Transcriptome and functional analysis of the eukaryotic-type serine/threonine kinase PknB in Staphylococcus aureus

Abstract

The function of the Staphylococcus aureus eukaryotic-like serine/threonine protein kinase PknB was investigated by performing transcriptome analysis using DNA microarray technology and biochemical assays. The transcriptional profile revealed a strong regulatory impact of PknB on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis. Functional activity of overexpressed and purified PknB kinase was demonstrated using the myelin basic protein as a surrogate substrate. Phosphorylation occurred in a time-dependent manner with Mn(2+) as a preferred cofactor. Furthermore, biochemical characterization revealed regulation of adenylosuccinate synthase (PurA) activity by phosphorylation. Phosphorylated PurA showed a 1.8-fold decrease in enzymatic activity compared to unphosphorylated PurA. Loss of PknB led to formation of larger cell clusters, and a pknB deletion strain showed 32-fold-higher sensitivity to the cell wall-active antibiotic tunicamycin. The results of this study strongly indicate that PknB has a role in regulation of purine biosynthesis, autolysis, and central metabolic processes in S. aureus.

FIG. 1.

Domain architecture for S. aureus PknB. TM, transmembrane domain. The domain library is available at SMART.

FIG. 2.

Phosphorylation of MBP by PknB. Purified PknB was incubated with the surrogate kinase substrate MBP and [γ-³²P]ATP in the presence or absence of Mn²⁺ or Mg²⁺. The reaction products were resolved on a 12% SDS-PAGE gel that was stained with Coomassie blue (not shown) and visualized by autoradiography. (A) Time-dependent phosphorylation of MBP. (B) Concentration-dependent phosphorylation with different Mn²⁺ or Mg²⁺ concentrations. The positions and masses (in kDa) of protein standards are indicated on the left. Besides phosphorylation of MBP autophosphorylation of PknB is visible.

FIG. 3.

Phosphorylation of PurA by PknB. The phosphorylation reaction was performed in the presence or absence of PknB as described in Materials and Methods. The His tag of both PknB and PurA was cleaved to exclude the possibility of a nonspecific in vitro phosphorylation effect. All reaction products were analyzed on a 12% SDS-PAGE gel, stained with Coomassie blue, and subjected to autoradiography. Lane 1 shows phosphorylation of PurA in the presence of PknB. Lane 2 contained the control without PknB. The positions and masses (in kDa) deduced from comparison with protein standards are indicated on the left.

FIG. 4.

Autolysis of whole cells of S. aureus wild-type strain 8325 (⧫) and the 8325 ΔpknB mutant (▪) during growth with Triton X-100. The results are expressed as decreases in the OD₆₀₀ over time, which represented increases in autolysis (for details see Materials and Methods). The data are the means ± standard deviations of three independent experiments.

FIG. 5.

Scanning electron microscopy of the 8325 ΔpknB mutant (A) and wild-type strain 8325 (B) grown on polystyrene surfaces. Cells of the ΔpknB strain formed larger cell clusters than cells of the wild-type strain formed.