Escherichia coli cyclic AMP receptor protein mutants provide evidence for ligand contacts important in activation.

The three-dimensional model of the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP) shows that several amino acids are involved as chemical contacts for binding cAMP. We have constructed and characterized mutants at four of these positions, E72, R82, S83, and R123. The mutations were made in wild-type crp as well as a cAMP-independent crp, crp*. The activities of the mutant proteins were characterized in vivo for their ability to activate the lac operon. These results provide genetic evidence to support that E72 and R82 are essential and S83 and R123 are important in the activation of CRP by cAMP.     

MetR and CRP bind to the Vibrio harveyi lux promoters and regulate luminescence

The induction of luminescence in Vibrio harveyi at the later stages of growth is controlled by a quorum-sensing mechanism in addition to nutritional signals. However, the mechanism of transmission of these signals directly to the lux promoters is unknown and only one regulatory protein, LuxR, has been shown to bind directly to lux promoter DNA. In this report, we have cloned and sequenced two genes, crp and metR, coding for the nutritional regulators, CRP (cAMP receptor protein) and MetR (a LysR homologue), involved in catabolite repression and methionine biosynthesis respectively. The metR gene was cloned based on a general strategy to detect lux DNA-binding proteins expressed from a genomic library, whereas the crp gene was cloned based on its complementation of an Escherichia coli crp mutant. Both CRP and MetR were shown to bind to lux promoter DNA, with CRP being dependent on the presence of cAMP. Expression studies indicated that the two regulators had opposite effects on luminescence: CRP was an activator and MetR a repressor. Disruption of crp decreased luminescence by about 1,000-fold showing that CRP is a major activator of luminescence the same as LuxR, whereas disruption of MetR resulted in activation of luminescence over 10-fold, confirming its function as a repressor. Comparison of the levels of the autoinducers involved in quorum sensing excreted by V. harveyi, and the crp and metR mutants, showed that autoinducer production was not significantly different, thus indicating that the nutritional signals do not affect luminescence by changing the levels of the signals required for quorum sensing. Indeed, the large effects of these nutritional sensors show that luminescence is controlled by multiple signals related to the environment and the cell density which must be integrated at the molecular level to control expression at the lux promoters.     

UV resistance of E. coli K-12 deficient in cAMP/CRP regulation.

Deletion of genes for adenylate cyclase (delta cya) or cAMP receptor protein (delta crp) in E. coli K-12 confers a phenotype that includes resistance to UV radiation (254 nm). Such mutations lead to UV resistance of uvr+, uvrA, lexA and recA strains which could partly be abolished by the addition of cAMP to delta cya but not to delta crp strain culture medium. This effect was not related to either inducibility of major DNA repair genes or growth rate of the bacteria. Enhanced survival was also observed for UV-irradiated lambda bacteriophage indicating that a repair mechanism of UV lesions was involved in this phenomenon.     

Involvement of cyclic AMP (cAMP) and cAMP receptor protein in anaerobic respiration of Shewanella oneidensis

Shewanella oneidensis is a metal reducer that can use several terminal electron acceptors for anaerobic respiration, including fumarate, nitrate, dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), nitrite, and insoluble iron and manganese oxides. Two S. oneidensis mutants, SR-558 and SR-559, with Tn5 insertions in crp, were isolated and analyzed. Both mutants were deficient in Fe(III) and Mn(IV) reduction. They were also deficient in anaerobic growth with, and reduction of, nitrate, fumarate, and DMSO. Although nitrite reductase activity was not affected by the crp mutation, the mutants failed to grow with nitrite as a terminal electron acceptor. This growth deficiency may be due to the observed loss of cytochromes c in the mutants. In contrast, TMAO reduction and growth were not affected by loss of cyclic AMP (cAMP) receptor protein (CRP). Fumarate and Fe(III) reductase activities were induced in rich medium by the addition of cAMP to aerobically growing wild-type S. oneidensis. These results indicate that CRP and cAMP play a role in the regulation of anaerobic respiration, in addition to their known roles in catabolite repression and carbon source utilization in other bacteria.     

