The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene

In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA^Glc, a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIA^Glc, CAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA^Glc. In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA^Glc were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA^Glc. In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.     

The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene

In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA^Glc, a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIA^Glc, CAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA^Glc. In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA^Glc were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA^Glc. In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.     

Modulation of Escherichia coli Adenylyl Cyclase Activity by Catalytic-Site Mutants of Protein IIAGlc of the Phosphoenolpyruvate:Sugar Phosphotransferase System

It is demonstrated here that in Escherichia coli, the phosphorylated form of the glucose-specific phosphocarrier protein IIA^Glc of the phosphoenolpyruvate:sugar phosphotransferase system is an activator of adenylyl cyclase and that unphosphorylated IIA^Glc has no effect on the basal activity of adenylyl cyclase. To elucidate the specific role of IIA^Glc phosphorylation in the regulation of adenylyl cyclase activity, both the phosphorylatable histidine (H90) and the interactive histidine (H75) of IIA^Glc were mutated by site-directed mutagenesis to glutamine and glutamate. Wild-type IIA^Glc and the H75Q mutant, in which the histidine in position 75 has been replaced by glutamine, were phosphorylated by the phosphohistidine-containing phosphocarrier protein (HPr~P) and were equally potent activators of adenylyl cyclase. Neither the H90Q nor the H90E mutant of IIA^Glc was phosphorylated by HPr~P, and both failed to activate adenylyl cyclase. Furthermore, replacement of H75 by glutamate inhibited the appearance of a steady-state level of phosphorylation of H90 of this mutant protein by HPr~P, yet the H75E mutant of IIA^Glc was a partial activator of adenylyl cyclase. The H75E H90A double mutant, which cannot be phosphorylated, did not activate adenylyl cyclase. This suggests that the H75E mutant was transiently phosphorylated by HPr~P but the steady-state level of the phosphorylated form of the mutant protein was decreased due to the repulsive forces of the negatively charged glutamate at position 75 in the catalytic pocket. These results are discussed in the context of the proximity of H75 and H90 in the IIA^Glc structure and the disposition of the negative charge in the modeled glutamate mutants.     

Regulation of glycerol and maltose uptake by the IIAGlc-like domain of IINag of the phosphotransferase system inSalmonella typhimurium LT2

InEnterobacteriaceae the nonphosphorylated form of IIA^G1c of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the Ma1K protein of the maltose transport system, respectively. In this report we show that the IIA^Glc-Iike domain of the membrane-bound II^{N-acetylglucosamine} (II^Nag) of the PTS can replace IIA^Glc in aSalmonella typhimurium crr mutant strain that lacks all soluble IIA^Glc. The inhibition was most severe in cells which were partially induced for the glycerol or maltose up take systems. TheStreptococcus thermophilus lactose transporter LacS, which also contains a IIA^Glc-like domain, could not replace IIA^Glc. Neither IINag nor LacS could replace IIA^Glc in activation of adenylate cyclase.     

Regulation of glycerol and maltose uptake by the IIAGlc-like domain of IINag of the phosphotransferase system inSalmonella typhimurium LT2

InEnterobacteriaceae the nonphosphorylated form of IIA^G1c of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the Ma1K protein of the maltose transport system, respectively. In this report we show that the IIA^Glc-Iike domain of the membrane-bound II^{N-acetylglucosamine} (II^Nag) of the PTS can replace IIA^Glc in aSalmonella typhimurium crr mutant strain that lacks all soluble IIA^Glc. The inhibition was most severe in cells which were partially induced for the glycerol or maltose up take systems. TheStreptococcus thermophilus lactose transporter LacS, which also contains a IIA^Glc-like domain, could not replace IIA^Glc. Neither IINag nor LacS could replace IIA^Glc in activation of adenylate cyclase.     

The role of cAMP in flagellation of Salmonella typhimurium

Summary A mutational alteration either in adenylate cyclase (cya ^-) or in cyclic-35-AMP (cAMP) receptor protein (crp ^-) rendered Salmonella typhimurium incapable of producing flagella. The amount of mRNA specific for flagellin in these mutants was almost negligible when assayed in an in vitro protein synthesizing system. A secondary mutation, cfs, partially suppressing the cya ^- mutation, was identified among the revertants of cya ^-. A mutation in the same cistron as cfs resulted in a non-flagellate phenotype either by itself or in combination with cfs. The cistron, which was given the gene symbol flaT, was located between flaE and flaL. It was suggested that cAMP receptor protein together with cAMP modulates the gene flaT, which in turn acts as a positive effector on the synthesis of active mRNA specific for flagellin.     

