Regulation of the pho regulon in Escherichia coli K-12

J. Mol. Biol. (1983) 168,477-488

Regulation of the pho Regulon in Escherichia coli K-12 Genetic and Physiological Regulation o f the Positive Regulatory Gene phoB HIDE() SHIXAGAWA'I',Kozo MAKINO AND ATSUO NAKATA

Department of Experimental Chemotherapy The Research Institute far Microbial Diseases, Osaka University 3-1, Yamadaoka, Suita, Osaka, Japan 565 (Received 22 February 1983) phoB is a positive regulatory gene for phoA, which codes for alkaline phosphatase, as well as for other genes belonging to the phosphate (pho) regulon whose expression is inducible by phosphate limitation in Escherichia coli. A hybrid plasmid that contains a phoB-lacZ fused gene was constructed in vitro. This plasmid enabled us to study phoB gene expression by measuring the ~galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the phosphate regulon, and phoB gene expression in these strains was studied under limited and excess phosphate conditions. It was found that the regulation of phoB expression was very similar to that of phoA expression. Expression of both genes was induced by phosphate starvation. Both genes were constitutively expressed in phoR, phoS, phoT and phoU mutants and were not expressed in a phoR-phoM double mutant. The implications of these findings for the regulatory mechanism of the pho regulon are discussed.

1. Introduction Alkaline p h o s p h a t a s e , which is encoded by the phoA gene in Escherichia coli, is a periplasmic e n z y m e whose synthesis is induced when the cell is s t a r v e d of p h o s p h a t e . Transcription of phoA is controlled in a complex m a n n e r b y three positive regulator genes (phoB, phoM and phoR), one of which (phoR) acts also as a negative regulator (Garen & Echols, 1962; W a n n e r & Latterell, 1980). Alkaline p h o s p h a t a s e is constitutively synthesized in phoR m u t a n t s a n d is not synthesized in either phoB m u t a n t s or phoM-phoR double m u t a n t s . I t was p r o p o s e d t h a t phoR encodes a bifunctional protein, which acts as a repressor and an a c t i v a t o r , under excess and limited p h o s p h a t e conditions, respectively, a n d its positive r e g u l a t o r y role can be a t least partially s u b s t i t u t e d for b y the phoM p r o d u c t (Wanner & Latterell, 1980). On the other hand, the phoB p r o d u c t is always required for phoA transcription (Brickman & Beekwith, 1975). I n addition, the t Author to whom all correspondence should be addressed. 477 0022-2836/83/230477-12 $03.00/0

9 1983 Academic Press Inc. (London) Ltd

478

H. SHINAGAWA, K. MAKINO AND A. NAKATA

phoU gene (defined by a mutation formerly designated phoT35), recently identified by us (Amemura et al., 1982), functions also as a negative regulator. Since the phoS, phoT and pstA genes are all involved in phosphate transport, and cells with a mutation in any of these genes are constitutive for alkaline phosphatase synthesis (Zuckier & Torriani, 1981), these genes probably regulate phoA gene expression indirectly by influencing the level or metabolism of phosphate in the cell. Evidence has been accumulated which shows t h a t the genes coding for several periplasmic proteins and an outer m e m b r a n e protein are also under the same physiological and genetic regulation as phoA, and they consitute a single pho regulon (Willsky & Malamy, 1976; Argast & Boos, 1980; Tommassen & Lugtenberg, 1980). Among the genes belonging to the pho regulon, regulation of expression of phoA, phoS and phoE was most extensively studied. Recently, the regulatory genes of the pho regulon, phoB and phoR, were cloned on plasmid vectors, and it was reported t h a t phoB and phoR code for proteins with molecular weights of approximately 30,000 and 47,000, respectively (Tommassen et al., 1982; Makino et al., 1982). To assign the role of phoB in the complex regulatory system of the pho regulon, we intended to s t u d y the physiological and genetic regulation of phoB gene expression. F o r this purpose, plasmids having a fused phoB-lacZ were constructed in vitro, which enabled us to quantify the level of phoB gene expression by measuring the activity of flgalactosidase in the cells. The hybrid plasmids were introduced into various phorelated m u t a n t s and the levels of fl-galactosidase in these strains grown under excess and limited phosphate conditions were measured. In this paper, we describe the construction of the fused gene in vitro, and the results of studies of the regulation of phoB gene expression and discuss the implications of our findings in terms of the regulatory mechanism of the pho regulon.

