Sequestration from Protease Adaptor Confers Differential Stability to Protease Substrate.

According to the N-end rule, the N-terminal residue of a protein determines its stability. In bacteria, the adaptor ClpS mediates proteolysis by delivering substrates bearing specific N-terminal residues to the protease ClpAP. We now report that the Salmonella adaptor ClpS binds to the N terminus of the regulatory protein PhoP, resulting in PhoP degradation by ClpAP. We establish that the PhoP-activated protein MgtC protects PhoP from degradation by outcompeting ClpS for binding to PhoP. MgtC appears to act exclusively on PhoP, as it did not alter the stability of a different ClpS-dependent ClpAP substrate. Removal of five N-terminal residues rendered PhoP stability independent of both the clpS and mgtC genes. By preserving PhoP protein levels, MgtC enables normal temporal transcription of PhoP-activated genes. The identified mechanism provides a simple means to spare specific substrates from an adaptor-dependent protease.

Involvement of WalK (VicK) phosphatase activity in setting WalR (VicR) response regulator phosphorylation level and limiting cross-talk in Streptococcus pneumoniae D39 cells.

WalRK (YycFG) two-component systems (TCSs) of low-GC Gram-positive bacteria play critical roles in regulating peptidogylcan hydrolase genes involved in cell division and wall stress responses. The WalRK (VicRK) TCSs of Streptococcus pneumoniae (pneumococcus) and other Streptococcus species show numerous differences with those of other low-GC species. Notably, the pneumococcal WalK sensor kinase is not essential for normal growth in culture, unlike its homologues in Bacillus and Staphylococcus species. The WalK sensor kinase possesses histidine autokinase activity and mediates dephosphorylation of phosphorylated WalR∼P response regulator. To understand the contributions of these two WalK activities to pneumococcal growth, we constructed and characterized a set of walK kinase and phosphatase mutants in biochemical reactions and in cells. We identified an amino acid substitution in WalK that significantly reduces phosphatase activity, but not other activities. Comparisons were made between WalRK regulon expression levels and WalR∼P amounts in cells determined by Phos-tag SDS-PAGE. Reduction of WalK phosphatase activity resulted in nearly 90% phosphorylation to WalR∼P, consistent with the conclusion that WalK phosphatase is strongly active in exponentially growing cells. WalK phosphatase activity was also shown to depend on the WalK PAS domain and to limit cross-talk and the recovery of WalR∼P from walK(+) cells.

The putative hydrolase YycJ (WalJ) affects the coordination of cell division with DNA replication in Bacillus subtilis and may play a conserved role in cell wall metabolism.

Bacteria must accurately replicate and segregate their genetic information to ensure the production of viable daughter cells. The high fidelity of chromosome partitioning is achieved through mechanisms that coordinate cell division with DNA replication. We report that YycJ (WalJ), a predicted member of the metallo-ß-lactamase superfamily found in most low-G+C Gram-positive bacteria, contributes to the fidelity of cell division in Bacillus subtilis. B. subtilis ΔwalJ (ΔwalJ(Bsu)) mutants divide over unsegregated chromosomes more frequently than wild-type cells, and this phenotype is exacerbated when DNA replication is inhibited. Two lines of evidence suggest that WalJ(Bsu) and its ortholog in the Gram-positive pathogen Streptococcus pneumoniae, WalJ(Spn) (VicX), play a role in cell wall metabolism: (i) strains of B. subtilis and S. pneumoniae lacking walJ exhibit increased sensitivity to a narrow spectrum of cephalosporin antibiotics, and (ii) reducing the expression of a two-component system that regulates genes involved in cell wall metabolism, WalRK (YycFG), renders walJ essential for growth in B. subtilis, as observed previously with S. pneumoniae. Together, these results suggest that the enzymatic activity of WalJ directly or indirectly affects cell wall metabolism and is required for accurate coordination of cell division with DNA replication.

Kinetic characterization of the WalRKSpn (VicRK) two-component system of Streptococcus pneumoniae: dependence of WalKSpn (VicK) phosphatase activity on its PAS domain.

