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The Streptococcus pneumoniae pezAT Toxin – Antitoxin System Reduces β -Lactam Resistance and Genetic Competence

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ABSTRACT

Chromosomally encoded Type II Toxin–Antitoxin operons are ubiquitous in bacteria and archaea. Antitoxins neutralize the toxic effect of cognate Toxins by protein–protein interactions and sequestering the active residues of the Toxin. Toxins target essential bacterial processes, mostly translation and replication. However, one class apart is constituted by the PezAT pair because the PezT toxin target cell wall biosynthesis. Here, we have examined the role of the pezAT toxin–antitoxin genes in its natural host, the pathogenic bacterium Streptococcus pneumoniae. The pezAT operon on Pneumococcal Pathogenicity Island 1 was deleted from strain R6 and its phenotypic traits were compared with those of the wild type. The mutant cells formed shorter chains during exponential phase, leading to increased colony-forming units. At stationary phase, the mutant was more resilient to lysis. Importantly, the mutant exhibited higher resistance to antibiotics targeting cell walls (β-lactams), but not to antibiotics acting at other levels. In addition, the mutants also showed enhanced genetic competence. We suggest that PezAT participates in a subtle equilibrium between loss of functions (resistance to β-lactams and genetic competence) and gain of other traits (virulence).

No MeSH data available.


Schematic organization of the pneumococcal pezAT operon. The scheme depicts the position of the single promoter sequence (line with arrowhead pointing to the direction of transcription), which includes the -35 and -10 regions, as well as the inverted repeated sequence (PS, convergent arrows) that is the binding site of PezA and PezT proteins. Ribosome-binding site sequences are denoted as RBS and the initial start site is also depicted with arrow. The pezA termination codon, and the overlapping pezT initiation codon are also indicated. Substitution of the entire operon by a gene cassette encoding resistance to kanamycin is shown below.
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Figure 1: Schematic organization of the pneumococcal pezAT operon. The scheme depicts the position of the single promoter sequence (line with arrowhead pointing to the direction of transcription), which includes the -35 and -10 regions, as well as the inverted repeated sequence (PS, convergent arrows) that is the binding site of PezA and PezT proteins. Ribosome-binding site sequences are denoted as RBS and the initial start site is also depicted with arrow. The pezA termination codon, and the overlapping pezT initiation codon are also indicated. Substitution of the entire operon by a gene cassette encoding resistance to kanamycin is shown below.

Mentions: The genome of S. pneumoniae R6wt (Tettelin et al., 2001) harbors a single copy of the pezAT operon (spr0951–spr0952; NCBI accession no. NC_003098), located within the PPI1 (Khoo et al., 2007). Other pneumococcal strains, like CGSP14, ATCC 700669, and P1031 have two copies of the same operon (Chan et al., 2012). The presence of the pezAT operon has been related to pneumococcal virulence (Brown et al., 2004). Like the typical Type II TAs, the pezA antitoxin gene is placed upstream of pezT (Figure 1), and both genes overlap by one nucleotide, suggestive of coupled translation (Chan et al., 2012). Co-transcription of both genes is directed by a single promoter that is located upstream of pezA. This region also includes a long inverted repeat which spans the -35 and -10 regions of the promoter and that is the region where PezA/PezT proteins bind to control their own synthesis: binding of the PezA:PezT to their target would hinder binding of the host RNA polymerase to the promoter, leading to transcriptional repression of the operon (Khoo et al., 2007; Chan et al., 2016). We have studied the influence of pezAT on the pneumococcal lifestyle without recurring to previous general approaches that consist of the overexpression of the toxin gene, either in the homologous or in a heterologous host, followed by analysis of the resulting effects (Christensen and Gerdes, 2003; Khoo et al., 2007; Nieto et al., 2007). Instead, we constructed a mutant strain (R6ΔPezAT) in which the entire pezAT operon, including the upstream intergenic region was deleted and replaced by a gene encoding kanamycin (Figure 1), confirming the observation that the operon is not essential for S. pneumoniae (Brown et al., 2001). Under these conditions, lack of the operon could be studied on a number of pneumococcal responses.


