTGC
gfp-N-F
GAAGGTGATGCAACATACGG
gfp-NT7R
TAATACGACTCACTATAGGGCGAGAAGGACCATGTGGTCACGC
nprE-N-F
GTCGCTGCTTCGTTTATGAG
nprE-NT7R
TAATACGACTCACTATAGGGCGATCTGGTGATTTGCCGATAGC
aprE-N-F
AATGAGTGCCATGAGTTCCG
aprE-NT7R
TAATACGACTCACTATAGGGCGACTTCCTGTTGAATCAAGCAC
AtBSMT1
-N-F
GTCTTGTGAGTGAGTCAAAG
AtBSMT1
-N-T7R
TAATACGACTCACTATAGGGCGATACTAGCTCCACATCATCAC
mCherryF1
AAATTCATCATATGGTCAGCAAGGGCGAGGAGGATAAC
mCherryR2
ACTCCTCGAGCTTGTACAGCTCGTCCATGC