Artic le Microbiology

The stress response factor RpoS is required for the naturaltransformation of Escherichia coli

Yan Zhang • Mengyue Guo • Ping Shen •

Zhixiong Xie

Received: 12 May 2013 / Accepted: 5 June 2013 / Published online: 30 January 2014

� Science China Press and Springer-Verlag Berlin Heidelberg 2014

Abstract The natural transformation of Escherichia coli

is a novel and recently developed system that has signifi-

cance for genetic studies and the biological safety of genetic

engineering. However, the mechanisms of transformation,

including development of competence and DNA uptake, are

not thoroughly understood. In this study, we demonstrated

the effect of the general stress response regulator RpoS,

which has been associated with E. coli transformation, on

natural transformation performed in an ‘‘open system’’. We

find that RpoS is required for natural transformation but not

to artificial transformation and RpoS mainly affect trans-

formation in the liquid culture prior to plating. In the liquid

culture, RpoS over-expression promotes natural transfor-

mation in early exponential phase and static incubation

accumulates RpoS and promotes transformation to a limited

extent. These findings provide detailed understanding of

RpoS function on natural transformation.

Keywords RpoS � Escherichia coli � Natural

transformation � Competence development

RpoS (or rS), encoded by the rpoS gene, is an alternative

sigma factor of RNA polymerase in Escherichia coli. This

factor can partially replace the ‘‘housekeeping’’ sigma factor

r70 (RpoD) in many stress conditions [1–3]. As a general

stress response factor, RpoS regulates the expression of

many stress response genes, including those necessary for

repairing DNA damage and maintaining homeostasis in

E. coli [4–7].

Open system E. coli natural transformation at 37 �C was

reported in 2006 [8], in which neither Ca2? nor heat shock

is required. In many naturally transformable bacteria, a

conserved DNA uptake machinery is used [9, 10]. Natural

transformation of E. coli is basically different from natural

transformation in other bacteria. Although Tsen’s [11]

work in 2002 ever suggested the existence of a recognition

sequence might be in E. coli for gene transfer, there had not

been any DNA uptake gene orthologs mediating DNA

transfer in previous studies [12]. In 2012, the rpoS gene

was shown to be related to transformation in another nat-

ural transformation system at 30 �C without static culture,

but its role was unclear [13].

In this study, we investigated the expression and effects

of RpoS in open system natural transformation of E. coli

and the stage RpoS plays a major role at the expression

level in detail. These findings would help us come to a

better understanding of RpoS function and the mechanism

of the natural transformation of E. coli.

1 Materials and methods

1.1 Strains and plasmids used

The E. coli strains and plasmids used in this study are listed

in Table 1. Primers used for cloning are listed in Table 2.

1.2 E. coli genetic transformation protocols

The natural transformation of E. coli was carried out as

previously described [8]. E. coli strains were grown in

Luria–Bertani (LB) medium [14] at 37 �C with shaking at

Y. Zhang � M. Guo � P. Shen � Z. Xie (&)

Key Laboratory of Analytical Chemistry for Biology and

Medicine (Ministry of Education), State Key Laboratory of

Virology, College of Life Sciences, Wuhan University,

Wuhan 430072, China

e-mail: zxxie@whu.edu.cn

123

Chin. Sci. Bull. (2014) 59(5–6):521–527 csb.scichina.com

DOI 10.1007/s11434-013-0014-7 www.springer.com/scp

200 r/min overnight. Overnight culture (50 lL) was used

to inoculate 5 mL of fresh LB broth in a tube, and the cells

were grown at 37 �C with shaking at 200 r/min. After 14 h

of incubation (stationary growth phase), 1 mL of culture

was transferred to an open system, namely, a beaker (4-cm

in diameter and 6-cm in height) covered by an air-perme-

able membrane. If the static cultivation was missed, cul-

tures were concentrated to 109 CFU mL-1 at 4,000 r/min.

After 10 h of static culture at 37 �C in the open system,

nearly 0.7 mL of the original 1 mL culture remained. Two

microgram of pDsRED plasmid DNA was then added to

each culture aliquot (50 lL), which was plated on 20-mL

LB-agar plates containing ampicillin (200 lg mL-1).

Transformation frequency was calculated by dividing the

number of transformants by viable cell counts.

Classical E. coli artificial transformation with Ca2? was

carried out using the protocol described by Sambrook et al.

