Isolation and Characterization of a Mutant of Escherichia coli Blocked ...

Vol. 101, No. 3 Printted in U.S.A.

JOURNAL OF BACTERIOLOGY, Mar. 1970, p. 725-730 Copyright © 1970 American Society for Microbiology

Isolation and Characterization of a Mutant of Escherichia coli Blocked in the Synthesis of Putrescine I. N. HIRSHFIELD,' H. J. ROSENFELD, Z. LEIFER, AND W. K. MAAS Departmenit of Microbiology, New York Uniiversity Sc/tool of Medlicilne, New York, New York 10016

Received for publication 2 September 1969

A mutant of Escherichia coli is described which is defective in the conversion of arginine to putrescine. The activity of the enzyme agmatine ureohydrolase is greatly reduced, whereas the activity of the other two enzymes of the pathway, the constitutive arginine decarboxylase and the inducible arginine decarboxylase, are within the normal range. The growth behavior of the mutant reflects the enzymatic block. It grows well in the absence of arginine, but only poorly in the presence of arginine. Under the former conditions, putrescine can be formed from ornithine as well as arginine, whereas under the latter conditions, because of feedback control, it can be formed only from arginine.

The polyamines putrescine and spermidine are formed in Escherichia coli by two routes, one from arginine via agmatine and the other from ornithine, a precursor of arginine. The reaction steps involved in these pathways are shown in Fig. 1. There are two ornithine decarboxylases and two arginine decarboxylases for each pair, one being induced at low pH and the other being constitutive. Thus far, only one agmatine ureohydrolase has been found (14). Putrescine and spermidine are present at high concentrations in E. coli (5), but their functions in metabolism have not been elucidated. A mutant blocked in putrescine synthesis might permit such an analysis because one can look for metabolic lesions under conditions of putrescine and spermidine starvation. At first sight, it seems difficult to screen for a putrescine-requiring mutant because of the existence of alternate pathways. However, when bacteria are grown in the presence of arginine, the ornithine pathway is virtually shut off because of repression and feedback inhibition of ornithine synthesis by arginine and also because the conversion of ornithine to arginine is largely irreversible. Thus, if either putrescine or spermidine is an essential metabolite, a mutant blocked between arginine and putrescine should require putrescine for growth in the presence of arginine but not in its absence. In the present paper, we describe the isolation and properties of such a mutant. I Present address: Huntington Laboratories, General Hospital, Boston, Mass. 02114.

MATERIALS AND METHODS

Media and culture methods. The minimal medium used was medium A of Davis and Mingioli (3) with 0.5% glucose as the carbon source, except for growth of cells used for extraction of the constitutive arginine decarboxylase, in which case 1 %, succinate was used as the carbon source. For growtlh of cells used for extraction of agmatine ureohydrolase, the minimal medium was supplemented with trace elements (2). For growth of cells used to determine the inducible arginine decarboxylase, M0ller's medium was used (12). This last reference also describes the conditions used for the "fermentation" test for arginine decarboxylase. Amino acid and polyamine supplements to minimal medium were made at a concentration of 100 ,g/ml; the thiamine supplement was l ,g/ml. Medium AF (arginine-free) is an enriched medium described by Novick and Maas (15). For solid media, Difco agar was added to a final concentration of 2%. Liquid cultures were grown with aeration at 37 C, the inocula being taken from fresh overnight cultures, unless indicated otherwise. Growth was measured by recording the optical density (OD) with a Lumetron colorimeter at 580 nm. Strains. The mutant strains used are derivatives of strain E. coli K-12, and their relevant properties are described in Table 1. Mutagenesis and mating methods. The procedure for treatment with ethyl methane sulfonate (EMS) has been described previously (6). For mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NG) the method of Adelberg et al. (1) was used. The methods used for carrying out crosses with F-prime donors have been described (6). Enzyme measurements. For the determination of the Massachusetts inducible arginine decarboxylase, overnight 5-ml cul725

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HIRSHFIELD ET AL.

