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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.


Deletion of the pezAT operon increases genetic competence. The development of spontaneous competence of both pneumococcal wt and pezAT mutant cells were assessed by measuring, at different pH, the transcriptional level of Photinus pyralis luc gene that was fused downstream of the promoter of ssbB, which is a late competent gene. Gray lines indicate growth curves at OD492, whereas black lines depict RLUs/OD492, ranging from pH 7.7–7.9. Vertical line joining both panels points the time differences in the onset of the main peaks of competence. Values correspond to individual cultures representative of three independent experiments (Caymaris et al., 2010).
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Figure 6: Deletion of the pezAT operon increases genetic competence. The development of spontaneous competence of both pneumococcal wt and pezAT mutant cells were assessed by measuring, at different pH, the transcriptional level of Photinus pyralis luc gene that was fused downstream of the promoter of ssbB, which is a late competent gene. Gray lines indicate growth curves at OD492, whereas black lines depict RLUs/OD492, ranging from pH 7.7–7.9. Vertical line joining both panels points the time differences in the onset of the main peaks of competence. Values correspond to individual cultures representative of three independent experiments (Caymaris et al., 2010).

Mentions: The above results showed that the transformability of S. pneumoniae increased in the strain devoid of the pezAT operon. However, they do not allow us to conclude that genetic competence was different in both strains. Consequently, we made use of the observation that induction of spontaneous competence strongly relies on the initial pH of the cultures (Chen and Morrison, 1987). To determine whether PezAT plays a role in regulation/development of competence, we used a gene cassette with a transcriptional fusion of the luciferase gene to the late competence gene ssb (Caymaris et al., 2010). The cassette was inserted into the chromosome of strains R6wt and R6ΔPezAT, thus constructing the two isogenic strains R6luc and the mutant R6ΔPezATluc. Media with initial pH ranging from 6.4 to 7.9 were used to investigate the time of occurrence and the level of competence of both strains. Under the same conditions, we observed that both R6wt and R6ΔPezAT mutant strains developed spontaneous competence only from pH 7.7 onward, and no prominent development of spontaneous competence was observed from pH 6.4 to 7.6 (only data from pH 7.6 to 7.9 are shown; Figure 6). Even though the R6luc and the R6ΔPezATluc strains developed spontaneous competence at the same initial pH (i.e., 7.7), the time of competence development and the magnitude of the competence peaks were different. At initial pH 7.7, the R6luc strain developed spontaneous competence at 140 min and peaked at 154 min with RLU/OD492∼8.9 × 104, whereas the R6ΔPezATluc mutant stain started at 112 min and reached the peak at 140 min with ∼6.1-fold higher magnitude. Similar results were observed at medium with initial pH 7.8, where R6luc parent strain started to develop spontaneous competence at 119 min and peaked at 140 min with RLU/OD492∼3.8 × 105; whereas the mutant began at 98 min and peaked at 119 min with ∼2.7-fold higher magnitude. For medium with initial pH 7.9, spontaneous competence started for the parent and the mutant strain at 98 and 77 min, respectively; they reached the competence peak at 119 and 98 min, respectively. Further, the ratio RLU/OD492 for the mutant was 1.3-fold higher than the parent strain (RLU/OD492∼1.0 × 106). In summary, the mutant strain developed spontaneous competence much earlier and with higher magnitude, even though the growth rate for both strains was similar in medium with initial pH 7.7–7.9. Combining all these observations, we conclude that the pezAT operon has a significant influence on the competence development and hence, transformability of S. pneumoniae.


The Streptococcus pneumoniae pezAT Toxin – Antitoxin System Reduces β -Lactam Resistance and Genetic Competence
Deletion of the pezAT operon increases genetic competence. The development of spontaneous competence of both pneumococcal wt and pezAT mutant cells were assessed by measuring, at different pH, the transcriptional level of Photinus pyralis luc gene that was fused downstream of the promoter of ssbB, which is a late competent gene. Gray lines indicate growth curves at OD492, whereas black lines depict RLUs/OD492, ranging from pH 7.7–7.9. Vertical line joining both panels points the time differences in the onset of the main peaks of competence. Values correspond to individual cultures representative of three independent experiments (Caymaris et al., 2010).
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Related In: Results  -  Collection

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Figure 6: Deletion of the pezAT operon increases genetic competence. The development of spontaneous competence of both pneumococcal wt and pezAT mutant cells were assessed by measuring, at different pH, the transcriptional level of Photinus pyralis luc gene that was fused downstream of the promoter of ssbB, which is a late competent gene. Gray lines indicate growth curves at OD492, whereas black lines depict RLUs/OD492, ranging from pH 7.7–7.9. Vertical line joining both panels points the time differences in the onset of the main peaks of competence. Values correspond to individual cultures representative of three independent experiments (Caymaris et al., 2010).
Mentions: The above results showed that the transformability of S. pneumoniae increased in the strain devoid of the pezAT operon. However, they do not allow us to conclude that genetic competence was different in both strains. Consequently, we made use of the observation that induction of spontaneous competence strongly relies on the initial pH of the cultures (Chen and Morrison, 1987). To determine whether PezAT plays a role in regulation/development of competence, we used a gene cassette with a transcriptional fusion of the luciferase gene to the late competence gene ssb (Caymaris et al., 2010). The cassette was inserted into the chromosome of strains R6wt and R6ΔPezAT, thus constructing the two isogenic strains R6luc and the mutant R6ΔPezATluc. Media with initial pH ranging from 6.4 to 7.9 were used to investigate the time of occurrence and the level of competence of both strains. Under the same conditions, we observed that both R6wt and R6ΔPezAT mutant strains developed spontaneous competence only from pH 7.7 onward, and no prominent development of spontaneous competence was observed from pH 6.4 to 7.6 (only data from pH 7.6 to 7.9 are shown; Figure 6). Even though the R6luc and the R6ΔPezATluc strains developed spontaneous competence at the same initial pH (i.e., 7.7), the time of competence development and the magnitude of the competence peaks were different. At initial pH 7.7, the R6luc strain developed spontaneous competence at 140 min and peaked at 154 min with RLU/OD492∼8.9 × 104, whereas the R6ΔPezATluc mutant stain started at 112 min and reached the peak at 140 min with ∼6.1-fold higher magnitude. Similar results were observed at medium with initial pH 7.8, where R6luc parent strain started to develop spontaneous competence at 119 min and peaked at 140 min with RLU/OD492∼3.8 × 105; whereas the mutant began at 98 min and peaked at 119 min with ∼2.7-fold higher magnitude. For medium with initial pH 7.9, spontaneous competence started for the parent and the mutant strain at 98 and 77 min, respectively; they reached the competence peak at 119 and 98 min, respectively. Further, the ratio RLU/OD492 for the mutant was 1.3-fold higher than the parent strain (RLU/OD492∼1.0 × 106). In summary, the mutant strain developed spontaneous competence much earlier and with higher magnitude, even though the growth rate for both strains was similar in medium with initial pH 7.7–7.9. Combining all these observations, we conclude that the pezAT operon has a significant influence on the competence development and hence, transformability of S. pneumoniae.

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.