[14] and a previously described procedure [15]. E. coli

strains were grown in LB medium at 37 �C with shaking at

200 r/min overnight. Overnight grown culture (50 lL) was

inoculated to 5 mL of fresh LB broth in a tube, and the

cells were grown at 37 �C with shaking at 200 r/min to an

A600 = 0.3–0.4. The cells were harvested by centrifugation

at 12,0009g for 30 s and washed twice with ice-cold

100 mmol L-1 CaCl2. The cell suspension was diluted

with ice-cold 100 mmol L-1 CaCl2 to yield 107–108 cells

in 50 lL (per tube). For performing transformation, 1 lg of

pDsRED DNA was added, and the tubes were mixed gently

and incubated on ice for 30 min. The tubes were placed in

a 42 �C water bath for exactly 90 s and then rapidly

transferred to an ice bath for 1–2 min. An additional

950 lL of LB medium was added to each tube, and the

cells were incubated for 45 min with shaking at 200 r/min

and 37 �C. The number of transformants was determined

by plating aliquots of the transformation mixtures onto LB

plates containing ampicillin (200 lg mL-1). Transforma-

tion frequency was calculated as described above.

Plate transformation with Ca2? was carried out according

to the protocol described by Chen [16]. The overnight cul-

ture from a single E. coli colony was diluted 1:100 in fresh

LB and incubated at 37 �C for 2 h. Bacteria in 1 mL of the

culture were collected by centrifugation at 12,000 r/min for

1 min at 4 �C and re-suspended in 200 lL fresh LB with

10 ng plasmid DNA. A total of 20 lL of the mixture was

spread immediately onto a pre-cold selective plate contain-

ing ampicillin (100 lg mL-1) and 100 mmol L-1 CaCl2.

The plate was incubated at 37 �C for 18–20 h to determine

the transformants.

1.3 Survival of transformants on plates with different

agar concentration

Transformants of E. coli strains were grown at 37 �C with

shaking at 200 r/min to a concentration of 109 CFU mL-1.

Cultures were diluted 106-fold and spread on 1.5 % and

5 % agar plates for viable cell counts.

1.4 Cloning of rpoS into a tac promoter vector

An 871-bp fragment carrying the rpoS structure gene was

recovered from strain ZK126 by the PCR with the primers

P1-r and P2-r listed in Table 2. After digestion with BamH

I and Xba I (Fermentas, CA, USA), the fragment was

ligated into plasmid vector pGZ0 treated with the same

restriction enzymes. Vector pGZ0 carries the p15A repli-

con, lacIq, the tac promoter, and a multiple cloning site.

The resulting plasmid (pGZ1) contained rpoS structure

gene under the control of the tac promoter.

Table 2 Primers used for cloning

Name Sequence Target gene Reference

P1-r ggTCTAGAatggccgaagaggaac rpoS This study

P2-r agGGATCCttactcgcggaacag rpoS This study

Table 1 Strains and plasmid used

Strains and

plasmid

Description Sources or

reference

Strains

ZK126 W3110 DlacU169 tna-2 Laboratory

reserve

ZK1000 ZK126 rpoS: kan [21]

MG1655 F- k- ilvG-rfb-50 rph-1 CCTCC

BL21

(DE3)

F- ompT hsdSB (rB- mB

-) gal dcm (DE3) CCTCC

DH5a fhuA2 lac(del)U169 phoA glnV44U 800

lacZ(del)M15 gyrA96 recA1 relA1

endA1 thi-1 hsdR17

CCTCC

Plasmid

pSXJ6130 p15A replicon, Cmr [12]

pSXJ6133 pSXJ6130 carrying the rpoS gene, Cmr [12]

pRL pSXJ6130 carrying the lacZ gene under

rpoS promoter control, CmrThis study

pGZ0 p15A replicon, lacIq, Ptac,Cmr This study

pGZ1 pGZ0 carrying the rpoS structure gene,

CmrThis study

pET-

26b(?)

Gene over-expression vector, Kanr Laboratory

reserve

pDsRED pUC19 carrying the red fluorescence

gene, Ampr[22]

CCTCC China Center for Type Culture Collection

522 Chin. Sci. Bull. (2014) 59(5–6):521–527

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1.5 SDS-PAGE and immunoblot analysis

SDS-PAGE and immunoblot analysis were carried out

mainly as described previously [17]. For immunoblot

analysis samples taken from different culture phase

were suspended in 200 lL of PBS (pH 7.4) and rup-

tured by ultrasonication. Samples corresponding to

15 lg of total cellular protein were boiled with 59

loading buffer for 10 min, separated on 10 % SDS–

polyacrylamide gels and directly electroblotted onto

nitrocellulose membranes. Blots were blocked 1 h in

TBSTM [50 mmol L-1 Tris-HC1 (pH 7.5), 150 mmol L-1

NaC1, 0.05 % Tween (Fluka, VA, USA), and 5 % non-

fat milk], probed with the polyclonal antiserum against

RpoS overnight, washed with TBST for 30 min, and

incubated with peroxidase-conjugated goat anti-rabbit

IgG (Protein Tech, China). The blots were developed

with a chemiluminescent luminol reagent (Millipore,

MA, USA).

