Dorit Amikam

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.

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.

Molecular and biological features of mollicutes (mycoplasmas)

The small size of the mollicute genome considerably restricts the amount of genetic information available to the organisms. This is reflected in the relatively small number of cell proteins synthesized, the lack of many biosynthetic pathways and the marked dependence on exogenous nutrients for growth. The protein synthesizing machinery of mollicutes resembles that of eubacteria and is sensitive to the same antibiotics, except for rifampicin, to which RNA polymerases of mollicutes appear resistant. The mollicute ribosomes are built of 50 S and 30 S subunits and contain about 50 different proteins and 5 S, 16 S and 23 S rRNA, as in eubacteria. However, the 5 S rRNA in mollicutes appears shorter (107-112 nucleotides) than in eubacteria (116-120 nucleotides). We hybridized restriction endonuclease-digested DNA from a variety of Mycoplasma, Ureaplasma, Acholeplasma and Spiroplasma species with nick-translated probes consisting of defined portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of M. capricolum. The results suggest the presence of only one or two sets (operons) of rRNA genes in the genome of Mollicutes, a number falling considerably below that of the eubacteria examined so far but resembling that found in archaebacteria. Our data also indicate a marked nucleotide sequence homology along the rrnB rRNA operon of E. coli and the rRNA operons of the various mollicutes, indicating that the rRNA genes in mollicutes are linked in the classical prokaryotic fashion 16 S-23 S-5 S. Each mollicute appeared to possess, on its genome, different flanking sequences adjacent to the rRNA operon(s), resulting in species-specific hybridization patterns.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated expression of the CD4 receptor and cell cycle arrest are induced in Jurkat cells by treatment with the novel cyclic dinucleotide 3′,5′-cyclic diguanylic acid

The effect of the novel, naturally occurring nucleotide cyclic diguanylic acid (c‐di‐GMP) on the lymphoblastoid CD4+ Jurkat cell line was studied. When exposed to 50 μM c‐di‐GMP, Jurkat cells exhibited a markedly elevated expression of the CD4 receptor of up to 6.3‐fold over controls. C‐di‐GMP also causes blockage of the cell cycle at the S‐phase, characterized by increased cellular thymidine uptake, reduction in G2/M‐phase cells, increase in S‐phase cells and decreased cell division. Additionally c‐di‐GMP naturally enters these cells and binds irreversibly to the P21ras protein. The effects described appear to be unique for c‐di‐GMP.