Hyogo University of Teacher Education Journal Vol. 15, Ser. 3, (1995)

Properties of the phoP-phoQ and phoQ Deletion Mutants ofEschenchia coli: Growth Curve and Protein Variability.

Megumi KASAHARA, Kazuyo MATSUDA, and Kozo INABA

(Received September 20, 1994)

Abstract

The phoP-phoQ operon of Escherichia coli is a

member of the family of two-component regulatory

systems which modulate expression of sets of genes

responding to environmental changes. To elucidate

the physiological roles of E. coli phoP and phoQ

genes, we constructed the phoP-phoQ and phoQ

deletion mutants and examined the growth curve

and protein spectra. The growth of the phoP-phoQ

deletion mutant was slow as compared with those

of wild type or phoQ mutant. In the protein spectra

obtained by SDS-PAGE of the phoP-phoQ or phoQ

mutant, there were some differences in protein

patterns as compared with that of the wild type

strain. It was implied that the phoP and phoQ

genes also might be associated with the regulation

of the expression of general genes in the growth of

E. coll.

Introduction

The phoP-phoQ operon of Salmonella typhi一

munum belongs to a large family of two-

component regulatory systems which modulate

expression of sets of genes responding to environ-

mental changes; PhoP seems to be a regulator

protein and PhoQ is a sensor protein (Groisman et

al. 1989). This operon exists also in E. coll, which

has been mapped at 25 min on the E. coli linkage

map, and its nucleotide sequence has been reported

previously (Groisman et al. 1992, Kasahara et al.

1992). The features of E. coli phoP-phoQ operon are

very similar to those of Salmonella typhimurium,

which are involved in the regulation of the expres-

sion of phoN gene which codes for a nonspecific

acid phosphatase (Kier et al. 1979, Kasahara et al.

1991). The expression of phoN gene is induced by

carbon, phosphate, nitrogen, and sulfer limitation

47

(Kier et al. 1977) as well as that by low pH

(Miller et al. 1989). The expression of phoP-phoQ

operon of E. coh is also induced by carbon, phos-

phate and nitrogen starvation, and is positively

autoregulated (Kasahara 1994). For the study on

the mechamsmes of signal transduction and physio-

logical roles of the phoP-phoQ regulon, we analyzed

the protein spectra profiles in the growth stages of

the phoP-phoQ and phoQ deletion strains of E. coll.

Materials and Methods

Bacterial strains.

The strain Escherichia coli K-12 CSH26 [F‾ am

A (.pro-lac) met thi] was used as wild type strain

in this study (Miller 1972). The phoP-phoQ and

phoQ deletion mutants, MKPQl and MKQl, which

were constructed from the CSH26 strain respec-

tively, were used in this study (Kasahara 1994).

Media.

LB broth and LB agar were as described by

Miller (1972). For the single colony isolation of

MKPQl and MKQl strains, kanamycin was added

to LB agar at final concentration of 20〟g/ml

(Sambrook et al. 1989).

Sodium dodecyl sulfate - polyacrylamide gel electro-

phoresis (SDS - PAGE).

The sample solution was electrophoresised

following the method of Laemmli (1970). The

spacer gel was 4 % polyacrylamide and the separa-

tion gel, 15 %. The running buffer was 45 mM

solution of Tris-glycine (pH 8.3). The gel was

stained with Coomassie brelliant blue R-250.

Results and Discussion

Growth curves of the CSH26, MKPQl and MKQl

strains.

BiologicalInstitute,HyogoUniversityofTeacherEducation,Yashiro,Kat0-gun,Hyogo673-14,Japan.

48

Table 1. Growth of the CHS26, MKPQl and MKQl strains in LB medium.Each 0.1 OD unit is roughly equivalent to lOs cells/ml.

Time OD ㈱ nm(hours) CSH26 MKPQ 1 MKQ 1

0.05

0.5 0.09

0.18

1.5 037

0.74

2.5 1.09

1.36

3.5 1.49

1.69

4.5 1.80

1.90

5.5 2Xp

2.21

6.5 2.42

2.50

7.5 2.58

2.75

8.5 2.86

3.05

9.5 3.26

10 3.33

0.5 3.45

1 1 3.63

1.5 3.70

12 3.77

13 3.88

14 4.20

1 5 4.22

0.05 0.05

0.07 0.08

0.13 0.15

0.27 0.3 1

0.56 0.65

0.82 1.00

1.03 1.28

.20 1.46

1.36.65

I.5】 1.79

1.57 1.92

1.72 2.02

1.77 2. 16

1.97 2.36

2. I 2.55

2.05 2.56

2.28 2.75

23 2 2.84

2.45 3.02

2.53 3. 14

2.64 333

2.70 3.45

2.73 3.49

2.82 3.62

2.84 3.55

2.97 3.86

3.(冶4.02

3. I 4.23

In order to obtain some clues on the role of

phoP and phoQ genes in the growth of E. coh, we

monitored the cell number from exponentially phase

to stationaly one. The cells were pregrown exponen-

tially in LB medium at 37 ℃ before the start of

the experiment. The pregrown cells were inoculated

to the fresh LB medium at OD�"0.05. These cul-

tures were grown with 250 ml of LB medium in

500-ml flasks with vigorous shaking. Samples were

taken at 30 mm intervals for measurement of cell

density. These results were shown in Table 1 and

Figure 1.

