Jeanne Cattanéo

GLYCOGEN ACCUMULATION BY WILD-TYPE AND URIDINE DIPHOSPHATE GLUCOSE PYROPHOSPHORYLASE-NEGATIVE STRAINS OF ESCHERICHIA COLI.

Abstract Several uridine diphosphate glucose-pyrophosphorylase (UDPG-PP)-negative mutants of Escherichia coli were found to accumulate intracellularly a polyglucose compound during the stationary phase caused by limiting nitrogen. The mutants accumulated as much or more of the polyglucose compound than their wild-type parents. The polyglucose compound was isolated and shown to be a true glycogen. No significant difference in structure was found between the glycogen synthesized by the mutants and that synthesized by their wild-type parents. These observations strongly suggest that the glycogen synthetase of E. coli utilizes as a substrate some nucleoside diphosphate glucose other than UDPG. Similarly, the results show that the inhibition of phosphorylase by UDPG is not a prerequisite for glycogen accumulation, although inhibition by other NDPG compounds such as TDPG and ADPG may play a role in vivo . One of the strains used in this study was a “leaky” threonine-requiring mutant. In the presence of exogenous threonine this mutant grows with a generation time of 70 minutes and a growth yield (based on glucose utilized) of about 48%. When threonine becomes limiting the growth rate and growth yield decrease markedly, but the specific rate of glucose utilization continues at a rate close to the original rate. During the first generation of this uncoupled growth phase, glycogen rapidly accumulates. This result is consistent with the idea that glycogen accumulation is a result of excess ATP production, over and above that necessary to maintain a given exponential growth rate (or macromolecule turnover rate for nitrogen-limited stationary cultures). The addition of chloramphenicol or p -fluorophenylalanine to nitrogen-limited stationary cultures strongly inhibits glycogen accumulation. The mechanism of this inhibition is unknown. A possible explanation is that some enzyme(s) involved in glycogen synthesis has an extremely high turnover rate, and that the steady-state level of the enzyme(s) decreases rapidly when protein synthesis is inhibited.

Characterization and partial purification of a branching enzyme from Escherichiacoli

Abstract Under conditions of growth limitation by either nitrogen or sulfur, Escherichia coli and Aerobacter aerogenes accumulate, in the presence of excess glucose, large intracellular amounts of a highly branched glycogen closely similar to animal and yeast glycogens ( Segel et al ., 1965 ; Sigal et al ., 1964 ). The (1 → 4) α-D-glycosidic linkages of this bacterial glycogen are formed in the presence of a primer by an ADP-glucose-transglucosylase which has only a very low activity with UDP-glucose as glucosyl donor ( Greenberg and Preiss, 1964 ). This finding is in agreement with the previous observation ( Sigal et al ., 1964 ) that normal or even larger amounts of glycogen are accumulated by UDPG-pyrophosphorylase negative mutants of E . coli . The present paper reports the isolation from E. coli and partial purification of a branching enzyme, or amylo-1,4 → 1,6-transglucosidase (systematic name: α-1,4 glucan: α-1,4 glucan, 6-glycosyl-transferase, EC 2.4.1.18). Found in plants, animal tissues and yeast, this enzyme had been little studied in bacteria, its presence being only reported in crude dialyzed extracts of Arthrobacter globiformis ( Zevenhuizen, 1964 ). The branching enzyme from E . coli is active with both amylose and amylopectin, and has been separated from an amylolytic enzyme producing reducing sugars from the same substrates.

Escherichia coli K-12 glycogen synthase: Ability to use UDPglucose and adpglucose as glucosyl donors in the absence of added primer

Abstract A mechanism of initiation of glycogen biosynthesis in Escherichia coli has been previously postulated: In a first step, the glucosyl groups would be transferred into an acceptor protein from UDPglucose or ADPglucose by two glucosyl transferases, distinct from the glycogen synthase. In this work, the activity of transfer from UDPglucose into a methanol-insoluble fraction could not be found in the crude extracts of six independently isolated glycogen synthase-deficient mutants of E. coli K-12. Purified E. coli K-12 glycogen synthase was able to catalyze the unprimed reaction from ADPglucose and UDPglucose but at a very low rate; the rate with UDPglucose is 6–7% the rate observed with ADPglucose. With these two substrates, the unprimed reaction was strongly stimulated by the simultaneous presence of salts and branching enzyme. However the activity with UDPglucose increased rapidly at low concentrations of branching enzyme and was inhibited at physiological concentrations whereas the activity with ADPglucose reached a maximum only at these concentrations. Consequently, the relative activities found with ADPglucose and UDPglucose varied with the branching enzyme concentration. Transfer from UDPglucose was inhibited by low concentrations of ADPglucose and high concentrations of glycogen. These results suggest that the same enzyme, namely the glycogen synthase, catalyzes the unprimed transfer from ADPglucose and UDPglucose and that ADPglucose is probably the most important physiological donor in glycogen biosynthesis in E. coli .

Unprimed glucan biosynthesis by a particulate ADP-glucose-glucan glucosyl transferase from an Escherichia coli mutant and its stimulation by a protein factor

Summary A particulate fraction of an E. coli mutant catalyses the transfert of glucose from ADP-glucose to glycogen but also to a methanol-insoluble product in the absence of primer. The last reaction requires the presence of albumin and either high concentrations of salts or a protein factor. This factor is present in the 158,000 x g supernatant of DF 2000 mutant and in the extracts of mutants lacking glycogen synthase.

PURIFICATION AND PROPERTIES OF THE β-ASPARTIC DECARBOXYLASE OF DESULFOVIBRIO DESULFURICANS

SUMMARY The β-aspartic decarboxylase of Desulfovibrio desulfuricans (strain El Agheila Z, NCIB 8380) has been purified 330-fold by ammonium sulfate fractionation and chromatography on CM-cellulose. The purified preparations contain per mg of protein, 330 mμg of PLP tightly bound to the enzyme. The enzyme is inactivated by several inorganic anions (SO 4 2− , S 2 0 3 2− , NO 3 -) and is reactivated by catalytic amounts of PLP, PL or α-keto acids. A postulated mechanism of enzyme inactivation, involving hydrolysis of a ketimine-PLP-enzyme complex, is discussed.