Related Experiment Video
Updated: Jun 22, 2026
Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins
Published on: September 30, 2014
[Recombinant expression of Streptococcus pneumoniae comD/E/C genes and correlation of ComD/C with beta-lactam
Huan Fan1, Ai-Hua Sun, Xiao-Ping Xia
1Department of Microbiology and Parasitology, College of Medicine, Zhejiang University, Hangzhou 310058, China.
Objective:
To construct prokaryotic expression systems of TCS genes comD/comE/comC of Streptococcus pneumoniae, and to determine the correlation of ComD and ComC with the drug resistance.
Methods:
The entire comD, comE and comC genes were amplified by PCR and their prokaryotic expression systems were established by routine genetic engineering technique. SDS-PAGE and Bio-Rad Agarose Image Analyzor was applied to measure the outputs of target recombinant proteins rComD, rComE and rComC. Rabbits were immunized with these recombinant proteins to prepare antisera. The resistance of S.pneumoniae strains to penicillin and cefotaxime was examined after ComD and ComC were blocked by antisera.
Result:
Compared with the reported sequences, similarities of nucleotide and amino acid sequences of the cloned comD, comE and comC genes were 98.4% approximately 99.3% and 99.1% approximately 100%, respectively. The constructed engineering bacteria E.coli BL21DE3(pET42a-comD), E.coli BL21DE3(pET42a-comE) and E.coli BL21DE3(pET42a-comC) were able to efficiently express the target recombinant proteins and the outputs of rComD, rComE and rComC were 28%, 25% and 35% of the total bacterial proteins, respectively. The double immunodiffusion titers of rabbit antisera against rComD, rComE or rComC were 1:4, 1:4 and 1:8, respectively. After the ComD and/or ComC were blocked by the antisera, the cefotaxime-sensitive S. pneumoniae strains became to resistant to antibiotics but there were no changes for cefotaxime-resistant strains and resistance to penicillin for all tested strains.
Conclusion:
The prokaryotic expression systems of S.pneumoniae comD/come/comC genes have been successfully constructed, and the study also indicates that both the ComD and ComC are involved in the drug resistance of S. pneumoniae to cefotaxime.
Insights
Researchers constructed prokaryotic expression systems for Streptococcus pneumoniae ComD, ComE, and ComC genes. ComD and ComC proteins are involved in cefotaxime resistance in S. pneumoniae.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Streptococcus pneumoniae is a significant human pathogen.
- Understanding the genetic mechanisms of antibiotic resistance is crucial for effective treatment.
Purpose of the Study:
- To construct prokaryotic expression systems for the comD, comE, and comC genes of S. pneumoniae.
- To investigate the role of ComD and ComC proteins in bacterial drug resistance.
Main Methods:
- Genes comD, comE, and comC were amplified using PCR.
- Prokaryotic expression systems were established in E. coli.
- Recombinant proteins (rComD, rComE, rComC) were expressed and quantified.
- Antisera were generated against recombinant proteins.
- Antibiotic resistance of S. pneumoniae strains was tested after blocking ComD and ComC with antisera.
Main Results:
- High sequence similarity was observed for the cloned genes compared to reported sequences.
- Efficient expression of recombinant proteins rComD, rComE, and rComC was achieved.
- Blocking ComD and ComC proteins rendered cefotaxime-sensitive S. pneumoniae strains resistant to cefotaxime.
Conclusions:
- Successfully constructed prokaryotic expression systems for S. pneumoniae comD/comE/comC genes.
- ComD and ComC proteins play a role in the cefotaxime resistance of S. pneumoniae.
Related Concept Videos
01:25Mechanism of Antibiotic Resistance in MRSA
01:25Clinical Significance of Antibiotic Resistance
01:29Coordination of Gene Expression Processes in Bacteria
00:57Antibiotic Selection
01:30Development of Antibiotic Resistance