Mechanism Investigation On Gene ComE Controlling The Pneumococcal Transformation Rate

Transformation is one of the most common manners invovled in gene recombination by integrating exogenous DNA to bacterial chromosome, thus causing changes in some biological features. This can be genetically inherited. An extensive researches had revealed that transformation had a close correlation with exhibition of some biological characters, such as evolution, virulence factors and drug-resistance. Also, transformation is a convenient and important approach for gene reconstruction of bacteria in laboratory.Streptococcus pneumoniae can initiate transformation by self generating competence at natural growth state, which called natural transformation. Also, its competence state can be induced by competence-stimulating peptide(CSP), which called induced-transformation. At present, it has been cleared about the basic molecular molecule of S. pneumoniae controling transformation, however, the transformation rates at different growth states and concentrations of CSP were quite distinct, yet molecular mechanism was still not investigated. Operon comCDE is the key gene which control pneumococcal transformation, and comE protein is believed to be the effect protein of this operon, so whether comE expression is directly responsible for the occurrence of different transformation rates? Our research schedule was: firstly, verifiy the key role of gene comE in transformation by insertion-mutation method; then investigate the correlation between this gene and transformation rates at natural and induced transformation state; lastly, try to reveal the control mechanism by proteome method. The research work included the following three parts:1. The effect of comE gene to pneumococcal transformation. We first constructed the comE-deficient strain, and then the two strains (wild type and mutant) were subjected to undergo transformation. Transformation rates of comE-deficient strain were 0 at natural and induced competence states, suggesting that comE gene was essential for pneumococcal transformation, both under natural and induced states.2. Correlation between natural transformation rates at different growth states and comE expression levels. mRNA expression levels of comE were assayed at different growth states by FQ-PCR, meanwhile, the corresponding transformation rates were calculated. The result showed that pnuemococcus had only one transformation peak during the whole growth process at the OD550 of about 0.3; surprisingly, the mRNA expression levels of comE had 2 to 3 peaks, which was not correlated well with the different transformation rates (r<0.3), which means that besides gene comE, there were other factors could control natural transformation rate directly.3. Correlation between comE expression levels and induced-transformation rates at different concentrations of CSP. mRNA expression levels of comE at different concentrations of CSP and different times after CSP treatment was detected by FQ-PCR, at the same time the corresponding transformation rates were also calculated. The result showed that, (1) levels of comE mRNA increased significantly after 10min treatment with CSP at the concentration of 100μg/L, followed by a sharp decrease. Importantly, transformation rates was correlated well with the basic expression levels of comE (r>0.99), which suggested that comE was responsible for the induced-transformation at CSP concentration of 100μg/L; (2) At the CSP concentrations of 10μg/L, 100μg/L and 1000μg/L, transformation rates did not enhance with the increase of CSP, and increased comE expression was only observed at the CSP concentration of 100μg/L, which indicated that CSP concentration was not accounted for the varied transformation rates and comE levels; moreover, our data suggested that, besides CSP, there must be other factors able to control comE expression and pneumococcal transformation, and maybe there were other factors could control transformation rate directly except comE.With an aim to reveal the potential control mechanism of pneumococcal transformation under protein-level, we compared the protein-expression models during the periods of competence occurrence-to-close by using proteome technology (2-DE combine MALDI-TOF-MS). Differential expression dots were noticed at different transformation states, we tried to get the exact imformation about those proteins but, due to technology, not success. The further work is ongoing.