RpoS Regulates a Novel Type of Plasmid DNA Transfer in Escherichia coli

Abstract

Spontaneous plasmid transformation of Escherichia coli is independent of the DNA uptake machinery for single-stranded DNA (ssDNA) entry. The one-hit kinetic pattern of plasmid transformation indicates that double-stranded DNA (dsDNA) enters E. coli cells on agar plates. However, DNA uptake and transformation regulation remain unclear in this new type of plasmid transformation. In this study, we developed our previous plasmid transformation system and induced competence at early stationary phase. Despite of inoculum size, the development of competence was determined by optical cell density. DNase I interruption experiment showed that DNA was taken up exponentially within the initial 2 minutes and most transforming DNA entered E. coli cells within 10 minutes on LB-agar plates. A half-order kinetics between recipient cells and transformants was identified when cell density was high on plates. To determine whether the stationary phase master regulator RpoS plays roles in plasmid transformation, we investigated the effects of inactivating and over-expressing its encoding gene rpoS on plasmid transformation. The inactivation of rpoS systematically reduced transformation frequency, while over-expressing rpoS increased plasmid transformation. Normally, RpoS recognizes promoters by its lysine 173 (K173). We found that the K173E mutation caused RpoS unable to promote plasmid transformation, further confirming a role of RpoS in regulating plasmid transformation. In classical transformation, DNA was transferred across membranes by DNA uptake proteins and integrated by DNA processing proteins. At stationary growth phase, RpoS regulates some genes encoding membrane/periplasmic proteins and DNA processing proteins. We quantified transcription of 22 of them and found that transcription of only 4 genes ( osmC , yqjC , ygiW and ugpC ) encoding membrane/periplasmic proteins showed significant differential expression when wildtype RpoS and RpoS K173E mutant were expressed. Further investigation showed that inactivation of any one of these genes did not significantly reduce transformation, suggesting that RpoS may regulate plasmid transformation through other/multiple target genes.

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

“…Furthermore, we found that DNA uptake gene orthologs required for other naturally transformable species were not involved in this transformation system (9), which implied that a different molecular mechanism of competence development and DNA uptake was being used. 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).…”

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

“…During transformation of E. coli on plates, OMPs should directly contact with the surface of agar plates where DNA uptake occurs [13]. Our previous work demonstrated that agar significantly promotes transformation on plates [18].…”

“…Our previous work showed that natural transformation of E. coli is regulated by the transcription regulator RpoS [13], which is able to compete with σ 70 [33], the principle regulator of ompA (Fig. S2).…”

“…heat shock and a high concentration of divalent cations) [6]–[9]. Recently, transformation of E. coli on agar plates without these artificial treatments has been repeatedly reported by several independent groups [10]–[13].…”

“…Ca 2+ , Mg 2+ , Mn 2+ ) contained in the agar. Further analysis showed that natural transformation of E. coli on plates was regulated by an alternative sigma factor RpoS [13]. However, it remains mysterious how DNA transfers across bacterial membranes and how RpoS regulates DNA transfer on the solid surface.…”