Molecular and Structural Characterization of the PezAT Chromosomal Toxin-Antitoxin System of the Human Pathogen Streptococcus pneumoniae

Abstract

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 ∼3 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 σ70-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 PezA2PezT2 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 PezA2PezT2 complex, PezA neutralizes the toxicity of PezT by blocking the nucleotide binding site through steric hindrance. 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 ∼3 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 σ70-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 PezA2PezT2 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 PezA2PezT2 complex, PezA neutralizes the toxicity of PezT by blocking the nucleotide binding site through steric hindrance. Toxin-antitoxin (TA) 4The abbreviations used are: TA, toxin-antitoxin; IPTG, isopropyl-1-thio-β-d-galactopyranoside; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; MES, 4-morpholineethanesulfonic acid; RACE, rapid amplification of cDNA ends; r.m.s.d., root mean square deviation; HTH, helix-turn-helix; Pez, pneumococcal epsilon-zeta; ITC, isothermal titration calorimetry. 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 (1Hayes F. Science. 2003; 301: 1496-1499Crossref PubMed Scopus (456) Google Scholar, 2Gerdes K. Christensen S.K. Lobner-Olesen A. Nat. Rev. Microbiol. 2005; 3: 371-382Crossref PubMed Scopus (867) Google Scholar). 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 (3Aizenman E. Engelberg-Kulka H. Glaser G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6059-6063Crossref PubMed Scopus (502) Google Scholar). 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 (3Aizenman E. Engelberg-Kulka H. Glaser G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6059-6063Crossref PubMed Scopus (502) Google Scholar). The MazF toxin was found to exert its lethality by functioning as a sequence-specific endoribonuclease (4Zhang Y. Zhang J. Hoeflich K.P. Ikura M. Qing G. Inouye M. 2003; PubMed Scopus Google Scholar). homologs of MazF from Zhang Y. Zhang J. H. Inouye M. J. PubMed Scopus Google and A. Microbiol. 2005; PubMed Scopus Google have been demonstrated to be and when in E. toxin that was found to as an endoribonuclease is the toxin of the TA system encoded on the E. coli was to as for and the of is dependent on translation S.K. K. Microbiol. 2003; PubMed Scopus Google Scholar). with that the cells form they and be from by of the cognate antitoxin K. Christensen S.K. K. Microbiol. PubMed Scopus Google Scholar). TA systems were therefore to be mediators of programmed cell death, as these a that cells to of K. Christensen S.K. Lobner-Olesen A. Nat. Rev. Microbiol. 2005; 3: 371-382Crossref PubMed Scopus (867) Google Scholar). of the of TA systems and these encoded TA systems be and epsilon-zeta K. Christensen S.K. Lobner-Olesen A. Nat. Rev. Microbiol. 2005; 3: 371-382Crossref PubMed Scopus (867) Google Scholar, K. 2005; PubMed Scopus Google Scholar). TA systems that to the and have been The epsilon-zeta system was initially identified as an encoded on plasmid of S. pyogenes with epsilon functioning as the cognate antitoxin to the zeta The and genes form an operon on with the gene product as a repressor of transcription for with other transcription on A. S. PubMed Scopus Google Scholar, A. S. PubMed Scopus Google Scholar). The of the epsilon and zeta genes were to form a stable A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, J. S. K. A. H. PubMed Scopus Google Scholar). The toxic protein zeta a highly conserved for ATP/GTP as as a phosphoryltransferase active site. neutralizes the toxicity of zeta by blocking to the phosphoryltransferase active site through of a complex with zeta A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). the of of the zeta toxin of zeta was found to be encoded in the of the Gram-positive pathogen S. pneumoniae which other TA A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). of these pneumococcal TA and were to be TA systems Christensen S.K. K. M. Microbiol. PubMed Scopus Google Scholar, S.K. de E. M. J. PubMed Scopus Google Scholar). In the of the pneumococcal epsilon-zeta designated pneumococcal and that is a TA with PezA as the cognate antitoxin to the PezT that pezAT is negatively autoregulated the PezA functions as a transcriptional repressor with PezT as a co-repressor. and coli was used as a for of the experiments and transcriptional of and for two was in E. coli E. coli was with and to of through transcriptional of the E. coli was used as the to the effect of the of the pezAT genes on growth and E. coli was used for of the PezA and PezT for were in When the growth was with the for of E. coli and for the of for Overexpression of PezA and PezT in E. pezT was from the S. pneumoniae the and these and other The was initially the The plasmid was to the pezT which was to a a the pezA and pezT genes was the and a which was and in a designated of PezA, the was by amplification with the and and the was in a protein the pezAT and pezA were the such that would in an The