| Natural transformation is characterized by a spontaneously competent cell taking up free DNA from environment and then integrating it to chromosome or stabilizing extrachromosomely as plasmid, which leads to a new phenotype. Natural transformation has great significance for obtaining new genetic traits, adapting to complicated and changing natural environment and the evolution of species. Escherichia coli is widely used as model organism for basic and applied research. However, the study of natural transformation in E. coli is rarely reported and focused on the phenomenon of transformation lacking study of systematic regulatory mechanism. Our previous research showed that when cultured to stationary phase and then cultivated statically in an open system E. coli can develop natural competence and take up plasmid DNA on agar plate. In initial study of the mechanism, it was found that DNA uptake gene orthologs are not involved in this transformation system and the general stress response regulator RpoS (?S) was shown to mediate natural transformation, which implies that a different molecular mechanism of competence development and DNA uptake was being used. Taking the factors of competence induction and stress on plate as starting points, we deeply investigated the effect of nutritional condition and oxidative stress on natural transformation and their mechanisms in this study.1. Eutrophic condition is beneficial to natural transformation. Additional carbon souces can promote natural transformation by inhibiting cAMP-CRP and then derepressing rpoS.Comparing the transformation frequencies under different nutritional condition, we found that competence development is more in favor of eutrophic condition than oligotrophic condition. Addition of 0.2% available carbon sources(glucose, fructose, trehalose, mannose, mannitol, N-acetyl-glucosamine, galactose, fucose, maltose, glycerol, N-acetyl-neuraminic acid) can increase transformation frequency by 2.6-15.3 fold, but nitrogen sources have no apparent influence on transformation. Through comparing the influence of carbon sources added in different stages, it is confirmed that the effect of carbon sources is mainly in shaking culture period rather than transformation on agar plate. It was found that phosphotransferase system (PTS) mutants exhibit higher transformation frequency and the promotional transformation frequencies caused by carbon sources return to the level of control when cAMP is added. Considering the close relationship among carbon source, activity of PTS and cAMP level, we infer that the positive effect of carbon sources on transformation is related to the reduction of cAMP level. Through detecting the transformation frequencies of crp and cyaA mutants, it is found that their transformation frequencies are higer than that of wild-type approximately by an order of magnitude. It illustrates cAMP-CRP plays an inhibiting role in transformation. Thus, carbon sources promote transformation by repressing cAMP-CRP.As the regulatory effect of cAMP-CRP on rpoS is controversial, we did initial research on the regulatory model to explore whether the effect of cAMP-CRP on transformation is related to rpoS. The results from both the promoter activity assay and RpoS content analysis suggested that cAMP-CRP plays a biphasic role in rpoS expression; in particular, cAMP-CRP has a negative effect on rpoS in the early log phase but a positive effect in the mid and late log and stationary phases. In addition, it is identified that the two CRP sites around the rpoS promoter both play positive roles in the expression of rpoS by the means of detecting the promoter activity and RpoS content of CRP-binding sites mutagenesis. It implies that cAMP-CRP represses rpoS in early log phase indirectly.By testing the transformation frequencies of double knockout mutants, it shows that both crp and cyaA mutants show no transformation superiority when rpoS is knocked out. It can be concluded that both the crp and cyaA mutants have the ability to derepress rpoS expression in early log phase, whereby they aid in the promotion of natural transformation. Moreover, it is found that addition of carbon sources have no effect on transformation of rpoS mutant. In the study of the influence of carbon sources on RpoS content, it is shown that after carbon sources added RpoS contents significantly increase in log phase and decrease subsequently, which is consistent with the biphasic regulation of cAMP-CRP on rpoS. The results above illustrate that accumulation of RpoS during early log phase can account for the enhanced transformation aroused by additional carbon sources. Our results thus demonstrated that the presence of additional carbon sources promotes competence development and natural transformation by reducing cAMP-CRP and thus derepressing rpoS expression during log phase.2. Oxidative stress can promote natural transformation. The cooperated effect of reactive oxygen species and intracellular iron is involved in the promotion of natural transformation.In order to explore the relevant pathway involved in transformation, we have screened downstream genes of rpoS, genes respond to stress on solid plate and other related genes. It is found that the deficiency of the genes related to oxidative stress lead to an improvement in transformation frequency. The deficiency of hydrogen peroxide-stress response oxyR and its downstream gene katG (encodes catalase I) can increase natural transformation frequency by 6.6-fold and 3.6-fold respectively. The mutant of katE which encodes catalase II and regulated by rpoS can also improve transformation frequency. However, the mutants of superoxide-stress response gene soxRS and its downstream gene sodA and sodB which encode superoxide dismutase have no effect on transformation. Moreover, addition of hydrogen peroxide and superoxide inducer in liquid culture can increase transformation frequency by approximately an order of magnitude, which illustrates that the intracellular oxidative stress induced by reactive oxygen species especially hydrogen peroxide has a promotional effect on transformation. Further study showed that the effect of reactive oxygen species on transformation can be diminished by adding iron chelator which can reduce cellular level of unincorporated iron, which implies a cooperated effect of reactive oxygen species and iron on transformation. It suggests that the DNA damage caused by hydroxyl radical produced from Fenton reaction between hydrogen peroxide and ferrous iron is the main factor for the promotional transformation induced by oxidative stress.It is reported that cells face oxidative stress when transferred from liquid condition to solid plate and oxyR as well as soxRS are responsive. Addition of catalase on agar plate can compensate the growth deficiency of oxyR mutant on agar plate, which also confirm the importance of hydrogen peroxide scavenge system in response to solid plate for cells. Nevertheless, addition of hydrogen peroxide or catalase on agar plate has no prominent influence on natural transformation. It suggests that the induction of transformation is due to the endogenous oxidative stress produced in early stage. So endogenous oxidative stress and exogenous stress on agar plate may corporately participate in the transformation improvement.Above all, in the study of the factors involved in competence induction and transformation on agar plate, we illustrate the effect of nutritional condition and oxidative stress on natural transformation in E. coli and the related mechanism, which is important for understanding the relationship between natural transformation of bacteria and environmental factors such as nutritional state and solid substrate. In our study, the mechanism of the effect of carbon sources and cAMP-CRP on transformation is different from the reported mechanisms in other bacteria, and the influence of oxidative stress in transformation has not been reported yet. It implies a different transformation mechanism in E. coli. Summarizing the current conclusions and clues, we conjecture that natural transformation of E. coli is regulated by various factors. Research on the regulatory mechanism of transformation is conductive to enrich the understanding of horizontal gene transfer and species evolution, to develop a theoretical foundation of resistant and pathogenic gene spread, as well as to provide a guidance for biological safety of engineering bacteria. |
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