2. Materials and M e t h o d s (a) Bacterial strains and plasmids E. coli K-12 strains and plasmids used in this study are listed in Tables 1 and 2, respectively. r + was obtained from B. L. Wanner. (b) Media and enzyme assay LB agar plates, which were supplemented with tetracycline (20 ~g/ml) or ampicillin (100~g/ml for multi-copy plasmids and 20~g/ml for low-copy plasmids), were used to select colonies carrying plasmids with the corresponding drug resistant marker. Tris-glucose (TG) medium was supplemented either with a high (6"4• 10-4M) or low (6-4• -5 M) concentration of phosphate, referred to as TGHP or TGLP, respectively (Nakata et al., 1971 ). These media were used to grow cells for alkaline phosphatase assay and fl-galactosidase assay. Ampicillin (300 ~g/ml) was added to the media to ensure the presence of the plasmid in the cells. Alkaline phosphatase phenotypes were scored on TGHP or TGLP plates by spraying colonies with a mixture of a-naphthylphosphate and tetrazotized o-dianisidine, and alkaline phosphatase assays of liquid cultures were performed as described (Makino et al., 1982). fl-Galactosidase phenotypes were scored on lactose-MacConkey indicator plates and the enzyme assays of liquid cultures were performed as described by Miller (1972).

REGULATION

OF phoB

EXPRESSION

479

TABLE l

Bacterial s t r a i n s Strain

Characteristicsf

ANC24

F - leu lacY trp his argG strA )Iv metA (or B) thi

ANCBI9 ANCC2 ANCC3 ANCC4

As As As As

ANCC36 ANCC75

As ANC24 except for phoR20 As ANC24 except for phoS64 pure ilv +

ANCCg0

As ANC24 except for phoT9 pure ilv +

ANCG206 ANCUI8 BW521 BW3630 ('36

As ANC24 except for phoB62 As ANC24 except for phoA F - lacZ524 phoR68 phoM451 rpsL thi F - lacZ524 phoA458 phoR68 rpsL lhi Hft(?(PO2A) pho R20 relA I pit- l O spoT l tonA22 T2 r F - ara del(lac-pro) thi F - ara leu lac Y p~rE gal trp his argC malA rpsL xyl metl ily metA (or B) thi HfrC(PO2A) phoB62 relA l pit-lO spoTl tonA22 T2 r

CSH26 CSH57 G206

ANC24 ANC24 ANC24 ANC24

except except except except

for for for for

phoB phoR68 phoR69 phoU pure )Iv +

Source or reference Makino etal. (1982) Makino etal. (1982) Makino el al. (1982) Amemura etal. (1982) Amemura etal. (1982) Hfr cross: C36 x CSH57 Amemura et al. (1982) Amemura etal. (1982) Hfr cross: G206 x CSH57 Makino etal. (1982) B . L . Wanner B . L . Wanner B . J . Bachmann Miller (1972) Miller (1972) B . J . Bachmann

t For the ANC strains, genetic markers of sugar fermentation, except for lac Y, were not tested.

TABLE 2

Plasmids Plasmid

Characteristics

Source/reference

pSN802

pBR322 dervative carrying the phoB + and phoR + genes

Makino et al. (1982)

pSN813

pBR322 derivative carrying the phoR + gene

This work

pM('1403

Vector for cloning of transcriptional and translational signals, Ap'

Casadaban etal. (1980)

pMF3

Mil~iF, Ap f

Manis & K l i n e (1977)