2 Results

2.1 RpoS is required for natural transformation

but not to artificial transformation

To examine RpoS function in open system natural trans-

formation of E. coli, we analyzed the transformation fre-

quencies of E. coli strains ZK126, an RpoS mutant

derivative ZK1000, and RpoS complementation derivatives

ZK126/pSXJ6133 and ZK1000/pSXJ6133 [8]. The trans-

formation of ZK1000 was nearly 10-fold less than that of

the wild type, but transformation was rescued by comple-

mentation with RpoS (Fig. 1a). To determine whether this

regulation was specific to natural transformation, we

investigated the effects of RpoS in another two E. coli

transformation systems: classical artificial transformation

with Ca2? [14] and rapid plate transformation with Ca2?

[16]. The transformation frequencies of ZK126 and

ZK1000 were similar in these two transformation systems

(Fig. 1b).

RpoS has little effect on transformants survival. Given

that RpoS is a stress response factor, its deletion might

change E. coli survival in stress conditions. To assess

whether the reduced ZK1000 transformation frequency

was due to cell survival, viable transformants were

counted on plates with 5 % agar and 100 lg mL-1

ampicillin (stress conditions) or LB plates with 1.5 %

agar and no antibiotic (normal conditions). We found that

RpoS had no effect on the survival of transformants

(Fig. 1c).

Fig. 1 The effect of RpoS on the natural transformation of E. coli.

The relative transformation frequency was calculated as the frequency

of the test sample divided by the wild type control frequency (10-8–

10-7), the data represent the averages ±SD from at least three

independent experiments. Cell suspensions contained 109–1010

CFU mL-1. a Wild type (ZK126), rpoS mutant (ZK1000), and rpoS

multi-copy strains (ZK126/pSXJ6133 and ZK1000/pSXJ6133) natu-

ral transformation with 2 lg plasmid DNA per 50 lL of competent

cells. The relative transformation frequency of ZK126 was used as the

‘‘Test Mean’’ in the student t test **P B 0.01, *0.01 \ P B 0.05.

b Three types of transformation of wild type (ZK126) and mutant

(ZK1000) E. coli. The transformation frequency of ZK126 in each

transformation system was used as the ‘‘Test Mean’’ in the student

t test **P B 0.01, *0.01 \ P B 0.05. c Transformant survival rates

were calculated as the viable cell counts on 5 % agar plates to those

on 1.5 % agar plates. The viable count represents 108–

109 CFU mL-1. The survival rate of ZK126 was used as the ‘‘Test

Mean’’ in the student t test **P B 0.01, *0.01 \ P B 0.05

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2.2 The effect of RpoS expression in liquid culture

on natural transformation

Open system natural transformation of E. coli [8] contains

two stages: natural competence development (including

shaking and static culture) and DNA uptake on plates. To

assess the stage in which RpoS is most important, we

artificially increased RpoS expression by inducing Ptac-

rpoS on plasmid pGZ1 in ZK1000 bacteria. As shown in

Fig. 2, the transformation frequency of induced bacteria

increased by 0.7-fold in shaking cultures, 0.2-fold in static

cultures, 1.4-fold for shaking plus static cultures, or did not

change on plate cultures (decrease less than 1 %). This

suggests that RpoS expression influences natural transfor-

mation of E. coli in the competence development stage.

2.3 RpoS over-expression promotes natural

transformation in early exponential phase

RpoS is very important and, consequently, highly expres-

sed during the stationary phase [18]. However, RpoS

expression is low in growing cells [17, 19, 20]. To deter-

mine time that RpoS affects transformation during culture,

the cultures shaken for different amounts of time were

transformed after 10 h of static cultivation. Although RpoS

protein levels were different in the initial cultures, all final

cultures had similar transformation frequencies (Fig. 3a).

This observation suggested that natural transformation

frequency was not affected by RpoS levels in late expo-

nential phase shaking cultures or stationary phase. Thus,

RpoS might be required during the early exponential phase.