Multiple regulation of the activity of adenylate cyclase in Escherichia coli

Summary We have studied the correlation between the activities of adenylate cyclase (ATP pyrophosphatelyase-(cyclizing); EC 4.6.1.1) and in vivo rates of synthesis and intracellular concentrations of adenosine 3,5 cyclic monophosphate (cAMP) under various growth conditions in wild-type Escherichia coli and in mutants lacking or overproducing the cAMP receptor protein (CAP). We showed that when wild-type bacteria are grown in the presence of a variety of carbon sources the intracellular concentrations of cAMP are inversely related to the adenylate cyclase activities determined in permeabilized cells, suggesting that the carbon source-dependent modulation of cAMP levels is not directly related to the regulation of adenylate cyclase activity. In mutants lacking functional CAP (crp) the in vivo rates of cAMP synthesis are several hundred-fold higher than in the wild-type parent without a parallel increase of adenylate cyclase activities. In a strain carrying multiple copies of the crp gene and overproducing CAP the activity of adenylate cyclase is severely inhibited, although the in vivo rate of cAMP synthesis is similar to the parental strain. We interpret these results as indicating that CAP controls mainly the activity rather than the synthesis of adenylate cyclase.     

Type 1 fimbriation is negatively regulated by cyclic AMP and its receptor proteinvia conjugative plasmid F inEscherichia coli K-12

Expression of fimbriation was studied inEscherichia coli K-12 CA8000 HfrH, and itscya, crp and MS2 resistant mutants. The cells of cya⁺ crp⁺ parent strain were observed to be flagellated bacilli, lacking fimbriae, unable to agglutinate erythrocytes and deficient in ability to produce surface pellicle during growth in stationary culture. The cells ofcya andcrp mutants were observed to be cocci or coccobacilli devoid of flagella, having haemagglutinating activity, fimbriated and capable of producing surface pellicle in stationary cultures. The fimbriation and haemagglutinating activities were lower incya mutants grown with cAMP supplementation. Thecya andcrp mutants produced relatively small, smooth and compact colonies consisting mostly of fimbriated cells, like those of earlier described Fimσ mutants. Thecya ⁺ crp⁺ MS2 resistant mutant produced large sized colonies like those of parent but was deficient in conjugal donor ability. It resembledcya andcrp mutants in haemagglutinating and fimbriation properties. Thecya andcrp mutants have been earlier shown to be deficient in several Tra functions including conjugal donor ability. It is concluded thatEscherichia coli K-12 cells express fimbriation when Tra functions of F-plasmid carried in them are not expressed either due to deficiency of active cAMP-receptor protein complex or mutation in F-plasmid or when F-plasmid is absent.     

Multiple regulation of nucleoside catabolizing enzymes in Escherichia coli: effects of 3:5'' cyclic AMP and CRP protein.

Summary The regulation of the synthesis of nucleoside metabolizing enzymes has been studied in cya and crp mutant strains of Escherichia coli.The synthesis of the cyt-enzymes, cytidine deaminase and uridine phosphorylase regulated by the cytR gene product, is activated by the cAMP-CRP complex. On the other hand the synthesis of the deoenzymes: deoxyriboaldolase, thymidine phosphorylase, phosphodeoxyribomutase and purine nucleoside phosphorylase, appears to be increased if an active cAMP-CRP complex cannot be formed.It also seems that nucleosides serve as poor carbon sources for cya and crp mutants; this could not solely be explained by low levels of nucleoside metabolizing enzymes nor by a deficiency in nucleoside uptake. Addition of casamino acids stimulated the growth of cya and crp mutants, with nucleosides as carbon sources. When grown on glucose and casamino acids growth could be stimulated by adenine and hypoxanthine nucleosides; these results suggest an impaired nitrogen metabolism in cya and crp mutants.Abbreviations and Symbols cAMP cyclic adenosine 3:5-monophosphate - CRP cAMP receptor protein. Genes coding for: adenyl cyclase - cya cAMP receptor protein - crp cytidine deaminase - cdd uridine phosphorylase - udp thymidine phosphorylase - tpp purine nucleoside phosphorylase - pup; cytR regulatory gene for cdd, udp, dra, tpp, drm, and pup - deoR regulatory gene for dra, tpp, drm, and pup     

Phosphorylation and Functional Properties of the IIA Domain of the Lactose Transport Protein of Streptococcus thermophilus