480

H. SHINAGAWA. K. MAKINO AND A. NAKATA

(c) DNA manipulations Methods of plasmid preparation, restriction enzyme digestion, DNA agarose and polyacrylamide gel electrophoresis, DNA ligation, and transformation of ceils with plasmid DNA were as described (Makino et al., 1982). Fractionation and recover)' of DNA fragments were performed by electro-elution. 3. Results

(a) Construction of phoB-lacZ fusion Plasmid pMC1403 was used to identify and clone the DNA fragment that initiates transcription and translation internally and yields transcriptional and translational read-through into lacZ (Casadaban et al., 1980). Plasmid pSN802 is a hybrid plasmid that carries the phoB and phoR genes on the pBR322 vector as shown in Figure l(a), and has been described (Makino et al., 1982). It has been also shown that phoB is borne on the PvuII2-MluI 2 fragment and covers the PvuII a site. The PvuIIs-PvuII 4 fragment was isolated by electro-elution and ligated into the Sinai site on pMC1403. CSH26 (/at-deletion) was transformed with this DNA and plated on lactose-MacConkey agar plates containing ampicillin at 100/~g/ml. About 10~/o of the transformant colonies showed a Lac + phenotype. PIasmids were prepared from the transformants and the restriction patterns were examined. Two kinds of plasmids, in which the PvuIIs-PvuII 4 fragment was inserted into the Sinai site on pMC1403 in two opposite orientations, pSN1852 and pSn1853, were obtained (Fig. 2). Since it has been shown that the PvuII 3 site splits the phoB gene (Makino et al., 1982), pSN1852 carries a phoB-lacZ fusion gene that synthesizes active fused fl-galactosidase, the product of transcriptional and translational read-through from the phoB gene. In

E. co//chromosome PsP4AMz

(a)

9

-

M,

i i 9

I 4

H E ~

*

'

T c ~

P,

I -I

I --2

=

Ps

phoR

I 3

I 2

I I

EP,,P~ P~ M, (b)

H

pBR 3 2 2 '

v

phoB I

-

| l

I

i

I 0

H

H E" ~ T c

~

P,

I

I

-5

-',.=------Ap

I

i

phoR "=

E. co//chromosome

~ ~

I

-4

pBR 3 2 2

Fro. 1. Restriction enzyme cleavage map of pSN802 and pSN813. (a) pSN802 (phoB + phoR +) was described by Makino et al. (1982). Transcriptional direction of the phoB and its neighboring unidentified genes are shown by thick arrows (for details, see the text). (b) pSN813 (phoR +) was constructed from pSN801 (Makino et al., 1982) by deleting the Pv~lIIs-PwlII7 fi'agmcnt. Abbreviations used for the restriction enzymes: A, AvaI; E, EcoRI; H, HincII; M, MluI; P, PvuII; Ps, PstI. The units in the scale are kb.

E

O F phoB E X P R E S S I O N

REGULATION

481

EcoRISmoI BomHI

5'-GAATTCCCGGGGATCCC-3' CTTAAGGG CCCCTAGGG

EcoRI

~k

/lPh~

G

~ Ap

8th codon

~

PRuIT24~"pSN'~O2

~

~,~pBR522'/ ~

Pvug 4

l

lac

........

plVl~,,~uo

'Z

1

So/I

IPvulI

~Smol

R

Soll

I EcoR[,SalI

Soll

I EcoRI~So/Z

EcoRISolI

~IEcoRI,SalI

FI(:. 2. Construction of the plasmids car~Ting the phoB-lacZ fused genes, pSN802 was digested with PvuII and the 1'7 kb PwtIIa-PvuII 4 fragment was isolated by electro-elution. The fragment was ligated with Sinai digested pMCl403 by using phage T4 DNA ligase. Two kinds of Lac + plasmids, pSNl852 and pSNl853 in which the fragment was inserted in opposite orientation, were obtained. pSNl852 carries the phoB-lacZ fused gene, and pSNI853 carries the fusion of an unidentified gene with lacZ (for details, see the text). The EcoRI-SalI fragments from pSNl852 and pSNI853 were ]igated with miniF plasmid pMF3 previously digested with EcoRI and SalI. The resultant plasmids, pSN2852 and pSN2853, conferred Lac + phenotype to the host strain CSH26 (/ac-deletion) and the physical maps of the plasmids were confirmed to be as shown.