The strain CSH26 was used as a wild type

strain for the phoP and phoQ genes. This strain

entered to the stationally phase at about 3 hours.

The growth curve of MKQl(^ phoQ) strain was

similar to that of CSH26. 0n the other hand, the

growth of MKPQKJ [phoP-phoQ]) was slow in

comparison with those of CSH26 and MKQl

strains, and the cell number of this strain at

stationally phase was decreased than CSH26. These

results show that the expression of phoP gene

affected the cell growth in the rich medium. It is

well known that the phoP gene is a regulatory gene

in the two-component regulatory systems, and it

may be control the expression of many genes and as

a result the lacking of phoP gene may affect the

cell growth.

Protein variability in the CSH26, MKPQl and

MKQl strains.

To elucidate the physiological roles of E. coh

phoP-phoQ regulon, we attempted to identify the

Running title: Properties of the E.coli phoP-phoQ and phoQ Deletion Mutants

UIUO09QO

49

Figure 1. Growth curves of the CSH26(wt), MKPQIU iphoP-phoQj) and MKQKJ phoQ) strains. Thesestrains were cultured at 37℃ in LB medium with vigorous shaking. Samples were taken at 30min intervals and the concentration of cells in a culture was determined with a spectropho-

tometer by measuring the absobance at 600 nm.

proteins and the genes regulated by the phoP-phoQ

system. The pregrown cells of CSH26, MKPQK』

[phoP-phoQ]) and MKQl(/f phoQ ) strains were

inoculate to the fresh LB medium and these cultures

were grown with vigorous shaking. Samples were

taken at 30 mm intervals and were measured cell

density at 600 nm. These samples were centrifuged

at 12,000 rpm for 5 min and cells were harvested.

Cells were diluted by distilled water corresponding

to the cell density and an equal volume of SDS

sample buffer were added. Ten microliters of the

sample solution was electrophoresed at 130 V for

1.5 hours. The protein profiles of three strains on

the cell growth were analyzed by SDS-polyacry-

lamide gel electrophoresis (Figure 2). In three

strains protein patterns were different in the

exponential and stationaly phases. These results

suggest that the gene expression of E. coh varies

between the exponential and stationaly phases. This

mechanism is not yet understood in detail, but

many regulater proteins are probably related.

To examin the genes regulated by the phoP and

phoQ genes in the rich medium, we attempted to

analyze the proteins by SDS-PAGE. The protein

spectrum of the phoP or phoQ mutant was com-

pared with the protein profile of wild type strain

CSH26. The total protein samples prepared from

the exponential and stationaly phases were analyzed

by SDS-PAGE. These results were shown in Figure

3. There were few differences among the proteins in

the CSH26, MKPQl and MKQl strains. Several

quantitative and qualitative changes were, however,

observed in these proteins. That is, cellular protein

spectrum by SDS-PAGE shows that 5 protein

species at least are affected by the phoP-phoQ or

phoQ deletion mutant. The expression of these

proteins may be regulated by the phoP or phoQ

gene. Further works will be necessary to identify

the genes positively or negatively regulated by the

phoP-phoQ operon.

50

B. MKPQl (LphoP -phoQ)

C. MKQl (AphoQ)

MONOO.5 1 1.522.533.54455

Figure 2. Protein spectra in the growth stages of the CSH26(wt), MKPQIGJ [phoP-phoQ]) and MKQIU phoQ) strains by SDS-PAGE. A, CSH26(wt); B, MKPQl(z) [phoP-phoQ]); C, MKQK/fphoQ). In each picture, numbers indicate the time at which cells were harvested. "M"indicates the molecular weight markers whose weights are shown in daltons Oll the left

margin. Symbols O/N indicate the overnight culture.

Running title: Properties of the E.coli phoP-phoQ and phoQ Deletion Mutants 51

Figure 3. Comparison of protein spectra obtained by SDS-PAGE among the CSH26(wt), MKPQKJtphoP-phoQY) and MKQl(A phoQ) strains, wt, CSH26(wt); PQ, MKPQKzl [phoP-phoQl);Q, MKQKJ phoQ). "M" indicates the molecular weight standards. Arrow heads indicate theuniqe bands among three strains.

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