Positive Effect of Carbon Sources on Natural Transformation in Escherichia coli: Role of Low-Level Cyclic AMP (cAMP)-cAMP Receptor Protein in the Derepression of rpoS

Abstract

Natural plasmid transformation of Escherichia coli is a complex process that occurs strictly on agar plates and requires the global stress response factor S . Here, we showed that additional carbon sources could significantly enhance the transformability of E. coli. Inactivation of phosphotransferase system genes (ptsH, ptsG, and crr) caused an increase in the transformation frequency, and the addition of cyclic AMP (cAMP) neutralized the promotional effect of carbon sources. This implies a negative role of cAMP in natural transformation. Further study showed that crp and cyaA mutations conferred a higher transformation frequency, suggesting that the cAMP-cAMP receptor protein (CRP) complex has an inhibitory effect on transformation. Moreover, we observed that rpoS is negatively regulated by cAMP-CRP in early log phase and that both crp and cyaA mutants show no transformation superiority when rpoS is knocked out. Therefore, it can be concluded that both the crp and cyaA mutations derepress rpoS expression in early log phase, whereby they aid in the promotion of natural transformation ability. We also showed that the 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. This finding could contribute to a better understanding of the relationship between nutrition state and competence, as well as the mechanism of natural plasmid transformation in E. coli. IMPORTANCEEscherichia coli, which is not usually considered to be naturally transformable, was found to spontaneously take up plasmid DNA on agar plates. Researching the mechanism of natural transformation is important for understanding the role of transformation in evolution, as well as in the transfer of pathogenicity and antibiotic resistance genes. In this work, we found that carbon sources significantly improve transformation by decreasing cAMP. Then, the low level of cAMP-CRP derepresses the general stress response regulator RpoS via a biphasic regulatory pattern, thereby contributing to transformation. Thus, we demonstrate the mechanism by which carbon sources affect natural transformation, which is important for revealing information about the interplay between nutrition state and competence development in E. coli. Horizontal gene transfer (HGT) is the transfer of genes between distantly related organisms. It is widely recognized that HGT contributes significantly to the evolution of bacterial genomes and the adaptation of bacteria to new environments (1, 2). In bacteria, the physical process of DNA transfer is accomplished by transduction, conjugation, and transformation. Natural transformation is characterized by the spontaneous uptake of free DNA from the environment by a competent cell, which then integrates said DNA into its chromosome or stabilizes the DNA extr...

Trouble is coming: Signaling pathways that regulate general stress responses in bacteria

“…cAMP is synthesized when the cell is using secondary carbon sources (rather than glucose), and the global transcriptional regulator CRP, in complex with cAMP, positively regulates the genes necessary for using these less-favored carbon sources. A number of studies show higher levels of RpoS early in exponential phase in the absence of cAMP and CRP (3,17,23,24), suggesting negative transcriptional regulation by CRP/cAMP, and based on these studies, EcoCyc currently shows the rpoS promoter as negatively regulated by CRP (25). However, other studies show positive effects of cAMP and CRP (26), and muta- JBC REVIEWS: Bacterial general stress responses tions in the suggested CRP-binding sites upstream and downstream of the major rpoS promoter significantly reduced RpoS expression (23), providing compelling evidence for an important positive role for CRP for RpoS transcription.…”

The carbon catabolite repressor CcpA mediates optimal competence development in Streptococcus oligofermentans through post‐transcriptional regulation

“…In a few Gram‐negative bacterial species, it was found that carbon catabolite repression (CCR) is involved in the regulatory network governing natural competence and transformation (Lo Scrudato and Blokesch, ; Guo et al , ; Yang and Lan, ). The CCR mechanism is widely employed by bacteria to repress the utilization of non‐preferred carbohydrates such as galactose and mannose (Gorke and Stulke, ; Rojo, ).…”

Pull in and Push Out: Mechanisms of Horizontal Gene Transfer in Bacteria

“…Second, an OM protein, OmpA, plays opposite roles in natural and chemical transformation of E. coli : it promotes chemical transformation but suppresses natural transformation ( Sun et al, 2013 ). Third, exponentially growing E. coli cells are often employed for preparing chemically competent cells with the highest efficiency, and chemical transformation occurs in a liquid, whereas the natural transformation of stationary-phase E. coli cells is regulated by the transcriptional regulator RpoS and the cyclic AMP (cAMP) – cAMP receptor protein (CRP), and these cells can acquire exogenous DNA exclusively on agar plates ( Zhang et al, 2012 ; Guo et al, 2015 ). The functions of RpoS or the cAMP-CRP complex in the chemical transformation of E. coli have not been found.…”