The WalRK two-component system plays important roles in maintaining cell wall homeostasis and responding to antibiotic stress in low-GC Gram-positive bacteria. In the major human pathogen, Streptococcus pneumoniae, phosphorylated WalR(Spn) (VicR) response regulator positively controls the transcription of genes encoding the essential PcsB division protein and surface virulence factors. WalR(Spn) is phosphorylated by the WalK(Spn) (VicK) histidine kinase. Little is known about the signals sensed by WalK histidine kinases. To gain information about WalK(Spn) signal transduction, we performed a kinetic characterization of the WalRK(Spn) autophosphorylation, phosphoryltransferase, and phosphatase reactions. We were unable to purify soluble full-length WalK(Spn). Consequently, these analyses were performed using two truncated versions of WalK(Spn) lacking its single transmembrane domain. The longer version (Delta35 amino acids) contained most of the HAMP domain and the PAS, DHp, and CA domains, whereas the shorter version (Delta195 amino acids) contained only the DHp and CA domains. The autophosphorylation kinetic parameters of Delta35 and Delta195 WalK(Spn) were similar [K(m)(ATP) approximately 37 microM; k(cat) approximately 0.10 min(-1)] and typical of those of other histidine kinases. The catalytic efficiency of the two versions of WalK(Spn) approximately P were also similar in the phosphoryltransfer reaction to full-length WalR(Spn). In contrast, absence of the HAMP-PAS domains significantly diminished the phosphatase activity of WalK(Spn) for WalR(Spn) approximately P. Deletion and point mutations confirmed that optimal WalK(Spn) phosphatase activity depended on the PAS domain as well as residues in the DHp domain. In addition, these WalK(Spn) DHp domain and DeltaPAS mutations led to attenuation of virulence in a murine pneumonia model.

Localization and cellular amounts of the WalRKJ (VicRKX) two-component regulatory system proteins in serotype 2 Streptococcus pneumoniae.

The WalRK two-component regulatory system coordinates gene expression that maintains cell wall homeostasis and responds to antibiotic stress in low-GC Gram-positive bacteria. Phosphorylated WalR (VicR) of the major human respiratory pathogen Streptococcus pneumoniae (WalR(Spn)) positively regulates transcription of several surface virulence genes and, most critically, pcsB, which encodes an essential cell division protein. Despite numerous studies of several species, little is known about the signals sensed by the WalK histidine kinase or the function of the WalJ ancillary protein encoded in the walRK(Spn) operon. To better understand the functions of the WalRKJ(Spn) proteins in S. pneumoniae, we performed experiments to determine their cellular localization and amounts. In contrast to WalK from Bacillus subtilis (WalK(Bsu)), which is localized at division septa, immunofluorescence microscopy showed that WalK(Spn) is distributed throughout the cell periphery. WalJ(Spn) is also localized to the cell surface periphery, whereas WalR(Spn) was found to be localized in the cytoplasm around the nucleoid. In fractionation experiments, WalR(Spn) was recovered from the cytoplasmic fraction, while WalK(Spn) and the majority of WalJ(Spn) were recovered from the cell membrane fraction. This fractionation is consistent with the localization patterns observed. Lastly, we determined the cellular amounts of WalRKJ(Spn) by quantitative Western blotting. The WalR(Spn) response regulator is relatively abundant and present at levels of approximately 6,200 monomers per cell, which are approximately 14-fold greater than the amount of the WalK(Spn) histidine kinase, which is present at approximately 460 dimers (920 monomers) per cell. We detected approximately 1,200 monomers per cell of WalJ(Spn) ancillary protein, similar to the amount of WalK(Spn).

Roles of rel(Spn) in stringent response, global regulation and virulence of serotype 2 Streptococcus pneumoniae D39.

