Change in the location of amylomaltase produced by mutation in Escherichia coli

Abstract

The utilization of sugars, for example lactose or maltose, can take place in E8cherichia coli only in the presence of two catalytic systems, namely permease and enzymes catalysing the breakdown of the sugars within the cell (Cohen & Monod, 1957).The present paper reports mutants that are not capable of utilizing maltose, although amylomaltase is present in the cell. The results, however, indicate that it is not crypticity to maltose that causes incapacity for utilization but a change in location that is connected with the binding of amylomaltase in the cell structure.a-Glucosidase in an inactive form bound to the cell structures of Saccharomyce8 cerevi8iae was found by Robertson & Halvorson (1957), and a similar observation was made by Axelrod (1962) in Saccharomycea oviformis. In the latter case it was shown that ribonuclease solubilizes bound aglucosidase from the cell structures.A preliminary account of this work was presented at the Third Congress of the Czechoslovak Biochemical Society, Olomouc, Czechoslovakia, 1-5 July 1963. METHODSBacterial cultures. All experiments were carried out with E. coli strain K12 from our collection numbered 3310 and with a mutant prepared from this strain denoted as 33101M.In one experiment (see Fig. 2) two further strains from our collection were used, HbL and K10-10.If not stated otherwise, cultures of the above strains were used in the early exponential phase ofgrowth, cultivated on broth [beef extract (Difoo), 0-3 %; peptone (Difco), 1% (w/v); NaCl, 1-5% (w/v); pH 7] with aeration. For the wild-type organism maltose (30 mm) was added to the medium. Growth was determined with a Pulfrich photometer at 550 mju. After growth, the bacteria were centrifuged at 00 and washed three times in O lM-phosphate buffer. The O ulM-phosphate buffer used in all experiments was prepared by adding 0l1m-Na2HPO4 to 0O1m-KH2PO4 to give a final pH of 7.For the isolation of the mutant and some other experiments, minimal medium M 63 (Pardee, Jacob & Monod, 1959) and the minimal medium described by Davis (1950), were used. These media are referred to below as minimal media.Preparation of the mutant. The mutants were obtained by essentially the same method as that used by Gorini & Kaufman (1960), with the differences that the concentration of glucose during cultivation before and after irradiation with ultraviolet light was 1% (w/v) and that sucrose was not added to the medium before subjecting the bacteria to the action of penicillin. Plating method. This was carried out to determine whether the examined strains could grow on a minimal medium, where the only carbon source was maltose. The bacteria were plated on the surface of a solid agar-medium gel, which contained minimal medium with maltose or glucose and 1-5 % (w/v) of agar (Difco). In some cases there was a soft agar layer, containing minimal medium with sugar and 0-6 % of agar (Difco) with the suspension of bacteria at 450 layered on solid agar-medium gel.Amylomaltase activity. This was determined by a modification of the method of Keston (1956...