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Ogawa and Inaba are two main serotypes of O1 V. cholerae and alternate among cholera epidemics. The rfbT gene encodes a methyltransferase and is required for Ogawa serotype. The Inaba serotype is the consequence of genetic alterations in rfbT gene which results in loss-of-function enzyme product. However, the expression and regulation of rfbT has not been understood yet. Here we demonstrated that a global regulator, cAMP receptor protein (CRP), positively regulates rfbT transcription through a non-canonical CRP binding site (CBS) in its promoter region. This finding is supported by the analyses of rfbT mRNA abundance, rfbT–lacZ fusions and electrophoretic mobility shift assay (EMSA). The analyses of rfbT mRNA level in wild type (WT), Δcrp, and lower or higher level of cAMP backgrounds revealed that CRP is required for rfbT expression in response to intracellular cAMP level. Subsequent rfbT–lacZ fusions and EMSA collectively displayed that cAMP-CRP complex transcriptionally activates rfbT through directly binding to CBS in rfbT promoter region. Consistently, serological microagglutination test showed that crp deletion resulted in at least 4-fold decrease in titer of Ogawa serum compared to its WT. These results expanded our knowledge of understanding the genetic determinants and probable regulatory mechanism of V. cholerae O1 serotype shift between Ogawa and Inaba.

Antibiotics have been one of the most successful forms of therapy in medicine. However, the efficiency of antibiotics is compromised by the emergence of antibiotic-resistant pathogens. To reduce antibiotic resistance, complete understanding of bacterial tactics to defend themselves against antibiotics is necessary. Small-noncoding RNAs (sRNAs) modulate gene expression by base-pairing with multiple target mRNAs. Cellular levels of Hfq-dependent sRNAs influence antibiotic resistance by modulating expression of specific target genes; therefore, such sRNAs could be a good tool to identify target mRNAs that modulate antibiotic susceptibility and may themselves be used as druggable molecules. Here, we report the identification of genes and pathways associated with OxyS RNA-mediated cephalothin resistance using phenotypic and expression analyses of OxyS-regulated genes identified by RNA-seq, literature mining, or predictions. From our studies we found that the differential expression of 27 OxyS-regulated genes was involved in cephalothin susceptibility. Among them, 17 gene knockouts showed resistance to the drug and nine from them is associated with cAMP receptor protein (CRP), a transcriptional dual regulator in E. coli. Moreover, levels of OxyS and OxyS-modulated genes (cycA and cysH) were also altered in multidrug-resistant (MDR) E. coli strains. Together, our data suggest that OxyS extensively modulates gene expression in multiple pathways to develop cephalothin resistance. In addition, OxyS and its regulated target genes, either individually or in combination, could be used as molecular markers and targets for the identification and eradication of cephalothin-resistant strains.

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infection (UTI), a common bacterial infectious disease. This bacterium invades the urinary tract cells, where it aggregates, and subsequently forms multicellular colonies termed intracellular bacterial communities (IBCs). The motility of the bacteria plays a key role in the mechanism of virulence in the host bladder. Here, we show that CytR is a modulator of bacterial internalization and aggregation within the bladder epithelial cells sustained by CRP in UPEC. Mutational analyses and gel-shift assays indicated that CytR represses the expression of flhD, thereby encoding a master regulator for flagellar expression that is responsible for bacterial motility when CRP is present, whereas CRP is an activator of flhD expression. Thus, elevated flagellar expression was involved in promoted virulence in the cytR mutant. These combined observations suggest another regulatory layer of flagellar expression and the role of CytR in UPEC virulence.

The chromosomal inactivation of the unique transcription factor of Streptomyces coelicolor that displays a cyclic-nucleotide-binding domain, Crp(Sco), led to a germination-defective phenotype similar to the mutant of the adenylate cyclase gene (cya) unable to produce cAMP. By means of cAMP affinity chromatography we demonstrate the specific cAMP-binding ability of Crp(Sco), which definitely demonstrate that a Cya/cAMP/Crp system is used to trigger germination in S. coelicolor. However, electromobility shift assays with the purified Crp(Sco)-cAMP complex and the CRP-like cis-acting element of its own promoter failed. Moreover, we were unable to complement an Escherichia coli crp mutant in trans with Crp(Sco). The fact that Vfr from Pseudomonas aeruginosa and GlxR from Corynebacterium glutamicum could complement such an E. coli mutant suggests that the way Crp(Sco) interacts with DNA should mechanistically differ from its most similar members. This hypothesis was further supported by homology modelling of Crp(Sco) that confirmed an unusual organisation of the DNA-binding domain compared to the situation observed in Crp(Eco).

