Molec. gen. Genet. 1 5 1 , 2 1 5 - 2 1 9 (1977) © by Springer-Verlag 1977

Altered Dihydrofolate Reduetase in fol Regulatory Mutants of Escherichia coli K12 Robert Sheldon* Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, England

Summary. Three spontaneous fol regulatory mutants contain dihydrofolate reductase molecules which differ in physical properties from enzymes of their parent strains. The enzymes were purified over 100-fold by affinity chromatography and were shown to differ in vitro in thermolability and in affinity for trimethoprim, a competitive inhibitor of the enzyme. These results indicate that some fol regulatory mutations occur in the structural gene for dihydrofolate reductase.

Introduction Mutation to trimethoprim-resistance in bacteria is often accompanied by a large increase in the level of the target enzyme, dihydrofolate reductase (Sirotnak and Hachtel, 1969; Sheldon and Brenner, 1976). Relatively little is known about either the mechanism of overproduction or of the normal control of the enzyme. Sheldon and Brenner (1976) demonstrated that in E. coli the mutations which caused overproduction (jol regulatory mutations) are cis-acting. Although such mutations usually occur in nondiffusible regulatory elements such as operators and promoters, some of the fol regulatory mutants were found to be temperature-sensitive for enzyme regulation. Earlier work by Sirotnak and collaborators had indicated that in Diplococcus pneumoniae some of the regulatory mutations occurred within the structural gene for dihydrofolate reductase. They based this conclusion on two findings: 1) Regulatory mutations map on both sides of a mutation known to be in the structural gene (Sirotnak and Hachtel, 1969); and *

Current address." Department of Medical Genetics, University

of Toronto, Toronto, Canada, M5S 1A8

2) Dihydrofolate reductase from some regulatory mutants had slightly altered kinetic properties from those of the wildtype enzyme when assayed in crude extracts (Sirotnak, Hachtel and Williams, 1969). From this it has been concluded that dihydrofolate reductase regulates its own synthesis (see for example Goldberger, 1974). In the light of our finding that in E. coli fol regulatory mutations act cis it seemed important to determine whether any of these mutations also affected the structure of dihydrofolate reductase itself. Accordingly we have screened the enzymes from several spontaneous regulatory mutants and their parents for altered behaviour in vitro. In three cases, enzymes from spontaneous regulatory mutants, purified over 100-fold by affinity chromatography, can be shown to differ from the parental enzymes in their affinity for trimethoprim and/or their thermolability at 51 °. This suggests that some fol regulatory mutations do, indeed, occur in the dihydrofolate reductase structural gene.

Materials and Methods Chemicals

Folic acid, trimethoprim, N A D P H , dithionite and 1-ethyl-3 (3dimethylaminopropyl)-carbodiimide HC1 were purchased from Sigma. Lederle Laboratories generously donated pure methotrexate. Dihydrofolic acid was prepared according to Sheldon and Brenner (1976). CNBr-activated Sepharose 4B was purchased from Pharmacia. Buffers utilized were: Buffer A, 0.05 M Tris-HCl pH 7.2 0.05 M KC1 - 0.0I M 2]?-Mercaptoethanol-0.001 M E D T A ; Buffer B, 0.05 M Tris-HC1 p H 7.2 0.05 M K C I ~ . 0 1 M 2/~-Mercaptoethanol - 0.001 M E D T A - 1 M NaC1; Buffer C, 0.10 M Tris-HCl p H 8.0 1 M NaC1 - 0.01 M 2/%Mercaptoethanol - 0.001 M EDTA. Media

All media used have been described by Sheldon and Brenner (1976).

R. Sheldon: fol Regulatory Mutations in Structural Gene

216 Table 1. Dihydrofolate reductase specific activity Strain

Origin

Allele

30 °

42 °

per ml, equivalent to 0.15 nmoles enzyme per ml. The sensitivity of enzymes to trimethoprim was determined according to Sheldon and Brenner (1976).

RS0

(a)

fol +

0.04

0.016

RS16 RS89

(a) (a)

fo138 fo138 73

0.04 0.42

-

Results

RS35 RS62

(a) (a)

fo160 fo 160rev 17

1.0 0.20

0.27 0.015

RS134 RS145

(b) (b)

fo138NG12 fo138NG12rev3

0.53 0.20

0.14 0.07

The three pairs of alleles described in this paper and their enzyme specific activities are presented in Table 1. Each consists of a parent strain and a spontaneously-occurring regulatory mutant of that strain. For example, RS89 is a spontaneous regulatory mutant of RS16.

The origins of the mutants are described in the text. (a) Sheldon and Brenner, 1976; (b) this work. Cells were grown overnight in B broth under aeration at the indicated temperatures and dihydrofolate reductase was assayed as described in Materials and Methods

1. fol38 and fol38-73 Bacterial Strains Strains used are described in Table 1. M u t a n t s were isolated as described by Sheldon and Brenner (1976).