the pezAT was the and for whereas the pezA designated was with the and for mutagenesis was as in A. J. 2003; PubMed Scopus Google coli cells were with either or and with with of E. coli the plasmids were to an of in with and and cells were to to an of plasmids were in the of of Overexpression of the genes was by of and were for to h the of the were to the number of experiments were with in Overexpression and of and coli were with plasmids or were in to an of to and protein was with was to J. J. PubMed Scopus Google Scholar, and induction was to of the protein complex, cells were by and the cell was in for were by and the was by was a with A. was by with A. The was with with the protein complex were and the of PezA was by in a for The was a the protein complex was with and the The were in a of to and were to a by with were to the type protein with the of of PezA, cells were in and the cells were by The cell was by and the was to to a of the was in and J. The were a with J. with the were from the of the cells were in E. cell by the was a with E. were in a with and were in a to The protein were with to on a with G. was in a to were with H. the protein was to were by the of the and of were by matrix-assisted laser was in the with and a and protein were from in of was used as a on the were with of the protein or of which the was with the was to the and the was with a of for after of the of was to the and was to The of or were the and an of was immediately to the protein protein was and a of the was to the was a were the by the to was by and protein were to were carried out in a were the cell and PezA in the and of the protein complex were from of of protein and of to in a K. was by to of a to the to a of in were with and the was to for and were the with the K. The were and J. Scopus Google Scholar). The were with the PubMed Scopus Google Scholar). and was with Scopus Google Scholar). was with the M. A. PubMed Scopus Google and J. M. PubMed Scopus Google in the and PubMed Scopus Google in the of The of the was by J. Google Scholar). In of a was to the to in PezA and Both pezA and pezT were as with the repressor as and in with the as In the of were the and and the for the The of pezA was by and and for the of and of an the after amplification with the and was the pezT was for the and for the The E. coli was with of the and and or and the protein by the and the protein by the was by growth of the on the RNA and RNA were from E. coli an to the The RNA were by and the was by was carried out of RNA with the to the The gene and were used in the of and to of the pezAT operon were and the J. Microbiol. PubMed Scopus Google to transcriptional for the of promoter the promoter region upstream from pezA was the and a which was in the the promoter region with pezA was the and as a which was to the pezAT operon with its promoter region was with the and for the was by of with a that a pezA that was the and E. coli was with the plasmids and were for to the of in and to the of RNA was used to out on the for the of the and RNA from E. coli the various were the transcription was carried out for and the was for of amplification were carried out the for for the of the and for The E. coli-encoded gene was used as a the and The of the was on its and the for an a the by PubMed Scopus Google Scholar). gene or cDNA were and used to the with the for the the were carried out from which the mean was the of the transcriptional site of the pezAT operon was the RNA was from E. coli the plasmid and was to cDNA synthesis of which was to of the pezA was to the of the cDNA and The cDNA was the which was to the and a of the pezA The product was and to the transcriptional which would be the nucleotide that is immediately after the palindrome sequence which is the protein binding was the and and with the were carried out the was for with and of protein PezA or the protein in binding K. were from by on a in and a The was a and the of the S. pyogenes plasmid pSM19035-encoded zeta toxin was found in the sequence of S. pneumoniae The PezT protein encoded by the shares sequence with zeta that have functions in species. a for the antitoxin to the epsilon protein in the sequence of S. pneumoniae any TA systems usually as the that were sequence of the upstream gene product PezA with epsilon that the C-terminal of PezA sequence with epsilon in a bicistronic the overlaps with by where the nucleotide of the of pezA is the of the of This is to that found in the other two S. pneumoniae that have been and found that the of PezA to sequence with transcriptional of the and pezAT was to the two genes for a toxin-antitoxin that is autoregulated by PezA, which functions as the cognate antitoxin to the PezT Overexpression of pezAT in E. pezAT is a TA system and pezT encodes for the toxic the pezAT genes were as as the The pezAT genes were by amplification from of S. pneumoniae as The plasmids were on for found that the pezAT from S. pneumoniae by when compared with the sequence for the of these sequence led to were the PezA and PezT by to with the of was found when the pezAT from the and in the were compared with the thus a of sequence The pezAT