Azide- or cyanide-induced methionine starvation in Escherichia coli

Biochimica et Biophysica Acta, 324 (1973) 218-225

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97807 AZIDE- OR CYANIDE-INDUCED METHIONINE STARVATION IN ESCHERICHIA

COLI

ROSS N. NAZAR* and J. TZE-FEI WONG Department of Biochemistry, Unit~ersity of Toronto, Toronto (Canada)

(Received May l lth, 1973)

SUMMARY In some strains of Escherichia coli methionine could effectively reverse the inhibition of cell growth brought about by moderate concentrations of azide or cyanide. Measurements of the intracellular amino acid pools indicated that in these strains the inhibitors induced a methionine deficiency.

INTRODUCTION In Escherichia coli moderate concentrations of azide (5 m M ) or cyanide (25/aM) induce a 4-5-fold decrease in cell growth accompanied by an extensive simulation of the nutrient-induced shift-down response 1. This response includes a strong preferential inhibition of R N A relative to D N A synthesis, and a transient decrease in protein synthesis. Since azide and cyanide are widely employed as inhibitors of cellular energy metabolism, intracellular nucleotide pools were measured during inhibitor-induced growth shifts in an effort to explain the rapid regulation of R N A synthesis. Surprisingly ,no decreases in nucleotide triphosphates were observed with cyanide and only gradual decreases were observed with azide 2 so that the rapid cessation of growth and R N A synthesis could not be accounted for on the basis of changes in energy-linked phosphorylations. An examination of various nutrient supplements during an azide-induced shiftdown, however, indicated that the inhibition of R N A synthesis in some strains of cells could be effectively reversed by methionine 1. In order to clarify this involvement of methionine, in the present study the uptake of methionine and its stimulatory effects on R N A synthesis and cell growth during azide- and cyanide-inhibited growth have been determined. In addition, a comparison between changes in the intracellular amino acid pools and changes in macromolecular syntheses has been carried out in inhibitor-treated cells.

* Present address: Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77025, U.S.A.

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MATERIALS AND METHODS

Chemicals [2-x4C]Uridine (25 Ci/mole) [2-14C]thymine (28 Ci/mole), L-[14C]leucine (235 Ci/mole), L-[35S]methionine (5 mCi/mole) and H235SO4 (carrier free) were obtained from Nuclear Chicago Corp. Millipore filters (HA, 0.45/am) were obtained from the Millipore Corp., Bedford, Mass. and chloramphenicol was a gift from Parke, Davis and Co. Bacterial strains and #rowth conditions The E. coli strains used, and their growth conditions have been reported earlier 1. Growth and cell mass were measured in terms of absorbance at 450 nm in a Gilford spectrophotometer with a 10-mm light path. 1 ml of bacterial culture with an absorbance of 1.0 corresponded to approximately 0.2 mg dry weight or 4 • l0 s cells. For studies in which amino acids were measured by the incorporation of 35SO4zcells were grown in a modified medium 63 containing 1 mM MgC12 and 50 #M MgSO 4. In all experiments sufficient amounts of inhibitor (5 mM azide or 25 #M cyanide) were added to exponentially growing cells to bring a 4-5-fold reduction in in the growth rate. Incorporation of radioactive precursors into soluble fractions and macromolecules The uptake of methionine into the intracellular acid-soluble fraction was measured essentially as described by Britten and McClure 3. Cells were incubated with L-[35S]methionine (1/aCi/nl); 0.1-ml samples were either directly filtered, dried and counted, or were first precipitated with 5 % trichloroacetic acid before filtration. The radioactivity of the directly filtered cells (corrected for filter-held medium) represented the total uptake of precursor and incorporation; the precipitated samples gave a measure of precursor incorporated into macromolecules. The difference between these was taken as a measure of methionine uptake into the intracellular soluble pool. The syntheses of protein, RNA or DNA were assayed by incorporation of radioactively labeled precursors into acid-insoluble materials. Cultures were supplemented with 20pg/ml L-[14C]leucine (5/aCi/mmole), 4/ag/ml [2-14C]uridine (50 /aCi/mmole+20/ag/ml cytidine) or 1/ag/ml [2-14C]thymine (500 pCi/mmole) and at various times 1.0-ml samples were pipetted directly into cold trichloroacetic acid and counted as previously described 1. The effects of inhibitors on the precursor pools have already been considered earlier ~. Analysis of amino acid pools The intracellular amino acid pools were estimated using a Spinco 120C amino acid analyzer 4 fitted with a high-sensitivity colorimeter cell of 18-mm path length (Evans Electroselenium Ltd.). A 150-cm column (Spherical Research Resin) was used for analysis of acidic and neutral amino acids at 51.5 °C. A 5-cm column was used to separate basic amino acids. 500-ml samples of cell culture (approx. A45 o ,m of 0.7) were collected on 165-ram Millipore filters (HA, 0.45/am), and the filters were quickly immersed in 55 ml of ice-cold 0.1 M HCI in large petri dishes 5. After 15 min of agitation (0-4 °C) the filter was discarded and 50 ml of the extract suspension was lyophilized. For analysis of the acidic and neutral amino acids, the lyo-

