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.

In this article, we describe the medaka mutant pale gray eyes (pge) that shows reduction of black, white, and silver pigmentation and lethality approximately a week after hatching. The pge mutation was mapped to the tip of linkage group 14 and no recombinations were observed between the mutation and medaka vps11 in 900 meioses. Vps 11 is one of the evolutionarily conserved class C vacuolar protein sorting genes (c-vps: vps11, vps16, vps18, and vps33), whose products physically associate to form the c-vps protein complex required for vesicle docking and fusion in the budding yeast. Mutations in vps16, vps18, and vps33 are known to result in decreased pigmentation in organisms such as Drosophila. We cloned the full-length medaka vps11 cDNA by rapid amplification of cDNA ends (RACE) and found no RACE products from the pge mutants. Similarly, no vps11 transcripts were detected from the pge mutants by Northern analysis. The injection of an antisense morpholino against vps11 phenocopied the pge mutant. Taken together, the results suggest that reduced expression of medaka vps11 causes pge and that medaka vps11 is indispensable for survival and normal pigmentation.