Vol. 122, No. 1 Printed in USA.
JOURNAL OF BACTERIOLOGY, Apr. 1975, p. 332-334 Copyright 0 1975 American Society for Microbiology
Analysis of Glutamate Exit in Escherichia coli YEHESKEL S. HALPERN1 AND HERBERT L. ENNIS* Roche Institute of Molecular Biology, Nutley, New Jersey 07110
Received for publication 2 October 1974
Escherichia coli B exhibits carrier-mediated first-order exit of glutamate with a half-time of less than 4 min, similar to that observed in K-12 strains. Glutamate exit in both B and K-12 strains is inhibited by arsenite. Practically all of the radioactivity lost during exit by K-12 cells has been accounted for as glutamate in the cell filtrate. A rapid first-order temperature-dependent reaction of glutamate exit in Escherichia coli K-12 has been described (3). The reaction was followed by measuring the rate of loss of radioactive glutamate by preloaded cells after dilution into glutamate-free medium. However, in these studies, glutamate was not determined directly in the filtrates of the exit mixtures. That the loss of radioactivity actually reflected efflux of glutamate rather than possible metabolic products was strongly supported by the finding that addition of nonradioactive glutamate greatly enhanced the rate of exit. In a later study of glutamate transport in E. coli B, Frank and Hopkins (2), using an arsenite-poisoned system, did not detect glutamate exit from cells exposed to media lacking the amino acid. However, addition of unlabeled glutamate caused a rapid release of radioactivity from the cells (2). The discrepant results of the two laboratories could be due to any of the following possibilities: (i) E. coli B, which was used by Frank and Hopkins (2), may lack the ability to perform carriermediated exit of glutamate characteristic of K-12 strains. (ii) The arsenite used in the study with E. coli B might have blocked exit by poisoning the carrier, although carriermediated glutamate entry was not greatly affected. A recent study on glutamate transport in E. coli K-12 points to the nonidentity of carriers mediating entry and exit (4). (iii) For lack of direct contrary evidence, one cannot completely rule out the possibility that in our experiments with K-12 strains the loss of radioactivity primarily reflected the efflux of metabolic products rather than direct efflux of a glutamate. The present paper demonstrates that, when tested in the absence of arsenite, E. coli B exhibits glutamate exit similar to that observed in K-12 strains. Moreover, glutamate exit in E. coli K-12 is also inhibited by arsenite. Furthermore, practically all of the radioactivity lost by I Permanent address: Hebrew University, Jerusalem, Israel.
K-12 cells in the exit reaction was accounted for as glutamate in the filtrate of the exit medium. Since E. coli K-12 strain CS 101 cannot utilize glutamate as carbon source, its glutamate-utilizing derivative CS7 (6) and wild-type E. coli B were used. Growth on glutamate and glutamate uptake were followed, and the radioactive materials in the intracellular pool and in culture filtrates were quantitatively determined as earlier described (5). The exit reaction was followed according to Halpern (3). The doubling time of strains CS7 and E. coli B on glutamate as carbon source was 2.9 h and 5.1 h, respectively; strain CS 101 could not grow on glutamage as the major carbon source. The kinetics of glutamate uptake by these strains are shown in Fig. 1. We have recently reported that in the presence of 25% sucrose the capacity of strain CS7 for glutamate uptake increases greatly due to inhibition of exit (4). We used this property to demonstrate exit in E. coli B. This experiment is presented in Fig. 2, which compares the effects of 25% sucrose on glutamate uptake by strains CS7 and B. At very short times of uptake sucrose does not significantly affect glutamate uptake by either strain. With increase in duration of incubation, there is a similar enhancement of glutamate uptake in the presence of sucrose in both strains. The greater increase in uptake at longer times of incubation in the presence of sucrose is due to inhibition of the exit component, which becomes important with increase of the intracellular glutamate concentration with time. Strain B exhibits rapid firstorder exit of radioactive glutamate in the presence of unlabeled glutamate in the medium at a similar rate to that found in strain CS7 ( T/2 < 4 min) (Fig. 3). Similar results, not shown here, were obtained in the presence of 0.01 M sodium azide instead of nonradioactive glutamate. In arsenite-free medium in the absence of cold glutamate and sodium azide, the rate of ["4C Jglutamate exit in both B and K-12 strains was approximately one-third of that observed upon
VOL. 122, 1975
addition of cold glutamate or sodium azide to the exit medium. However, when potassium arsenite was used, glutamate exit from strain CS7 into medium lacking unlabeled glutamate and sodium azide was virtually abolished (Fig. 4). Similar results were obtained with the B strain. Arsenite also markedly inhibited [14C]-
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