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philized material was made up in 2 ml of pH 2.2 analyzer buffer containing 0.25 /~mole/ml of norleucine as an internal standard and centrifuged for 10 min at 12 000 ×9. The supernatant was then applied to the 150-cm column. For analysis of the basic amino acids, only 1 ml of pH 2.2 buffer was used and 0.5 ml was applied to the 5-cm column. Methionine, cystine and S-adenosylmethionine were measured using H235504. The cells were grown for at least three generations in low-sulphate medium containing H235SO4 (100-400 Ci/mole). 1-ml samples of culture were filtered on 25-mm Millipore filters (HA, 0.45/~m) and the filters were quickly immersed in 5.5 ml of ice-cold 0.1 M HCl in small beakers. After 15 min of agitation (0-4 °C), the filter was discarded and 5 ml of the extract was lyophilized. The lyophilized material was made up in 1 ml of pH 2.2 buffer, centrifuged at 12 0 0 0 × # for l0 min, and 0.5 ml of the supernatant was applied to a 10-cm amino acid analyzer column (ChromoBeads Type A, Technicon Chemical Co.). The column was eluted using pH 3.25 analyzer buffer for l h followed by 1 M sodium citrate (70 ml/h) for a second hour. The output of the column was directly connected to a Nuclear Chicago Liquid Scintillation Flow System which was used to measure the ass-labeled amino acids. A typical elution pattern is shown in Fig. 1.

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Fig. 1. The separation of 35S-labeled amino acids by c h r o m a t o g r a p h y on Chromo-Beads Type A. The amino acids were labeled, extracted and separated on a 10-cm column o f Chromo-Beads Type A as described in Materials and Methods. SAM, S-adenosylmethionine. RESULTS

Possible effects of azide or cyanide on methionine biosynthesis were first suggested when a methionine supplement (1 mg/ml) in the growth medium effectively reversed the inhibition of R N A synthesis caused by moderate concentrations of these inhibitors 1. As shown in Figs 2 and 3, low concentrations of methionine greatly stimulated the incorporation of [14C]uridin e into RNA in both azide- and cyanidetreated cells, although the time courses differed. The uptake of methionine into the intracellular pool was very rapid in cyanide-treated cells (Fig. 2B) resulting in an immediate increase in R N A synthesis (Fig. 2A). In contrast the uptake was more gradual in azide-treated cells (Fig. 3B) with a correspondingly more gradual increase in R N A synthesis (Fig. 3A). When limited amounts of methionine (0.1/~g/ml) were added to cyanide-treated cells the rate of synthesis decreased again after about 12 min as the methionine supplement was depleted (Fig. 2). These stimulatory effects

