Cyclic Adenosine 3′,5′-Monophosphate in Escherichia coli

The concentration of cyclic adenosine 3',5'-monophosphate (c-AMP) in Escherichia coli growing on different sources of carbon was studied. Cultures utilizing a source of carbon that supported growth relatively poorly had consistently higher concentrations of c-AMP than did cultures utilizing sugars that supported rapid growth. This relationship was also observed in strains defective in c-AMP phosphodiesterase and simultaneously resistant to catabolite repression; in such strains the c-AMP concentration was slightly higher for several sources of carbon tested. Cultures continued to synthesize c-AMP and secreted it into the medium, under conditions that brought about an inhibition of the intracellular accumulation of the cyclic nucleotide. Transient repression of the synthesis of beta-galactosidase was not associated with an abrupt decrease in the cellular concentration of c-AMP.     

Cyclic 3′,5′-Adenosine Monophosphate Phosphodiesterase of Escherichia coli

The cyclic 3',5'-adenosine monophosphate (c-AMP) phosphodiesterase from Escherichia coli has been partially purified. The enzyme has an apparent molecular weight of 30,000, a Michaelis constant of 0.5 mM c-AMP, and a pH optimum of 7. The partially purified enzyme requires for activity the presence of a reducing compound and of either iron or a protein which seemingly acts as iron carrier.     

3′,5′-Cyclic Adenosine Monophosphate-Requiring Mutants of Escherichia coli

Mutants that require exogenous 3',5'-cyclic adenosine monophosphate (cAMP) for exponential growth were isolated from strains deficient in adenyl cyclase. Studies of one strain showed that cAMP is not incorporated into macromolecules; instead, it seems to have a regulatory function, i.e., in media lacking cAMP, cells form ribonucleic acid (RNA) and protein at linear rather than exponential rates. The exact lesion is not known; ribosomes, messenger RNA, and the beta and beta' subunits of RNA polymerase continue to be made in absence of added cAMP.     

5-Bromouridylyl-(3′ → 5′)-Adenosine Isolated from 5-Bromouracil-Induced Filaments of Escherichia coli

A dinucleoside monophosphate was isolated from 5-bromouracil-induced filaments of a thymine auxotroph of Escherichia coli K-12. The dinucleoside monophosphate was fractioned from a [(14)C]5-bromouracil-labeled perchloric acid extract using Dowex-1-formate ion-exchange chromatography. Sephadex chromatography revealed its molecular weight to be 710. Snake venom phosphodiesterase digest of the dinucleoside monophosphate yielded [(14)C]5-bromouridine and adenosine 5'-monophosphate. The presence of [(14)C]5-bromouracil in bacterial ribonucleic acid indicates that ribonucleic acid, which had incorporated 5-bromouracil, was the probable source of this dinucleoside monophosphate, 5-bromouridylyl-(3' --> 5')-adenosine.     

Requirement of Cyclic Adenosine 3′,5′-Monophosphate for the Thermosensitive Effects of Rts1 in a Cyclic Adenosine 3′,5′-Monophosphate-Less Mutant of Escherichia coli

Previous publications showed that a covalently closed circular (CCC) Rts1 plasmid deoxyribonucleic acid (DNA) that confers kanamycin resistance upon the host bacteria inhibits host growth at 42 degrees C but not at 32 degrees C. At 42 degrees C, the CCC Rts1 DNA is not formed, and cells without plasmids emerge. To investigate the possible role of cyclic adenosine 3',5'-monophosphate (cAMP) in the action of Rts1 on host bacteria, Rts1 was placed in an Escherichia coli mutant (CA7902) that lacks adenylate cyclase or in E. coli PP47 (a mutant lacking cAMP receptor protein). Rts1 did not exert the thermosensitive effect on these cells, and CCC Rts1 DNA was formed even at 42 degrees C. Upon addition of cAMP to E. coli CA7902(Rts1), cell growth and formation of CCC Rts1 DNA were inhibited at 42 degrees C. The addition of cAMP to E. coli PP47(Rts1) did not cause inhibitory effects on either cell growth or CCC Rts1 DNA formation at 42 degrees C. The inhibitory effect of cAMP on E. coli CA7902(Rts1) is specific to this cyclic nucleotide, and other cyclic nucleotides such as cyclic guanosine 3',5'-monophosphate did not have the effect. For this inhibitory effect, cells have to be preincubated with cAMP; the presence of cAMP at the time of CCC Rts1 DNA formation is not enough for the inhibitory effect. If the cells are preincubated with cAMP, one can remove cAMP during the [(3)H]thymidine pulse and still observe its inhibitory effect on the formation of CCC Rts1 DNA. The presence of chloramphenicol during this preincubation period abolished the inhibitory effect of cAMP. These observations suggest that cAMP is necessary to induce synthesis of a protein that inhibits CCC Rts1 DNA formation and cell growth at 42 degrees C.     