Activation or inhibition of the cyclic AMP (cAMP)—protein kinase A (PKA) pathway can ultimately regulate the transcription of a variety of genes. In vertebrates, the best characterized nuclear targets of PKA are the ‘cAMP response element’ (CRE) binding proteins (CREB). Differences in the transcriptional response to this pathway between cells and tissues can be based on the presence of distinct CREB isoforms. In this context, we have now investigated the presence of different dCREB transcripts in a stable, embryonic insect cell line, i.e., Drosophila Schneider 2 (S2) cells. In addition, we have studied the possible effect of cellular cAMP- and Ca2+ increases on the expression of a luciferase reporter in cells transfected with a CRE-containing reporter gene construct. In combination with recent data from the literature, our results indicate that the regulation of CRE-dependent gene expression shows some important differences between insects and vertebrates.

Highlights: •Escherichia coli contains two L-asparaginase isozymes with distinct localization, kinetics and regulation. •Mutant strains were used to examine the roles of these enzymes in L-asparagine utilization. •We report that L-asparaginase II permits growth on asparagine and glycerol under anaerobic conditions. •We propose that this enzyme is the first step in a co-regulated pathway leading to fumarate. •The pathway is regulated by anaerobiosis and cAMP and provides a terminal elector acceptor. -- Abstract: Escherichia coli expresses two L-asparaginase (EC 3.5.1.1) isozymes: L-asparaginse I, which is a low affinity, cytoplasmic enzyme that is expressed constitutively, and L-asparaginase II, a high affinity periplasmic enzyme that is under complex co-transcriptional regulation by both Fnr and Crp. The distinct localisation and regulation of these enzymes suggest different roles. To define these roles, a set of isogenic mutants was constructed that lacked either or both enzymes. Evidence is provided that L-asparaginase II, in contrast to L-asparaginase I, can be used in the provision of an anaerobic electron acceptor when using a non-fermentable carbon source in the presence of excess nitrogen.

A study on the sequence dependent DNA binding mode of DAPI has been carried out on pUC8 and the βgal promoter region by restriction endonuclease and DNAase I protection experiments. A molecular model depicting drug interaction at the level of selected palyndromes has also been constructed that confirms the A-T sequence specificity of the compound. Experimental data indicate that the binding sites for RNA polymerase and cyclic AMP receptor protein (CRP) in the βgal gene are privileged locales for DAPI interaction, a feature that explains impairment of transcription at this level. From a stereochemical view point, DAPI binding to DNA minor groove, while being incompatible with promoter unwinding in the open complex, may also disturb optimal contacts with proteins regulating RNA polymerase activity.

Using the tertiary structure of the globular domain of H5 (GH5) and based on an alternative sequence homology between GH5 and DNA-binding proteins containing the helix-turn-helix motif, a model for H5-DNA interaction is proposed. From molecular graphics it follows that helix II recognizes the major groove of the DNA, as does the second helix of the helix-turn-helix motif, while helix III makes minor groove contacts, in agreement with the hypothesis of Turnell et al. (FEBS letters 232, 263–268). In the resulting model GH5 makes contact with a full turn of DNA.

Inactivation of iscS encoding cysteine desulfurase results in a slow growth phenotype associated with the deficiency of iron–sulfur clusters, thiamine, NAD, and tRNA thionucleosides in Escherichia coli . However, the other roles of iscS in vivo are unknown. By using differential screening strategies, we identified 2 pyrimidine salvage enzymes, namely, uridine phosphorylase and cytidine deaminase, which were down-regulated in the iscS mutant. Both enzymes are positively regulated by the cAMP receptor protein (CRP). We also identified a novel protein complex, namely, YeiT–YeiA, whose expression level was decreased in the iscS mutant. The recombinant YeiT–YeiA complex exhibited NADH-dependent dihydropyrimidine dehydrogenase activity, indicating its role in pyrimidine metabolism. The presence of a CRP-binding consensus sequence on the 5′-upstream of the yeiT–YeiA gene suggests its regulation by CRP. These results provide a clue to the possible role of iscS in pyrimidine metabolism by gene regulation.

