The regulatory region of sinI contains both the activator and operator sites for 0A∼P. Shown are nucleotide sequences of the regulatory regions of sinI in B. subtilis, B. amyloliquefaciens, and B. licheniformis. In B. subtilis, the −35 and −10 motifs of the σ^A-dependent promoter (shown in lactic letters), the transcription start of sinI (indicated with arrow), the consensus 0A-box, and four imperfect 0A-boxes (underlined and noted as 0A-box, O1, O2, O3, and O4) were annotated accordingly. In B. amyloliquefaciens and B. licheniformis, the predicted −35 and −10 motifs of the σ^A-dependent promoter were also shown in lactic letters. A consensus 0A-box was identified immediately upstream of the predicted −35 region in both promoters whereas five and three imperfect 0A-boxes (underlined) respectively, were also identified in the sinI promoters in B. amyloliquefaciens and B. licheniformis. The matched sequences in the imperfect 0A-boxes to the consensus 0A-box (‘TTCGACA') were shown in capital letters.

Site-directed mutagenesis of 0A∼P binding sites in the sinI promoter. (A) Substitutions in the nucleotide sequences of the consensus and imperfect 0A-boxes were shown below each labelled box and the name for each mutant allele was designated on the left. (B) β-Galactosidase activities of 3610 containing either a wild-type P_sinI-lacZ fusion (▪) or P_sinI-lacZ with mutations in the consensus 0A-box (♦, 0A^mut) were shown in the left panel. Reporter fusions were integrated at amyE on the chromosome of 3610 and cells were grown in MSgg medium. Shown in the middle and right panels are β-galactosidase activities of cells containing the same fusion but with indicated mutations in the imperfect 0A-boxes. Cells were grown in DS medium. Each strain contains wild type (▪), M1 allele (•), M2 allele (♦), M3–4 allele (▴), Δ(2–4) allele (Δ), or random sequence shuffle of O(2–4) (*) in the promoter region of P_sinI-lacZ. (C) Block of transcription read-through into sinI from the upstream yqhHG operon impairs biofilm formation. A schematic drawing shows insertion of spectinomycin resistance gene (spec) into two different loci upstream of sinI to block transcription read-through from the upstream yqhHG operon. Regions of the two DNA fragments that were used in complementation of ΔsinI sinR mutations were indicated by horizontal lines.

C-terminal DNA-binding domain of 0A binds to the operators in the sinI promoter. (A) Gel mobility shift assays of the C-terminal DNA-binding domain of 0A (0A-CTD) binding to the promoter sequences of sinI. DNA probes used in the assays include a wild-type sinI promoter sequence (panel a), a sinI promoter sequence with mutations in the consensus 0A-box (panel b), a sinI promoter sequence with mutations in the consensus 0A-box and with deletions from the putative 0A∼P operators 2–4 (panel c), and a mixture of DNA probes used in (b, c) (panel d). 0A-CTD proteins were added at 0, 6.2, 12.5, 25, 50, and 100 ng per lane, respectively. (B) DNase I footprinting assays of 0A-CTD to the wild-type sinI promoter sequence. Proteins were added at 800, 400, and 200 ng per lane, respectively. Arrows on the left point to protected DNA regions while stars indicate hypersensitive DNA regions in DNase I reactions.

Transplanting or increasing the copy number of sinI sinR blocked biofilm formation. (A) A circular map showing various chromosomal loci that were used for ΔsinI sinR complementation. (B) Colony and pellicle formation of ΔsinI sinR strains that were complemented with yqhHG sinI sinR at various chromosomal loci (indicated at the top of each panel). Shown in panels on the right-most are biofilms formed by 3610 with a second copy of yqhHG sinI sinR integrated at 213° position (bkdB) on the chromosome. (C) A cartoon model shows B. subtilis cells at three different stages. Among them, growing cells are a partial diploid, but probably contain only one copy of sinI sinR, whereas cells at the start of sporulation or early during asymmetric division contain two complete copies of the chromosome and hence two copies of sinI sinR. We propose those cells (shown in the middle and bottom) as being restrictive in matrix production.

Cells with mispositioned sinI sinR or containing a second copy of yqhHG sinI sinR are blocked in matrix gene expression and appearance of SlrR. (A) β-Galactosidase activities of a wild-type strain (YC686, open diamonds) and a ΔsinI sinR deletion mutant in which yqhHG sinI sinR was complemented at the origin-proximal amyE locus (YC687, open squares), and a wild-type strain with a second copy of yqhHG sinI sinR at 213° (bkdB) (YC688, open triangles). The strains contained P_yqxM-lacZ integrated at the thrC locus. (B) Western immunoblots of SlrR and SinR. Shown in lanes from left to right in each panel are cell lysates prepared from strains corresponding to those in (A) but without bearing the reporter fusion (3610, YC270, and YC562, respectively). The left-hand and right-hand images were from the same samples but differed in exposure time during development of the chemiluminescent signal (10 and 60 s, respectively). The appearance of SlrR was blocked in cell lysates from both YC270 and YC562. The σ^A protein was used as a control and shown in bottom panels. (C) Gel mobility shift assays for SinR binding to the DNA probe containing the epsA-O promoter in the absence or presence of SinI. Concentrations of SinR and SinI are indicated above each lane. In the absence of SinI, SinR showed cooperative binding to the DNA probe (six lanes on the left). SinI antagonized SinR binding to the DNA in a dose-dependent manner (four lanes in the middle) but did much less so when concentrations of both SinR and SinI doubled (next four lanes on the right).

Block of matrix gene expression by transplanting sinI sinR to an origin-proximal locus in a strain engineered to induce sinI during growth. (A) Fluorescent microscopic analysis of cells harbouring both a P_yqxM-yfp reporter and an IPTG-inducible copy of spo0A-sad67. Panels on the left represent cells with sinI and sinR genes at the native locus (origin distal, YC716) and those on the right are cells with transplanted yqhHG sinI sinR at the amyE locus (origin proximal, YC717). The operators for 0A∼P were also deleted (Δ2–4) from the sinI regulatory region in both strains to avoid repression by high levels of 0A-Sad67. (B) Flow cytometric analysis of the same cell samples from (A). In panel b, upon treatment of IPTG (10 μM) for 60 min, about half of the YC716 cells were strongly turned on the matrix reporter, whereas under the same condition, the YC717 cells were largely blocked in expression of the matrix reporter (panel d).

Matrix genes were shut off at the start of sporulation. (A) Luciferase activities (blue line with filled diamonds) of a strain (YC734) that harbours both a P_yqxM-lux luciferase reporter and a P_spoIIA-gfp reporter. Red line with filled squares represents growth curve of YC734 cultured in MSgg medium at 37°C. At 8.5 h after inoculation, activities of the luciferase reporter peaked and then started to decline. (B) Fluorescent microscopic analysis of YC734 cells at 7.5 h (panel a), 8.5 h (panel b), and 9.5 h (panel c) after inoculation. The P_spoIIA-gfp reporter in YC734 was turned on at 9.5 h in a large proportion of cells, indicating that cells started to enter sporulation pathway.