Cell Growth and Enzyme Preparation Ceils were grown under aeration at 30 ° in B broth and harvested by centrifugation. They were washed twice with 0.1 M Tris-HC1 pH 7.4 at 4 °, once with buffer A, then resuspended in buffer A and broken at 4 ° in a French pressure cell at 12,000 psi (Poe et al., 1972). Cell debris were removed by centrifugation for 20 rain at 25,000 x g at 4 °, and the supernatant fluid fractionated by a m m o nium sulfate precipitation at 4 °. The 38-82% fraction was dialysed for 18 h against buffer B (Kaufman, 1974). Dihydrofolate reductase was further purified by methotrexateagarose affinity chromatography. The methotrexate-agarose was prepared according to K a u f m a n (1974) and used essentially according to Poe et al. (1972). The enzyme in buffer B was shaken for 30 min at 4 ° with the affinity resin, then filtered under v a c u u m through W h a t m a n No. 1 filter paper which had been previously wetted with buffer B. The cake was resuspended in Buffer B and the procedure repeated until the A28 o of the eluate was less than 0.03. The cake was then suspended in buffer C and repeatedly washed until the eluate had an Azso less than 0.03. To elute the enzyme, the washed resin was shaken for 30 rain at 4 ° with 4 times its volume of Buffer C containing 2 m M dihydrofolate, then filtered on W h a t m a n No. 1 paper. The eluate, which contained about 10 15% of the initial enzyme activity, was loaded on a G-75 Sephadex column (1 x 55 cm) and eluted with buffer A as described by Poe et al. (1972). At least 50% of the initial enzyme activity was recovered from the column, but only the most active fractions were kept. These were stored tightly capped at 4 ° after being equilibrated with N2 gas. Overall yield was about 5%, and 100-200 x purification was usually achieved. The enzyme fractions studied in this paper were 15-20% pure, based upon specific activity (Sheldon and Brenner, 1976).

fol38-73 was selected from fo138 on the basis of increased resistance in vivo to trimethoprim. (fol38 itself is weakly resistant to trimethoprim because it contains a mutant dihydrofolate reductase with a reduced affinity for trimethoprim, compared to the wild-type enzyme.) It arose spontaneously at a frequency of about 1 × 10-9, suggesting that it is a single mutational event. The data in Table 1 record that it specifies about 10-fold more enzyme than either fol + or fo138, and it has been shown to act cis (Sheldon and Brenner, 1976). Preliminary experiments with crude extracts indicated that enzymes from fol +, fol38, and fol3~73 cells were half-maximally inhibited by 4 nM, 80-100 nM and 43 nM trimethoprim, respectively. This suggested that the presumably single event which caused a 10-fold increase in enzyme level also changed the structure of the enzyme. To confirm this, both fol38 and fo138-73 enzymes were purified over 100-fold and their sensitivity to trimethoprim determined. The results in Figure 1 show that the I5o of partially purified fo138 enzyme is 74 nM trimethoprim, while the I5o of partially purified fol 38-73 enzyme is 42 nM. Both figures are in good agreement with those obtained from crude extracts and demonstrate a clear difference in affinity for trimethoprim between enzymes specified by fol38 and

fol38-73. 2. fol60 and fol60 revl7 fol6Orevl7 is a spontaneous mutant of fol60 which

Enzyme Assays Dihydrofolate reductase was assayed by the spectrophotometric method of Poe et al. (1972) as described by Sheldon and Brenner (1976). One unit of enzyme is defined as the a m o u n t of enzyme which causes a decrease of 1 Aa4o/min under standard conditions. This corresponds to a reduction of 86 nmoles of dihydrofolate

has a radically different pattern of enzyme expression from its parent, fol60 is a temperature-sensitive, cisacting regulatory mutant which is resistant to trimethoprim at 30 ° and fails to grow at 42 ° on minimal medium, even in the absence of trimethoprim. Although fol60 grows at 42 ° in the presence of Difco

R. Sheldon:

fol

Regulatory Mutations in Structural Gene

217

>~

100

60 -~ 40

S 100

~°~° •

V

8o -~

)

"

fo138-73 ~°'~"o~o

40

{- 2o C3

-o

¢-

I 20

I 40

I 60

I 80

60

20

_

fo138 NG12 r e v 3

100

nM Trimethoprim

I

I

I

t

I

20

40

60

80

100

nM Trimethoprim

Fig. 1. Trimethoprim inhibition of dihydrofolate reductase from

Fig. 3. Trimethoprim inhibition of dihydrofolate reductase from

fo138 and fol38 73

fo138NG12 and fo138NG12rev3

>100 o 100 0

Altered dihydrofolate reductase in fol regulatroy mutants of Escherichia coli K12.

Molec. gen. Genet. 1 5 1 , 2 1 5 - 2 1 9 (1977) © by Springer-Verlag 1977 Altered Dihydrofolate Reduetase in fol Regulatory Mutants of Escherichia co...
414KB Sizes 0 Downloads 0 Views