RelA/SpoT homologue (RSH) proteins have (p)ppGpp synthetase and hydrolase activities that mediate major global responses to nutrient limitation and other stresses. RSH proteins are conserved in most bacteria and play diverse roles in bacterial pathogenesis. We report here that the RSH protein of Streptococcus pneumoniae, Rel(Spn), can be deleted and is the primary source of (p)ppGpp synthesis in virulent strain D39 under some conditions. A D39 Deltarel(Spn) mutant grew well in complex medium, but did not grow in chemically defined medium unless supplemented with the metals copper and manganese. Transcriptome analysis of D39 rel(+)(Spn) and Deltarel(Spn) strains treated with mupirocin revealed rel(Spn)-independent (translation stress), rel(Spn)-dependent (stringent response) and Deltarel(Spn)-dependent changes, suggesting that rel(Spn) and (p)ppGpp amount play wide-ranging homeostatic roles in pneumococcal physiology, besides adjusting macromolecular synthesis and transport in response to nutrient availability. Notably, the rel(Spn)-dependent response included significant upregulation of the ply operon encoding pneumolysin toxin, whereas the Deltarel(Spn)-dependent response affected expression linked to the VicRK and CiaRH two-component systems. Finally, a D39 Deltarel(Spn) mutant was severely attenuated and displayed a significantly altered course of disease progression in a mouse model of infection, which was restored to normal by an ectopic copy of rel(+)(Spn).

Physiological Roles of the Dual Phosphate Transporter Systems in Low and High Phosphate Conditions and in Capsule Maintenance of Streptococcus pneumoniae D39.

Unlike most bacteria, Streptococcus pneumoniae (pneumococcus) has two evolutionarily distinct ABC transporters (Pst1 and Pst2) for inorganic phosphate (Pi) uptake. The genes encoding a two-component regulator (PnpRS) are located immediately upstream of the pst1 operon. Both the pst1 and pst2 operons encode putative PhoU-family regulators (PhoU1 and PhoU2) at their ends. This study addresses why S. pneumoniae contains dual Pi uptake systems and the regulation and contribution of the Pst1 and Pst2 systems in conditions of high (mM) Pi amount and low (µM) Pi amount. We show that in unencapsulated mutants, both pst1 and pst2 can be deleted, and Pi is taken up by a third Na(+)/Pi co-transporter, designated as NptA. In contrast, either pst1 or pst2 is unexpectedly required for the growth of capsule producing strains. We used a combination of mutational analysis, transcript level determinations by qRT-PCR and RNA-Seq, assays for cellular PnpR~P amounts by SDS-PAGE, and pulse-Pi uptake experiments to study the regulation of Pi uptake. In high Pi medium, PhoU2 serves as the master negative regulator of Pst2 transporter function and PnpR~P levels (post-transcriptionally). ΔphoU2 mutants have high PnpR~P levels and induction of the pst1 operon, poor growth, and sensitivity to antibiotics, possibly due to high Pi accumulation. In low Pi medium, Pst2 is still active, but PnpR~P amount and pst1 operon levels increase. Together, these results support a model in which pneumococcus maintains high Pi transport in high and low Pi conditions that is required for optimal capsule biosynthesis.

Dynamic distribution of the SecA and SecY translocase subunits and septal localization of the HtrA surface chaperone/protease during Streptococcus pneumoniae D39 cell division.