J. BACTERIOL.

spermidine agmatine

putrescine

4

inducible

glutamic acid

of

{constitutive

inducible{

arginine

> ornithine

-p

FIG. 1. Schenmatic representation

constitutive

putrescine alId spermidine biosylithesis

arid

its relatiolnship

to

acrgininze biosyntthesis.

tures were harvested by centrifugation, washed once with 5 ml of saline, and resuspended in I ml of 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer containing 10-3 M ethylenediaminetetraacetic acid and 10-3 M dithiothreitol (standard Tris buffer). Before assay, each suspension was treated with two drops of chloroform and one drop of 0.25% sodium dodecyl sulfate. The suspensions were then agitated for a few seconds with a Vortex mixer and incubated with shaking for 10 min at 37 C (11). After that they were kept on ice, and samples were taken for enzyme and protein determinations. Enzyme activity was determined by measuring CO2 released from uniformly labeled '4C-arginine with a Nuclear Chicago Mark 1 scintillation counter, by using a modification of a method described by Tabor and Tabor (17). The reaction was carried out in a scintillation vial, and the released CO2 was absorbed onto a filter-paper disc fitted into the cap of the vial and impregnated with 0.1 ml of NCS (Nuclear Chicago Corp.), a C02-trapping reagent. The reaction mixture contained, in a volume of 0.5 ml, 0.8 ,ug of pyridoxal phosphate, 1.2 ,moles of MgSO4, 1.6 jumoles of L-_4C-arginine (0.025 c/mole), 40 jMmoles of sodium acetate, and 0.1 ml of enzyme preparation. The pH of the reaction mixture was 5.2. After 10 min at 37 C, the reaction was terminated by the addition of five to seven drops of I N H2SO4. After that incubation was continued for an additional 30 min to permit the complete absorption of the released CO2. For counting, the discs were transferred to a second scintillation vial containing 10 ml of scintillation fluid [prepared by adding 42 ml of Liquifluor (New England Nuclear Corp.) to I liter of toluene]. A blank containing water instead of extract was run in each assay and used as correction for nonenzymatic release of CO.

For the determination of the constitutive arginine decarboxylase, 500-ml cultures were grown in 2-liter flasks, harvested by centrifugation, and washed twice with cold saline. The washed bacteria were centrifuged, and the pellet was resuspended in 8 ml of standard Tris buffer. The bacteria were disrupted in the cold by sonic treatment for four periods of 30 sec each with a Branson Sonifier at setting 6. The resulting extract was centrifuged, and the supernatant fluid was used for enzyme measurements.

The assay procedure was the same as that described for the inducible decarboxylase, except that the specific activity of arginine was 10 times higher and the buffer in the reaction mixture was 0.1 M Tris-hydrochloride at pH 7.5. For determination of agmatine ureohydrolase, the bacteria from a I-liter culture, after growth and harvesting by centrifugation, were washed once with normal saline and centrifuged again; the pellet was weighed. An extract was prepared by grinding with an amount of powdered Pyrex glass (Corning, 325 mesh) equal to twice the pellet weight. The resulting paste was suspended in about 6 ml of standard Tris buffer and centrifuged at 10,000 rev/min for 30 min in a Sorvall SS I centrifuge in the cold. The supernatant fluid was used for determinations of enzyme activity. The reaction mixture consisted of 0.3 ml of 0.044 M agmatine sulfate, 0.3 ml of 0.3 M Tris-hydrochloride buffer (pH 7.5), and 0.3 ml of extract containing 8 to 10 mg of protein. Incubation was at 37 C for 10 min. The reaction was stopped by the addition of 0.3 ml of 7%/ perchloric acid. The mixture was centrifuged, and 0.1-ml samples of the supernatant fluid were assayed for urea by the method of Hunninghake and Grisolia (9). Protein determinations. Protein was measured by

TABLE 1. List of strains used" Nutritional requirements

Response to Mating type

Strain

MA 98 MA 131 MA 3020 MA 135 MA 138

Arg

Orn

br br -

br br -

His

Pro

Trp

Cys

Thi

Can

Arg

-

+ + -

+ -

+ _ + + +

-

S R S R R

R F S F R F'Iac+ SF sF-

-

-

-

-

"The following abbreviations are used to describe phenotypic characteristics: Arg, arginine; Orn ornithine; His, histidine; Pro, proline; Trp, tryptophan; Cys, cysteine; Thi, thiamine; Can, canavanine. +, prototrophic; -, auxotrophic; br, bradytrophic. S, sensitive; R, resistant.

