1 Supplementary Material
2
3 Stripping Bacillus : ComK auto-stimulation is responsible for the bistable response
4 in competence development
5
6
7
Smits et al .

8 Results
9
10
PcomG-comK uncouples comK-transcription from transcription factors.
DegU. Due to the distance between the two ComK-dimer binding sites, the affinity of
11 ComK for its own promoter is relative low, and the formation of a stable ComK-tetramer
12 requires binding of the two-component response regulator DegU in between two ComK-
13 dimers (Hamoen et al.
, 2000) . As shown in Fig. 7A , when the comK pomoter was
14 replaced by the comG promoter, deletion of degU did not significantly affect the
15 expression of comG-lacZ .
16 AbrB and ComS.
When the transition state regulator AbrB was mutated in the PcomG-
17 comK Pxyl-comK background, the expression of comG-lacZ dropped dramatically ( Fig.
18 7A ). Although AbrB is a repressor of the comK promoter (Hamoen et al.
, 2003;Hahn et
19
20 al.
, 1995), the regulation of competence by this transcription factor is more complicated.
Expression of ComS requires the presence of AbrB, for reasons yet unknown, and
21 deletion of abrB results in a strong reduction of competence (Hahn and Dubnau, 1991).
22 Figure 6A shows that the deletion of comS in a PcomG-comK Pxyl-comK mutant
23 abolishes comK induction, consistent with the fact that the MecA-dependent regulation
24 of comK expression is still present in such a background. Thus, the failure to induce
25 comK expression in a PcomG-comK Pxyl-comK background when abrB is mutated is
26
27 likely a result of insufficient ComS production.
Rok.
In wild-type B. subtilis cells, disruption of rok results in overproduction of ComK
28
29
(Hoa et al.
, 2002). In a PcomG-comK Pxyl-comK background, disruption of rok led to a somewhat delayed expression of comG-lacZ ( Fig. 7A ), but eventually, the

-
30
31 galactosidase activities reached the same levels as in a PcomG-comK Pxyl-comK background strain with an intact rok gene. To verify that this moderate influence was not
32 due to binding of Rok to the promoter of comG , an electrophoretic mobility shift assay
33 (EMSA) with a fragment of the promoter of comG was performed, using purified Rok

34 protein ( Fig. 7B ). The presence of Rok did not lead to an altered mobility of the promoter
35 fragment, even at high concentrations, confirming that PcomG is not a direct target for
36 Rok. Addition of Rok to a promoter fragment of comK did lead to a change in mobility
37 (data not shown), as was also demonstrated before (Hoa et al.
, 2002).
38 SinR. It was shown that sinR is positively required for competence development,
39 however its mode of action is still unclear. In a recent study, it was suggested that SinR
40 represses the expression of rok , and therefore is required for comK expression (Hoa et
41 al.
, 2002). The introduction of a sinR mutation in a PcomG-comK Pxyl-comK background
42 gave no appreciable difference in comG lacZ expression ( Fig. 7A ). Since a deletion of
43 sinR still does exert an influence in a PwtcomK P xyl comK strain (Hahn et al, 1996), this
44 suggests that SinR influences competence via the comK promoter under wild-type
45
46 conditions, and thus supports the hypothesis that the effect of SinR on competence is due to repression of rok .
47 CodY.
A third repressor that binds to the promoter of comK is CodY (Ratnayake-
48 Lecamwasam et al.
, 2001;Serror and Sonenshein, 1996) .
When a codY mutation was
49 introduced in a PcomG-comK Pxyl-comK background, the expression of comG-lacZ
50 seemed to be about 1 hr delayed compared to an isogenic strain in which codY was not
51 mutated, but as was the case with Rok, we obtained no evidence for specific binding of
52
53 this CodY to PcomG from EMSA experiments ( Fig. 7B ). Also in this case, an EMSA experiment using PcomK and CodY demonstrated specific binding, as demonstrated
54 before (Serror and Sonenshein, 1996).
55 From these results, we conclude that none of the transcription factors tested exerts an
56 effect on PcomG -driven comKexpression through binding to the comG promoter region.
57
58

59
60
Experimental procedures
β-galactosidase assays and electrophoretic mobility shift assays
β-galactosidase assays were performed as described in the Materials and Methods of 61
62 the main text. Gel retardations were performed essentially as described (Hamoen et al.
,
63 1998). A promoter fragments of comG (Susanna et al.
, 2004) was amplified by PCR,
64
65
66 using Expand polymerase (Roche). The obtained fragment was end-labeled with T4 polynucleotide kinase (Roche) and

32 P ATP. The proteins used were a kind gift of D.
Dubnau. Protein and probe were mixed on ice in a 20
 l volume, and subsequently incubated for 20 minutes at 37°C. Samples were loaded onto a 6% non-denaturing 67
68 polyacrylamide gel (prepared with 1xTAE) and run in a 0.5-2.0x gradient of TAE (40mM
69 Tris acetate, pH 8.0, 2mM EDTA) at 100V for 1 hour in a BioRad MiniProtean system
70
71
(BioRad). Gels were dried in a vacuumdryer and signals were recorded using Phosphor
Screens and a Cyclone Phosphoimager (Packard).
72
73

74 References
75
76 Hahn,J., and Dubnau,D. (1991) Growth stage signal transduction and the
77 requirements for srfA induction in development of competence. J Bacteriol 173 :
78 7275-7282.
79 Hahn,J., Roggiani,M., and Dubnau,D. (1995) The major role of Spo0A in genetic
80 competence is to downregulate abrB, an essential competence gene. J Bacteriol
81 177 : 3601-3605.
82 Hamoen,L.W., Kausche,D., Marahiel,M.A., van Sinderen,D., Venema,G., and
83 Serror,P. (2003) The Bacillus subtilis transition state regulator AbrB binds to the -
84 35 promoter region of comK . FEMS Microbiol Lett 218 : 299-304.
85 Hamoen,L.W., Van Werkhoven,A.F., Bijlsma,J.J., Dubnau,D., and Venema,G.
86 (1998) The competence transcription factor of Bacillus subtilis recognizes short
87 A/T-rich sequences arranged in a unique, flexible pattern along the DNA helix.
88 Genes Dev 12 : 1539-1550.
89 Hamoen,L.W., Van Werkhoven,A.F., Venema,G., and Dubnau,D. (2000) The
90 pleiotropic response regulator DegU functions as a priming protein in
91 competence development in Bacillus subtilis . Proc Natl Acad Sci U S A 97 : 9246-
92 9251.

93 Hoa,T.T., Tortosa,P., Albano,M., and Dubnau,D. (2002) Rok (YkuW) regulates
94 genetic competence in Bacillus subtilis by directly repressing comK . Mol
95 Microbiol 43 : 15-26.
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et al.
(2004) Mechanism of transcription activation at the comG
103 promoter by the competence transcription factor ComK of Bacillus subtilis . J
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105
106