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Fig. 2. Methionine stimulation o f RNA synthesis in E. coli K12 leu- during growth with 25/~M cyanide. (A) The effect of methionine concentration. E. coli K12 leu- were grown in medium 63 with 0.25 % glucose and 20/~g ofleucine per ml and subjected to cyanide shift-down. The rate of RNA synthesis was measured in terms o f [laC]uridine (added to zero time) incorporation as described in Materials and Methods and the results are given in cpm/ml of cell culture (about 2 • l0 s cells). Different concentrations of methionine, as indicated below, were added to replicate cultures at 30-s intervals after treatment with cyanide at time zero. Symbols: RNA synthesis in untreated cells (C)), cells treated with cyanide ( • ) ; or cyanide plus methionine at 100/~g/ml (r-l); 10/~g/ml ( • ) ; 0.5/~g/ml (C)); 0.25/~g/ml (ll); 0.1/tg/ml (A). (B) The uptake of methionine into intracellular pools. Cells were grown in medium 63 with 0.25 % glucose and 20/~g of leucine per ml and subjected to shift-down. [3SS]Methionine (0. I #g/ml)was added 0 min and later and at the different times indicated samples were removed and analyzed for total incorporation; open circles, and incorporation into acid-insoluble materials; closed circles, as described in Materials and Methods. The results are recorded in cpm/ml of cell culture (about 2 • 108 cells) and the solid line which represents a continuous subtraction of the acid-precipitable plot from the total incorporation indicates the uptake of methionine into the intracellular acid-soluble pool.

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Fig. 3. Methionine stimulation of RNA synthesis in E. coli K12 leu- during growth with 5 mM azide. (A) The effect o f methionine concentration. The cell growth and experimental conditions were the same as described in Fig. 2A. Symbols: R N A synthesis in untreated cells (C)); ceils treated with azide ( 0 ) ; or azide plus methionine at 10#g/ml (ll); 5 mg/ml (A); 1 mg/ml ([]); 50 mg/ml (A). (B) The uptake o f methionine into intracellular pools. The experimental conditions and symbols were the same as described in Fig. 2B. [aSS]Methionine (50 #g/ml) was added 15 min after the cells were treated with azide.

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Fig. 4. The effect o f methionine on macromolecular syntheses and cell growth in E. coli 15T- during a shift-down induced with 25 # M cyanide. Cell growth a n d labeling conditions are described in Materials a n d Methods. 15 m i n after the addition o f cyanide 1 m g o f m e t h i o n i n e per ml was added. Symbols: A4so nm ( 0 ) ; D N A ( A ) , protein (C)), a n d R N A (11); a control for R N A synthesis to which m e t h i o n i n e was n o t a d d e d is represented by the dotted line.

were not limited to R N A synthesis but applied also to D N A and protein syntheses as well as well as to cell growth in general (Fig. 4). Because inhibited cell growth was influenzed by methionine, measurements were made of the intracellular amino acid pools after treatment with inhibitors. Tables I and II show the results with 5 mM azide and 25 pM cyanide, respectively. TABLE I C H A N G E S I N T H E I N T R A C E L L U L A R A M I N O A C I D P O O L L E V E L S IN E. COLI K12 leuDURING GROWTH SHIFT-DOWN INDUCED WITH 5 mM AZIDE Cells were g r o w n a n d their a m i n o acid pools were analyzed as described in Materials a n d M e t h o d s . Samples were taken j u s t prior to a n d 2.5, 5, 10, 20 a n d 30 m i n after the addition o f azide. C h l o r a m p h e nicol (CM, 50/~g/ml) was then a d d e d to the r e m a i n i n g cells a n d pool m e a s u r e m e n t s were again m a d e 5 a n d 10 m i n later. T h e zero time values represent the average o f six determinations with an average s t a n d a r d error o f a b o u t 3 ~ . T h e results are recorded as the q u a n t i t y o f a m i n o acid per g dry weight o f cell culture.

Amino acid Quantity (#moles/#) Time (min): Control 2.5 Alanine Arginine Aspartate Cystine Glutamate Histidine Isoleucine Leucine Lysine Phenylalanine Serine Threonine Tryptophan Tyrosine Valine

0.80 0.09 6.94 0.04 108 0.04 0.25 15.7 0.51 0.25 5.32 0.86 0.06 0.28 1.01

2.66 6.78 0.09 45.7 0.22 15.9 0.26 2.71 0.83 0.20 2.80

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2.70 0.12 8.08 0.19 43.2 0.01 0.34 14.1 0.61 0.33 2.57 0.70

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5.14 0.16 7.91 0.11 88.1 0.60 1.15 18.9 7.27 1.18 4.97 ! .99 0.02 1.23 7.10

5.31 0.24 8.22 0.04 105 0.66 0.93 11.1 8.94 1.42 5.47 2.07 0.02 1.39 5.17

7.60 0.26 59.6

0.35 3.48 0.71 0.18 2.75

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TABLE II CHANGES IN THE INTRACELLULAR AMINO ACID POOL LEVELS IN E. COLI Kl2 leuD U R I N G A GROWTH SHIFT-DOWN I N D U C E D WITH 25/zM CYANIDE Cell growth and experimental conditions were the same as described in Table I. Samples were taken just prior to and 5 and 15 min after the addition of cyanide. Chloramphenicol (CM ,50 #g/ml) was then added to the remaining cells and pool measurements were again made 5 and 15 rain later.

