Purification and activity testing of the full-length YycFGHI proteins of Staphylococcus aureus

Abstract

Background: The YycFG two-component regulatory system (TCS) of Staphylococcus aureus represents the only essential TCS that is almost ubiquitously distributed in gram-positive bacteria with a low G+C-content. YycG (WalK/VicK) is a sensor histidine-kinase and YycF (WalR/VicR) is the cognate response regulator. Both proteins play an important role in the biosynthesis of the cell envelope and mutations in these proteins have been involved in development of vancomycin and daptomycin resistance.

Methodology/principal findings: Here we present high yield expression and purification of the full-length YycG and YycF proteins as well as of the auxiliary proteins YycH and YycI of Staphylococcus aureus. Activity tests of the YycG kinase and a mutated version, that harbours an Y306N exchange in its cytoplasmic PAS domain, in a detergent-micelle-model and a phosholipid-liposome-model showed kinase activity (autophosphorylation and phosphoryl group transfer to YycF) only in the presence of elevated concentrations of alkali salts. A direct comparison of the activity of the kinases in the liposome-model indicated a higher activity of the mutated YycG kinase. Further experiments indicated that YycG responds to fluidity changes in its microenvironment.

Conclusions/significance: The combination of high yield expression, purification and activity testing of membrane and membrane-associated proteins provides an excellent experimental basis for further protein-protein interaction studies and for identification of all signals received by the YycFGHI system.

Figure 1. Gram-positive bacteria with orthologous yyc (wal/vic) genes.

The upper part of the figure shows the structural organization of orthologous yyc operons in Gram-positive bacteria with low G+C-content adopting the classification into class I and II proposed by Szurmant et al. . To distinguish between genera that contain 5 or 6 cistrons, the class I operon was divided into two subclasses. For a clear overview, orthologous genes are named according to the corresponding yyc gene in Bacillus subtilis in all genera and synonymous terms commonly used in a genus are given in brackets. The lower part of the figure illustrates the organization of the mtrAB/lpqB operon in Gram-positive Actinobacteria with high G+C-content. The operons are drawn to scale from representative species: Bacillus subtilis 168 (NC_000962), Staphylococcus aureus N315 (NC_002745), Streptococcus pneumoniae R6 (NC_003098), Lactococcus lactis Il1403 (NC_002662), Corynebacterium glutamicum ATCC 13032 (NC_006958). Essential genes are highlighted. TM coding regions were determined utilizing the TMHMM 2.0 server 2.0 web interface (http://www.cbs.dtu.dk/services/TMHMM/) and marked as black bars. Functions are indicated below the arrows: RR = response regulator, SK = sensor kinase, NR = negative regulator of kinase function, ‘β-lac’ = similarities to an enzyme super-family containing metallo-β-lactamases, protease = serine protease, LP = conserved lipoprotein. Within the class II of yyc operons, the YycG kinase of Lactococcus lactis is an exception of the rule, because it possesses two transmembrane domains (instead of one), which flank a very short extracytoplasmic loop comprising four amino acids , .

Figure 2. Oligomeric state analysis via BN-PAGE.

(A) Multimeric organization of all C-terminal His₆-tagged full-length Yyc proteins in this work as observed in blue native polyacrylamide gel electrophoresis (BN-PAGE). Marker proteins in BN-PAGE (kDa): Conalbumin (dimer) 154, BSA (dimer) 132, conalbumin (monomer) 77, BSA (monomer) 66 and ovalbumin 45. The molecular weights (M_W) of the Yyc-C-His₆ proteins on basis of their amino acid sequence are shown in brackets. As a control, the separation of all purified His₆-tagged full-length Yyc proteins by SDS-PAGE is shown in (B). (C)–(E): The apparent discrepancy between the molecular weights and migration distances of the Yyc-C-His₆ constructs in BN-PAGE can be solved by application of a conversion factor, which is deduced by linear regression from the correlation of the M_W as calculated by amino acid sequence (M_W ^AA) and the apparent molecular weight observed in BN-PAGE (M_W ^BPN), respectively. Symbols: YycF (open square), YycG (closed diamond), YycH (closed circle) and YycI (closed triangle).