The lactose-H⁺ symport protein (LacS) of Streptococcus thermophilus has a carboxyl-terminal regulatory domain (IIA^LacS) that is homologous to a family of proteins and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in various organisms, of which IIA^Glc of Escherichia coli is the best-characterized member. On the basis of these similarities, it was anticipated that IIA^LacS would be able to perform one or more functions associated with IIA^Glc, i.e., carry out phosphoryl transfer and/or affect other catabolic functions. The gene fragment encoding IIA^LacS was overexpressed in Escherichia coli, and the protein was purified in two steps by metal affinity and anion-exchange chromatography. IIA^LacS was unable to restore glucose uptake in a IIA^Glc-deficient strain, which is consistent with a very low rate of phosphorylation of IIA^LacS by phosphorylated HPr (HPr~P) from E. coli. With HPr~P from S. thermophilus, the rate was more than 10-fold higher, but the rate constants for the phosphorylation of IIA^LacS (k₁ = 4.3 × 10² M⁻¹ s⁻¹) and dephosphorylation of IIA^LacS~P by HPr (k₋₁ = 1.1 × 10³ M⁻¹ s⁻¹) are still at least 4 orders of magnitude lower than for the phosphoryltransfer between IIA^Glc and HPr from E. coli. This finding suggests that IIA^LacS has evolved into a protein domain whose main function is not to transfer phosphoryl groups rapidly. On the basis of sequence alignment of IIA proteins with and without putative phosphoryl transfer functions and the known structure of IIA^Glc, we constructed a double mutant [IIA^LacS(I548E/G556D)] that was predicted to have increased phosphoryl transfer activity. Indeed, the phosphorylation rate of IIA^LacS(I548E/G556D) by HPr~P increased (k₁ = 4.0 × 10³ M⁻¹ s⁻¹) and became nearly independent of the source of HPr~P (S. thermophilus, Bacillus subtilis, or E. coli). The increased phosphoryl transfer rate of IIA^LacS(I548E/G556D) was insufficient to complement IIA^Glc in PTS-mediated glucose transport in E. coli. Both IIA^LacS and IIA^LacS(I548E/G556D) could replace IIA^Glc, but in another function: they inhibited glycerol kinase (inducer exclusion) when present in the unphosphorylated form.     

The cya gene region of Erwinia chrysanthemi B374: organisation and gene products

Summary A DNA fragment containing the cya gene region of Erwinia chrysanthemi, B374 was cloned in vivo and transferred into cells of E. coli using a plasmid pULB113 derived from RP4 followed by subcloning in vitro into the vector pBR322. The cya gene encodes a 95 kDal protein that complemented E. coli cya mutants. Apparently, cya genes truncated at the 3 end could still produce proteins complementing cya-defective strains, thus showing that adenylate cyclase truncated at its carboxy-terminal end could synthesise cAMP. A protein of unknown function (40 kDal) is encoded by a gene that is transcribed divergently from the control region of the adenylate cyclase gene.     

Interaction of the CRP-cAMP complex with the cea regulatory region

Summary Analysis of the induction of expression of cea-lacZ fusions in cya and crp mutants showed that catabolite repression affects the kinetics of induction and the rate of induced synthesis. In a cya mutant, addition of cAMP reduced the induction lag and increased the amount of -galactosidase produced. The CRP-cAMP complex was found to bind to two sites 5 to the cea promoter, but deletion analysis showed that only one of these was involved in the control of cea. Deletion of this site resulted in a loss of the stimulatory effects of cAMP in a cya mutant.     

Resistance to mecillinam produced by the co-operative action of mutations affecting iipopolysaccharide, spoT, and cya or crp genes of Salmonella typhimurium

Lipopolysaccharide (LPS), spoT, and cya or crp mutations individually do not affect the minimum inhibitory concentration of mecillinam on Salmonella typhimurium. However, when mutations of two of these types were combined in the same strain, high-level resistance appeared, and increased even further when all three types of mutations were present. Most mutations affecting LPS (rfa, rfb, rfc) showed this behaviour, although to different degrees. The highest resistance to mecillinam was caused by galE and rfc mutations whereas almost no effect was noticed with rfaB or rfaK mutations. This phenomenon appears to be specific for mecillinam since none of several other antibiotics elicited it. Reduction of guanosine tetraphosphate (ppGpp) levels by introduction of a relA mutation did not significantly affect the MIC of mecillinam on strains carrying different combinations of SpoT, galE, and cya or crp mutations. All the strains produced spherical cells in medium with a low concentration (0.05 μg ml^?1) of the antibiotic. These results suggest that the antibacterial action of mecillinam on S. typhimurium is somehow dependent on the interaction of LPS, cyclic AMP/cyclic AMP receptor protein (cAMP/CRP), and SpoT. The reported resistance to mecillinam of cya and crp mutants of Escherichia coli K-12 is probably due to the natural LPS defectiveness of this strain.