482

H. SHINAGAWA, K. MAKINO AND A. NAKATA

pSN1853, lacZ is fused to some unidentified gene, which must be transcribed in a direction opposite to that of phoB. Both of the fused lacZ genes are "in-phase", since when the plasmids were digested with BamHI and then the staggered region was either removed by S1 nuclease or filled with phage T4 DNA polymerase before ligation, which produced a - 1 or + l coding frame-shift, the treated plasmids became Lac-. The fused gene products were identified by SDSt/polyacrylamide gel electrophoresis of the cell iysates. The fused peptide encoded by pSN1852 had a molecular weight of 158 x l0 a, which is larger than native fl-galaetosidase (135x l0 a) by 23x l0 a, equivalent to a 0"6 kb coding sequence of DNA, and the one encoded by pSN1853 was 170 x l0 a, which is larger by 35 x 10 a, equivalent to 1-0 kb DNA (data not shown). These results were also confirmed by the maxicell method (data not shown), which specifically identifies the plasmid coded proteins (Sancar et al., 1981). Thus, the coding regions of the two genes identified here occupies about 1"6 kb of DNA oil the 1"7 kb PvuII 3PvuII a fragment, and it is unlikely that the fragment carries another gene or promoter other than those described above. The fused protein produced by pSN1852 was more abundant in the cells grown in the low phosphate medium (TGLP) than in those grown in the high phosphate medium (TGHP), while the fused protein coded by pSNl853 was produced equally under both conditions (data not shown). Since pSN1852 and pSN1853 are multi-copy plasmids whose replicons were derived from pBR322, the regulation of gene expression may be different from the single gene per chromosome state. In order to minimize this effect, the fused genes from pSN1852 and pSN1853 were transferred to a low-copy miniF plasmid vector, pMF3, as shown in Figure 2. The resultant plasmids were pSN2852 and pSN2853, whose EcoRI-SalI fragments were derived from pSNl852 and pSN1853, respectively. (b) Phosphate starvation induces phoB gene expression CSH26 was used as host strain for cloning the fused genes on the plasmids, since it has a deletion of the lacZ gene. However, the presence of a chromosomal lacZ + gene was found not to interfere with the measurement of fl-galactosidase produced from the fusion genes, since inclusion of 0"2~o {w/v) glucose in the growth media reduced fl-galactosidase synthesis from the authentic chromosomal lac promoter to negligible levels. Therefore, in the subsequent experiments, we employed strain ANC24, which has an intact lacZ and pho +, and its nearly isogenic pho mutant strains. ANC24 strains carrying the plasmids that beax the phoB-lacZ fusion genes were assayed for alkaline phosphatase and fl-galactosidase after the cells were grown for 12hours in the high and low phosphate media (Table 3). As shown, ANC24/pSN1852 produced more fl-galactosidase encoded by the phoB-lacZ fused gene under the low phosphate condition than under high phosphate. Repression by phosphate was more apparent in ANC24/pSN2852, which carries the phoBt Abbreviationsused: SDS, sodiumdodecylsulphate; kb, 10a base-pairs; Apr, ampicillinresistance; Tcr, tetracyclineresistance.

REGULATION

OF

phoB

EXPRESSION

483

TABLE 3

Phosphate regulation of phoB-lacZ expression Plasmid carrying

Alkaline phosphatase~f TGHP TGLP

phoB-lacZ fused gene None pSN 1852 pSN2852 pSN1853 pSN2853

0"01 0"01 0"01 0-01 0"01

~-Galactosidase:~ TGHP TGLP

7-6 2-8 7.8 2.8 7.2

0"5 1500 180 1700 41

0'3 2700 2200 1200 44

ANC24 was the host strain for each plasmid. The cells were grown in TGHP overnight and 0.01 volumes of the cultures were inoculated into TGHP or TGLP. After 12 h incubation, alkaline phosphatase and ~-galactosidase activities were measured as described in Materials and Methods. The data are the averages of 2 experiments. t Units of activity per unit of absorbance at 540 nm. :~ Units of activity per unit of absorbance at 600 nm.