UNLABELLED: The Sec translocase pathway is the major route for protein transport across and into the cytoplasmic membrane of bacteria. Previous studies reported that the SecA translocase ATP-binding subunit and the cell surface HtrA protease/chaperone formed a single microdomain, termed "ExPortal," in some species of ellipsoidal (ovococcus) Gram-positive bacteria, including Streptococcus pyogenes. To investigate the generality of microdomain formation, we determined the distribution of SecA and SecY by immunofluorescent microscopy in Streptococcus pneumoniae (pneumococcus), which is an ovococcus species evolutionarily distant from S. pyogenes. In the majority (≥ 75%) of exponentially growing cells, S. pneumoniae SecA (SecA (Spn)) and SecY (Spn) located dynamically in cells at different stages of division. In early divisional cells, both Sec subunits concentrated at equators, which are future sites of constriction. Further along in division, SecA(Spn) and SecY(Spn) remained localized at mid-cell septa. In late divisional cells, both Sec subunits were hemispherically distributed in the regions between septa and the future equators of dividing cells. In contrast, the HtrA (Spn) homologue localized to the equators and septa of most (> 90%) dividing cells, whereas the SrtA(Spn) sortase located over the surface of cells in no discernable pattern. This dynamic pattern of Sec distribution was not perturbed by the absence of flotillin family proteins, but was largely absent in most cells in early stationary phase and in cls mutants lacking cardiolipin synthase. These results do not support the existence of an ExPortal microdomain in S. pneumoniae. Instead, the localization of the pneumococcal Sec translocase depends on the stage of cell division and anionic phospholipid content. IMPORTANCE: Two patterns of Sec translocase distribution, an ExPortal microdomain in certain ovococcus-shaped species like Streptococcus pyogenes and a spiral pattern in rod-shaped species like Bacillus subtilis, have been reported for Gram-positive bacteria. This study provides evidence for a third pattern of Sec localization in the ovococcus human pathogen Streptococcus pneumoniae. The SecA motor and SecY channel subunits of the Sec translocase localize dynamically to different places in the mid-cell region during the division cycle of exponentially growing, but not stationary-phase, S. pneumoniae. Unexpectedly, the S. pneumoniae HtrA (HtrA(Spn)) protease/chaperone principally localizes to cell equators and division septa. The coincident localization of SecA(Spn), SecY (Spn), and HtrA (Spn) to regions of peptidoglycan (PG) biosynthesis in unstressed, growing cells suggests that the pneumococcal Sec translocase directs assembly of the PG biosynthesis apparatus to regions where it is needed during division and that HtrA(Spn) may play a general role in quality control of proteins exported by the Sec translocase.

Polymorphism and regulation of the spxB (pyruvate oxidase) virulence factor gene by a CBS-HotDog domain protein (SpxR) in serotype 2 Streptococcus pneumoniae.

spxB-encoded pyruvate oxidase is a major virulence factor of Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes H2O2 and acetyl phosphate, which play roles in metabolism, signalling, and oxidative stress. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen for spontaneous mutations that caused colonies of strain D39 to change from a semitransparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency approximately 3 x 10(-5)) were impaired for SpxB function or expression. Two mutations changed amino acids in SpxB likely required for cofactor or subunit binding. One mutation defined a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three spontaneous mutations created the same frameshift near the start of the trans-acting spxR regulatory gene. The SpxR protein contains helix-turn-helix, CBS and HotDog domains implicated in binding DNA, adenosyl compounds, and CoA-containing compounds respectively, and suggest that SpxR positively regulates spxB transcription in response to energy and metabolic state. Microarray analyses unexpectedly demonstrated that SpxR also positively regulates the strH exoglycosidase gene, which, like spxB, has been implicated in colonization. Finally, SpxR is required for full virulence in a murine model of infection.

Genome sequence of Avery's virulent serotype 2 strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory strain R6.

Streptococcus pneumoniae (pneumococcus) is a leading human respiratory pathogen that causes a variety of serious mucosal and invasive diseases. D39 is an historically important serotype 2 strain that was used in experiments by Avery and coworkers to demonstrate that DNA is the genetic material. Although isolated nearly a century ago, D39 remains extremely virulent in murine infection models and is perhaps the strain used most frequently in current studies of pneumococcal pathogenesis. To date, the complete genome sequences have been reported for only two S. pneumoniae strains: TIGR4, a recent serotype 4 clinical isolate, and laboratory strain R6, an avirulent, unencapsulated derivative of strain D39. We report here the genome sequences and new annotation of two different isolates of strain D39 and the corrected sequence of strain R6. Comparisons of these three related sequences allowed deduction of the likely sequence of the D39 progenitor and mutations that arose in each isolate. Despite its numerous repeated sequences and IS elements, the serotype 2 genome has remained remarkably stable during cultivation, and one of the D39 isolates contains only five relatively minor mutations compared to the deduced D39 progenitor. In contrast, laboratory strain R6 contains 71 single-base-pair changes, six deletions, and four insertions and has lost the cryptic pDP1 plasmid compared to the D39 progenitor strain. Many of these mutations are in or affect the expression of genes that play important roles in regulation, metabolism, and virulence. The nature of the mutations that arose spontaneously in these three strains, the relative global transcription patterns determined by microarray analyses, and the implications of the D39 genome sequences to studies of pneumococcal physiology and pathogenesis are presented and discussed.