Repression of Alkaline Phosphatase in Salmonella typhimurium ...

JOURNAL OF BACTERIOLOGY, Nov. 1976, p. 661-664 Copyright X) 1976 American Society for Microbiology

Vol. 128, No. 2 Printed in U.S.A.

Repression of Alkaline Phosphatase in Salmonella typhimurium Carrying a phoA + phoR - Episome from Escherichia coli EZRA YAGIL* AND EFRAT HERMONI Department of Biochemistry, The George S. Wise Center for Life Sciences, Tel-Aviv University, Tel-Aviv, Israel

Received for publication 27 May 1976

Salmonella typhimurium does not produce alkaline phosphatase (nor f3galactosidase). Nevertheless, it has the function of the phoR+ regulatory gene but lacks the function of the lacI+ regulatory gene. Several periplasmic proteins are derepressed when cells of S. typhimurium are starved for inorganic phosphate. The role of phoR is discussed. Starvation of Escherichia coli cells for inorganic phosphate (Pi) results in strong derepression of several periplasmic proteins. These include: alkaline phosphatase (EC 3.1.3.1), which is the product of the phoA gene (min 9 on the E. coli linkage map) (1); a Pi-binding protein, which is the product of the phoS gene (min 82); and one or two additional, as yet unidentified, proteins (14, 21, 23; Fig. 1A and B). Cells mutated in phoS synthesize alkaline phosphatase constitutively, probably because of impaired transport of Pi (11, 20). Mutations in another locus, phoR, which is linked to phoA, also result in the constitutive synthesis of alkaline phosphatase and the other periplasmic proteins (14, 23). The activity of the phoR+ gene products is trans dominant, and it was proposed that it is a regulatory protein controlling the formation of alkaline phosphatase (6, 7, 12, 14, 19). Salmonella typhimurium does nqt make alkaline phosphatase and thus would not be expected to have a regulatory gene that specifically controls the synthesis of this enzyme. The F'13 episome of E. coli carries phoA+, phoR+, and the lac operon (4, 15), and its insertion into cells of S. typhimurium results in a merodiploid hybrid that can make alkaline phosphatase (and the lac gene products) (16). To determine whether S. typhimurium contains the phoR + function, we introduced into Salmonella cells the E. coli F'13 episome carrying a phoR mutation and assayed alkaline phosphatase activity in these merodiploids grown either in a low concentration of Pi (low Pi) or in excess Pi (high Pi). Table 1 shows the low-Pi/high-Pi ratio of enzyme activity in the interspecific F'phoR Salmonella merodiploid (line 9, Table 1), in the

analogous "wild-type" F'phoR + merodiploid

(line 8), and in several other diploid and haploid controls. A comparison of the last two lines of the table clearly shows that S. typhimurium carrying a phoA + phoR - episome is not constitutive; i.e., the Salmonella genome can compensate for the missing phoR- function. Although the derepression value (low Pi/high Pi) in the interspecific merodiploids is lower than in the E. coli strains (compare lines 8 and 9 to lines 2, 4, and 6), this lower value clearly is not affected by the phoR genotype of the episome. The low enzyme activity found in Salmonella (line 1) could be due to other phosphatases (5). The data of line 6 confirm that phoR - is recessive to phoR +. It should also be noted that all of the strains carrying an episome (lines 4 through 9) have a relatively higher derepressed enzyme activity (low Pi) owing to the presence of more than one episome per cell (8). To determine whether S. typhimurium might contain a regulatory gene related to another E. coli system not present in S. typhimurium, we performed an analogous test of the lad gene (a recessive regulatory gene of the lac operon). The structural genes of the operon are induced by isopropyl-,f-n-thiogalactoside, but lac- mutants synthesize them constitutively (2). As is the case for alkaline phosphatase, the structural genes of the lac operon are missing in Salmonella but can be introduced by an F' episome (16). The question asked was whether Salmonella has the lacI+ function. Table 2 shows the activity of f8-galactosidase (specified by lacZ, a structural gene of the lac operon) in induced and uninduced cultures of Salmonella (line 1), wild-type E. coli (line 2), and lachaploid cells of E. coli (line 3) in which the lacmutation is carried on an F' episome. Line 4 in Table 2 shows that when the F'lac- episome 661

662

J. BACTERIOL.

NOTES

N

U

A;

._a o,w ;|r

p..P-.

__~I

In--_

F

[ *_r,, _

_ a. _r~ ax

FIG. 1. Electrophoretic patterns ofperiplasmic proteins. (A) Strain K10 (HfrC derivative of strain K-12); (B) strain W3747 (the F'13 donor); (C) Salmonella LT2; and (D) W3747/Salmonella heterogenotes. +, HighPi medium; -, low-Pi medium. AP, Alkaline phosphatase; P4, Pi-binding protein; P2 and P3, two unknown proteins. Growth conditions, extraction of periplasmic proteins, and acrylamide slab-gel procedure were as described previously (23).

