Abnormally high rate of cyclic AMP excretion from an Escherichia coli mutant deficient in cyclic AMP receptor protein

By labeling adenosine 3′, 5′-cyclic monophosphate (cyclic AMP) with [³²P] phosphate and chromatographing it on a thin-layer alumina plate, we have determined the extra- and intracellular amounts of cyclic AMP in an $\text{Escherichia coli}$ CRP^? mutant (deficient in a cyclic AMP receptor protein) and its isogenic CRP⁺ cell. The CRP^? cell was found to excrete cyclic AMP at an abnormally high rate as compared to the CRP⁺ cell when growing on glucose or glycerol, which can be correlated with the abnormally high intracellular levels of cyclic AMP in the CRP^? cell.     

A monoclonal antibody that inhibits cyclic AMP binding by the Escherichia coli cyclic AMP receptor protein

The monoclonal antibody (mAb) 64D1 was found to inhibit cAMP binding by the cAMP receptor protein (CRP) from Escherichia coli (Li, X.-M., and Krakow, J. S. (1985) J. Biol. Chem. 260, 4378-4383). CRP is relatively resistant to attack by the Staphylococcus aureus V8 protease, chymotrypsin, trypsin, and subtilisin whereas both mAb 64D1-CRP and cAMP-CRP are attacked by these proteases yielding N-terminal core fragments. The fragment patterns resulting from proteolysis of mAb 64D1-CRP and cAMP-CRP differ indicating that the CRP in each complex is in a different conformation. The data presented indicate that the preferred conformation of the antigenic site for mAb 64D1 is present in unliganded CRP. Binding of mAb 64D1 to CRP is inhibited at high cAMP concentration. Formation of a stable cAMP-CRP-lac P+-RNA polymerase open promoter complex resistant to dissociation by mAb 64D1 occurs at a much lower cAMP concentration. The observed increase in resistance to mAb 64D1 may reflect a possible conformational change in CRP effected by contact with RNA polymerase in the open promoter complex.     

Involvement of cyclic AMP and its receptor protein in filamentation of an Escherichia coli fic mutant

Cyclic AMP (cAMP) inhibited septum formation in Escherichia coli PA3092 and induced cell filamentation at elevated temperatures. This phenomenon was first observed in E. coli PA3092 and is due to a temperature-sensitive mutation. We tentatively named this mutation fic (filamentation induced by cAMP). The fic gene was located near rpsL (formerly strA) on the E. coli K-12 map. the inhibitory effect of cAMP on cell division and filamentation in a fic mutant was not observed in a crp mutant. When cAMP was removed from the culture medium, filaments were divided into rods as the intracellular cAMP level decreased. These results suggest that the cAMP-cAMP receptor protein complex causes filamentation in the fic mutant, E. coli PA3092.     

Regulation of cyclic AMP levels in Arthrobacter crystallopoietes and a morphogenetic mutant.

The extracellular levels of cyclic AMP (cAMP), cAMP phosphodiesterase activity, and adenylate cyclase activity were measured at various intervals during growth and morphogenesis of Arthrobacter crystallopoietes. There was a significant rise in the extracellular cAMP level at the onset of stationary phase, and this rise coincided with a decrease in intracellular cAMP. The phosphodiesterase activity measured in vitro increased in the early exponential phase of growth as intracellular cAMP decreased, and, conversely, prior to the onset of stationary phase the phosphodiesterase activity decreased as the intracellular cAMP levels increased. Adenylate cyclase activity was greater in cell extracts prepared from cells grown in a medium where morphogenesis was observed. Pyruvate stimulated adenylate cyclase activity in vitro. A morphogenetic mutant, able to grow only as spheres in all media tested, was shown to have altered adenylated cyclase activity, whereas no significant difference compared to the parent strain was detectable in either the phosphodiesterase activity or the levels of extracellular cAMP. The roles of the two enzymes, adenylate cyclase and phosphodiesterase, and excretion of cAMP are discussed with regard to regulation of intracellular cAMP levels and morphogenesis.     