The Streptococcus pneumoniae pezAT Toxin – Antitoxin System Reduces β -Lactam Resistance and Genetic Competence
Schematic organization of the pneumococcal pezAT operon. The scheme depicts the position of the single promoter sequence (line with arrowhead pointing to the direction of transcription), which includes the -35 and -10 regions, as well as the inverted repeated sequence (PS, convergent arrows) that is the binding site of PezA and PezT proteins. Ribosome-binding site sequences are denoted as RBS and the initial start site is also depicted with arrow. The pezA termination codon, and the overlapping pezT initiation codon are also indicated. Substitution of the entire operon by a gene cassette encoding resistance to kanamycin is shown below.
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Related In: Results  -  Collection

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Figure 1: Schematic organization of the pneumococcal pezAT operon. The scheme depicts the position of the single promoter sequence (line with arrowhead pointing to the direction of transcription), which includes the -35 and -10 regions, as well as the inverted repeated sequence (PS, convergent arrows) that is the binding site of PezA and PezT proteins. Ribosome-binding site sequences are denoted as RBS and the initial start site is also depicted with arrow. The pezA termination codon, and the overlapping pezT initiation codon are also indicated. Substitution of the entire operon by a gene cassette encoding resistance to kanamycin is shown below.
Mentions: The genome of S. pneumoniae R6wt (Tettelin et al., 2001) harbors a single copy of the pezAT operon (spr0951–spr0952; NCBI accession no. NC_003098), located within the PPI1 (Khoo et al., 2007). Other pneumococcal strains, like CGSP14, ATCC 700669, and P1031 have two copies of the same operon (Chan et al., 2012). The presence of the pezAT operon has been related to pneumococcal virulence (Brown et al., 2004). Like the typical Type II TAs, the pezA antitoxin gene is placed upstream of pezT (Figure 1), and both genes overlap by one nucleotide, suggestive of coupled translation (Chan et al., 2012). Co-transcription of both genes is directed by a single promoter that is located upstream of pezA. This region also includes a long inverted repeat which spans the -35 and -10 regions of the promoter and that is the region where PezA/PezT proteins bind to control their own synthesis: binding of the PezA:PezT to their target would hinder binding of the host RNA polymerase to the promoter, leading to transcriptional repression of the operon (Khoo et al., 2007; Chan et al., 2016). We have studied the influence of pezAT on the pneumococcal lifestyle without recurring to previous general approaches that consist of the overexpression of the toxin gene, either in the homologous or in a heterologous host, followed by analysis of the resulting effects (Christensen and Gerdes, 2003; Khoo et al., 2007; Nieto et al., 2007). Instead, we constructed a mutant strain (R6ΔPezAT) in which the entire pezAT operon, including the upstream intergenic region was deleted and replaced by a gene encoding kanamycin (Figure 1), confirming the observation that the operon is not essential for S. pneumoniae (Brown et al., 2001). Under these conditions, lack of the operon could be studied on a number of pneumococcal responses.

View Article: PubMed Central - PubMed

ABSTRACT

Chromosomally encoded Type II Toxin–Antitoxin operons are ubiquitous in bacteria and archaea. Antitoxins neutralize the toxic effect of cognate Toxins by protein–protein interactions and sequestering the active residues of the Toxin. Toxins target essential bacterial processes, mostly translation and replication. However, one class apart is constituted by the PezAT pair because the PezT toxin target cell wall biosynthesis. Here, we have examined the role of the pezAT toxin–antitoxin genes in its natural host, the pathogenic bacterium Streptococcus pneumoniae. The pezAT operon on Pneumococcal Pathogenicity Island 1 was deleted from strain R6 and its phenotypic traits were compared with those of the wild type. The mutant cells formed shorter chains during exponential phase, leading to increased colony-forming units. At stationary phase, the mutant was more resilient to lysis. Importantly, the mutant exhibited higher resistance to antibiotics targeting cell walls (β-lactams), but not to antibiotics acting at other levels. In addition, the mutants also showed enhanced genetic competence. We suggest that PezAT participates in a subtle equilibrium between loss of functions (resistance to β-lactams and genetic competence) and gain of other traits (virulence).

No MeSH data available.