lacZ fusion on the low number copy plasmid than in ~NC24/pSN1852, which carries it on the multi-copy plasmid. Alkaline phosphatase synthesis was repressed similarly in both strains by phosphate but the maximal derepressed levels were reproducibly lower in the cells carrying the multi-copy phoB-lacZ gene. This phenomenon is equivalent to that observed when the cells carrying multi-copy intact phoB gene were induced for alkaline phosphatase synthesis by phosphate limitation (Makino et al., 1982). The multi-copy phoB + gene may interfere somehow with phoA gene expression, probably as a result of the multiple copies of the phoB promoter.

x~X~x----...._x~ B ~

"r-6

~ o

4

/~

,

~00

~=

1000

"~o

J~

_

9 500

i

<

0

4

8

7--7=o.7:-16 20

12

Time (h) F.~. 3. Kh~etics of induction of the phoB-lacZ fusion protein and the alkaline phosphatase syntheses in ANC24/pSN2852 after phosphate starvation. The culture of ANC24/pSN2852 was grown in TGHP overnight and 0'01 volumes of the culture were inoculated into TGHP and TGLP. Alkaline phosphatase and ~-galactosidase activities of the cells were measured at intervals after inoculation as described in Materials and Me~hods. (- - O - - O - -} Alkaline phosphatase activities of the cells grown in TGHP and ( - - O - - O - - ) in TGLP; ( - - x - - x - - ) fl-galactosidase activities of the cells grown in TGHP and ( - - x - - x - - ) in TGLP. |7

484

H. S H I N A G A W A , K. MAKINO AND A. NAKATA TABLE 4

Effect of the p h o mutations on p h o B - l a c Z expression Host strain

Relevant genotype

ANC24 ANCUI8 ANCB 19 ANCG206 ANCC2 ANCC36 ANCC3 ANCC75 ANCC90 ANCC4

Alkaline phosphatase t TGHP TGLP

Wild type

phoA phoB phoB62 phoR68 phoR20 phoR69 phoS64 phoT9 phoU

0'01 0"01 0"01 0"01 0-70 0"18 0"71 1'8 4-7 l" l

7"8 0"01 0"01 0-01 0"51 0-78 7"7 6'7 17 9' I

fl-Galactosidaset TGHP TGLP 180 150 250 220 870 430 720 1000 1700 930

2200 2700 2200 1300 1100 1300 2400 2700 2400 2600

Alkaline phosphatase and fl-galactosidase activities of the pho mutants that harbour pSN2852 (phoB-lacZ) were measured after the cells were grown in TGHP or TGLP as described in the legend to Table 3. t Units as for Table 3.

As a control experiment, ANC24/pSN1853 and ANC24/pSN2853, which carry a n u n i d e n t i f i e d n e i g h b o r i n g g e n e fused to lacZ in a m u l t i - c o p y s t a t e a n d low copy s t a t e , r e s p e c t i v e l y , were a s s a y e d for t h e t w o e n z y m e s ( T a b l e 3). A l t h o u g h a l k a l i n e p h o s p h a t a s e was i n d u c e d b y p h o s p h a t e s t a r v a t i o n , t h e level of fl-galactosidase e n c o d e d b y t h e fused gene was n o t affected b y p h o s p h a t e in t h e s e s t r a i n s . T h e levels of fl-galactosidase in A N C 2 4 / p S N 1 8 5 3 were 30 t o 40-fold h i g h e r t h a n t h o s e in A N C 2 4 / p S N 2 8 5 3 , which m i g h t reflect t h e gene c o p y n u m b e r s in t h e t w o strains.