To test this hypothesis, the disturbance of 10 h of static

culture should be eliminated. Cultures shaken for different

amounts of time (1, 2, 3, 4, or 6 h) were concentrated to

109 CFU mL-1 at 4,000 r/min and transformed without

static culture. Because transformants were detected in all

samples (data not shown), the 2-h time point was chosen

for subsequent experiments. The transformation frequency

of ZK1000/pGZ1 with or without 1 mmol L-1 IPTG

induction was measured. As shown in Fig. 3b, the cells in

early exponential phase with high RpoS levels exhibited

higher transformation frequencies. This result indicates that

RpoS content influences the natural competence develop-

ment mainly in early exponential phase.

2.4 Static incubation accumulates RpoS and promotes

transformation a limited extent

In previously published work, static cultivation was con-

sidered unnecessary in 30 �C natural transformation [13].

And in Fig. 3b we also proved that E. coli could naturally

transformed without static culture period when RpoS was

over-expressed. However, we still wondered if RpoS has

any influence on transformation during static cultivation.

The RpoS content, number of viable cells, and transfor-

mation frequency of wild type strain ZK126 were mea-

sured every 2 h during static cultivation. From 0 to 6 h, the

RpoS content of cells was consistently high, while the

transformation frequency gradually increased (Fig. 4a).

After 6 h, the RpoS content decreased, and the transfor-

mation frequency stabilized at a high level. The viable

counts during this process were nearly stable (108–

109 CFU mL-1). However, small decreases (up to fivefold)

were observed in longer culture times. To determine which

factor increased transformation frequency—RpoS expres-

sion or length of the culture period—we measured the same

parameters at the same culture stage using strain ZK1000/

pGZ1, which over-expresses RpoS after 1 mmol L-1 IPTG

induction, before static cultivation. Although RpoS levels

during the whole phase were high, 6 h of static culture was

needed for increased transformation frequency (Fig. 4a).

However, the peak transformation frequency occurred

earlier than in the wild type bacteria.

Fig. 2 The effect of RpoS changes on the natural competence

development of E. coli. The data represent the averages ±SD from at

least three independent experiments. Cell suspensions contained 109–

1010 CFU mL-1. RpoS expression was induced by IPTG different

times during the development of competence in ZK1000/pGZ-1 and

the controls, ZK126/pGZ-0 and ZK1000/pGZ-0. RpoS expression

was not induced (IPTG-) or was induced at the beginning of shaking

culture and again at the beginning of static cultivation again

(IPTG ? shaking ? static), at the beginning of shaking culture only

(IPTG ? shaking), at the beginning of static cultivation only

(IPTG ? static), or just before plating (IPTG ? plate). The relative

transformation frequency is the absolute transformation frequency of

the sample divided by that of the control, ZK126/pGZ-1 without

IPTG induction (10-8–10-7). The relative transformation frequency

of (IPTG-) sample was used as the ‘‘Test Mean’’ in the student t test

**P B 0.01, *0.01 \ P B 0.05

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3 Discussion

3.1 RpoS is required for natural transformation

but not to artificial transformation

In this study, we demonstrated that RpoS increases open system

natural transformation of E. coli frequency (Fig. 1a) but not

artificial transformation frequency (Fig. 1b). The key time points

when RpoS promotes transformation were intensively studied.

The E. coli transformation includes three steps: com-

petence development, DNA uptake, and transformant sur-

vival on the selection. Perturbation in any step will affect

the final transformation frequency. Because, RpoS has little

effects on the transformant survival (Fig. 1c), RpoS might

Fig. 3 The effect of RpoS content during early exponential phase during shaking culture. a ZK126 cultures from different shaking times (1, 2, 3,

4, 6, 8, 11, or 14 h) were transferred to static cultivation for 10 h then transformed with pDsRED plasmid. The relative transformation frequency

is the frequency of the sample divided by that of the 14-h culture, which was (10-8–10-7). The relative transformation frequency of 14 h sample

was used as the ‘‘Test Mean’’ in the student t test **P B 0.01, *0.01 \ P B 0.05. b In the transformation which static culture was omitted, the

transformation frequencies of different RpoS level were measured at early exponential phase. The RpoS expression was induced at 3 h of shaking

culture. The relative transformation frequency is the frequency of the sample divided by that of the IPTG-control, which was 10-7 to 10-6. The

relative transformation frequency of (IPTG-) sample was used as the ‘‘Test Mean’’ in the student t test **P B 0.01, *0.01 \ P B 0.05