Cyclic AMP receptor protein (CRP) binds to DNA and induces DNA bending. DNA bending plays an important role in transcription activation of many genes in Escherichia coli. To know DNA helical change by CRP, we used molar cyclization factor (j(M)), which is sensitive to helical twist or torsional alignment. Six consecutive DNA fragments were constructed that contained phasing sequence at left arm and CRP binding site in center. j(M) values measured in this study were approximately 10 times increased by CRP relative to that measured for DNA only. The analysis of set of j(M) values indicated that CRP induced DNA bending without the helical change like helical twisting or torsional alignment change.

Regulation of the Escherichia coli thioredoxin gene (trxA) was studied using trxA-lac translational fusion constructed in the vector pMC1403. Synthesis of beta-galactosidase from the trxA-lac fusion was found to be repressed in the presence of lactose. A switch of carbon source from glucose to lactose and an addition of cyclic AMP (cAMP) caused a decrease in beta-galactosidase synthesis from the trxA-lac fusion. The repression effect of exogenous cAMP was not observed in the crp mutant strain. The beta-galactosidase synthesis from the trxA-lac fusion lacking a plausible cAMP-CRP binding site was not lowered in the presence of lactose or in the addition of cAMP. Expression of the chromosomal trxA gene was reduced by exogenous cAMP. These findings indicate that the expression of the trxA gene is controlled by cAMP in a negative manner.

Summary The activation-associated markers, p47b and p71/73, have been recognized as minor proteins in peritoneal and bone marrow culture-derived macrophages activated for tumor cell killing. Proteins with identical characteristics of migration on 2-dimensional gels and comparable Cleveland peptide maps are described here in macrophage cell lines that could be activated for tumor cell killing (J774A.1, RAW 264, UNC-2). Macrophage cell lines that could not be activated (P388D 1 and PU5-1.8) did not express the markers. The expression of these markers in activatable macrophage cell lines strengthens their association with the activation process and provides a bulk source of the proteins for purification studies.

The formation of closed covalent circular DNA of R factor, Rts 1, does not take place at non-permissive temperature, 42°C, in E. coli 20SO. However, when Rts 1 was placed in mutants having a low level of cyclic AMP or lacking cyclic AMP receptor protein, the thermosensitive lesion is overcome. Addition of cyclic AMP caused inhibition of the formation of ccc DNA in mutants with low cyclic AMP level, but not in mutants lacking cyclic AMP receptor protein.

Summary A highly sensitive radioimmunological assay has been set up to determine the amount of the cyclic AMP receptor protein (CAP) in crude Escherichia coli extracts. This method enables to detect cross-reacting material in mutants carrying a deletion in the crp gene (coding for CAP) and to measure the amount of CAP under various growth conditions. We present results showing that the synthesis of CAP is affected neither by the growth conditions of bacteria nor by the carbon source or by cyclic AMP.

An unusual cAMP binding protein of 50 000 Da previously found in human tumors was isolated from HeLa cells in the presence of protease inhibitors. The protein was neutralized by anti-bovine R II antibodies but not by anti-R I . It was able to form a dimer, and to inhibit HeLa C kinase in a dose-dependent manner. The HeLa R II 50 000 was also subject to limited proteolysis and it could be phosphorylated by C kinase. HeLa cells contain two R I proteins, a predominant 49 000 Da and a minor 51 000 Da isoprotein. In addition, large amounts of a protein consisting of 19 000 and 20 000 Da subunits were isolated by 8-thio-CAMP affinity chromatography that was not immunologically related to the R proteins.

By labeling adenosine 3′, 5′-cyclic monophosphate (cyclic AMP) with [ 32 P] phosphate and chromatographing it on a thin-layer alumina plate, we have determined the extra- and intracellular amounts of cyclic AMP in an 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.

The occurrence of a cytosolic cAMP-binding protein of an approximate molecular weight of 41,000 daltons was monitored in vegetative and developing amoebae of Dictyostelium discoideum by the use of the photoaffinity probe (32P) 8N3-cAMP. There was a large apparent increase in the amount of this binding protein during development; its molecular weight remained constant, if appropriate methods were employed for the disruption of the amoebae. Comigration during electrophoresis on two-dimensional gels identifies this cAMP-binding protein, photoaffinity-labeled in crude extracts, as the regulatory subunit of the cAMP-dependent protein kinase of D. discoideum .

