A thin-layer-chromatographic method for the separation of sugar phosphates.

nutrient-agar plate was replica-plated on to nitrogen-limiting, 1 % lactose minimal medium. Several replicas of each plate were made and were incubated at 30°C for 3 days to guarantee maximal glycogen accumulation. The temperature was then increased to 40°C and an adequate nitrogen source added. Being lactose temperature-sensitive, strain LM 136 is incapable of utilizing lactose at 40°C and the consequent apparent carbon deficiency triggers glycogen mobilization. The replica plates were stained in series with I/KI at suitable time-intervals after transfer to 40°C. I/KI reagent specifically interacts with glycogen to give a characteristic brown stain. Monitoring the I stain provides a visual method for following intracellular glycogenolysis. Mutants with altered rates of iodine-stain disappearance as compared with the parent strain were picked off the master plate and cloned, and their phenotypic identity confirmed. The selection procedure is summarized in the flow diagram shown in Fig. 1 . Strain LM 136, the parent, exhibited the same characteristics as described for strain 5928 by Palmer et ul. (1973), in that intracellular glycogen was rapidly degraded under conditions of carbon deficiency. The mutants isolated showed significantly altered rates of glycogen catabolism, sufficient to indicate that they possess altered enzyme titres and/or regulatory control. In conclusion, the techniques described have permitted us to isolate mutants deficient or otherwise altered in one or more of the four enzymes of the proposed glycogen catabolic pathway. Study of such mutants will provide insight into the pathway of glycogen catabolism in E. coli and its regulation in vivo.