Amino acid Time (rain):

Quantity (l~moles/g)

Alanine Arginine Aspartate Cystine Glutamate Histidine lsoleucine Leucine Lysine Phenylalanine Serine Threonine Tryptophan Tyrosine Valine

0.80 0.09 6.94 0.04 108 0.04 0.25 15.7 0.51 0.25 5.32 0.86 0.06 0.28 1.02

Control

5 1.95 0.178 8.16 0.08 93.8 0.06 0.35 12.7 0.38 2.48 5.61 0.98 0.06 2.41 2.47

15 2.26 0.84 12.8 0.06 147 0.02 1.09 16.5 2.40 3.25 1.16 1.76 0.03 3.59 2.86

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A l t h o u g h s o m e rise o r fall in t h e c o n c e n t r a t i o n o f i n d i v i d u a l a m i n o a c i d s w a s recorded, no large decreases were detected. M e t h i o n i n e , h o w e v e r , c o u l d n o t be m e a -

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Fig. 5. Changes in the intracellular pool levels of aSS-labeled amino acids in E. coli K12 leu- during a growth shift-down induced with 5 mM azide. Cells were labeled with H2asSO4 as described in Materials and Methods. Chloramphenicol (CM, 50 #g/ml) was added 30 rain after azide. The control (zero time) values represent the average of three replicate determinations. The results are recorded in/zmoles of amino acid per g dry wt of cells. Methionine , ( ~ ) , cystine (--), S-adenosylmethionine (...). Fig. 6. Changes in the intracellular poolilevels of aSS-labeled amino acids in E. coli K12 leu- during a growth shift-down induced with 25 # M cyanide. Experimental conditions and symbols are described in Fig. 5.

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sured with the amino acid analyzer because diaminopimelic acid, a common cell wall component in E.coli, eluted with methionine. For methionine, cystine and Sadenosylmethionine, the levels were measured in terms of their synthesis from 35SO42-. The results, Figs 5 and 6, showed major drops in the intracellular levels of methionine in inhibitor-treated cells. These sharp drops could be directly correlated with the rapid inhibition in protein and R N A syntheses reported earlier in inhibitor-induced growth shifts 1. The decrease in S-adenosylmethionine, however, was more gradual and did not correlate well with the inhibition in cell syntheses while the cystine pool generally increased. When chloramphenicol was added to the inhibitor treated cells to stimulate R N A synthesis 6, this increase in synthesis correlated with an increase in S-adenosylmethionine while the intracellular methionine level remained relatively constant. Although the stimulatory effects of methionine were observed in strains K I2 leu- and 15 T - , they were not evident in either strain B or K12W6. Furthermore with a methionine supplement in the growth medium (100 pg/ml) a 4-5-fold inhibition in growth could still be induced in the sensitive strains K12 leu- and 15 T - (ref. 6) using much higher concentrations of azide (50 raM) and cyanide (2 mM). DISCUSSION Moderate concentrations of azide and cyanide are able to cause strong inhibitions in bacterial growth without significant changes in the intracellular pools of nucleoside triphosphates 2. The present study indicates that in some strains this is due to a drop in the intracellular methionine pools. The last step in the biosynthesis of methionine in E. coli is the transfer of a methyl group from NS-methyltetrahydropteryltriglutamate to homocysteine. Two transmethylase enzymes are known to catalyze these reactions; one contains a cobamide (vitamin B12) prosthetic group (B12 transmethylase), and utilizes either folate derivative; the other is a non-cobamide enzyme (non-B12 transmethylase) that uses the triglutamate folate derivative ~. Since cyanide and azide (K. G. Scrimgeour, personal communication) are able to form a complex with vitamin B12 (a co-factor in the major pathway for methionine biosynthesis in E. coli), this is a probable explanation for the methionine deprivations observed in this study. Normally cyanideinhibited K12 leu- cells recover spontaneously after about 40 min 6. An induction or switch to the non-B~2 transmethylase may be occurring in this case. The present results easily explain why cell growth in strains K12 leu- and 15 T - is inhibited by azide or cyanide in the absence of significant drops in ATP (ref. 2). They also explain the increases in MSI (ppGpp) and II (ref. 8) which have been reported in cyanide-treated E. coli (refs 6 and 9). A similar observation has also been made in azide-treated K12 leu- (ref. 6). Nevertheless the mechanism for growth inhibition in E. coli K12W6 and B remains unclear and is probably due to yet another unexpected effect in bacterial cells. The gradual uptake of the methionine supplement (Fig. 1B) and the eventual cell death of azide-treated K12 leu- after several hours of reduced growth 6 may be related to this. Alternately the gradual drop in nucleoside triphosphates 2 may bear a relationship to the uptake problem. With respect to the regulation of macromolecular syntheses in growth shiftdown this study indicates that limited amino acid starvation may fully mimic the