the from the sequence was for the PezA sequence is with the sequence whereas the PezT sequence and of these and were found in the PezT of the and for sequence the PezT with PezT and zeta a for of pezA and pezT on a any of the pezT was and was after been for the U. J. 2005; PubMed Scopus Google Scholar). In cell of PezT in growth inhibition with a of in cell for the The in cell growth was for the h where the by compared with the of cell growth was with a in the as as cell were in the as as the cell of the E. coli cells PezT when compared with its and the This growth is to been for the of zeta in E. coli U. J. 2005; PubMed Scopus Google Scholar). The was by the cells in of which an growth of plasmid from cells that have recovered from any in the pezT gene in the promoter region of the This inhibition in cell growth was when pezA was with pezT in E. coli the of PezT toxicity by PezA in a plasmid was with E. coli and the were to induction for protein expression. growth was that PezA was to PezT toxicity when the pezA was in these that PezT functions as a toxin that the growth of E. coli cells for the h of and toxicity is by of PezA, which functions as its cognate antitoxin. PezA with PezT and a plasmid was such that the PezA protein is as a protein from the promoter of the In PezT was with on two protein were on to the of the protein and PezT that PezA and PezT were demonstrated that PezA and PezT the the binding of PezA and PezT in experiments were The bind to other with an This low is the of by ITC, because the to the low When PezA was with the to to of PezA PezA is to bind to This is by where PezA a that PezA to The for the complex is that in form with a of In the PezA and PezT were a system where growth of the E. coli on the was for that were with and when the was and growth of the E. coli was on the of of upstream of pezA the of a promoter sequence upstream of the pezA as as an sequence that is by from the palindrome designated overlaps the and region and is upstream of site be found upstream of the on the of the promoter designated transcriptional with a gene as a in the E. coli plasmid were carried The of was when E. coli cells of the pezA upstream region upstream of in mean of compared with a of for cells the be in cells in the region upstream of the pezT promoter the pezA immediately upstream of The pezA and pezT by nucleotide and would to a bicistronic as in the of other TA systems (1Hayes F. Science. 2003; 301: 1496-1499Crossref PubMed Scopus (456) Google Scholar, 2Gerdes K. Christensen S.K. Lobner-Olesen A. Nat. Rev. Microbiol. 2005; 3: 371-382Crossref PubMed Scopus (867) Google Scholar). RNA was from E. coli the which the pezAT as as of upstream of pezA the promoter. was used to that were to pezAT and the were two and that would to the of pezA and the of was to the a RNA pezAT were of product its The pezA and pezT genes were that to the as be were in the a the RNA RNA from E. coli was to to the transcriptional site. of the from of RNA that the pezAT the upstream of the pezA in these that pezAT is a bicistronic operon that is from a σ70-like the upstream of the pezA The PezA from and the PezT as a of E. coli cells and the pezA with the upstream cells an in that PezA functions as a In the the upstream and pezA and pezT were by when compared with in the a in the of the of the and to any by was carried out to the in the E. coli of were to and as of to as the from the the of the pezA in in a in compared with the in and by in the of the pezAT the PezA antitoxin functions as a transcriptional repressor with the PezT toxin as a and therefore in the of the pezAT in other TA systems (1Hayes F. Science. 2003; 301: 1496-1499Crossref PubMed Scopus (456) Google Scholar, 2Gerdes K. Christensen S.K. Lobner-Olesen A. Nat. Rev. Microbiol. 2005; 3: 371-382Crossref PubMed Scopus (867) Google Scholar). the sequence upstream of pezAT is the binding site for the PezA and PezT were PezA and PezT were and as a and a protein complex from E. coli as The PezA protein and the protein complex were with a were carried out in the of of as a Results that the of was when of PezA was in the binding were with the complex where the of the with of the protein complex was with PezA The PezA2PezT2 the system in the of the protein complex a of by the and in and the in and cell in to the in to the in to the in to the in to the is the mean of and is the in to the in to the is the as on of the from from from in to the is the mean of and is the is the as on of the from from in a of the and of region was as a of region was as a of region was as a in a The of the TA system is a PezA2PezT2 which a with an The is by a of two PezA antitoxin that two PezT toxin to the PezA is to the in the of the epsilon-zeta system A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). the sequence the two antitoxin and toxin conserved on the of the PezA2PezT2 to region which was to be the binding of the phosphoryltransferase zeta A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). conserved that would of of these two TA systems be identified on the of the protein The of conserved is either the protein complex or is for a protein