Fig. 4 RpoS content, viable cell counts, and transformation frequency during the static cultivation. The relative transformation frequency is the

frequency of the samples divided by that of the 10-h static culture, which was 10-8–10-7. The data represent the averages ±SD from at least

three independent experiments. Cell suspensions contained 109–1010 CFU mL-1. The relative transformation frequency of 10 h sample was used

as the ‘‘Test Mean’’ in the student t test **P B 0.01, *0.01 \ P B 0.05. a ZK126 placed in static culture for different amounts of time (0, 2, 4, 6,

8, 10, or 12 h) was transformed with pDsRED plasmid. Total protein (30 lg) was assayed by Western blot. The relative band intensity is

compared with the sample of 0 h in ZK126. b A 14-h shaking culture of ZK1000/pGZ-1 was induced by 1 mmol L-1 IPTG. Static cultures of

various times (0, 2, 4, 6, 8, 10, or 12 h) were transformed with pDsRED plasmid. Total protein (15 lg) was assayed by Western blot. The relative

band intensity is compared with the sample of 0 h in ZK1000/pGZ1 ? IPTG which is more than 20-fold of that in ZK126

Chin. Sci. Bull. (2014) 59(5–6):521–527 525

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regulate the natural transformation of E. coli via compe-

tence development or DNA uptake.

3.2 The intracellular RpoS during throughout

the transformation process facilitates the natural

transformation of E. coli

RpoS content in the early exponential phase determines the

transformation frequency of the competent cells. To

determine which stage of open system natural transfor-

mation requires RpoS, RpoS expression was artificially

increased at different culture phases via plasmid pGZ1

(Fig. 2). When RpoS expression was induced at the

beginning of shaking cultivation only, the transformation

frequency increased more than in static culture only. This

result indicated that RpoS played a more important role in

shaking culture. Although the effect was not obvious,

increased RpoS also positively regulated static culture, and

the RpoS regulations of these two phases increased the

transformation frequency.

RpoS content in early exponential phase is more

important to natural transformation. RpoS expression is the

highest during the late exponential phase and stationary

phase [17]. To ensure that high levels of RpoS were

present, shaking cultures were usually incubated for 14 h.

However, the results shown in Fig. 3a suggest that

although the original situation was different, the transfor-

mation frequency after 10 h of static culture was the same.

Thus, a shaking culture with high levels of RpoS expres-

sion had less effect on natural transformation than expec-

ted. To verify RpoS effects in the early exponential phase,

static culture was investigated in the following test. First,

cells from different shaking times (1, 2, 3, 4, or 6 h) were

found to develop competence (data not shown, 10-7 to

10-6 transformant per competence). Because all cultures

from early exponential phase developed competence, the

transformation frequencies from 2 h of shaking culture

were measured in E. coli strains with different RpoS

expression levels. High levels of RpoS expression

increased the transformation frequency in early exponential

phase. Thus, RpoS plays a decisive role in transformation

in early exponential phase.

Static incubation accumulates RpoS and promotes

transformation a limited extent. Static cultivation is unique

to open system natural transformation of E. coli, as it is not

performed in artificial transformation. The time of static

culture was reported to influence natural transformation

[8].To detect the effect of RpoS in this phase, we measured

the transformation frequencies of strains with different

RpoS contents at certain culture time points. Regardless of

RpoS expression, a certain amount of static culture was

necessary for maximum transformation frequency. How-

ever, higher levels of RpoS throughout the culture period

could bring the transformation peak forward (Fig. 4). As

shown in Fig. 2, the transformation frequency was

increased by higher levels of RpoS expression in static

culture. Thus, static culture also played a part in RpoS

regulation, although this influence was not as important as

it was in early exponential phase. Furthermore, this result

explained why competence could be developed without

static culture in a previous study [13].

4 Conclusions

RpoS is required for natural transformation but not to

artificial transformation. It mainly affects transformation in

the liquid culture prior to plating. In the liquid culture

period, RpoS over-expression promotes natural transfor-

mation in early exponential phase. Although the effect of

RpoS in static culture to natural transformation is weaker

than that in early exponential phase, static incubation

accumulates RpoS and promotes transformation a limited

extent. These findings provide detailed understanding of

RpoS function on natural transformation.

Acknowledgments This work was supported by the National Basic

Research Program of China (2013CB933904), the National Natural

Science Foundation of China (30971573, 21272182), and the Science

Fund for Creative Research Groups of NSFC (20921062).

