Identification of genes controlled by the essential YycG/YycF two-component system of Staphylococcus aureus

Abstract

The YycG/YycF essential two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low-G+C gram-positive bacteria, including several pathogens such as Staphylococcus aureus. By studying growth of S. aureus cells where the yyc operon is controlled by an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter, we have shown that this system is essential in S. aureus during growth at 37 degrees C and that starvation for the YycG/YycF regulatory system leads to cell death. During a previous study of the YycG/YycF TCS of B. subtilis, we defined a potential YycF consensus recognition sequence, consisting of two hexanucleotide direct repeats, separated by five nucleotides [5'-TGT(A/T)A(A/T/C)-N(5)-TGT(A/T)A(A/T/C)-3']. A detailed DNA motif analysis of the S. aureus genome indicates that there are potentially 12 genes preceded by this sequence, 5 of which are involved in virulence. An in vitro approach was undertaken to determine which of these genes are controlled by YycF. The YycG and YycF proteins of S. aureus were overproduced in Escherichia coli and purified. Autophosphorylation of the YycG kinase and phosphotransfer to YycF were shown in vitro. Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified YycF to the promoter region of the ssaA gene, encoding a major antigen and previously suggested to be controlled by YycF. YycF was also shown to bind specifically to the promoter regions of two genes, encoding the IsaA antigen and the LytM peptidoglycan hydrolase, in agreement with the proposed role of this system in controlling virulence and cell wall metabolism.

FIG. 1.

IPTG-dependent growth of the conditional RN4220Pspac-yycF mutant strain. An overnight culture in TSB plus 1 mM IPTG was diluted to an OD₆₀₀ of 0.005 in TSB medium with (circles) or without (squares) 1 mM IPTG. (A) OD₆₀₀ values (solid symbols) and numbers of CFU per milliliter (open symbols) were measured in TSB medium. For measurement of the number of CFU per milliliter, cells were appropriately diluted in TSB liquid medium and plated on TSB plates with 1 mM IPTG. (B) OD₆₀₀ was measured with (circles) or without (squares) 1 mM IPTG. At the indicated times, 1 mM IPTG was added to the culture without IPTG. Each experiment was repeated three times, and the values obtained did not vary significantly.

FIG. 2.

Amino acid sequence alignment of the B. subtilis and S. aureus YycF proteins. Conserved residues are shaded, and the DNA-binding winged helix-turn-helix domains are boxed.

FIG. 3.

Purification and phosphorylation of ′YycG and YycF. (A) SDS-PAGE analysis of crude extracts from E. coli BL21/λDE3 carrying pET28/16 (lane 2), pETyycF (lane 3), or pETyycG (lane 5). Purified proteins (approximately 50 pmol) were loaded in lane 4 (YycF) and lane 6 (′YycG). Molecular size standards were loaded in lane 1. (B) In vitro phosphorylation of ′YycG and YycF. Lane 1, ′YycG incubated with [γ-³²P]ATP for 1 min at room temperature. ′YycG and YycF were incubated for various times following the addition of [γ-³²P]ATP. Lanes 2 to 7, 0.5, 1, 5, 10, 15, and 20 min, respectively.

FIG. 4.

Gel mobility shift assay and DNase I footprinting analysis of YycF binding to the ssaA promoter region. (A) DNA-binding reactions were performed with 0.1 pmol of a radiolabeled DNA fragment corresponding to the ssaA promoter region (−340 to +40 with respect to the translation initiation site) and purified YycF. Lanes: 1, no protein; 2, 26 pmol; 3, 34 pmol; 4, 53 pmol; 5, 68 pmol; 6, 106 pmol. (B) DNase I footprinting of YycF bound to the ssaA promoter region. Lanes contain approximately 0.5 pmol (5 × 10⁴ cpm per reaction mixture) of labeled nontemplate or template strand (NTS and TS, respectively) of ssaA (−340 to +40). Lanes: 1, no protein; 2, 140 pmol; 3, 212 pmol; 4, A+G Maxam and Gilbert reaction. Brackets indicate proximal and distal regions protected by YycF from DNase I cleavage (I and II, respectively). (C) Nucleotide sequences of the ssaA promoter region. Regions protected by YycF from DNase I cleavage are shown by bars, and the conserved repeats are indicated by arrows.

FIG. 5.

DNase I footprinting assay of YycF bound to the isaA and lytM promoter regions. Lanes contain approximately 0.7 pmol (5 × 10⁴ cpm per reaction mixture) of labeled nontemplate strands of isaA (−250 to +59 with respect to the translation initiation codon) (A) or lytM (−321 to +60 with respect to the translation initiation codon) (B). Fragments were incubated with increasing amounts of purified YycF. (A) Lanes: 1, no protein; 2, 34 pmol; 3, 68 pmol; 4, 136 pmol; 5, 204 pmol; 6, A+G Maxam and Gilbert reaction. (B) Lanes: 1, no protein; 2, 136 pmol; 3, 272 pmol; 4, 408 pmol; 5, A+G Maxam and Gilbert reaction. The brackets indicate regions protected by YycF from DNase I cleavage. (C) Nucleotide sequence of the isaA and lytM promoter regions. Regions protected by YycF are indicated by bars, and the conserved direct repeats are indicated by arrows.