NOTES

VOL. 128, 1976

663

TABLE 1. Alkaline phosphatase activity in haploid and merodiploid strains of E. coli and S. typhimuriuma Characteristics of the tested strain Sp act Low Pi/ Line High Low high Pi Episome Chromosome Ploidy Full genotype Pi

P1

1 2 3 4

W3747 (wild type)

5

W61 (phoR-)

6

W61 (phoR-)

7

W61 (phoR-)

8

W3747 (wild type) W61 (phoR-)

Salmonella ALE25 (wild type) RLA6 (phoR)

Haploid Haploid

Deleted for episomal genes (15) Deleted for episomal genes (3) ALE25 (wild type) RLA6 (phoR-)

0.040 0.021

0.13 1.08

3.2 51.4

1.61

1.40

0.9

Haploid

F-trp F-lac proC thi trp str F- lac proC thi trp str F'+/met AF'13

0.07

6.01

85.8

Haploid

F'phoRlmet

4.30

2.79

0.6

0.10

5.29

52.9

3.76

7.53

1.9

Haploid

AF'13

Merodiploid

Merodiploid

Salmonella

F'phoRllac proC thi trp str F'phoRlphoR lac proC thi str F' +Itrp

Interspecific 0.23 4.22 18.3 merodiploid 9 0.12 2.01 Salmonella 16.7 Interspecific F'phoR/trp merodiploid a Strains W3747 and W61 carry the F'13 episome; their chromosome is deleted (A) for the episomal genes (15). The origin of the E. coli strains used is given in reference 3. S. typhimurium is a trp- derivative of LT2. F-duction was carried out as described previously (22). In all cases, Lac+ Met+ F-ductants were selected (haploid S. typhimurium does not grow on lactose). Merozygotes always gave Lac- segregants on MacConkey plates, and they maintained the trp- mutation. For enzyme activity, cells were grown overnight in a tris(hydroxymethyl)aminomethane (Tris)-buffered minimal medium (18) supplemented as described previously (22). In low-Pi medium, KH2PO4 was supplemented to 0.05 mM. In high-Pi medium, KH2PO4 was supplemented at 1 mM and glucose was reduced to 0.05%. Thus, in both media the cells reached approximately the same density (-5 x 108 cells per ml). The cells were washed twice, suspended in 0.1 M Trischloride buffer (pH 8.0), and treated with 0.2% toluene. Alkaline phosphatase was avssayed with 1 mg of pnitrophenyl phosphate per ml as described previously (22). The reaction was stopped with Na2HPO4 (final concentration, 1 M). One enzyme unit is defined as AA410 = 1.0/min (whereA410 is the absorbance at 410 nm). Specific activity is expressed as enzyme units divided by the culture absorbance at 540 nm. TABLE 2.

f3Galactosidase activity in haploid and merodiploid strains of E. coli and S. typhimuriuma Characteristics of the tested strains

Sp act

Episome

Chromosome

CSH36 (lac-)

Salmonella K10 (wild type) Deleted for episomal genes (13) Salmonella

1 2 3 4

+ IPTG Idcd (in- repressed (repressed) duced)

-IPTG

Line

CSH36 (lac-)

Ploidy

Full genotype

Haploid Haploid

F-trp HfrC

0.04 1.24

0.05 0.10

12.4

Haploid

F'lacI/A(lac pro)

1.04

0.96

1.1

2.36

1.93

1.2

supE thyA (13)

Interspecific merodiploid

F'lacdltrp

a A thyA - mutation was introduced into the chromosome of strain CSH36 by selection on plates containing trimethoprim (17), and the interspecific merozygote was constructed by selection for Lac+ Thy+ sexductants (fooonote a, Table 1). Cells growing exponentially in supplemented, Tris-buffered minimal medium (18) were treated during one generation of growth with 1 mM isopropyl-,j-D-thiogalactoside (IPTG), washed, treated with toluene (footnote a, Table 1), and assayed with o-nitrophenyl-,8-D-galactoside as a substrate as described by Kepes (10). Enzyme units and specific activity were the same as for alkaline phosphatase (footnote a, Table 1).

664

NOTES

has been introduced to Salmonella cells the interspecific hybrids synthesize /3-galactosidase constitutively; i.e., the Salmonella genome cannot compensate for the lacI- mutation and thus, in contrast to its expression of the phoR + phenotype, it does not exhibit the lacI+ function. This finding suggests that the phoR+ activity of Salmonella is specific for this regulatory gene and is not an artifact of the interspecific hybrid. This somewhat unexpected finding suggests that phoR, despite being linked to phoA (in E. coli), does not specify a regulatory function of the phoA gene. The same deduction was made with regard to phoS: over a decade ago it was proposed that phoS specified a repressor of alkaline phosphatase synthesis (6, 7, 19). Later, Schlesinger and Olsen (16) questioned this proposal when they have found that Salmonella exhibited the function of the phoS+ gene. Indeed, it has recently been shown that phoS specifies a periplasmic Pi-binding protein (9, 21, 23). This protein corresponds to the P4 band shown in Fig. 1A and B and appears only in cells starved for Pi. Also, in Salmonella grown under Pi starvation, two to three additional periplasmic proteins can be detected (Fig. 1C), one of which (P4) probably corresponds to the Pi-binding protein. Figure 1D shows the periplasmic proteins of the Salmonella merodiploid carrying the E. coli phoA + phoR + episome (line 8, Table 1). Alkaline phosphatase appears here in addition to the other periplasmic proteins. Our finding that Salmonella cells have a phoR+ function can therefore be interpreted in two ways. (i) phoR+ is not involved directly in the regulation of alkaline phosphatase but, like phoS+, has a role in Pi metabolism or transport. (ii) phoR+ is a regulatory gene and in Salmonella regulates the synthesis of the other periplasmic proteins derepressed by Pi starvation. The excellent technical assistance of Nava Silberstein is

kindly acknowledged. We thank Annamaria Torriani and

J. BACTERIOL.

5.

6. 7.

8. 9.

10.

11. 12.

13. 14.

15. 16.

17. 18.

19.

20.

Moshe Bracha for their comments. This work was supported by the United States-Israel Binational Science Foundation, Jerusalem, Israel. 1. 2.

3. 4.

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