A new enzymic defect of galactose metabolism in $\text{Escherichia coli}$ K-12 mutants☆

A UTP-glucose-1-phosphate uridylyltransferase (SpGalU) and a galactokinase (SpGalK) were cloned from Streptococcus pneumoniae TIGR4 and were successfully used to synthesize UDP-galactose (UDP-Gal), UDP-glucose (UDP-Glc), and their derivatives in an efficient one-pot reaction system. The reaction conditions for the one-pot multi-enzyme synthesis were optimized and nine UDP-Glc/Gal derivatives were synthesized. Using this system, six unnatural UDP-Gal derivatives, including UDP-2-deoxy-Galactose and UDP-GalN₃ which were not accepted by other approach, can be synthesized efficiently in a one pot fashion. More interestingly, this is the first time it has been reported that UDP-Glc can be synthesized in a simpler one-pot three-enzyme synthesis reaction system.

The chapter discusses the biogenesis of the wall in bacterial morphogenesis. The characteristic shapes of bacterial cells can only be maintained by a contribution from all of the polymers in the walls, rather than by any one of them. Among the simplest and most easily defined changes in the shape of microorganisms is one from rods to spherical-shaped bacteria or from spherical protoplasts to rod-shaped bacilli. Several mutations and physiological conditions can bring about this change in different species of bacteria. Wall composition, biosynthetic mechanisms, and the physical properties of walls all refer to large populations of whole bacteria or broken preparations made from them. Changes in surface location of teichoic acids, or even more of the D-alanine substituent on it, within the wall of growing organisms could possibly have profound effects upon the morphology of the wall and upon cell shape. A complication of work with morphological mutants is the plethora of molecular and physiological changes that arise from single mutations. This difficulty can be overcome by finding two or more different sorts of mutations that have some of the biochemical changes in common, but which result in the same morphology.

The cessation of growth in a culture of E. coli W4597(K) which occurs when the nitrogen source (NH₄⁺) is exhausted in the presence of excess glucose is accompanied by a 4.17-fold increase in the rate of glycogen synthesis and the cellular ATP level increases 50% while the cellular FDP level decreases 76%. These data provide the first experimental evaluation concerning certain elements of the hypotheses of previous investigators to account for the increased accumulation of glycogen which occurs when bacterial growth is limited in the presence of an excess of a carbon and energy source.

The excision of prophage φ80 and the resultant changes in the regions of the host chromosome adjacent to the prophage were investigated in bacteria lysogenic for phage mutants with altered integration and excision functions. Lysogens for normal (wild-type) φ80 showed normal excision and host chromosome sealing after uv induction, and the standardized recovery of chromosomal genes increased about 3-fold on one side (Trp) but not on the other (UDPG) of the prophage genome. Lysogens for the mutant phages φ80b₂, φ80 int3 and φ80 del showed impaired prophage excision, and no prophage detachment or host chromosome sealing were observed. However, the standardized recovery of host chromosomal genes bordering the prophage on both sides (Trp and UDPG) increased approximately 10-fold over that for the wildtype lysogen. This specific marker increase, which was confirmed by DNA-DNA hybridization techniques, is difficult to understand since it did not result from the production and multiplication of transducing phage carrying the corresponding portions of the host chromosome. The mechanism of this host chromosome amplification, and the relationship between prophage excision and DNA synthesis are discussed.

This chapter discusses uridine diphosphoryl glucose pyrophosphorylase (UTP:α-D-glucose-l-phosphate uridylyltransferase. EC 2.7.7.9). This enzyme catalyzes the formation of nucleoside diphosphate sugars from nucleoside triphosphates and sugar-1-phosphates. The enzyme appears to be ubiquitous in nature, and since its discovery it has been detected and purified from a variety of sources. Several different assay procedures have been developed to detect and quantify uridine diphosphate (UDP) glucose pyrophosphorylase. In different studies, UDPglucose pyrophosphorylase was successfully crystallized from calf, human, lamb, goat, and rabbit livers. The procedure utilized an alkaline extraction of homogenized liver followed by protamine sulfate treatment. The enzyme was precipitated from the supernate fraction with ammonium sulfate, and after dialysis was treated with calcium phosphate gel and adsorbed on a diethylaminoethanol (DEAE)-cellulose column. After elution, the enzyme was again concentrated with ammonium sulfate, dissolved, and crystallized from ammonium sulfate solution. Various procedures have been used successfully in partially purifying the enzyme from other sources. The enzyme has been used to effect the synthesis of [¹⁴C]UDPglucose, using labeled glucose, glucose 6-P, or glucose–fructose mixtures as starting materials. The enzymic synthesis of UDPglucosamine has also been described that probably utilizes UDPglucose pyrophosphorylase.

The chapter summarizes the progress that has been made in the area of “sugar nucleotide” metabolism. The chapter discusses the enzymic synthesis of glycosyl esters of nucleoside 5'-pyrophosphates. Some sugar nucleotides are produced by “cross reactions” of biosynthetic enzymes that have broad substrate- specificity. Synthesis of oligosaccharides, homopolysaccharides, heteropolysaccharides, and other compounds such as glycolipids, glycoproteins, blood group substances, teichoic acids etc are also discussed. The biosynthesis of most sugar nucleotides is under efficient feed-back control so that, in some cases, little accumulation of these compounds takes place, even when their utilization is blocked. Several new types of enzyme that degrade “sugar nucleotides” are reported.