The chromosomal pezT gene of the Gram-positive pathogen Streptococcus pneumoniae encodes a protein that is homologous to the zeta toxin of the Streptococcus pyogenes plasmid pSM19035-encoded epsilon-zeta toxin-antitoxin system. Overexpression of pezT in Escherichia coli led to severe growth inhibition from which the bacteria recovered ϳ3 h after induction of expression. The toxicity of PezT was counteracted by PezA, which is encoded immediately upstream of pezT and shares weak sequence similarities in the C-terminal region with the epsilon antitoxin. The pezAT genes form a bicistronic operon that is co-transcribed from a 70 -like promoter upstream of pezA and is negatively autoregulated with PezA functioning as a transcriptional repressor and PezT as a co-repressor. Both PezA and the non-toxic PezA 2 PezT 2 protein complex bind to a palindrome sequence that overlaps the promoter. This differs from the epsilon-zeta system in which epsilon functions solely as the antitoxin and transcriptional regulation is carried out by another protein designated omega. Results from site-directed mutagenesis experiments demonstrated that the toxicity of PezT is dependent on a highly conserved phosphoryltransferase active site and an ATP/GTP nucleotide binding site. In the PezA 2 PezT 2 complex, PezA neutralizes the toxicity of PezT by blocking the nucleotide binding site through steric hindrance. Toxin-antitoxin (TA)4 systems were initially discovered on low copy number plasmids where they function as post-segregational killing systems that help ensure the segregational stability of plasmids. TA systems usually consist of two genes: the toxin gene encodes a stable protein whereas the antitoxin gene encodes either a labile protein or an untranslated, antisense RNA species. The toxic effect is neutralized by inhibition of toxin translation when the antitoxin is an RNA (type I), or by strong binding of the cognate antitoxin when the antitoxin is a protein (type II). When encoded on plasmids, TA systems were known as "addiction modules," because cells which lose these plasmids would be killed, thus causing the cells to be "addicted" to the short-lived antitoxin product because its de novo synthesis is essential for cell survival (1, 2).Over the past few years, homologs of these plasmid-borne TA systems have been identified on the chromosomes of Escherichia coli and various other bacteria. The discovery of the E. coli-encoded mazEF TA system led to the postulation that TA modules may function as mediators of a type of programmed cell death in bacteria, because the transcription of mazEF was found to be inhibited by various environmental stresses such as nutrient starvation (3). The MazE antitoxin is relatively unstable compared with the MazF toxin and any inhibition of mazEF transcription would "activate" MazF and lead to cell death. E. coli was therefore hypothesized to undergo altruistic cell death, thus helping to ensure the survival of the population during adverse conditions (3). The MazF toxin was found to exert its lethality by...

Toxin-antitoxin loci belonging to the yefM-yoeB family are located in the chromosome or in some plasmids of several bacteria. We cloned the yefM-yoeB locus of Streptococcus pneumoniae, and these genes encode bona fide antitoxin (YefM Spn ) and toxin (YoeB Spn ) products. We showed that overproduction of YoeB Spn is toxic to Escherichia coli cells, leading to severe inhibition of cell growth and to a reduction in cell viability; this toxicity was more pronounced in an E. coli B strain than in two E. coli K-12 strains. The YoeB Spn -mediated toxicity could be reversed by the cognate antitoxin, YefM Spn , but not by overproduction of the E. coli YefM antitoxin. The pneumococcal proteins were purified and were shown to interact with each other both in vitro and in vivo. Far-UV circular dichroism analyses indicated that the pneumococcal antitoxin was partially, but not totally, unfolded and was different than its E. coli counterpart. Molecular modeling showed that the toxins belonging to the family were homologous, whereas the antitoxins appeared to be specifically designed for each bacterial locus; thus, the toxin-antitoxin interactions were adapted to the different bacterial environmental conditions. Both structural features, folding and the molecular modeled structure, could explain the lack of crosscomplementation between the pneumococcal and E. coli antitoxins.The gram-positive, spherical bacterium Streptococcus pneumoniae (pneumococcus) is the cause of many human diseases, such as pneumonia, bacterial blood poisoning (bacteremia), inflammation of the membranes surrounding the brain and spinal cord (meningitis), middle-ear infection (otitis media), osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, and sinusitis; pneumonia is the most severe disease (15,28). Although the pneumococcus can normally be found in the noses and throats of healthy individuals, it can grow and cause infection when the immune system is weakened. The people who are most at risk of developing pneumococcal pneumonia have a weakened immune system. These people include the elderly, infants, cancer patients, AIDS patients, postoperative patients, alcoholics, and people with diabetes. The global rate of mortality is more than 1,000,000 people per year, and this figure represents about 15 to 20% of the people infected.

Type II (proteic) toxin-antitoxin systems (TAS) are ubiquitous among bacteria. In the chromosome of the pathogenic bacterium Streptococcus pneumoniae, there are at least eight putative TAS, one of them being the yefM-yoeB Spn operon studied here. Through footprinting analyses, we showed that purified YefM Spn antitoxin and the YefM-YoeB Spn TA protein complex bind to a palindrome sequence encompassing the ؊35 region of the main promoter (P yefM2 ) of the operon. Thus, the locus appeared to be negatively autoregulated with respect to P yefM2 , since YefM Spn behaved as a weak repressor with YoeB Spn as a corepressor. Interestingly, a BOX element, composed of a single copy (each) of the boxA and boxC subelements, was found upstream of promoter P yefM2 . BOX sequences are pneumococcal, perhaps mobile, genetic elements that have been associated with bacterial processes such as phase variation, virulence regulation, and genetic competence. In the yefM-yoeB Spn locus, the boxAC element provided an additional weak promoter, P yefM1 , upstream of P yefM2 which was not regulated by the TA proteins. In addition, transcriptional fusions with a lacZ reporter gene showed that P yefM1 was constitutive albeit weaker than P yefM2 . Intriguingly, the coupling of the boxAC element to P yefM1 and yefM Spn in cis (but not in trans) led to transcriptional activation, indicating that the regulation of the yefM-yoeB Spn locus differs somewhat from that of other TA loci and may involve as yet unidentified elements. Conservation of the boxAC sequences in all available sequenced genomes of S. pneumoniae which contained the yefM-yoeB Spn locus suggested that its presence may provide a selective advantage to the bacterium.