Soluble expression of a strong thrombolytic pro-urokinase mutant in Escherichia coli

A recombinant pro-urokinase mutant GZ5-sPA was successfully constructed by fusion of a high fibrin-affinity fragment GZ5 to the N-terminus of the serine protease domain of pro-urokinase (sPA). The fragment GZ5 contains a tetrapeptide GPRP and a tripeptide RGD, and was synthesized in bacterial preferred expressing codons. The mutant was then fused to the C-terminus of maltose binding protein (MBP) carried by pMAL-C2x vector, and expressed in Escherichia coli strain Origami (DE3). The produced fusion protein was highly soluble in the cytoplasm of the bacteria. After being cleaved with PreScission Protease to remove MBP tag, GZ5-sPA showed a molecular weight of 31 kDa on SDS-PAGE. GZ5-sPA maintained the same epitope as wild-type pro-urokinase and possessed a thrombolytic activity three times higher than standard urokinase did after being activated as two-chain form. The results could be a clue to other complicated heterogenous proteins similar to pro-urokinase.     

Effect of bacteriophage P1 lysogeny on lipopolysaccharide composition and the lambda receptor of Escherichia coli.

The outer membrane of Escherichia coli was altered as a consequence of lysogeny by bacteriophages P1 and P1 cmts. The predominant change was a reduction in the size of lipopolysaccharide to a heptose-deficient form. P1 cmts lysogens were still sensitive to several bacteriophages but were resistant to lambda vir. Neither whole cells nor solubilized outer membranes from P1 cmts lysogens were able to inactivate lambda vir, and 32P-labeled lambda vir was unable to adsorb to P1 cmts lysogens. P1 cmts lysogens were also affected in maltose transport. The level of periplasmic maltose-binding protein was reduced somewhat, but there was no significant reduction in the level of the outer membrane lambda receptor (LamB). These membrane abnormalities were all corrected in strains cured of P1 cmts. It is suggested that P1 cmts affects lipopolysaccharide biosynthesis by a phage conversion mechanism and consequently the function of the lambda receptor.     

Identification of a membrane protein induced concurrently with cell filamentation by cyclic AMP in an Escherichia coli K-12 fic mutant.

A membrane protein with a molecular weight of 40,000 (40K protein) was induced concurrently with cell filamentation by cyclic AMP (cAMP) in a fic mutant. In the crp mutant and the wild-type strain, cell filamentation by cAMP was not observed, and the 40K protein was not induced. Induction of the 40K protein is regulated by the cAMP-cAMP receptor protein complex and is closely related to cell filamentation by cAMP in the fic mutant.     

Stimulation of cytochrome synthesis in Escherichia coli by cyclic AMP

A cyclic AMP-requiring mutant of Escherichia coli K12 which grows slowly on glucose was found to contain reduced levels of cytochrome b₁ and cytochrome oxidase o. The addition of exogenous cyclic AMP stimulated the synthesis of these cytochrome components and restored growth on glucose to the normal rate observed with the parental strain. Cytochrome synthesis in the parental strain was also stimulated by exogenous cyclic AMP. These studies have provided evidence that cyclic AMP participates in regulating cytochrome synthesis in E. coli, and, coupled with other observations, have suggested a role for this cyclic nucleotide in controlling the construction and operation of the organism's membrane system.     

The effect of cyclic AMP on anaerobic growth of Escherichia coli

Adenosine 3′,5′-cyclic phosphate (cyclic AMP) stimulated a cyclic AMP-deficient mutant strain of $\text{Escherichia coli}$ to grow anaerobically on glucose in a minimal medium and in media supplemented with nitrate or casein hydrolysate. Cyclic AMP was found to stimulate the production of the formic hydrogenlyase system in this mutant strain, but had no effect on its ability to carry out anaerobic reductions of nitrate or nitrite. It was also observed that CO₂ stimulated the anaerobic growth of the mutant in the absence of cyclic AMP.     

Effect of gamma-irradiation of Escherichia coli on cyclic AMP binding activity

Cyclic AMP binding in the extract of E. coli was taken as a measure of cAMP receptor protein (CRP) present in the cells. The gamma-irradiation of cells caused a dose-dependent inhibition of cAMP binding activity indicating that CRP gene is affected by gamma-irradiation. The binding activity in cells irradiated with 240 Gy gamma-ray dose remained unaltered by post-irradiation incubation. This supports previous finding that the enhanced inhibition of the L-arabinose isomerase synthesizing capacity, following incubation of gamma-irradiated cells at higher doses, must be due to a different cause than the catabolite repression.