Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of inoculum size on plasmid transformation.Overnight grown (13 hr) E. coli MC4100 was inoculated into three triangle flasks each containing 100 ml of fresh LB at ratios of 1∶100 (triangle), 1∶1, 000 (circle) and 1∶100, 000 (rectangle). During incubation, OD600 of the cultures was measured periodically (open symbols). Transformation was performed at intervals as described in Materials and Methods and transformation frequencies were shown (solid symbols). Representative data from three independent experiments were shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3306417&req=5

pone-0033514-g001: Effect of inoculum size on plasmid transformation.Overnight grown (13 hr) E. coli MC4100 was inoculated into three triangle flasks each containing 100 ml of fresh LB at ratios of 1∶100 (triangle), 1∶1, 000 (circle) and 1∶100, 000 (rectangle). During incubation, OD600 of the cultures was measured periodically (open symbols). Transformation was performed at intervals as described in Materials and Methods and transformation frequencies were shown (solid symbols). Representative data from three independent experiments were shown.

Mentions: To know competence development during cell growth, we examined transformation patterns as a function of culture time with different inoculum sizes. To prepare recipient cells for plasmid transformation, overnight grown culture in LB broth was inoculated to 100 ml of 1.5× LB (containing yeast extract 7.5 g/L, tryptone 15 g/L and NaCl 7.5 g/L) with a ratio of 1∶100, 1∶1, 000 and 1∶ 10, 000 followed by incubation at 30°C with a low speed (150 rpm). At intervals, 500 µl of the culture was recovered by centrifugation and 450 µl of the supernatant was discarded. Cell pellets were resuspended in the remaining 50 µl supernatant with the addition of plasmid DNA. Transformation was performed by plating the above mixture onto selective plates containing 5% agar. We observed that few exponentially growing E. coli cells were transformed despite of the inoculum sizes (Figure 1). This is in contrast to artificial transformation which requires exponentially growing recipient cells. Instead, competence began to develop at the transition of exponential phase to stationary phase, reached the maximum at the entry of stationary phase and then moderately decreased (Figure 1). Competence development with three different inoculum sizes consistently showed that the highest transformation frequency (1∼2×10−7) occurred when the optical density at 600 nanometer (OD600) reached ∼1.5, no matter how long the cells were cultured (Figure 1). In the following experiments, we investigated plasmid transformation when cells grew to an OD600 of ∼1.5 unless otherwise indicated.


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)

Effect of inoculum size on plasmid transformation.Overnight grown (13 hr) E. coli MC4100 was inoculated into three triangle flasks each containing 100 ml of fresh LB at ratios of 1∶100 (triangle), 1∶1, 000 (circle) and 1∶100, 000 (rectangle). During incubation, OD600 of the cultures was measured periodically (open symbols). Transformation was performed at intervals as described in Materials and Methods and transformation frequencies were shown (solid symbols). 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-g001: Effect of inoculum size on plasmid transformation.Overnight grown (13 hr) E. coli MC4100 was inoculated into three triangle flasks each containing 100 ml of fresh LB at ratios of 1∶100 (triangle), 1∶1, 000 (circle) and 1∶100, 000 (rectangle). During incubation, OD600 of the cultures was measured periodically (open symbols). Transformation was performed at intervals as described in Materials and Methods and transformation frequencies were shown (solid symbols). Representative data from three independent experiments were shown.
Mentions: To know competence development during cell growth, we examined transformation patterns as a function of culture time with different inoculum sizes. To prepare recipient cells for plasmid transformation, overnight grown culture in LB broth was inoculated to 100 ml of 1.5× LB (containing yeast extract 7.5 g/L, tryptone 15 g/L and NaCl 7.5 g/L) with a ratio of 1∶100, 1∶1, 000 and 1∶ 10, 000 followed by incubation at 30°C with a low speed (150 rpm). At intervals, 500 µl of the culture was recovered by centrifugation and 450 µl of the supernatant was discarded. Cell pellets were resuspended in the remaining 50 µl supernatant with the addition of plasmid DNA. Transformation was performed by plating the above mixture onto selective plates containing 5% agar. We observed that few exponentially growing E. coli cells were transformed despite of the inoculum sizes (Figure 1). This is in contrast to artificial transformation which requires exponentially growing recipient cells. Instead, competence began to develop at the transition of exponential phase to stationary phase, reached the maximum at the entry of stationary phase and then moderately decreased (Figure 1). Competence development with three different inoculum sizes consistently showed that the highest transformation frequency (1∼2×10−7) occurred when the optical density at 600 nanometer (OD600) reached ∼1.5, no matter how long the cells were cultured (Figure 1). In the following experiments, we investigated plasmid transformation when cells grew to an OD600 of ∼1.5 unless otherwise indicated.

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