Mutation Analysis of the 5′ Untranslated Region of the Cold Shock cspA mRNA of Escherichia coli

The mRNA for CspA, a major cold shock protein in Escherichia coli, contains an unusually long (159 bases) 5' untranslated region (5'-UTR), and its stability has been shown to play a major role in cold shock induction of CspA. The 5'-UTR of the cspA mRNA has a negative effect on its expression at 37 degrees C but has a positive effect upon cold shock. In this report, a series of cspA-lacZ fusions having a 26- to 32-base deletion in the 5'-UTR were constructed to examine the roles of specific regions within the 5'-UTR in cspA expression. It was found that none of the deletion mutations had significant effects on the stability of mRNA at both 37 and 15 degrees C. However, two mutations (Delta56-86 and Delta86-117) caused a substantial increase of beta-galactosidase activity at 37 degrees C, indicating that the deleted regions contain a negative cis element(s) for translation. A mutation (Delta2-27) deleting the highly conserved cold box sequence had little effect on cold shock induction of beta-galactosidase. Interestingly, three mutations (Delta28-55, Delta86-117, and Delta118-143) caused poor cold shock induction of beta-galactosidase. In particular, the Delta118-143 mutation reduced the translation efficiency of the cspA mRNA to less than 10% of that of the wild-type construct. The deleted region contains a 13-base sequence named upstream box (bases 123 to 135), which is highly conserved in cspA, cspB, cspG, and cspI, and is located 11 bases upstream of the Shine-Dalgarno (SD) sequence. The upstream box might be another cis element involved in translation efficiency of the cspA mRNA in addition to the SD sequence and the downstream box sequence. The relationship between the mRNA secondary structure and translation efficiency is discussed.     

Surface Localization of Escherichia coli 5′-Nucleotidase by Electron Microscopy

The 5'-nucleotidase of Escherichia coli was shown to be located at the cell wall surface by histochemical techniques utilizing the deposition of inorganic phosphate. Penetration of the 5'-nucleotidase in the periplasmic space was seen only in cells treated with ethylenediaminetetraacetic acid (EDTA)-tris(hydroxymethyl)aminomethane (Tris). The 3'-nucleotidase of E. coli was also found to have a surface location, and periplasmic precipitation of inorganic phosphate was seen only after EDTA-Tris-sucrose exposure.     

Requirement of Adenosine 3′,5′-Cyclic Phosphate for Formation of the Phage Lambda Receptor in Escherichia coli

Adenosine 3',5'-cyclic phosphate is indispensable for formation of the phage lambda receptor in Escherichia coli K-12.     

Regulatory Role of the Conserved Stem-Loop Structure at the 5′ End of Collagen α1(I) mRNA

Three fibrillar collagen mRNAs, alpha1(I), alpha2(I), and alpha1(III), are coordinately upregulated in the activated hepatic stellate cell (hsc) in liver fibrosis. These three mRNAs contain sequences surrounding the start codon that can be folded into a stem-loop structure. We investigated the role of this stem-loop structure in expression of collagen alpha1(I) reporter mRNAs in hsc's and fibroblasts. The stem-loop dramatically decreases accumulation of mRNAs in quiescent hsc's and to a lesser extent in activated hsc's and fibroblasts. The stem-loop decreases mRNA stability in fibroblasts. In activated hsc's and fibroblasts, a protein complex binds to the stem-loop, and this binding requires the presence of a 7mG cap on the RNA. Placing the 3' untranslated region (UTR) of collagen alpha1(I) mRNA in a reporter mRNA containing this stem-loop further increases the steady-state level in activated hsc's. This 3' UTR binds alphaCP, a protein implicated in increasing stability of collagen alpha1(I) mRNA in activated hsc's (B. Stefanovic, C. Hellerbrand, M. Holcik, M. Briendl, S. A. Liebhaber, and D. A. Brenner, Mol. Cell. Biol. 17:5201-5209, 1997). A set of protein complexes assembles on the 7mG capped stem-loop RNA, and a 120-kDa protein is specifically cross-linked to this structure. Thus, collagen alpha1(I) mRNA is regulated by a complex interaction between the 5' stem-loop and the 3' UTR, which may optimize collagen production in activated hsc's.     

