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RpoS regulates a novel type of plasmid DNA transfer in Escherichia coli.

Zhang Y, Shi C, Yu J, Ren J, Sun D - PLoS ONE (2012)

Bottom Line: Normally, RpoS recognizes promoters by its lysine 173 (K173).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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Zhejiang Academy of Agricultural Sciences, Hangzhou, PR China.

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.

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Kinetics of DNA entry on LB-agar plates.E. coli MC4100 cell pellets from the culture at OD600 of 1.5 were resuspended in the supernatant and mixed with plasmid pDsRED at a concentration of 67 µg/ml. The liquid mixture was plated on selective LB-agar plates (50 µl per plate). At intervals, excessive DNase I was spread on one of these plates. The sample without DNase I treatment was set as the control. Percentages of remaining transformants (which indicates DNA uptake kinetics) were calculated by dividing the number of transformants with DNase I treatment by the number of transformants without DNase I treatment. Representative data from three independent experiments were shown.
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pone-0033514-g002: Kinetics of DNA entry on LB-agar plates.E. coli MC4100 cell pellets from the culture at OD600 of 1.5 were resuspended in the supernatant and mixed with plasmid pDsRED at a concentration of 67 µg/ml. The liquid mixture was plated on selective LB-agar plates (50 µl per plate). At intervals, excessive DNase I was spread on one of these plates. The sample without DNase I treatment was set as the control. Percentages of remaining transformants (which indicates DNA uptake kinetics) were calculated by dividing the number of transformants with DNase I treatment by the number of transformants without DNase I treatment. Representative data from three independent experiments were shown.

Mentions: Our previous study and the others showed that plasmid transformation of E. coli occurred on agar plates at 37°C [7], [8], [9]. To test whether DNA uptake occurred prior to plating, excessive DNase I was added after co-incubation of cells from the culture of OD600 of 1.5 and pDsRED in the liquid culture. For both MC4100 and BW25113, no transformants were detected (Table 1). To know when DNA entered competent cells on plates, immediately after plating the mixture of E. coli culture and plasmid DNA, excessive DNase I was spread on the LB-agar plates. No transformants were detected in repeated experiments (n>3). This result indicates that plasmid transformation should occur on LB-agar plates after plating. To explore the kinetics of DNA uptake on agar plates, following the plating of the mixture of competent cells and plasmid DNA, we interrupted DNA uptake by spreading excessive DNase I on LB-agar plates at intervals. We found that DNA was taken up exponentially after spreading competent cells and plasmid DNA on the surface of LB-agar plates. At the start time point (0 minute), no transformants were detected (Figure 2), implying that DNA had not entered cells at that time. Within the first 2 minutes, more than one third of the competent cells acquired plasmid DNA which was not sensitive to DNase I degradation (Figure 2). Five minutes later, the addition of DNase I reduced transformation frequency less than a half (Figure 2). Within the initial 10 minutes on agar plates, 71.9% of the transforming plasmid DNA entered the DNase I insensitive state (Figure 2). The rapid uptake of DNA implies that the route for dsDNA entry might be assembled in the liquid culture before plating but quickly activated on agar plates. Alternatively, stresses introduced by exposure of planktonic cells on plates may contribute to DNA entry. We investigated potential effects of physical stress by plating and oxidative/anti-oxidative stress on plasmid transformation and failed to detect obvious change of transformation frequency by these stresses (see Discussion S1).


RpoS regulates a novel type of plasmid DNA transfer in Escherichia coli.

Zhang Y, Shi C, Yu J, Ren J, Sun D - PLoS ONE (2012)

Kinetics of DNA entry on LB-agar plates.E. coli MC4100 cell pellets from the culture at OD600 of 1.5 were resuspended in the supernatant and mixed with plasmid pDsRED at a concentration of 67 µg/ml. The liquid mixture was plated on selective LB-agar plates (50 µl per plate). At intervals, excessive DNase I was spread on one of these plates. The sample without DNase I treatment was set as the control. Percentages of remaining transformants (which indicates DNA uptake kinetics) were calculated by dividing the number of transformants with DNase I treatment by the number of transformants without DNase I treatment. Representative data from three independent experiments were shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3306417&req=5

pone-0033514-g002: Kinetics of DNA entry on LB-agar plates.E. coli MC4100 cell pellets from the culture at OD600 of 1.5 were resuspended in the supernatant and mixed with plasmid pDsRED at a concentration of 67 µg/ml. The liquid mixture was plated on selective LB-agar plates (50 µl per plate). At intervals, excessive DNase I was spread on one of these plates. The sample without DNase I treatment was set as the control. Percentages of remaining transformants (which indicates DNA uptake kinetics) were calculated by dividing the number of transformants with DNase I treatment by the number of transformants without DNase I treatment. Representative data from three independent experiments were shown.
Mentions: Our previous study and the others showed that plasmid transformation of E. coli occurred on agar plates at 37°C [7], [8], [9]. To test whether DNA uptake occurred prior to plating, excessive DNase I was added after co-incubation of cells from the culture of OD600 of 1.5 and pDsRED in the liquid culture. For both MC4100 and BW25113, no transformants were detected (Table 1). To know when DNA entered competent cells on plates, immediately after plating the mixture of E. coli culture and plasmid DNA, excessive DNase I was spread on the LB-agar plates. No transformants were detected in repeated experiments (n>3). This result indicates that plasmid transformation should occur on LB-agar plates after plating. To explore the kinetics of DNA uptake on agar plates, following the plating of the mixture of competent cells and plasmid DNA, we interrupted DNA uptake by spreading excessive DNase I on LB-agar plates at intervals. We found that DNA was taken up exponentially after spreading competent cells and plasmid DNA on the surface of LB-agar plates. At the start time point (0 minute), no transformants were detected (Figure 2), implying that DNA had not entered cells at that time. Within the first 2 minutes, more than one third of the competent cells acquired plasmid DNA which was not sensitive to DNase I degradation (Figure 2). Five minutes later, the addition of DNase I reduced transformation frequency less than a half (Figure 2). Within the initial 10 minutes on agar plates, 71.9% of the transforming plasmid DNA entered the DNase I insensitive state (Figure 2). The rapid uptake of DNA implies that the route for dsDNA entry might be assembled in the liquid culture before plating but quickly activated on agar plates. Alternatively, stresses introduced by exposure of planktonic cells on plates may contribute to DNA entry. We investigated potential effects of physical stress by plating and oxidative/anti-oxidative stress on plasmid transformation and failed to detect obvious change of transformation frequency by these stresses (see Discussion S1).

Bottom Line: Normally, RpoS recognizes promoters by its lysine 173 (K173).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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Zhejiang Academy of Agricultural Sciences, Hangzhou, PR China.

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.

Show MeSH
Related in: MedlinePlus