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nutrient-induced shift-down. Previously we reported 1 that azide- and cyanide-induced shifts were qualitatively identical in synthetic events to physiological shifts. Since in strain K12 leu- the mechanism in azide- and cyanide-induced shifts appears to be through methionine starvation, it is attractive to postulate that this simple partial starvation of an amino acid can induce a normal shift-down response. This does not, however, suggest that all nutrient-induced shift-downs are basically amino acid starvations since relaxed strains do undergo growth rate shifts in an identical fashion ~° and clearly the relaxed strain K12W6 employs an alternate mechanism. Furthermore methionine utilization, unlike most of the other amino acids, is complex. Aside from being a direct substrate in protein synthesis, it is involved in the initiation of protein synthesis as N-formylmethionine 1° and it is a major methyl donor t~ in most bacterial transmethylation reactions. As S-adenosylmethionine, it is also involved in the biosynthesis of polyamines ~2 which have recently been linked with R N A regulation 13. The inhibition of RNA synthesis may, therefore, be the result of changes in methylation or polyamines rather than amino acid starvation per se. The increase in S-adenosylmethionine after chloramphenicol treatment, for example, was well correlated with the stimulation of RNA synthesis (Figs 5 and 6) (ref. 6). A great deal more must be clarified about these components before a real understanding of the overall response is realized; however, it is clear that the mechanisms by which azide and cyanide inhibit cell growth are complex and that caution must be exercised when these inhibitors are used strictly as "inhibitors of oxidative phosphorylation" in bacteria. ACKNOWLEDGEMENT

The authors are grateful to Dr T. Hofmann for providing the amino acid analyzer. This study was supported by the Medical Research Council of Canada. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12

Nazar, R. N. and Wong, J. T. (1969) J. BacterioL 100, 956-961 Nazar, R. N. and Wong, J. T. (1972) J. BioL Chem. 247, 790-797 Britten, R. J. and McClure, F. T. (1962) BacterioL Rev. 26, 292-335 Spackman, D. H., Stein, W. H. and Moore, S. (1970) AnaL Chem. 30, 1190-1206 Hancock, R. (1958) Biochim. Biophys. Acta 28, 402-412 Nazar, R. N. (1971) P h . D . Thesis, University of Toronto, Toronto, Canada Milner, L., Whitefield, C. and Weissbach, H. (1969) Arch. Biochem. Biophys. 133, 413-419 Cashel, M. and Gallant, J. (1969) Nature 221,838-841 Edlin, G. and Donini, P. (1971) J. BioL Chem. 246, 4371-4373 Adams, J. M. and Capecchi, M. R. (1966) Proc. NatL Acad. Sci. U.S. 55, 147-155 Webster, R.E., Engelhardt, D. L. and Zinder, N. D. (1966) Proc. Natl. Acad. Sci. U.S. 55, 155-161 Tabor, C. W. (1962) in Methods in Enzymology (Colowick, S. P. and Kaplan, N. O., eds), Vol. V, pp. 756-765, Academic Press, New York 13 Boyle, S. M. and Cohen, P. S. (1968) J. Bacteriol. 96, 1266-1272