Termination of Deoxyribonucleic Acid in Escherichia coli by 2′,3′-Dideoxyadenosine

2′,3′-Dideoxyadenosine was previously shown to be lethal to Escherichia coli and to inhibit deoxyribonucleic acid (DNA) synthesis irreversibly in this organism. It was also shown that triphosphate of this analogue terminates DNA chains in an in vitro system. Data presented here show that the nucleoside is relatively insensitive to E. coli adenosine deaminase and is converted intracellularly into the dideoxynucleotide, including the triphosphate. Thymine nucleotide pools were not reduced in inhibited bacteria, nor did preformed DNA break down. Some adenine was liberated from the dideoxyadenosine on incubation, and the latter was incorporated into ribonucleic acid. Nevertheless, about 4,000 molecules of the dideoxynucleoside were incorporated into DNA per cell. The dideoxynucleotide occurred in DNA chains in a terminal position, liberated selectively by venom phosphodiesterase. The possible nature of the lethal event is discussed.     

Localization of an Amikacin 3′-Phosphotransferase in Escherichia coli

A plasmid-encoded enzyme reported by us to phosphorylate amikacin in a laboratory strain of Escherichia coli has been localized in the bacterial cell. More than 88% of this amikacin phosphotransferase (APH) activity was retained in spheroplasts formed by ethylenediaminetetraacetate-lysozyme treatment of an APH-containing E. coli transconguant known to form spheroplasts readily. By comparison, the spheroplasts retained 94% of deoxyribonucleic acid polymerase I and 98% of glutamyl-transfer ribonucleic acid synthetase, two internal markers, whereas less than 10% of the activity of a periplasmic marker, acid phosphatase, was present in spheroplasts. Treatment of whole cells of the transconjugant with chemical probes incapable of crossing the plasma membrane obliterated acid phosphatase activity, whereas the internal markers deoxyribonucleic acid polymerase I, glutamyl-transfer ribonucleic acid synthetase, and β-galactosidase were virtually unaffected after treatment for 5 min; more than 60% of the APH activity remained. As a control, similar chemical treatment of sonic extracts, in which enzymes were not protected by bacterial compartmentalization, produced more extensive reduction in the activities of all test enzymes, including APH. Spheroplasts preincubated with adenosine triphosphatase were shown by thin-layer chromatography to phosphorylate amikacin. Spheroplasts of cells grown in the presence of H₃³²PO₄ were shown to utilize internally generated adenosine 5′-triphosphate in the phosphorylation of amikacin. The absence of ³²P-phosphorylated amikacin after incubation of [γ-³²P]adenosine 5′-triphosphate with spheroplasts confirmed that exogenous adenosine 5′-triphosphate was not used in the reaction. These results suggest an internal location for APH. This conclusion has implications for the role of such enzymes in aminoglycoside resistance of gram-negative bacteria.     

Phosphorolysis of 5-Fluoro-2′-Deoxyuridine in Escherichia coli and Its Inhibition by Nucleosides

The effect of ribonucleosides and deoxyribonucleosides on the phosphorolysis of 5-fluoro-2'-deoxyuridine (FUdR) was examined in cells of Escherichia coli. All nucleosides tested except guanosine and deoxyguanosine inhibited phosphorolysis. By using genetically marked strains it was found that in vivo FUdR is a specific substrate of thymidine phosphorylase.     

Role of Cyclic Adenosine 3′,5′-Monophosphate in the In Vivo Expression of the Galactose Operon of Escherichia coli

Studies of levels of galactokinase in Escherichia coli with mutations affecting synthesis of, or response to, cyclic adenosine 3',5'-monophosphate show that this nucleotide does not play a major role in expression of the galactose operon, causing at most a twofold stimulation. The discrepancy between our in vivo results and the marked stimulation by cyclic adenosine 3',5'-monophosphate in in vitro systems indicates that current cell-free systems lack a factor which allows efficient expression of the galactose operon even in the absence of cyclic adenosine 3',5'-monophosphate or of the binding protein for this nucleotide.     

Cyclic 3′,5′-Adenosine Monophosphate and N-Acetyl-glucosamine-6-Phosphate as Regulatory Signals in Catabolite Repression of the lac Operon in Escherichia coli

When an Escherichia coli mutant lacking the enzyme N-acetyl-glucosamine-6-phosphate (AcGN6P) deacetylase is grown in a succinate-mineral salts medium and exposed to an exogenous source of N-acetylglucosamine, approximately 20 to 30 pmoles of AcGN6P per mug of cell dry weight will accumulate in these cells. This accumulation occurs within 2 to 4 min after the addition of N-acetylglucosamine and is coincident with the production of a severe permanent catabolite repression of beta-galactosidase synthesis. This repression does not occur if adenosine 3',5'-cyclic phosphate (cyclic AMP) is added to the cells before AcGN6P accumulates. An immediate derepression occurs when cyclic AMP is added to cells that have already accumulated a large AcGN6P pool. These findings are consistent with the view that low-molecular-weight carbohydrate metabolites and cyclic AMP play key roles in the catabolite repression phenomenon, and that metabolites such as AcGN6P may participate in the represion mechanism by influencing either the formation or degradation of cyclic AMP in E. coli.