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

Yanmei Zhang,Chunyu Shi,Jingjing Ren,Dongchang Sun,Jiafei Yu,Alain Charbit

doi:10.1371/journal.pone.0033514

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 RpoSK173E 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.

Highlights

Gene transfer through plasmid conjugation or transformation is one of the major cause of antibiotic resistance in bacteria [1]
We investigated the effect of a higher cell density by concentration and plated competent E. coli cells on smaller agar plates
We investigated several basic questions in spontaneous plasmid transformation of E. coli on agar plates, which involves competence development and regulation and double-stranded DNA (dsDNA) transfer across two cell membranes

Summary

Introduction

Gene transfer through plasmid conjugation or transformation is one of the major cause of antibiotic resistance in bacteria [1]. Together with the work from several other groups, showed that E. coli is able to acquire naked plasmid DNA on agar plates at 37uC without the addition of Ca2+ or heat shock [7,8,9]. None of the DNA uptake gene homologs were found to be involved in mediating spontaneous plasmid transformation of E. coli [10]. Entry of dsDNA in E. coli is different from that in other naturally transformable bacteria, which often use the DNA uptake machinery for single-stranded DNA (ssDNA) uptake [11] and DNA binding proteins for processing and integrating the incoming ssDNA [12]. In plasmid transformation of Streptococcus pneumoniae, two strands of ssDNA from two plasmid molecules are taken up into the cytoplasm to re-establish a new plasmid with the assistance from recombinase RecA in the cytoplasm [13]

Methods

Results

Conclusion