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gave positive results, was found to be heavily contaminated with putrescine (20 to 40%). In the of other observations described in this paper, light RESULTS it is most likely that the reversal of the inhibition Isolation and growth characteristics of the condi- observed with agmatine was caused by contamitional putrescine-requiring mutant. This mutant nating putrescine. These findings suggested that was isolated in an experiment designed for anstrain MA 131 may not be able to convert arginine other purpose, namely to look for arginyl-transfer to putrescine. ribonucleic acid synthetase mutants. We have Nature of the biochemical block in strain MA been working on such mutants for some time and 131. To find out which reaction, if any, between have described a procedure for isolating them by arginine and putrescine is affected by the mutaplating cells of an arginine bradytroph onto an tion, the activities of the three enzymes of this enriched but arginine-free medium (AF) contain- pathway were measured in extracts of strains ing a growth-inhibitory concentration of cana- MA 98 (parent) and MA 131 (mutant). Results vanine (8). In the present experiment, the arginine of such an experiment are shown in Table 2. The bradytroph MA 98 was treated with EMS before mutant is clearly deficient in the activity of agmaplating. During the routine testing of the cana- tine ureohydrolase, and the mutation seems to vanine-resistant mutants isolated in this experi- have therefore affected this enzyme. The level of ment, one mutant was found which grew well on the inducible arginine decarboxylase is also lower AF medium containing canavanine and on AF in the mutant than in the wild type. However, the medium alone but only poorly on AF medium level of this enzyme, whose formation is induced containing arginine (AFA). On plates, growth was when cells are grown at an acid pH (7), has been slight after 24 hr, but the colonies reached normal found to vary widely in different experiments size in 48 to 72 hr. This unusual behavior merited (from 0.21 to 1.3 ,umoles per min per mg of profurther testing, and the mutant was therefore tein), and we consider the level shown in Table 2 preserved and assigned the number MA 131. to be within the normal range. Moreover, we Before describing further experiments concern- have studied a mutant which, according to "fering the inhibition by arginine, we would like to mentation" tests (12), was unable to decarboxylpoint out that the canavanine resistance of the ate arginine at an acid pH, and the level of the strain seems to be unrelated to its arginine sensi- inducible arginine decarboxylase in this strain was tivity. In addition, on further testing it was found found to be in the range of 0.01 ,umole per min that, in contrast to the parent strain, strain MA per mg of protein (Becker et al., unpublished data), 131 also requires cysteine for growth. In genetic about 5% that of the lowest value seen in strains experiments to be described below and in a subse- with a normal "fermentation" reaction. quent publication, the three properties have been Isolation of secondary, more stringent pufound to segregate among recombinants. Forma- trescine-requiring mutants. To obtain strains with tion of strain MA 131 from strain MA 98 seems a more stringent inhibition of growth by arginine, therefore to have involved three discrete muta- we introduced into strain MA 131 a mutation tions. resulting in a block in ornithine synthesis. This In further studies on the inhibition by arginine, was done by mating the ornithine-requiring muit was found that citrulline also inhibited growth, tant MA 3020 with strain MA 131 and isolating though not as markedly as arginine, whereas ornithine-requiring, arginine-sensitive recomornithine had no effect. This suggested that the binants, as exemplified by strain MA 135. The degree of inhibition is related to the intracellular rationale behind this experiment was that represconcentration of arginine. This suggestion is based on the observation that in AF medium canavanine resistance and arginine pathway repression, which TABLE 2. Enizyme levels int a putrescine auxotroph are presumed to be functions of the size of the Amt (pmoles) per min per mg of internal arginine pool, are enhanced by arginine Per cent protein Product mutant measured more than by citrulline and not at all by ornithine Enzyme mutant (16). In addition, the lack of inhibition by orniParent Mutant MA 98 MA 131 thine may be due to direct decarboxylation to putrescine via ornithine decarboxylase. A number 4 of compounds structurally or physiologically Agmatine ureohy- Urea 0.025 0.001 drolase related to arginine were tested for their ability to Arginine decarboxyl- CO2 0.0185 0.0171 93 overcome the inhibition by arginine. Positive ase (constitutive) results were obtained with putrescine, spermidine, Arginine 36 decarboxyl- CO2 0.78 0.28 and spermine, whereas lysine and cadaverine did ase (inducible) not affect the inhibition. Agmatine, which also the method of Lowry et al. (10) by using crystalline bovine serum albumin as a standard.

