Reaction of 2-azido-ATP with beta subunits in the F1-adenosine triphosphatase of Escherichia coli

The three beta subunits of the Escherichia coli F1-ATPase react independently with chemical reagents (Stan Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 248, 116-120). Thus, one beta subunit is readily cross-linked to the epsilon subunit, another reacts with N,N'-dicyclohexylcarbodiimide (DCCD), and the third one is modified by 4-chloro-7-nitrobenzofurazan (NbfCl). The relationship of the binding site for 2-azido-ATP to the three types of beta subunit recognized by chemical labeling was examined. The binding site for 2-azido-ATP was not associated with a specific type of beta-subunit. There was no relationship between the site of nucleotide and the association of the epsilon subunit with a particular beta subunit. It is concluded that the presence of the epsilon subunit (possibly in association with the other minor subunits) does not determine the position of the catalytic site. The possibility that the lack of a specific relationship between the 2-azido-ATP binding site and a specific beta subunit was due to turnover of the enzyme, making each beta a catalytic site in turn, could not be entirely rejected. However, the rate of hydrolysis of 2-azido-ATP by the DCCD-modified ATPase was very low in the presence of EDTA, and was likely due to catalysis at single sites.     

Frequency of recombination exchanges in Escherichia coli K-12: genetic determinants controlling this frequency

RecF, recQ, ruv, recJ and recN genes of so called RecF pathway of recombination appear to be not silent on the RecBCD pathway also. These genes are responsible for the frequency of recombination exchanges per unit length of DNA. The list: recF::Kmr greater than recQ::Tn3 greater than ruv54 greater than recJ::Tn9 demonstrated the efficiency of inhibition of recombination exchanges by these mutations. The recN262 mutation gives a feeble contrary effect. It slightly increases the frequency of recombination exchanges.     

Lysophospholipase L1 from Escherichia coli K-12 overproducer

After screening 900 E. coli strains of the Clarke and Carbon collection for by lysophospholipase L1 activities, we isolated a clone bearing the plasmid pLC6-34, which showed an increased level of lysophospholipase L1 activity. Strains bearing the plasmid pC124, a subclone of pLC6-34 in plasmid vector pUC8, showed approximately 11.4 times higher lysophospholipase L1 activity than that of the parental strain. Starting from those overproducing strains, the lysophospholipase L1 was purified to near homogeneity by sequential use of ammonium sulfate fractionation, Sephacryl S-300, DEAE-cellulose, hydroxyapatite and Sephacryl S-200 column chromatographies. The apparent molecular weight of the purified lysophospholipase L1 was estimated to be 20,500-22,000 both by SDS-polyacrylamide gel electrophoresis and by gel permeation chromatography. The specific activity of the homogeneous lysophospholipase L1 was 10,400 nmol/min/mg protein when 1-acyl-sn-glycero-3-phosphoethanolamine was used as the substrate. The amino acid sequence of the amino-terminal portion of purified lysophospholipase L1 was determined and was different from that of lysophospholipase L2, which had previously been purified from the envelope fraction of E. coli strains bearing its cloned structural gene, pldB [Karasawa, K., Kudo, I., Kobayashi, T., Sa-eki, T., Inoue, K., & Nojima, S. (1985) J. Biochem, 98, 1117-1125]. The gene responsible for overproduction of lysophospholipase L1 activity was designated as pldC (phospholipid degradation C). Its restriction enzyme map was also different from that of cloned pldB. These results further confirmed that, in E. coli, there are two lysophospholipases with distinct characteristics.     

Reaction of membrane-bound F1-adenosine triphosphatase of Escherichia coli with chemical ligands and the asymmetry of beta subunits

The three beta subunits of the isolated Escherichia coli F1-ATPase react independently with chemical reagents (Stan-Lotter, H. and Bragg, P.D. (1986) Arch. Biochem. Biophys. 284, 116-120). Thus, one beta subunit is readily cross-linked to the epsilon subunit, Another reacts with N,N'-dicyclohexylcarbodiimide (DCCD), and the third one is modified on a lysine residue by 4-chloro-7-nitrobenzofurazan (NbfCl). The binding site for the ATP analog, 2-azido-ATP, was not associated with a specific type of beta subunit (Bragg, P.D. and Hou, C. (1989) Biochim. Biophys. Acta 974, 24-29). We now show that this binding site is a catalytic site as opposed to a noncatalytic nucleotide-binding site. NbfCl reacted with a tyrosine residue on the DCCD-reacting beta subunit in contrast to the different subunit location of the lysine residue labeled by the reagent. Thus, O to N transfer of the Nbf group in the free F1-ATPase involves transfer between subunits. The chemical labelling pattern of membrane-bound F1-ATPase differed from that of free F1. The strict asymmetry of labeling of the free F1-ATPase was not observed. Thus, double labeling of beta subunits by several reagents was found. This suggests that the asymmetry was not induced by chemical modification, but is inherent in the structure of the ATPase.     

gamma-Glutamyltranspeptidase from Escherichia coli K-12: purification and properties.

gamma-Glutamyltranspeptidase (GGT) (EC 2.3.2.2) was purified from the periplasmic fraction of Escherichia coli K-12 to electrophoretic homogeneity. The final purification step, chromatofocusing, gave two protein peaks showing GGT activity (fractions A and B). The major heavy fraction (fraction A) consisted of two different subunits, with molecular weights of 39,200 and 22,000. The minor light fraction (fraction B) consisted of those with molecular weights of 38,600 and 22,000. Fraction A catalyzes the hydrolysis and transpeptidation of all gamma-glutamyl compounds tested, but it prefers basic amino acids and aromatic amino acids as acceptors. The apparent Km values for glutathione and gamma-glutamyl-p-nitroanilide as gamma-glutamyl donors in the transpeptidation reaction were both 35 microM, and those for glycylglycine and L-arginine as acceptors were 0.59 and 0.21 M, respectively. The enzyme was inhibited by some amino acids and by protease inhibitors and affinity-labeling reagents for GGT. The temperature stability of the purified GGT supports our hypothesis that E. coli GGT is synthesized only at lower temperature rather than that the synthesized GGT is degraded or inactivated at higher temperature.     

