Block and Boost DNA Transfer: Opposite Roles of OmpA in Natural and Artificial Transformation of Escherichia coli

Abstract

Our previous work established that DNA is naturally transferable on agar plates through a new transformation system which is regulated by the stationary phase master regulator RpoS in Escherichia coli. In this transformation system, neither additional Ca2+ nor heat shock is required. Instead, transformation is stimulated by agar. The membrane protein OmpA, a gated pore permeable to ions and larger solutes, serves as a receptor for DNA transfer during bacteriophage infection and conjugation. However, it remains unknown how DNA transfers across membranes and whether OmpA is involved in transformation of E. coli. Here, we explored potential roles of OmpA in natural and chemical transformation of E. coli. We observed that ompA inactivation significantly improved natural transformation on agar plates, indicating that OmpA blocks DNA transfer. Transformation promotion by ompA inactivation also occurred on soft plates, indicating that OmpA blocks DNA transfer independent of agar. By contrast, compared with the wild-type strain, chemical transformation of the ompA mutant was lower, indicating that OmpA has a role in DNA transfer. Inactivation of ompA also reduced chemical transformation in solution containing less Ca2+ or with a shortened time for heat shock, suggesting that the promotion effect of OmpA on DNA transfer does not solely rely on Ca2+ or heat shock. We conclude that OmpA plays opposite roles in natural and chemical transformation: it blocks DNA uptake on agar plates but promotes DNA transfer in the liquid Ca2+ solution. Considering that no single factor was identified to reverse the function of OmpA, we propose that multiple factors may cooperate in the functional reversal of OmpA during natural and artificial transformation of E. coli. Finally, we observed that ompA transcription was not affected by the expression of RpoS, excluding the possibility that RpoS regulates DNA transfer by suppressing ompA transcription.

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

“…As reported by our group and others, the general stress response regulator RpoS ( S ) was shown to mediate natural transformation (10), and transformation frequencies were apparently increased when rpoS was induced in the liquid culture stage but did not change significantly when rpoS was induced on solid medium (11). Moreover, OmpA, a membrane protein, has been reported to block DNA transfer, and the null mutation leads to a boost in transformation on agar plates (12).…”

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

“…It remains unclear whether osmotic pressure and/or any other biological/physical factor(s) contribute to the increase of transformation on plates with a high concentration of agar. 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 ).…”

“…To identify the OM pore used for DNA transport during natural transformation of E. coli , the pore-forming protein OmpA was evaluated, considering that OmpA performs functions in bacteriophage infection and bacterial conjugation. Inactivation of ompA increases natural transformation by 7- to 60-fold while decreasing chemical transformation by ∼10-fold, suggesting that OmpA blocks DNA transfer during natural transformation but promotes DNA transfer in artificial transformation ( Sun et al, 2013 ). OmpA is unlikely to form an open gate under natural conditions, but can be switched to the open state with the molecular force of electrostatic interaction (i.e., salt-bridge), that drives structural transition of a protein under different conditions ( Hong et al, 2006 ).…”

“…In the default “gate-closed” state, OmpA traps the transforming DNA, making it unable to reach the right channel for completing transformation. By contrast, during chemical transformation or electroporation, a high concentration of Ca 2+ or an electric current helps open the gate, allowing DNA to pass across the channel formed by OmpA ( Sun et al, 2013 ).…”

“…One of these methods has been shown to be independent of the conserved proteins for the transport of ssDNA during natural transformation or bacterial conjugation. Instead, double-stranded DNA (dsDNA) is taken up into the cytoplasm and internalized by E. coli cells on solid agar plates ( Sun et al, 2006 , 2009 , 2013 ; Sun, 2011 , 2016 ; Zhang et al, 2012 ). The other method of DNA transfer is dependent on cell-to-cell contact and DNA transfer occurs within a colony on agar plates ( Maeda et al, 2004 , 2006 ; Etchuuya et al, 2011 ; Sobue et al, 2011 ; Kurono et al, 2012 ; Matsuda et al, 2012 ; Matsumoto et al, 2016 ).…”