Article Figures & Data
Figures
- FIG. 1.
The essential yycFG two-component system is organized in either of two different operons. Essential genes are in gray. Sequences encoding for putative transmembrane regions are in black. RR, response regulator; HK, histidine kinase; ?, unknown; ‘β-lac’, homologous to enzyme family which includes β-lactamases; protease, serine protease.
- FIG. 2.
YycF-dependent expression in yycH, yycI, yycJ, and yycK mutant strains. Shown are growth in optical density (OD) units (solid lines and solid symbols) and β-galactosidase activity in Miller units (broken lines and open symbols) of strains expressing lacZ from the amyE locus under control of the YycF-dependent yocH promoter. Strains are wild type (squares), yycH (triangles), yycI (circles), yycJ (diamonds), and yycK (stars).
- FIG. 3.
YycF-dependent expression in JH25002 (yycH::pJM103) versus JH25011 (yycH::pJM117) strains. In strain JH25011, the genes downstream of yycH within the same operon are placed under control of the IPTG inducible PSpac promoter. Shown are growth in optical density (OD) units (solid lines and solid symbols) and β-galactosidase activity in Miller units (broken lines and open symbols) of strains expressing lacZ from the amyE locus under control of the YycF-dependent yocH promoter. Strains are wild type (squares), JH25002 (triangles), and JH25011 in the presence of 1 mM IPTG (circles) or in its absence (diamonds).
- FIG. 4.
Cell wall defect in yycH strains is independent of yocH expression. Whole-cell protein extract after treatment with or without lysozyme, separated by SDS-PAGE and visualized by Coomassie staining. Extract was from wild type (lane 1), JH25002 (yycH) (lane 2), JH25012 (yocH) (lane 3), JH25013 (yych yocH) (lane 4), and JH25011 (yycH::pJM117) in the presence (lane 5) or absence (lane 6) of 1 mM IPTG.
- FIG. 5.
Time-dependent expression of YycF and YycG in wild-type and yycH strains. Shown are immunoblots visualizing expression levels of YycF and YycG in wild-type and yycH liquid cultures at indicated times before or after the onset of stationary phase.
- FIG. 6.
YycH gets exported to the extracellular space. MH3402 (phoA phoB) harboring pMA5-yycH′-′phoA or pMA5-′phoA were streaked over a plate containing 50 μg/ml of the alkaline phosphatase substrate 5-bromo-4-chloro-3-indolyl-phosphate. Blueish-green indicates a Pho+ phenotype.
- FIG. 7.
Model of the YycG-dependent balancing act. YycH (dark gray) gets exported to the extracellular space, where it down-regulates YycG (black) activity in order to keep growth and cell wall levels at the optimum (middle). In the absence of YycG, cells die because of a shift of the equilibrium toward the unphosphorylated form of the response regulator YycF (white) and, therefore, reduced expression of the YycF regulon (left). Cells show cell wall and growth defects in the absence of YycH, likely due to a shift in the equilibrium toward the phosphorylated form of YycF and, therefore, overexpression of the YycF regulon (right).
Tables
- TABLE 1.
Plasmids and strains used in this study
Plasmid or strain Description or genotype Source or reference Plasmids pIC133 mini-Tn10-OS delivery vector, Ermr, Spcr 22 pJM103 Ampr, Cmr 20 pJM115 amyE integration vector for lacZ fusions, Ampr Kanr 2 pJM117 pMUTIN 4 derivative, Ampr Cmr M. Perego, unpublished pJM134 Spcr gene in pBluescript (Stratagene) Ampr Spcr M. Perego, unpublished pMA5 B. subtilis/E.coli shuttle vector 3 pJV211 pJM103-′phoA 3 pJV217 pMA5-kapBC26P′-′phoA 3 pJC11 pJM103-′yycH′ 5 pJC12 pJM103-′yycI′ 5 pJC13 pJM103-′yycJ′ 5 pJC14 pJM103-′yycK′ 5 pYOCHP pJM115-PYocH-lacZ This work pJS01 pJM103-yycH′-′phoA This work pJS02 pMA5-yycH′-′phoA This work pJS03 pJM117-′yycH′ This work pJS04 pJM117-′yycH′ ΔlacZ This work pJS05 pJM134-′yocH′ This work Strains E. coli DH5α Cloning host Lab stock TG1 Cloning host Lab stock B. subtilis MH3402 pheA1 trpC2 phoA::cat phoB::mTn10 F. M. Hulett JH642 pheA1 trpC2 Lab stock JH17038 pheA1 yycFH215P This work JH17041 leu-8 met-5 yycFH215P 5 JH25001 amyE::(PyocH-lacZ aph3-A) This work JH25002 yycH::pJC11 amyE(PyocH-lacZ aph3-A) This work JH25003 yycI::pJC12 amyE(PyocH-lacZ aph3-A) This work JH25004 yycJ::pJC13 amyE(PyocH-lacZ aph3-A) This work JH25005 yycK::pJC14 amyE(PyocH-lacZ aph3-A) This work JH25011 yycH::pJS03 amyE(PyocH-lacZ aph3-A) This work JH25012 yocH::pJM134 amyE(PyocH-lacZ aph3-A) This work JH25013 yocH::pJM134 yycH::pJC11 amyE(PyocH-lacZ aph3-A) This work JH25014 phoA::cat phoB::mTn10 pJS04 This work