Transport of antibiotics and metabolite analogs by systems under cyclic AMP control: positive selection of Salmonella typhimurium cya and crp mutants.

Mutants in the cyclic AMP (cAMP) control system in Salmonella typhimurium (cya = adenyl cyclase, crp = cAMP receptor protein) were partially resistant to growth inhibition by 22 antibiotics (including fosfomycin, nalidixic acid, and streptomycin) and 29 inhibitory analogs of normal bacterial fuel/carbon sources. This resistance was used as the basis for an efficient positive selection of cya and crp mutants. We propose that these antibiotics and analogs enter the bacteria through transport systems normally used for transporting fuel/carbon sources and that this is accomplished because of a structural similarity between the antibiotic and the natural substrate of the particular transport system involved. We propose that these transport systems are all under positive control by cAMP and that cAMP acts as a signal molecule (alarmone) for fuel/carbon deprivation. Evidence is provided for a hierarchy within operons controlled by cAMP. The methodology is shown to be useful for analyzing both antibiotic transport systems and the cAMP super-control system.     

Cyclic AMP and its receptor protein are required for expression of transfer genes of conjugative plasmid F in Escherichia coli

A number of cya and crp mutants of Escherichia coli HfrH were analyzed for several Tra functions of the F plasmid. The mutants were observed to be deficient in conjugal donor ability, absorption of phages MS2 and Q beta and surface exclusion. These defects were suppressed in cya mutants grown with cAMP supplementation. A cAMP concentration of 3 X 10(-4) M produced maximal suppression of donor ability defect in a cya strain. cAMP did not suppress the Tra- phenotype of crp mutants. Latent periods of MS2 were shorter in cya and crp bacteria. Phage T7 development appeared similar in wild type, cya, and crp cells. It is concluded that tra genes of F plasmid are expressed only to a small extent in cya and crp mutants and that cAMP and its receptor protein are required for the normal expression of tra genes.     

Cloning and DNA sequence analysis of the wild-type and mutant cyclic AMP receptor protein genes from Salmonella typhimurium.

The crp gene from Salmonella typhimurium, as well as two mutant adenylate cyclase regulation genes designated crpacr-3 and crpacr-4, were cloned into the EcoRI site of plasmid pUC8. Initially cloned on 5.6-kilobase fragments isolated from EcoRI digests of chromosomal DNA, these genes were further subcloned into the BamHI-EcoRI site of plasmid pBR322. When tested, Escherichia coli crp deletion strains harboring the clones regained their ability to pleiotropically ferment catabolite-repressible sugars. Also, the crpacr-containing strains displayed sensitivity to exogenous cyclic AMP (cAMP) when grown on eosin-methylene blue medium with xylose as the carbon source. The proteins encoded by the S. typhimurium wild-type and mutant crp genes were found to have similar molecular weights when compared with the wild-type cAMP receptor protein (CRP) from E. coli. DNA sequence analysis of the wild-type crp gene showed only a three-nucleotide difference from the E. coli sequence, suggesting little divergence of the crp gene between these organisms. The crpacr sequences, however, each contained single nucleotide changes resulting in amino acid substitutions at position 130 of the CRP. Based on the site at which these substitutions occur, the crpacr mutations are believed to affect CRP-cAMP interactions.     

Characterization of Escherichia coli adenylate cyclase mutants with modified regulation

In Escherichia coli there is a large increase of cAMP synthesis in crp strains, which are deficient in the catabolite gene activator protein. In this work it was shown that this increase in cAMP synthesis does not occur in crp crr strains, deficient in both the catabolite gene activator protein and enzymeIII-glucose, a component of the phosphotransferase system. It was also shown that the other components of the phosphotransferase system are required to obtain the increase of cAMP synthesis in a crp background. Adenylate cyclase mutants were obtained, by random mutagenesis, which had partial adenylate cyclase activity but which did not exhibit increased levels of cAMP in a crp background. For three mutants the mutation was identified as a single point mutation. This allowed the identification of residues arginine 188, aspartic acid 414 and glycine 463 which could be involved in the catabolite gene activator protein dependent activation process.     