Figure 3. Autophosphorylation activity in the detergent-micelle-model.

(A) Activity of the YycG and the YycG(Y306N) kinase after incubation for 60 min at RT in presence of 8.5 mM Triton X-100, 25 µM ATP, 4 µCi [γ-³³P]ATP and various KCl concentrations. (B) Activity of YycG in presence of diverse alkali salts in various concentrations under the same conditions.

Figure 4. Influence of temperature/fluidity in the detergent-micelle-model.

Activity of the YycG and the YycG(Y306N) kinases after incubation for 60 min in presence of 8.5 mM Triton X-100, 25 µM ATP, 4 µCi [γ-³³P]ATP and 500 mM KCl at different temperatures to induce changes in microviscosity of the membrane mimicking surfactant Triton X-100.

Figure 5. Phosphoryl group transfer to YycF in the detergent-micelle-model.

(A) The phosphorylation activities of both kinase proteins in presence of their cognate response regulator YycF were observed in dependence of time. All phosphorylation assays were performed in presence of 8.5 mM Triton X-100, 25 µM ATP, 4 µCi [γ-³³P]ATP and 500 mM KCl at RT. As a control the autophosphorylation activity of the YycG and the YycG(Y306N)-C-His₆ kinase after 1 min incubation is shown. (B) Influence of YycH and YycI in the detergent-micelle-model. Phosphorylation activity of YycG in presence of various combinations of YycF, YycH and YycI is shown. After starting the reaction, the incubation was stopped for all samples after 60 s. All phosphorylation assays were performed in presence of 8.5 mM Triton X-100, 25 µM ATP, 4 µCi [γ-³³P]ATP and 500 mM KCl at RT.

Figure 6. Comparison of the YycG-C-His₆ and the YycG(Y306N)-C-His₆ kinase activities.

The experiments were performed in presence or absence of the cognate response regulator YycF in the detergent-micelle-model and the phospholipid-liposome-model, respectively. The activities of the kinases within the same model system can be compared directly. In the detergent-micelle-model 1.5 µg of the His-tag constructs of YycG, YycG(Y306N) and YycF were employed. In the phospholipid-liposome-model, equal amounts of YycG-C-His₆ and YycG(Y306N)-C-His₆ ULVs as determined by the concentration of the inorganic phosphate (in triplicate) were used and 1.5 µg of YycF-C-His₆ were added to the ULVs. All phosphorylation reactions were started by the addition of 4 µCi [γ-³³P]ATP and stopped after 60 min.

Figure 7. Influence of temperature/fluidity in the phospholipid-liposome-model.

Equal amounts of YycG-C-His₆ and YycG(Y306N)-C-His₆ ULVs as determined by the concentration of the inorganic phosphate (in triplicate) were incubated at different temperatures to observe changes in activity due to changes in microviscosity of the ULVs. All phosphorylation reactions were started by the addition of 4 µCi [γ-³³P]ATP and stopped after 60 min.

Figure 8. Model for the control of the YycG kinase activity by the membrane fluidity in its microenvironment.

The organization of the growing septum in S. aureus and the localization of enzymes involved in peptidoglycan degradation and synthesis are depicted according to who showed by cryo transmission electron micrography that the murein in the center of the nascent cell wall is hydrolyzed already during biosynthesis of the septum. Normally, the peptidoglycan (PG) ensures structural integrity of the cell by maintaining a counter pressure to the intracellular turgor which is symbolized by arrows. The turgor pressure of the cytoplasm and the counter pressure of the peptidoglycan also affect the tension, fluidity and stability of the plasma membrane (PM). A local weakness of the PG caused e.g. by an excess of degradation by peptidoglycan hydrolases (PGH) would also affect the state of the membrane and might be recognized via the TM domains of YycG, leading to a decrease of kinase activity and a concomitant decrease of the expression of the autolysin genes.