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 approximately 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 sigma(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.
Highlights
Gational killing systems that help ensure the segregational stability of plasmids
The epsilon-zeta system was initially identified as an addiction module encoded on plasmid pSM19035 of S. pyogenes with epsilon functioning as the cognate antitoxin to the zeta toxin
Sequence Analysis—A homolog of the S. pyogenes plasmid pSM19035-encoded zeta toxin was found in the genome sequence of S. pneumoniae TIGR4
Summary
Bacterial Strains and Growth Conditions—E. coli DH5␣ was used as a host strain for most of the cloning experiments and transcriptional analyses using lacZ fusions. A DNA fragment spanning both the pezA and pezT genes was PCR-amplified using the primers PezA-F and PezT-R, producing a single 1236-bp PCR product, which was digested and cloned into pET11a resulting in a construct designated pET11a-PezAT. Total RNA was extracted from E. coli DH5␣ harboring the pQF-PezAT recombinant plasmid and was subjected to first strand cDNA synthesis using 20 ng of GSP1 primer, which was designed to anneal 399-bp downstream of the pezA start codon. Electrophoretic Mobility Shift Assays—A 203-bp DNA fragment containing palindrome sequence PS, which is the putative regulatory protein binding site, was PCR-amplified using the primer pair PS-F and PS-R, purified and labeled with biotin using the Biotin 3Ј-End DNA Labeling kit (Pierce). The biotin end-labeled DNA was detected using a streptavidin-horseradish peroxidase conjugate and the supplied chemiluminescent substrate
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