YycH regulates the activity of the essential YycFG two-component system in Bacillus subtilis

Abstract

Of the numerous two-component signal transduction systems found in bacteria, only a very few have proven to be essential for cell viability. Among these is the YycF (response regulator)-YycG (histidine kinase) system, which is highly conserved in and specific to the low-G+C content gram-positive bacteria. Given the pathogenic nature of several members of this class of bacteria, the YycF-YycG system has been suggested as a prime antimicrobial target. In an attempt to identify genes involved in regulation of this two-component system, a transposon mutagenesis study was designed to identify suppressors of a temperature-sensitive YycF mutant in Bacillus subtilis. Suppressors could be identified, and the prime target was the yycH gene located adjacent to yycG and within the same operon. A lacZ reporter assay revealed that YycF-regulated gene expression was elevated in a yycH strain, whereas disruption of any of the three downstream genes within the operon, yycI, yycJ, and yycK, showed no such effect. The concentrations of both YycG and YycF, assayed immunologically, remained unchanged between the wild-type and the yycH strain as determined by immunoassay. Alkaline phosphatase fusion studies showed that YycH is located external to the cell membrane, suggesting that it acts in the regulation of the sensor domain of the YycG sensor histidine kinase. The yycH strain showed a characteristic cell wall defect consistent with the previously suggested notion that the YycF-YycG system is involved in regulating cell wall homeostasis and indicating that either up- or down-regulation of YycF activity affects this homeostatic mechanism.

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 P_Spac 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).