complex were in the the PezA and PezT have a and on is for toxin and for the antitoxin the a be used during because such the to PezT be to to and of the the of PezT in the and the be for the The of the that the the region of the PezA found that a of for PezA were to the In for other PezA these were and for for these is that the that the PezA experiments of a to the of PezA, any that would to a and have been the The PezT the of and the as been for the zeta toxin A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google PezT sequence that for ATP/GTP binding such as a or In to PezT and zeta have the toxic that were to be in zeta toxicity A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google in PezT and these in cell of or PezT known from to be in ATP/GTP binding and A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, J. J. PubMed Scopus Google Scholar). an was in the to the and of the in a non-toxic of of the of toxicity was in the zeta toxin A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). toxic to a of ATP/GTP and The of the in PezT and the in zeta that a from ATP/GTP to an is for toxicity a on the epsilon-zeta the binding site be any be the of the toxin a of with that to the ATP/GTP binding site and conserved PezT and zeta in a that is by and that region is in two conserved and two the and in to form with the Both PezT in a non-toxic that a growth when in E. coli the led to a in the growth which h induction for The in which growth was inhibited in the was to that of the The PezA a the the and the TA systems is found the two antitoxin the C-terminal region of the PezA a to that of that form the of the whereas in PezA conserved of PezA conserved The of of the antitoxin the of the that region be for PezA of PezT by the PezA2PezT2 protein complex, the nucleotide binding site of PezT is by two of PezA and of the antitoxin PezA and with the nucleotide binding site of PezT and nucleotide binding in a to the system. and the active site of PezT and by steric hindrance. The in the is by the of in PezA that is by with the of in in which to be in is by a to the of of PezA of PezA from the nucleotide binding site of the that the of conserved that an essential in toxin PezT and This that the is to the of the in The in have an for the in the of PezA, the to a the of The identified in the is in of the of the PezA2PezT2 In the of the form strong with other or PezT and therefore that the of the PezA2PezT2 complex binding to the the two the of the complex and in bind to a sequence such as found for the promoter. In that the encoded pezAT bicistronic operon of the pathogen S. pneumoniae is a TA with pezA the cognate antitoxin to the toxic product of TA the pezAT is autoregulated with the PezA antitoxin functioning as a transcriptional repressor by binding to a sequence that overlaps the promoter and the PezT toxin as a co-repressor. in and in were by the where the of PezA the of a binding with a to the and the system differs from the of the S. pyogenes plasmid where as a is the transcriptional for the epsilon-zeta bicistronic functions solely as the antitoxin for zeta A. S. PubMed Scopus Google Scholar, U. J. 2005; PubMed Scopus Google Scholar, S. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and been to bind to a that to the of transcriptional K. de J. PubMed Scopus Google Scholar, A. F. PubMed Scopus Google Scholar). the for transcription of the system and the systems and systems have transcriptional to TA epsilon was to have a zeta J. S. K. A. H. PubMed Scopus Google Scholar). the which a of epsilon for functioning of the TA system In of the regulation immediately because the transcriptional repressor and the be found a in of antitoxin would be by its transcriptional repressor function and PezA that the toxin PezT and The of the PezT toxin is to zeta and in of PezT which have been to be in zeta toxicity PezT is therefore that PezT and zeta have for ATP/GTP for to an by site-directed mutagenesis that a binding site of zeta A. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google is in of two that from the nucleotide binding site to is by the growth of E. coli cells of growth was and of growth h after induction of to the in cells that of during growth inhibition induction of the system was U. J. 2005; PubMed Scopus Google Scholar). of that induction of zeta in E. coli cells led to the inhibition of RNA and protein was hypothesized that zeta a effect on the of the cells H. M. E. Y. PubMed Scopus Google Scholar). 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In to were discovered in the of S. pneumoniae number of genes and of the PubMed Scopus Google Scholar). was demonstrated that of pezT in growth in or the function of the pezAT was the that pezT is in a of is to be an essential gene for may the of that PubMed Scopus Google Scholar). of TA systems and was for the system of that a the and an of K. M. J. PubMed Scopus Google Scholar). The TA systems and the for the of TA systems in bacteria where they have been in through to F. Y. E. and J. for and and for to J. K. F. M. K. and K. for J. H. for help with for of the and the the for F. and Y. F. for of S. with