Inhibition of E. coli growth by cyclic adenosine monophosphate is observed in wild type strains cultured in glucose as carbon source, but not in a cyclic AMP receptor protein deficient mutant. A deletion mutant of the adenylate cyclase gene requires cyclic adenosine monophosphate for optimal growth. Using glucose as carbon source, 2 mM cyclic AMP promotes maximal rates of cell multiplication in this mutant; however higher concentrations of the nucleotide inhibit growth. Cell multiplication of wild type strains grown in glycerol is not affected by cyclic adenosine monophosphate. Nevertheless, in this carbon source the growth rate of the adenylate cyclase mutant is strongly inhibited by concentrations of this nucleotide beyond 0.1 mM. This suggests that growth inhibition by exogenous cyclic adenosine monophosphate is highly dependent on the intracellular levels of the nucleotide.

A cAMP binding site present on isolated plasma membranes of aggregation-competent D. discoideum cells has been solubilized with the nonionic detergent Emulphogene BC-720. An assay has been developed based on the principle of hydrophobic chromatography, in which the detergent solubilized cAMP binding protein is immobilized on alkyl-agarose beads at low detergent concentration. This allows the necessary rapid separation of bound and free [ 3 H]-cAMP by filtration of the beads. The kinetics and nucleotide specificity of the detergent solubilized cAMP binding protein are comparable to those of the cAMP chemotactic receptor on intact cells and plasma membranes. The alkyl-agarose bead assay may have general utility for the assay of detergent solubilized membrane receptors.

Incubation of nuclei from hormone-dependent rat mammary tumors with its cytosol activated with 5 nM 17β-estradiol resulted in a 4-fold increase of nuclear estrogen binding activity over the control nuclei. The presence of 100 nM cAMP in the activated cytosol inhibited this nuclear uptake of estrogen receptor by 50%. Conversely, incubation of the nuclei with cytosol activated with 100 nM cAMP increased nuclear cAMP binding and cAMP-dependent protein kinase activity 4-fold, while the presence of 5 nM 17β-estradiol in the activated cytosol inhibited the nuclear cAMP binding and the protein kinase activity by 50%. No competition was found between estrogen and cAMP for each other's cytoplasmic binding proteins or the nuclear acceptor sites. These data suggest that a mutual antagonism exists between the cAMP-binding protein and estrogen receptor during their nuclear translocation.

Incubation of the particulate fraction of cell extract prepared from NIH3T3 mouse fibroblasts resulted in preferential proteolytic degradation of a cAMP-binding protein. The proteolysis was inhibited by human c-Ha- ras gene products produced by Escherichia coli . The proteolysis was observed at pH 6 to 7, and inhibited by antipain and leupeptin. These results suggest that cAMP-binding proteins might be cleaved by thiol proteinases. In fact, c-Ha- ras gene products were proved to inhibit the cathepsin B-like activity present in the particulate fraction.

Using [ 32 P]-8-N 3 -cAMP, a photoaffinity analog of cAMP, we have established that nuclear binding of cAMP is preferentially localized in the “nuclear matrix”. Two major radioactive bands corresponded to proteins of M r 40 K and 50 K, and three minor bands to proteins of M r 55, 150 and 200 K. Even though the molecular weight of the major nuclear binding proteins in the matrix are similar to those of the cytosolic cAMP binding proteins, the characteristics of the binding reaction in the nucleus were markedly different from those in the cytosol.

Summary A cAMP-binding protein isolated from the cytosol of Dictyostelium discoideum was partially purified. The protein binds cAMP maximally at pH 5–7; its K D for cAMP is in the nanomolar range and the specificity for cAMP is high; the molecular weight is approximately 42,000. This cAMP-binding protein is not found in vegetative amoebae; it appears during the first two hours of development. The protein inhibits the activity of the purified catalytic subunit of bovine cAMP-dependent protein kinase in the absence, but not in the presence, of cAMP. It is suggested that the cAMP-binding protein, isolated from Dictyostelium , is either the regulatory subunit of cAMP-dependent protein kinase or the product of the partial degradation of such a regulatory subunit.

We examined the effects of agents/conditions that raise intracellular cAMP concentration in the induction of the 47,000-dalton R I cAMP-binding protein in the “neurite-minus” N1A-103 mouse neuroblastoma cells. The amount of R I in cell extracts was quantitated by photoaffinity labeling with 8-N 3 -[ 32 P]cAMP and by ELISA and Western blot. Our results showed that treatment of the N1A-103 neuroblastoma cells with 20 μM forskolin or 1 mM dibutyryl cAMP, or reducing the serum concentration in the culture medium from 10% to 1% caused a 3–4 fold increase in the amount of R I . Our results suggest that the induction of R I can occur independent of morphological differentiation in the mouse neuroblastoma cells.