A Eukaryotic Type Serine/Threonine Kinase and Phosphatase inStreptococcus agalactiae Reversibly Phosphorylate an Inorganic Pyrophosphatase and Affect Growth, Cell Segregation, and Virulence

Abstract

Protein phosphorylation is essential for the regulation of cell growth, division, and differentiation in both prokaryotes and eukaryotes. Signal transduction in prokaryotes was previously thought to occur primarily by histidine kinases, involved in two-component signaling pathways. Lately, bacterial homologues of eukaryotictype serine/threonine kinases and phosphatases have been found to be necessary for cellular functions such as growth, differentiation, pathogenicity, and secondary metabolism. The Gram-positive bacteria Streptococcus agalactiae (group B streptococci, GBS) is an important human pathogen. We have identified and characterized a eukaryotic-type serine/threonine protein kinase (Stk1) and its cognate phosphatase (Stp1) in GBS. Biochemical assays revealed that Stk1 has kinase activity and localizes to the membrane and that Stp1 is a soluble protein with manganese-dependent phosphatase activity on Stk1. Mutations in these genes exhibited pleiotropic effects on growth, virulence, and cell segregation of GBS. Complementation of these mutations restored the wild type phenotype linking these genes to the regulation of various cellular processes in GBS. In vitro phosphorylation of cell extracts from wild type and mutant strains revealed that Stk1 is essential for phosphorylation of six GBS proteins. We have identified the predominant endogenous substrate of both Stk1 and Stp1 as a manganese-dependent inorganic pyrophosphatase (PpaC) by liquid chromatography/tandem mass spectrometry. These results suggest that these eukaryotictype enzymes regulate pyrophosphatase activity and other cellular functions of S. agalactiae.Protein phosphorylation is a principal mechanism of signal transduction governing various cellular processes such as physiology, growth, and development. Regulation of protein function or enzyme activity by covalent and reversible phosphorylation is crucial for the regulation of cellular responses of both prokaryotes and eukaryotes to dynamic internal and external environmental conditions. Signal transduction in prokaryotes was thought to occur primarily by histidine kinases that activate transcription by phosphorylation of cognate response regulators at aspartate residues (1). However, phosphorylation by serine kinases have also been described in prokaryotes. Examples of signal transduction by serine kinases, specifically in Gram-positives, include the well characterized and novel HPr system with bifunctional kinase/phosphatase activity necessary for carbon catabolite repression (for review, see Ref.2) and the isocitrate dehydrogenase kinase/phosphatase system (3, 4). Other examples of serine phosphorylation involves cognate pairs of kinases and phosphatases that regulate stress responses in Bacillus subtilis (5). Interestingly, although these serine kinases are homologous to the two-component histidine kinases (6), SpoIIE, which regulates sporulation in B. subtilis, is homologous to eukaryotic-type protein phosphatases (7).In eukaryotes, reversible protein phosphorylation via serine, threonin...

Role of Serine/Threonine Phosphatase (SP-STP) in Streptococcus pyogenes Physiology and Virulence

“…7B). We therefore hypothesized that this observed attenuation in the virulence despite complementation could be either due to the instability of the plasmid in the host, as reported earlier (16), or due to the overexpression of STP from a multicopy plasmid, resulting in the aberrant dephosphorylation.…”

A eukaryotic-type Ser/Thr kinase in Enterococcus faecalis mediates antimicrobial resistance and intestinal persistence

“…Homologs of PrkC in some pathogenic streptococci are required for full virulence in animal models of infection (21,23), suggesting that the streptococcal PrkC homologs regulate the expression or activity of virulence factors in their respective hosts. These streptococcal PrkC homologs each exhibit a PrkClike bipartite domain architecture including PASTA domains, suggesting that, like E. faecalis PrkC, they detect perturbations of the cell envelope as sensory input.…”

Evolution of the PPM-family protein phosphatases in Streptomyces: duplication of catalytic domain and lateral recruitment of additional sensory domains

“…The functions of the microbial PPMs that have been characterized include regulation of spore formation, stress response, cell density during stationary phase, carbon and nitrogen assimilation, vegetative growth, development of fruiting bodies and cell segregation (Beuf et al, 1994;Duncan et al, 1995;Gaidenko et al, 2002;Irmler & Forchhammer, 2001;Rajagopal et al, 2003;Shi et al, 1999;Treuner-Lange et al, 2001;Yang et al, 1996). It is notable that most bacterial species containing relatively large numbers of PPMencoding genes, such as Anabaena sp.…”