Threonine phosphorylation prevents promoter DNA binding of the Group B Streptococcus response regulator CovR

All living organisms communicate with the external environment for their survival and existence. In prokaryotes, communication is achieved by two-component systems (TCS) comprising histidine kinases and response regulators. In eukaryotes, signalling is accomplished by serine/threonine and tyrosine kinases. Although TCS and serine/threonine kinases coexist in prokaryotes, direct cross-talk between these families was first described in Group B Streptococcus (GBS). A serine/threonine kinase (Stk1) and a TCS (CovR/CovS) co-regulate toxin expression in GBS. Typically, promoter binding of regulators like CovR is controlled by phosphorylation of the conserved active site aspartate (D53). In this study, we show that Stk1 phosphorylates CovR at threonine 65. The functional consequence of threonine phosphorylation of CovR in GBS was evaluated using phosphomimetic and silencing substitutions. GBS encoding the phosphomimetic T65E allele are deficient for CovR regulation unlike strains encoding the non-phosphorylated T65A allele. Further, compared with wild-type or T65A CovR, the T65E CovR is unable to bind promoter DNA and is decreased for phosphorylation at D53, similar to Stk1-phosphorylated CovR. Collectively, we provide evidence for a novel mechanism of response regulator control that enables GBS (and possibly other prokaryotes) to fine-tune gene expression for environmental adaptation.     

A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversibly phosphorylate an inorganic pyrophosphatase and affect growth,cell segregation,and virulence

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 eukaryotic-type 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 eukaryotic-type enzymes regulate pyrophosphatase activity and other cellular functions of S. agalactiae.     

Regulation of purine biosynthesis by a eukaryotic-type kinase in Streptococcus agalactiae

Group B streptococci (GBS) are the principal causal agents of human neonatal pneumonia, sepsis and meningitis. We had previously described the existence of a eukaryotic-type serine/threonine kinase (Stk1) and phosphatase (Stp1) in GBS that regulate growth and virulence of the pathogen. Our previous results also demonstrated that these enzymes reversibly phosphorylated an inorganic pyrophosphatase. To understand the role of these eukaryotic-type enzymes on growth of GBS, we assessed the stk1-mutants for auxotrophic requirements. In this report, we describe that in the absence of the kinase (Stk1), GBS are attenuated for de novo purine biosynthesis and are consequently growth arrested. During growth in media lacking purines, the intracellular G nucleotide pools (GTP, GDP and GMP) are significantly reduced in the Stk1-deficient strains, while levels of A nucleotides (ATP, ADP and AMP) are marginally increased when compared with the isogenic wild-type strain. We provide evidence that the reduced pools of G nucleotides result from altered activity of the IMP utilizing enzymes, adenylosuccinate synthetase (PurA) and IMP dehydrogenase (GuaB) in these strains. We also demonstrate that Stk1 and Stp1 reversibly phosphorylate and consequently regulate PurA activity in GBS. Collectively, these data indicate the novel role of eukaryotic-type kinases in regulation of metabolic processes such as purine biosynthesis.     

The two faces of Janus: virulence gene regulation by CovR/S in group A streptococci

The group A streptococcus (GAS) causes a variety of human diseases, including toxic shock syndrome and necrotizing fasciitis, which are both associated with significant mortality. Even the superficial self-limiting diseases caused by GAS, such as pharyngitis, impose a significant economic burden on society. GAS can cause a wide spectrum of diseases because it elaborates virulence factors that enable it to spread and survive in different environmental niches within the human host. The production of many of these virulence factors is directly controlled by the activity of the CovR/S two-component regulatory system. CovS acts in one direction as a kinase primarily to activate the response regulator CovR and repress the expression of major virulence factors and in the other direction as a phosphatase to permit gene expression in response to environmental changes that mimic conditions found during human infection. This Janus-like behaviour of the CovR/S system is recapitulated in the binding of CovR to the promoters that it directly regulates. Interactions between different faces of the CovR DNA binding domain appear to depend upon DNA sequence, leading to the potential for differential regulation of virulence gene expression.     

