Haim Weinhouse

c-di-GMP-binding protein, a new factor regulating cellulose synthesis in Acetobacter xylinum.

A protein which specifically binds cyclic diguanylic acid (c‐di‐GMP), the reversible allosteric activator of the membrane‐bound cellulose synthase system of Acetobacter xylinum, has been identified in membrane preparations of this organism. c‐di‐GMP binding is of high affinity (K D 20 nM), saturable and reversible. The equilibrium of the reaction is markedly and specifically shifted towards the binding direction by K+. The c‐di‐GMP binding protein, structurally associated with the cellulose synthase, appears to play a major role in modulating the intracellular concentration of free c‐di‐GMP and thus may constitute an essential factor in regulating cellulose synthesis in vivo.

Control of cellulose synthesis Acetobacter xylinum. A unique guanyl oligonucleotide is the immediate activator of the cellulose synthase

Abstract The mechanism of GTP-specific activation of the membrane-bound cellulose synthase system of Acetobacter xylinum has been further elucidated. The activation by GTP was previously attributed to the presence of a soluble protein factor derived from washed membranes. The protein factor has now been shown to be an enzyme that forms from GTP a low-molecular-weight, heat-stable compound which is highly effective in activating the cellulose synthase. The activator-forming enzyme has been isolated by affinity chromatography on an immobilized GTP column. The heat-stable activator has been purified by ion-exchange chromatography and characterized by labeling experiments, t.l.c., and spectral, chemical, and enzymic analyses. The compound could be labeled with [1- 32 P]GTP and [8- 3 H]GTP but not with [3- 32 P]GTP. The compound contains guanine, ribose, and phosphate in a 1:1:1 ratio, is labile to mild alkali and snake venom phosphodiesterase, but is resistant to alkaline phosphatase, mild acid hydrolysis, and the periodate-β-elimination reaction. The results indicate that the activator is an unusual, cyclic guanyl oligonucleotide composed of GMP residues. The cellulose synthase-containing membranes of A. xylinum exhibit a phosphodiesterase-like activity which rapidly degrades the nucleotide activator into 5′-GMP. This activity, however, is strongly inhibited by calcium. It is suggested that intracellular levels of the nucleotide activator, in conjunction with calcium ions, may regulate the rate of cellulose synthesis in vivo .

Cloning of a gene involved in cellulose biosynthesis in Acetobacter xylinum: complementation of cellulose-negative mutants by the UDPG pyrophosphorylase structural gene.

SummaryThree cellulose-negative (Cel-) mutants of Acetobacter xylinum strain ATCC 23768 were complemented by a cloned 2.8 kb DNA fragment from the wild type. Biochemical analysis of the mutants showed that they were deficient in the enzyme uridine 5′-diphosphoglucose (UDPG) pyrophosphorylase. The analysis also showed that the mutants could synthesize β(1-4)-glucan in vitro from UDPG, but not in vivo from glucose. This result was expected, since UDPG is known to be the precursor for cellulose synthesis in A. xylinum. In order to analyze the function of the cloned gene in more detail, its biological activity in Escherichia coli was studied. These experiments showed that the cloned fragment could be used to complement an E. coli mutant deficient in the structural gene for UDPG pyrophosphorylase. It is therefore clear that the cloned fragment must contain this gene from A. xylinum. This is to our knowledge the first example of the cloning of a gene with a known function in cellulose biosynthesis from any organism, and we suggest the gene be designated celA.

Regulation of gluconeogenesis in Acetobacter xylinum by hexoses

Abstract Cellulose formation in Acetobacter xylinum occurs at a similar rate when either glucose, fructose or succinate are added as the source of carbon. When either glucose or fructose are added in addition to succinate, cellulose formation from succinate is decreased by 80%. A similar inhibition by added hexoses is obtained when the conversion of pyruvate to cellulose is followed. Possible mechanisms correlating these observations with the properties of the enzyme systems involved in the interconversion of the various substrates are discussed.