PMID:22438941

Citation	Zhang, Y, Shi, C, Yu, J, Ren, J and Sun, D (2012) RpoS regulates a novel type of plasmid DNA transfer in Escherichia coli. PLoS ONE 7:e33514
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.
Links	PubMed PMC3306417 Online version:10.1371/journal.pone.0033514
Keywords	Amino Acid Substitution; Bacterial Load; Bacterial Proteins/antagonists & inhibitors; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; DNA, Bacterial/genetics; DNA, Bacterial/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/antagonists & inhibitors; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Gene Expression Regulation, Bacterial; Genes, Bacterial; Kinetics; Mutagenesis, Site-Directed; Plasmids/genetics; Plasmids/metabolism; Sigma Factor/antagonists & inhibitors; Sigma Factor/genetics; Sigma Factor/metabolism; Transformation, Genetic
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOLI:RPOS	Contributes to	GO:0045809: positive regulation of establishment of competence for transformation	ECO:0000315:		P		Figure 4A shows the transformation frequency was greater in the rpoS+ strain than in the rpoS- strain at similar optimal culture densities.	complete CACAO 4025

References

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