JOURNAL OF BACTERIOLOGY, Feb. 1978, p. 1047-1049

Vol. 133, No. 2

0021-9193/78/0133-1047$02.00/0 Copyright X 1978 American Society for Microbiology

Printed in U.S.A.

Cyclic AMP Regulation of the Hexose Phosphate Transport System in Escherichia colit JOHN W. EZZELL AND WALTER J. DOBROGOSZ* Department ofMicrobiology, North Carolina State University, Raleigh, North Carolina 27607 Received for publication 19 July 1977

Synthesis ofthe hexosephosphate transport system in Escherichia coli required the cyclic AMP-receptor protein regulatory complex. The apparent Km value for hexosephosphate activity was affected by the level of phosphate in the uptake environment. Mutant strains of Escherichia coli K-12 lacking functional adenylate cyclase (cya) or the cyclic AMP (cAMP) receptor protein (crp) are able to grow on most substrates transported into the cells by the phosphoenolpyruvate:phosphotransferase system; they are unable to grow on most substrates transported via non-phosphoenolpyruvate:phosphotransferase system routes (1, 5, 6). We were unable to ascertain which of these categories applied when glucose 6-phosphate (G6P) was used as a growth substrate. Under certain conditions G6P supported growth of cya and crp strains; under other conditions a functional cAMP-receptor protein complex seemed absolutely required for the cells to grow on this substrate. The research reported here was undertaken to determine the extent to which G6P utilization was under cAMP-receptor protein control. It was reasonable to assume that if such control existed it would involve the inducible, catabolite-repressible hexosephosphate transport (HPT) system. This system has been well characterized (2-4, 7-11), and a report exists that there may be more than one type of HPT system (11). Also, a wide range of apparent Km values for G6P transport, ranging from 14 to 500 ,uM, have been reported in the literature (2, 7, 8, 11). The experiments described below shed some light on these matters as well. A preliminary account of some of these findings was reported earlier (J. W. Ezzell and W. J. Dobrogosz, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, K56, p. 156). The E. coli strains used in this study have been described elsewhere (1, 5); they include a wild-type E. coli K-12, a cya mutant derivative, strain C57, and a crp mutant derivative, strain C51. Also used were E. coli strain 1100 and its cya (strain 5336) and crp (strain 5333) mutant derivatives. All cultures were grown with vigort Paper no. 5330 of the Joumal Series of the North Carolina Agricultural Experiment Station, Raleigh, NC 27607.

ous shaking at 370C in minimal medium (5) containing 20 mM glucose or 5 mM G6P, and, when added, 5 mM cAMP. The HPT system was induced as follows: log-phase cells were harvested, washed, and resuspended in fresh medium containing 5 mM G6P with and without 5 mM cAMP. These cells were then incubated for 80 min under growth conditions. At this time they were harvested, washed twice with 10 ml of cold 10 mM HEPES (N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid)-20 mM potasium phosphate buffer (pH 7.2), and then resuspended in prewarmed (370C) HEPES-potassium phosphate buffer at a density of approximately 80 ,ug of dry weight per ml. These cells were used for the HPT assay which was conducted as previously described for a-methylglucoside uptake (5), except that the uptake reaction mixture contained0.40 mM [ U- 14C]G6P (0.87

Co., Arlington Heights, Ill.). [14C]G6P taken up during the initial 30 s was measured as previously described (5), and the results were recorded as nanomoles of G6P taken up per 30 s per milligram of cell dry weight. Corrections were made for nonspecific binding of label to the filters. Apparently all batches of [14C]G6P contain contaminating [14C]glucose. This was true in our case, and we found it to be absolutely essential to remove the contaminating ["4C]glucose. This was done chromatographically using AG x 8 (Cl; 100 to 200 mesh) anion-exchange resin from which the free glucose was eluted with water or a 0 to 0.15 M HCl gradient. The [14C]G6P was eluted with 0.15 M HCI, neutralized, and used shortly thereafter. Failure to thus purify the G6P can lead to some erroneous results. The data summarized in Fig. 1 show that E. coli C57 (cya) required cAMP to grow on G6P when the potassium phosphate concentration of the culture medium was 0.22 M or higher. However, when lower levels of this salt were used, cAMP was no longer essential for growth, although it still greatly stimulated growth. The

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Cyclic AMP regulation of the hexose phosphate transport system in Escherichia coli.

JOURNAL OF BACTERIOLOGY, Feb. 1978, p. 1047-1049 Vol. 133, No. 2 0021-9193/78/0133-1047$02.00/0 Copyright X 1978 American Society for Microbiology...
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