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HIRSHFIELD ET AL.

sion and feedback inhibition in strain MA 131 may not completely shut off ornithine synthesis and that the slow growth found in medium AFA is due to putrescine formation via ornithine. However, this explanation turned out to be incorrect, because inhibition of growth by arginine of strain MA 135 was no greater than that of strain MA 131. In addition, it should be noted that in strain MA 135 the mutation for cysteine requirement is no longer present. The strain is, however, still canavanine resistant. In a further search for mutants more sensitive to arginine, a culture of strain MA 135 was treated with the mutagen NG, grown subsequently overnight in medium AF containing ornithine (AFO), and plated on medium AF containing arginine and putrescine (AFAP). Colonies arising on these plates were tested by a replica technique for sensitivity to arginine by printing master plates onto plates containing medium AFA and medium AFAP. Of 300 colonies tested, 34 showed an enhanced inhibition by arginine compared to strain MA 135. One of these, strain MA 138, was selected for further studies. With this strain, only slight growth was observed on AFA plates after 72 hr of incubation. The levels of activity of the enzymes described in Table 2 were the same for the two arginine decarboxylases as in strain MA 131, but the activity of agmatine ureohydrolase was reduced by at least 50%. The second mutation in strain MA 138 appeared to have affected, as did the original mutation in strain MA 131, a gene responsible for the production of agmatine ureohydrolase. Inhibition of growth by arginine in liquid media. When the effect of arginine on the growth of strains MA 131, MA 135, and MA 138 was first tested in liquid AF medium, inhibition was observed but the effect was less marked than on plates. Thus, for strain MA 138, the growth rate in medium AFA was only 20 to 30% less than that in medium AFAP. In these experiments, the bacteria were pregrown overnight in medium AFO, washed with saline, and inoculated into medium AFA and medium AFAP at a low but measurable turbidity. One possible explanation for the smaller inhibition in liquid medium was thought to be the presumably large intracellular concentrations of putrescine and spermidine present in the bacteria after growth in medium AFO. As shown in the accompanying paper by Morris and Jorstad (13), a 100-fold decrease in the intracellular concentration of putrescine results in only a 10% reduction of the growth rate. Therefore, we tried to exhaust the intracellular pools of putrescine and spermidine before testing for inhibition by arginine in liquid medium. The procedure we finally adopted was to

J. B,.N(- i.10.

starve the cells on plates, spreading 10( to 10' AFO-grown bacteria on each of' a number of plates containing medium AFA and incubating fot 24 hr. The bacteria were harvested into a small volume of AFA medium, and this suspensioni was used as inoculum for growth experiments. As shown in Fig. 2, the inhibition by arginine wals now clear-cut, the doubling time of straLin MA 138 in AFA medium being 200 min as compalred to a (ldl1bling time of 65 min in medium AFAP. The figLure also shows the effect of other polyamincs on the inhibition of growth by arginine. Spermidine is somewhat less effective than putrescine in alccelerating growth, and spermine is even less effective than spermidine. Cadaverine has pr.actically no effect. The lowest concentraLtion of' putrescine required to restore growth maxillm\ff was 0.1 ,g/ml. In these experiments, v zbility of' the bacteria was determined after growth on AL'A plates, and no significant difference was olserved in the ratio of OD to viable count between lhecteria grown overnight in liquid AFAP mediimLn and bacteria grown on AFA plates. However, microscopic observation revealed the presence of many long "snake-like" forms alfter prolongedi growth in AFA medium but not after growth in AFAP medium. DISCUSSION We have described mutants which grow only slightly in the presence of arginine, uLnless poLItrescine, spermidine, or spermine is supplived in the culture medium. They do grow normally in

O

100

200 300 MINUTES

400

FIG. 2. Growth responise of mutanl7t A11 13 to argilnilie antd to various polyamilnes in ll/prhcocu ol arginliine. Tlhe ordinate is optical densitv (logaritf/omic scale). Curve 1, medium1 AFA; curve 2. mncdioumn AFA with cadaverinie; curve 3, inediunm AFA witld .sperm/cin ; curve 4, medium AFA wit/i spermidine; curve 5, inedium AFA with puttrescinle.