TABLE 5

Effect of the p h o M and p h o R mutations on p h o B - l a c Z expression

Host strain

Plasmid/ phage

BW3630

pSN5852

Relevant genetic backgroundt

Alkaline phosphatase t TGHP TGLP

fi-Galactosidase:~ TGHP TGLP

Wild type

0.01

1-2

74

1800

phoA phoR

0.01 l'l

0"01 0-95

84 850

1700 1500

phoAphoR phoR phoM phoM

0-01

0-01 0.01 0.85

960 210 79

1600 270 1600

~80phoA + BW3630 pSN5852 BW3630 pSN2852 _

$80phoA +

BW3630 pSN2852 BW52I pSN2852 BW521 p S N 5 8 5 2

0.04 0.01

Alkaline phosphatase and fl-galactosidase activities of the cells grown in TGHP and TGLP were measured as described in the legend to Table 3. t Nearly isogenic strains except for phoM, phoR and/or phoA were constructed by introducing these genes in the forms of transducing phage and hybrid plasmids. :~Units as for Table 3.

REGULATION OF phoB EXPRESSION

485

The kinetics of alkaline phosphatase and fl-galaetosidase syntheses in ANC24/pSN2852 were studied. The cells were grown in the high phosphate medium overnight and 0"01 volumes of the cultures were inoculated into the high and low phosphate media, and then the enzyme activities in the cells were measured at the indicated intervals after inoculation. As shown in Figure 3, both alkaline phosphatase and fl-galactosidase were induced by phosphate starvation and the kinetics of synthesis were very similar. Since fl-galactosidase transcription and translation were initiated from the phoB gene part of the fused gene, the results show that phoB gene expression is also induced by phosphate starvation. (c) Effect of the pho regulatory mutations on phoB expression Since it has been shown that a functional phoB product is required for phoA expression, phoA expression may be regulated positively by the level of the phoB product in the cell. To test this hypothesis, we examined the effects of various pho regulatory mutations on phoB expression. The levels of fl-galactosidase and alkaline phosphatase in the various pho mutants carrying pSN2852 were measured after the cells were cultured for 12 hours in excess phosphate medium or limited phosphate medium. As shown in Table4, alkaline phosphatase constitutive mutants, phoR, phoS, phoT and phoU strains, all synthesized fl-galactosidase constitutively. Among the three phoR strains, ANCC3/pSN2852, which synthesized the maximal derepressed level of alkaline phosphatase in TGLP, also synthesized the maximal level of fl-galactosidase observed in the wild type strain, while the low-level constitutive strain ANCC2/pSN2852 and partially inducible strain ANCC36/pSN2852 synthesized corresponding levels of fl-galactosidase in TGHP and TGLP media. The phoA, phoB and wild type strains synthesized similar levels of fl-galactosidase. Thus, the phoB gene function itself seems not to be related to the regulation of the phoB gene expression. It has been shown that alkaline phosphatase synthesis is much reduced in phoR-phoM double mutants (Wanner & Latterell, 1980). Using the fused phoBlacZ gene, we examined phoB expression in the phoR-phoM double mutant BW521/pSN2852. The levels of fl-galactosidase were very low in the mutant cells grown either in TGHP or TGLP, corresponding to the low levels of alkaline phosphatase (Table 5). In order to measure the level of the phoB gene expression in phoM-phoR + background, a low-copy plasmid carrying the phoR + gene in addition to the phoB-lacZ fusion (pSN5852) was constructed as shown in Figure 4. The induced levels of fl-galactosidase as well as alkaline phosphatase in BW521/pSN5852 were substantially elevated, and were nearly the same as the levels in the control strain, BW3630 (~80phoA+)/pSN5852 (Table 5). These results are consistent with the hypothesis t h a t the phoMand phoR gene products activate the phoB gene, and the phoB gene product, in turn, activates the phoA gene. The phoM and phoR products can, at least partially; replace each other as positive regulators of the phoB gene, since the induced levels of fl-galactosidase in the phoR mutant (BW3630 (r the phoM mutant (BW521/pSN5852) were similar to the level in the pho + strain (BW3630 (q~8OphoA +)/pSN5852).

