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Updated: May 2, 2026
Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43
Published on: September 11, 2020
Differences in virulence of pneumolysin and autolysin mutants constructed by insertion duplication mutagenesis and
Esther Yip-Mei Liu, Feng-Yee Chang, Jen-Chang Chang
1Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei 11217, Taiwan. cpfung@vghtpe.gov.tw.
Background:
Insertion duplication mutagenesis (IDM) and in-frame deletion (IFD) are common techniques for studying gene function, and have been applied to pneumolysin (ply), a virulence gene in Streptococcus pneumoniae (D39). Discrepancies in virulence between the two techniques were observed in both the previous and present studies. This phenomenon was also observed during mutation analysis of autolysin (lytA).
Results:
Our data showed that target gene restoration (TGR) occurred in IDM mutants, even in the presence of antibiotics, while the IFD mutants were stable. In PCR result, TGR occurred later in IDM-ply and -lytA mutants cultured in non-supplemented medium (4-5 h) compared with those grown in medium supplemented with erythromycin (erm)/chloramphenicol (cat) (3-4 h), but plateaued faster. Real-time PCR for detecting TGR had been performed. When compared with 8-h culture, TGR detection increased from Day 1 and Day 2 of IDM mutant's culture. erm-sensitive clones from IDM mutant were found. Southern blot hybridization and Western blotting also confirmed the phenomenon of TGR. The median survival of mice following intraperitoneal (IP) injection with a 3-h culture of IDM-mutants was significantly longer than that with an 8-h culture, irrespective of antibiotic usage. The median survival time of mice following IP injection of a 3-h culture versus an 8-h culture of IDM-ply in the absence of antibiotics was 10 days versus 2 days (p = 0.031), respectively, while in the presence of erm, the median survival was 5 days versus 2.5 days (p = 0.037), respectively. For an IDM-lytA mutant, the corresponding values were 8.5 days versus 2 days (p = 0.019), respectively, for non-supplemented medium, and 2.5 versus 2 days (p = 0.021), respectively, in the presence of cat. A comparable survival rate was observed between WT D39 and an 8-h IDM culture.
Conclusion:
TGR in IDM mutants should be monitored to avoid inconsistent results, and misinterpretation of data due to TGR could lead to important biological meaning being overlooked. Therefore, based on these results, IFD is preferable to IDM for disruption of target genes.
Insights
Target gene restoration (TGR) in insertion duplication mutagenesis (IDM) mutants of Streptococcus pneumoniae can lead to inconsistent results. In-frame deletion (IFD) is a more reliable method for gene disruption studies.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Insertion duplication mutagenesis (IDM) and in-frame deletion (IFD) are standard techniques for gene function analysis.
- Previous and current studies noted virulence discrepancies between IDM and IFD methods in Streptococcus pneumoniae, specifically for pneumolysin (ply) and autolysin (lytA) genes.
Purpose of the Study:
- To investigate the phenomenon of target gene restoration (TGR) in IDM mutants.
- To compare the stability and reliability of IDM versus IFD for gene disruption in S. pneumoniae.
- To assess the impact of TGR on experimental outcomes, particularly virulence studies.
Main Methods:
- Utilized IDM and IFD techniques to create mutants of S. pneumoniae (D39) targeting ply and lytA genes.
- Employed Polymerase Chain Reaction (PCR) and real-time PCR to detect and quantify TGR.
- Confirmed TGR using Southern blot hybridization and Western blotting.
- Assessed mutant virulence through median survival time in mice following intraperitoneal injection.
Main Results:
- Target gene restoration (TGR) was observed in IDM mutants, even in the presence of antibiotics, while IFD mutants remained stable.
- TGR occurred earlier in IDM mutants cultured with antibiotics (3-4 h) compared to non-supplemented media (4-5 h).
- Mice injected with early-stage IDM mutants (3-h culture) showed significantly longer survival times than those injected with later-stage mutants (8-h culture), indicating a loss of mutant phenotype over time due to TGR.
Conclusions:
- Target gene restoration (TGR) in IDM mutants necessitates careful monitoring to prevent inconsistent experimental results.
- Misinterpretation of data due to undetected TGR can obscure significant biological findings.
- In-frame deletion (IFD) is recommended as a more stable and preferable method for target gene disruption compared to IDM.
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