A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro

The pneumococcal serine threonine protein kinase (StkP) acts as a global regulator in the pneumococcus. Bacterial mutants deficient in StkP are less virulent in animal models of infection. The gene for this regulator is located adjacent to the gene for its cognate phosphatase in the pneumococcal genome. The phosphatase dephosphorylates proteins phosphorylated by StkP and has been shown to regulate a number of key pneumococcal virulence factors and to modulate adherence to eukaryotic cells. The role of StkP in adherence of pneumococci to human cells has not previously been reported. In this study we show StkP represses the pneumococcal pilus, a virulence factor known to be important for bacterial adhesion. In a serotype 4 strain regulation of the pilus by StkP modulates adherence to human brain microvascular endothelial cells (HBMEC) and human lung epithelial cells. This suggests that the pneumococcal pilus may play a role in adherence during infections such as meningitis and pneumonia. We show that regulation of the pilus occurs at the population level as StkP alters the number of pili-positive cells within a single culture. As far as we are aware this is the first gene identified outside of the pilus islet that regulates the biphasic expression of the pilus. These findings suggest StkPs role in cell division may be linked to regulation of expression of a cell surface adhesin.     

Role of serine/threonine phosphatase (SP-STP) in Streptococcus pyogenes physiology and virulence

Reversible phosphorylation is the key mechanism regulating several cellular events in prokaryotes and eukaryotes. In prokaryotes, signal transduction is perceived to occur primarily via the two-component signaling system involving histidine kinases and cognate response regulators. Although an alternative regulatory pathway controlled by the eukaryote-type serine/threonine kinase (Streptococcus pyogenes serine/threonine kinase; SP-STK) has been shown to modulate bacterial growth, division, adherence, invasion, and virulence in group A Streptococcus (GAS; S. pyogenes), the precise role of the co-transcribing serine/threonine phosphatase (SP-STP) has remained enigmatic. In this context, this is the first report describing the construction and characterization of non-polar SP-STP mutants in two different strains of Type M1 GAS. The STP knock-out mutants displayed increased bacterial chain lengths in conjunction with thickened cell walls, significantly reduced capsule and hemolysin production, and restoration of the phenotypes postcomplementation. The present study also reveals important contribution of cognately regulated-reversible phosphorylation by SP-STK/SP-STP on two major response regulators of two-component systems, WalRK and CovRS. We also demonstrate a distinct role of SP-STP in terms of expression of surface proteins and SpeB in a strain-specific manner. Further, the attenuation of virulence in the absence of STP and its restoration only in the complemented strains that were generated by the use of a low copy plasmid and not by a high copy one emphasize not only the essential role of STP in virulence but also highlight the tightly regulated SP-STP/SP-STK-mediated cognate functions. SP-STP thus is an important regulator of GAS virulence and plays a critical role in GAS pathogenesis.     

A eukaryotic-type serine/threonine protein kinase is required for biofilm formation, genetic competence, and acid resistance in Streptococcus mutans

We report an operon encoding a eukaryotic-type serine/threonine protein kinase (STPK) and its cognate phosphatase (STPP) in Streptococcus mutans. Mutation of the gene encoding the STPK produced defects in biofilm formation, genetic competence, and acid resistance, determinants important in caries pathogenesis.     

CovS/CovR of group B streptococcus: a two-component global regulatory system involved in virulence

In this study, we carried out a detailed structural and functional analysis of a Streptococcus agalactiae (GBS) two-component system which is orthologous to the CovS/CovR (CsrS/CsrR) regulatory system of Streptococcus pyogenes. In GBS, covR and covS are part of a seven gene operon transcribed from two promoters that are not regulated by CovR. A DeltacovSR mutant was found to display dramatic phenotypic changes such as increased haemolytic activity and reduced CAMP activity on blood agar. Adherence of the DeltacovSR mutant to epithelial cells was greatly increased and analysis by transmission electron microscopy revealed the presence at its surface of a fibrous extracellular matrix that might be involved in these intercellular interactions. However, the DeltacovSR mutant was unable to initiate growth in RPMI and its viability in human normal serum was greatly impaired. A major finding of this phenotypic analysis was that the CovS/CovR system is important for GBS virulence, as a 3 log increase of the LD(50) of the mutant strain was observed in the neonate rat sepsis model. The pleiotropic phenotype of the DeltacovSR mutant is in full agreement with the large number of genes controlled by CovS/CovR as seen by expression profiling analysis, many of which encode potentially secreted or cell surface-associated proteins: 76 genes are repressed whereas 63 were positively regulated. CovR was shown to bind directly to the regulatory regions of several of these genes and a consensus CovR recognition sequence was proposed using both DNase I footprinting and computational analyses.