Sites of allosteric shift in the structure of the cyclic AMP receptor protein

We have characterized crp mutations in E. coli that allow CRP to function without cAMP. crp* mutants carrying a deletion of the gene encoding adenylate cyclase (cya) show significant lac expression. Cyclic GMP, normally an ineffective activator of CRP+, can stimulate these mutant CRP*s to permit greater lac expression in vivo. Cyclic AMP binding to the amino-terminal domain of CRP+ induces an allosteric transition that changes the DNA-binding property of the carboxy domain. The CRP* phenotype is caused by substitution of amino acids with bulkier side chains in the D alpha-helix of the protein's carboxy domain, near the hinge connecting the two domains. These results are consistent with a model in which the mutant CRP*s assume, in part, a conformation normally evoked only by cAMP binding: one in which the relative orientation of the C, D, and F alpha-helices is altered. We define precisely the amino acids of these alpha-helices that interact to cause the allosteric shift.     

A novel catecholamine-activated adenosine cyclic 3',5'-phosphate independent pathway for beta-adrenergic receptor phosphorylation in wild-type and mutant S49 lymphoma cells: mechanism of homologous desensitization of adenylate cyclase

Virtually all known biological actions stimulated by beta-adrenergic and other adenylate cyclase coupled receptors are mediated by cAMP-dependent protein kinase. Nonetheless, "homologous" or beta-adrenergic agonist-specific desensitization does not require cAMP. Since beta-adrenergic receptor phosphorylation may be involved in desensitization, we studied agonist-promoted receptor phosphorylation during homologous desensitization in wild-type S49 lymphoma cells (WT) and two mutants defective in the cAMP-dependent pathway of beta-agonist-stimulated protein phosphorylation (cyc- cannot generate cAMP in response to beta-adrenergic agonists; kin- lacks cAMP-dependent kinase). All three cell types demonstrate rapid, beta-adrenergic agonist-promoted, stoichiometric phosphorylation of the receptor which is clearly not cAMP mediated. The amino acid residue phosphorylated is solely serine. These data demonstrate, for the first time, that catecholamines can promote phosphorylation of a cellular protein (the beta-adrenergic receptor) via a cAMP-independent pathway. Moreover, the ability of cells with mutations in the adenylate cyclase-cAMP-dependent protein kinase pathway to both homologously desensitize and phosphorylate the beta-adrenergic receptors provides very strong support for the notion that receptor phosphorylation may indeed be central to the molecular mechanism of desensitization.     

Phenotype, virulence and immunogenicity of Edwardsiella ictaluri cyclic adenosine 3',5'-monophosphate receptor protein (Crp) mutants in catfish host

Edwardsiella ictaluri is an Enterobacteriaceae that causes lethal enteric septicemia in catfish. Being a mucosal facultative intracellular pathogen, this bacterium is an excellent candidate to develop immersion-oral live attenuated vaccines for the catfish aquaculture industry. Deletion of the cyclic 3',5'-adenosine monophosphate (cAMP) receptor protein (crp) gene in several Enterobacteriaceae has been utilized in live attenuated vaccines for mammals and birds. Here we characterize the crp gene and report the effect of a crp deletion in E. ictaluri. The E. ictaluri crp gene and encoded protein are similar to other Enterobacteriaceae family members, complementing Salmonella enterica Δcrp mutants in a cAMP-dependent fashion. The E. ictaluri Δcrp-10 in-frame deletion mutant demonstrated growth defects, loss of maltose utilization, and lack of flagella synthesis. We found that the E. ictaluri Δcrp-10 mutant was attenuated, colonized lymphoid tissues, and conferred immune protection against E. ictaluri infection to zebrafish (Danio rerio) and catfish (Ictalurus punctatus). Evaluation of the IgM titers indicated that bath immunization with the E. ictaluri Δcrp-10 mutant triggered systemic and skin immune responses in catfish. We propose that deletion of the crp gene in E. ictaluri is an effective strategy to develop immersion live attenuated antibiotic-sensitive vaccines for the catfish aquaculture industry.