References

1. Tanaka K, Takayanagi Y, Fujita N et al (1993) Heterogeneity of

the principal sigma factor in Escherichia coli: the rpoS gene

product, r38, is a second principal sigma factor of RNA poly-

merase in stationary-phase Escherichia coli. Proc Natl Acad Sci

USA 90:8303

2. Hengge-Aronis R (2002) Signal transduction and regulatory

mechanisms involved in control of the rs (rpoS) subunit of RNA

polymerase. Microbiol Mol Biol Rev 66:373–395

3. Maciag A, Peano C, Pietrelli A et al (2011) In vitro transcription

profiling of the sigmas subunit of bacterial RNA polymerase: re-

definition of the sigmas regulon and identification of sigmas-

specific promoter sequence elements. Nucleic Acids Res 39:

5338–5355

4. Battesti A, Majdalani N, Gottesman S (2011) The rpoS-mediated

general stress response in Escherichia coli. Annu Rev Microbiol

65:189–213

5. Stasic AJ, Wong AC, Kaspar CW (2012) Osmotic and desicca-

tion tolerance in Escherichia coli O157: H7 requires rpoS (r38).

Curr Microbiol 65:660–665

6. Claverys JP, Prudhomme M, Martin B (2006) Induction of

competence regulons as a general response to stress in gram-

positive bacteria. Annu Rev Microbiol 60:451–475

7. Robey M, Benito A, Hutson RH et al (2001) Variation in resis-

tance to high hydrostatic pressure and rpoS heterogeneity in

natural isolates of Escherichia coli O157: H7. Appl Environ

Microbiol 67:4901–4907

8. Sun D, Zhang Y, Mei Y et al (2006) Escherichia coli is naturally

transformable in a novel transformation system. FEMS Microbiol

Lett 265:249–255

526 Chin. Sci. Bull. (2014) 59(5–6):521–527

123

9. Claverys JP, Martin B, Polard P (2009) The genetic transforma-

tion machinery: composition, localization, and mechanism.

FEMS Microbiol Rev 33:643–656

10. Chen I, Dubnau D (2004) DNA uptake during bacterial trans-

formation. Nat Rev Microbiol 2:241–249

11. Tsen SD, Fang SS, Chen MJ et al (2002) Natural plasmid

transformation in Escherichia coli. J Biomed Sci 9:246–252

12. Sun D (2009) The study on natural transformation in Escherichia

coli. Doctor Thesis, Wuhan University, Wuhan

13. Zhang Y, Shi C, Yu J et al (2012) RpoS regulates a novel type of

plasmid DNA transfer in Escherichia coli. PLoS ONE 7:e33514

14. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a

laboratory manual, 2nd edn. Cold Spring Harbor Laboratory

Press, Cold Spring Harbor

15. Cohen SN, Chang AC, Hsu L (1972) Nonchromosomal antibiotic

resistance in bacteria: genetic transformation of Escherichia coli

by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114

16. Chen X, Guo P, Xie Z et al (2001) A convenient and rapid

method for genetic transformation of E. coli with plasmids. An-

tonie Van Leeuwenhoek 80:297–300

17. Lange R, Hengge-Aronis R (1994) The cellular concentration of

the rs subunit of RNA polymerase in Escherichia coli is con-

trolled at the levels of transcription, translation, and protein sta-

bility. Genes Dev 8:1600–1612

18. Lange R, Hengge-Aronis R (1991) Identification of a central

regulator of stationary-phase gene expression in Escherichia coli.

Mol Microbiol 5:49–59

19. Mand TD, Dopfer D, Ingham B et al (2013) Growth and survival

parameter estimates and relation to rpos levels in serotype O157:

H7 and non-o157 shiga toxin-producing Escherichia coli. J Appl

Microbiol 114:242–255

20. Loewen PC, Hu B, Strutinsky J et al (1998) Regulation in the

rpoS regulon of Escherichia coli. Can J Microbiol 44:707–717

21. Wang L, Hashimoto Y, Tsao CY et al (2005) Cyclic AMP

(cAMP) and cAMP receptor protein influence both synthesis and

uptake of extracellular autoinducer 2 in Escherichia coli. J Bac-

teriol 187:2066–2076

22. Tolker-Nielsen T, Brinch UC, Ragas PC et al (2000) Develop-

ment and dynamics of Pseudomonas sp. biofilms. J Bacteriol

182:6482–6489

Chin. Sci. Bull. (2014) 59(5–6):521–527 527

123