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the presence of ornithine. We have shown that the activity of one of the enzymes of the arginine to putrescine pathway, agmatine ureohydrolase, is present in greatly reduced amounts in the mutants. Since putrescine and spermidine are normal cellular constituents, this finding suggests that spermidine and possibly its precursor putrescine are essential for growth. Spermine is not found in E. coli cells, but it is conceivable that there is an enzyme system for converting spermine to spermidine. The specificity of the requirement is underscored by the observation that cadaverine does not accelerate growth in AFA medium. The accelerating effect of agmatine can be ascribed to contaminating putrescine. There is, however, another possible explanation for the conditional polyamine requirement. As shown in the accompanying paper by Morris and Jorstad (13), agmatine accumulates in mutants blocked in the agmatine ureohydrolase reaction. It is conceivable that this accumulated agmatine inhibits a reaction necessary for growth and unrelated to the normal function of polyamines and that this inhibition is overcome by putrescine, spermidine, or spermine. In that case, it is not necessary to assume that these polyamines are metabolites essential for growth. An answer to this problem, however, awaits the availability of conditions under which agmatine does not accumulate, such as would be provided by a mutant unable to convert arginine to agmatine. Starting with a strain defective in the formation of the inducible arginine decarboxylase, we are now trying to isolate a mutant of this kind. The results presented here indicate that whatever the mechanism for the reduced growth in the presence of arginine may be, it does not result in death of the cells but does seem to interfere with cell division. Furthermore, whether putrescine and spermidine are essential for growth, mutants with stringent blocks in putrescine synthesis, such as MA 138, should facilitate investigation of the physiological role of these polyamines. In the course of these studies, we found that the formation of agmatine ureohydrolase shows a curious requirement for trace minerals. In earlier experiments, we were unable to obtain reproducible results in the formation of this enzyme in the parent strain MA 98 and many preparations had very low activities. In these experiments, we did not add trace elements to our minimal medium. When this was done, the inability to form enzyme was overcome. That the presence of trace elements is actually necessary for enzyme synthesis is shown by the findings that adding trace elements just before harvesting the cells or adding trace elements to the extract does not increase the activity of low-level enzyme preparations. In testing

729

individual components of the trace element mixture, it was found that the single addition of Fe3+, MnW+, Caa+, or B033- ions produced the same effect as adding the complete trace element mixture. The underlying mechanism for this unusual phenomenon has thus far remained obscure. In searching for mutants with greater arginine sensitivity, NG mutagenesis of strain MA 135 yielded many, and the ones that were examined were affected in the same enzyme reaction as strain MA 135. We assume that the second mutation occurred in the same gene as the original mutation, and presumably this gene is the structural gene for agmatine ureohydrolase. Our reason for making this assumption is that once an alteration has occurred in a gene resulting in a partially defective enzyme, a second change in the same gene has a better chance of producing an effect than if the same second mutation occurs in the wild-type gene. It is known that a large fraction of mutational alterations in wild-type alleles do not lead to observable phenotypic changes (4), but they may do so in a gene that is already defective. This principle of stepwise isolation of stringently blocked mutants should be generally applicable in the search for mutants with welldefined requirements. ACKNOWLEDGMENTS We thank Helen McKeon for expert technical assistance. This investigation was supported by Public Health Service grant 5 RO GM 06048 from the National Institute of General Medical Sciences. I. H. was a trainee in Genetics, Public Health Service grant 5 TI HE 5307 from the National Heart Institute. H. R. is the recipient of a postdoctoral fellowship from the Public Health Service. Z. L. is a predoctoral trainee in Microbiology, Public Health Service grant GM 01290 from the National Institute o1 General Medical Sciences. W. K. M. is the recipient of Research Career Award K6 GM-15,129.

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