Figure 1.
Diagram of the genetic organization of the chromosomal regions surrounding mstX and yugO.
Sequence homology was identified by BLAST.
Figure 2.
Alterations in colony morphology and biofilm formation related to mstX expression in the domesticated strain PY79.
Images show colony morphology after 1 days of growth on MsGG medium at 30°C. Scale bar corresponds to approximately 3 mm. (A) Colony morphology of B. subtilis PY79. (B) Colony morphology of B. subtilis PY79 ΔmstX (MEL64). (C) Colony morphology of B. subtilis PY79 domesticated strain after IPTG induction of mstX (lacA::Pspac-mstX-erm; MEL66). (D) Colony morphology of Bacillus subtilis PY79 after IPTG induction of mstX (M75A) (lacA::Pspac-mstX (M75A)-erm; MEL67). (E) Microtitre crystal violet staining assay for WT, ΔmstX, lacA::Pspac –mstX-erm, and lacA::Pspac -mstX (M75A)-erm strains (strains PY79, MEL64, MEL66 and MEL67). Error bars represent standard error calculated from three independent experiments.
Figure 3.
The mstX and yugO genes regulate biofilm formation in undomesticated Bacillus subtilis NCIB3610.
Top rows of images show colony morphology after 3 days of growth on MsGG medium at 22°C; bottom rows show pellicle formation in MsGG medium after 72 hours of growth at 22°C. Strains used include NCIB3610 (wt), MEL240 (ΔmstX::kan), MEL239 (ΔyugO::kan), MEL422 (ΔmstX::kan, amyE::Pxyl –mstX-spc), MEL421 (ΔyugO::kan, amyE::Pxyl –mstX-spc), MEL218 (ΔsinR::spc), MEL425 (ΔmstX::kan, ΔsinR::spc), MEL424 (ΔyugO::kan, ΔsinR::spc), MEL430 (NCIB3610 Pxyl-yugO-spc, Δyugo::kan), and MEL431 (NCIB3610 Pxyl-yugO-spc, ΔmstX::kan). The ΔmstX and ΔyugO mutations reduce colony architecture and pellicle formation, which is rescued by the sinR mutation. Microtitre wells measure approximately 3 cm in diameter.
Figure 4.
MstX negatively regulates parallel antirepressors involved in biofilm formation.
(A) Quantitative RT-PCR analysis of abbA, sinI, epsE, kinC, and tasA in wild type, ΔmstX, ΔsinR, and ΔsinR ΔmstX double mutants (strains MEL65, MEL240, MEL423 and MEL 425) were grown in MsGG medium at 30°C and collected at 0.5–0.8 OD600. Error bars represent standard error calculated from three independent experiments. (B) Model for mstX activation of kinC with corresponding increases in abbA and sinI transcription.
Figure 5.
Temporal expression of mstX and regulation by the transcriptional repressor SinR.
(A) Comparison of mstX expression during log growth and biofilm growth using RT-PCR analysis in strain MEL63. The veg gene (BSU00440) is a constitutively expressed gene frequently used as a positive control for RT-PCR. (B) Comparison of mstX gene expression in the presence and absence of the transcriptional repressor SinR using RT-PCR in strains MEL63 and MEL73. Cultures were collected during log growth. (C) Chromatin immunoprecipitation assay of strain MEL102 shows that SinR-FLAG binds near the mstX promoter during exponential growth.
Figure 6.
Addition of 150 mM Potassium chloride or the kinC deletion abrogates pellicle formation after mstX overexpression and in the sinR strain in LB medium that does not normally support biofilm formation in the NCIB3610 background strain.
Strains used includes MEL65 (wt NCIB3610), MEL423 (ΔsinR) and MEL428 (ΔsinR, ΔkinC), MEL422 (amyE::Pxyl-mstX-spc) and MEL429 (amyE::Pxyl-mstX-spc, ΔkinC::cm). Xylose-inducible strains were grown in the presence of 0.5% xylose.
Figure 7.
A positive autoregulatory loop involving MstX, YugO, potassium, and biofilm formation in B. subtilis.
Our data suggest that MstX and YugO both positively regulate biofilm formation by inhibiting SinR (in a manner dependent on KinC and influenced by potassium) and that the expression of mstX and yugO are negatively regulated by SinR. This suggests that MstX and YugO participate in a positive feedback loop to lock a subpopulation of cells in the biofilm assembling state. We propose that MstX mediates the assembly of YugO, a putative potassium efflux channel, and that potassium leakage activates KinC [15], [35], [44].