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-basepair 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.Streptococcus pneumoniae (pneumococcus) is a major human respiratory pathogen that causes several serious diseases, including pneumonia, otitis media (ear infection), sinusitis, meningitis, and septicemia (reviewed in references 81, 112, and 114). Invasive pneumococcal diseases result in high rates of mortality and morbidity, especially among young, elderly, debilitated, and immunosuppressed individuals (54, 55). It is estimated that more than 1 million people die each year from pneumococcal infections worldwide, especially in developing countries (59, 80). In the United States and elsewhere, resistance to a range of antibiotics is increasing at an alarming rate among clinical isolates of S. pneumoniae (6, 60). As part of its life cycle, pneumococcus exists as a commensal bacterium that inhabits and colonizes the nasopharynx of up to 20 and 50% of healthy adults and children, respectively, at any time (81, 113). The transition from commensal bacterium to opportunistic pathogen often occurs after another respiratory tract infection.For example, pneumococcal pneumonia has been a leading secondary infection and cause of death during influenza pandemics (9).Strains of S. pneumoniae are categorized into seroty...

Summary 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 transacting 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 ª 3 ¥ 10

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Summary RelA/ SpoT homolog (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 S. pneumoniae, RelSpn, can be deleted and is the primary source of (p)ppGpp synthesis in virulent strain D39 under some conditions. A D39 ΔrelSpn 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 ΔreSpn strains treated with mupirocin revealed relSpn-independent (translation stress), relSpn-dependent (stringent response), and ΔrelSpn-dependent changes suggesting that relSpn 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 relSpn-dependent response included significant up-regulation of the ply operon encoding pneumolysin toxin, whereas the ΔrelSpn-dependent response affected expression linked to the VicRK and CiaRH two component systems. Finally, a D39 ΔrelSpn 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.

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 Ϸ6,200 monomers per cell, which are Ϸ14-fold greater than the amount of the WalK Spn histidine kinase, which is present at Ϸ460 dimers (920 monomers) per cell. We detected Ϸ1,200 monomers per cell of WalJ Spn ancillary protein, similar to the amount of WalK Spn .