H. S H I N A G A W A ,

486

K. M A K I N O

E c o R I ~ O C

A N D A. N A K A T A

Z

~ EcorRI

Ap

~.:2r 7"5/0-0 kb

pBR522

W

EcoRI " ~ p N

/ I EcoRI

SolI I EcoRI

EcoR I

EcoRI ['}

~

pSN5852

/

FI(L 4. Construction of pSN5852 (phoB-lacZ, phoR+). Plasmids pSN2852 (phoB-lacZ) and pSNSI3 (phoR+) were digested with EcoRI and subsequently ligated using phage T4 [igase. ANCC2 (phoR-) was transformed by the DNA mixture. Plasmid was isolated from the Ap r, Tc ~, PhoR + transformant and the restriction map of the plasmid (pSN5852) shown was confirmed. A more detailed restriction map of pSN813 is shown in Fig. l(b).

4. Discussion

In this report, we have shown that the patterns of physiological and genetic regulation of phoA and phoB expression are very similar. Both are induced by phosphate starvation. Both are constitutively expressed in phoR, phoS, phoT and phoU mutants, and expressed at a much lower level in the phoR-phoM double mutant. It has been shown that the functional phoB product is required for phoA expression, and proposed that the phoB gene product promotes transcription of phoA by interacting with the regulatory region of the phoA gene (Wanner & Latterell, 1980; Inouye et al., 1982). These results, together with the present

phoB EXPRESSION 487 findings, are consistent with the hypothesis that phoA transcription is positively regulated by the phoB gene product, and phoB gene expression, in turn, is regulated positively by the phoR and phoM gene products and negatively by the phoR, phoS, phoT and phoU gene products, directly or indirectly. At first sight it would seem t h a t the regulation would be the same if phoR, phoM, etc. act directly on phoA. However, the induced level ofphoA expression is about 1000-fold more than the repressed level, while the induced level of phoB expression is only tenfold more than the repressed level, phoB may act as a kind of amplifier allowing a stronger signal to act on the pho regulon. As the phoS and phoT genes are involved in phosphate transport, they may regulate phoB gene expression by influencing the level of the hypothetical corepressor. Since phoU is located next to the phoS-phoT region (Amemura et al., REGULATION OF

1982), whose functions are required for phosphate transport, the gene may also be involved in phosphate metabolism. Its product may be required for the formation of the corepressor. Alternatively, it may code for the repressor or a subunit of the repressor of the phoB gene. It may form an active repressor together with the phoR gene product when the cells are grown under 'the excess phosphate condition. The levels of alkaline phosphatase in the three phoR mutants qualitatively corresponded to the levels of phoB expression in the same strains. The results are consistent with the hypothesis that the phoR product functions as a repressor of phoB under the excess phosphate condition and as an activator under the limited phosphate condition. Since different phoR mutants showed different levels of phoB expression depending on whether the cells were grown in excess of limiting phosphate, the mutation sites may affect different domains of the phoR protein, either that interacting with the regulatory region of phoB or that interacting with the effector. Since there are several positive and negative regulators of phoB, and several physiological inducing conditions for phoA expression, such as phosphate deprivation and thymine starvation (Wilkins, 1972), it is possible that the different regulatory proteins interact with different effectors to regulate phoB expression. The evidence presented here suggests that phoA and presumably phoS, phoE and some unidentified genes under phosphate regulation as well, are regulated positively by the levels of the phoB product, and phoB expression, in turn, is regulated by phosphate and several positive and negative regulators. However, the possibility that the phoB gene product activates phoA expression indirectly via another regulator remains to be examined. The question is raised as to whether phoB expression is controlled by the quantity or quality of these regulatory gene products, which may respond to physiological signal(s). Construction of the fused genes of phoR, phoM and phoU with lacZ is in progress, and this approach will give some clues to the answer. We thank Haruo Omori for advice on the construction of the fused gene. We are grateful to B. L. Wanner and M. J. Casadaban for generously supplying the bacterial strains and pMC1403, respectively. We thank M.R. Moynihan for correcting the English of this manuscript.

488

H. SHINAGAWA, K. MAKINO AND A. NAKATA

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