Recombination and the Escherichia coli K-12 sex factor F.

Recombination between two Flac tra minus elements to give Flac tra plus recombinants was measured in Rec plus and Rec minus strains of Escherichia coli K-12. Polar tra mutations were used to increase the proportion of tra plus recombinants among the parental Flac tra minus elements transferred by complementation. The kinetics, measured in a rec plus strain, showed that recombination began about 1 h after the initiation of mating and was completed about 1 h later. Recombination was abolished in a recA minus strain, reduced by two-thirds in a recF minus strain, and unaffected in recB minus and recC minus strains. It is proposed that the part not due to the RecF pathway results from a RecBC- and RecF-independent system for formation of single-stranded joins. One such join could be followed either by transfer and a site-specific recombination event, or by a second single-stranded join and then transfer: in either case replication and inheritance of the recombinant molecule would be dependent upon the F transfer replication system. Chromosome mobilization by an F' element was normal in a recB plus recF minus strain, and was reduced only fourfold in a recB minus recF plus strain: in the latter strain, both the RecF pathway and the system for single-stranded joins may have contributed to mobilization. Measurement of post-conjugational chromosomal recombination in exponential-phase recipient cells carrying surface exclusion-deficient Flac mutants indicated that F does not itself determine a generalized recombination system able to replace the RecA plus product or the RecBC and RecF pathways.     

Purification and properties of inosine-guanosine phosphorylase from Escherichia coli K-12.

A xanthosine-inducible enzyme, inosine-guanosine phosphorylase, has been partially purified from a strain of Escherichia coli K-12 lacking the deo-encoded purine nucleoside phosphorylase. Inosine-guanosine phosphorylase had a particle weight of 180 kilodaltons and was rapidly inactivated by p-chloromercuriphenylsulfonic acid (p-CMB). The enzyme was not protected from inactivation by inosine (Ino), 2'-deoxyinosine (dIno), hypoxanthine (Hyp), Pi, or alpha-D-ribose-1-phosphate (Rib-1-P). Incubating the inactive enzyme with dithiothreitol restored the catalytic activity. Reaction with p-CMB did not affect the particle weight. Inosine-guanosine phosphorylase was more sensitive to thermal inactivation than purine nucleoside phosphorylase. The half-life determined at 45 degrees C between pH 5 and 8 was 5 to 9 min. Phosphate (20 mM) stabilized the enzyme to thermal inactivation, while Ino (1 mM), dIno (1 mM), xanthosine (Xao) (1 mM), Rib-1-P (2 mM), or Hyp (0.05 mM) had no effect. However, Hyp at 1 mM did stabilize the enzyme. In addition, the combination of Pi (20 mM) and Hyp (0.05 mM) stabilized this enzyme to a greater extent than did Pi alone. Apparent activation energies of 11.5 kcal/mol and 7.9 kcal/mol were determined in the phosphorolytic and synthetic direction, respectively. The pH dependence of Ino cleavage or synthesis did not vary between 6 and 8. The substrate specificity, listed in decreasing order of efficiency (V/Km), was: 2'-deoxyguanosine, dIno, guanosine, Xao, Ino, 5'-dIno, and 2',3'-dideoxyinosine. Inosine-guanosine phosphorylase differed from the deo operon-encoded purine nucleoside phosphorylase in that neither adenosine, 2'-deoxyadenosine, nor hypoxanthine arabinoside were substrates or potent inhibitors. Moreover, the E. coli inosine-guanosine phosphorylase was antigenically distinct from the purine nucleoside phosphorylase since it did not react with any of 14 monoclonal antisera or a polyvalent antiserum raised against deo-encoded purine nucleoside phosphorylase.     

luxS-dependent gene regulation in Escherichia coli K-12 revealed by genomic expression profiling

The bacterial quorum-sensing autoinducer 2 (AI-2) has received intense interest because the gene for its synthase, luxS, is common among a large number of bacterial species. We have identified luxS-controlled genes in Escherichia coli under two different growth conditions using DNA microarrays. Twenty-three genes were affected by luxS deletion in the presence of glucose, and 63 genes were influenced by luxS deletion in the absence of glucose. Minimal overlap among these gene sets suggests the role of luxS is condition dependent. Under the latter condition, the metE gene, the lsrACDBFG operon, and the flanking genes of the lsr operon (lsrR, lsrK, tam, and yneE) were among the most significantly induced genes by luxS. The E. coli lsr operon includes an additional gene, tam, encoding an S-adenosyl-l-methionine-dependent methyltransferase. Also, lsrR and lsrK belong to the same operon, lsrRK, which is positively regulated by the cyclic AMP receptor protein and negatively regulated by LsrR. lsrK is additionally transcribed by a promoter between lsrR and lsrK. Deletion of luxS was also shown to affect genes involved in methionine biosynthesis, methyl transfer reactions, iron uptake, and utilization of carbon. It was surprising, however, that so few genes were affected by luxS deletion in this E. coli K-12 strain under these conditions. Most of the highly induced genes are related to AI-2 production and transport. These data are consistent with the function of LuxS as an important metabolic enzyme but appear not to support the role of AI-2 as a true signal molecule for E. coli W3110 under the investigated conditions.     

Purification and properties of orotate phosphoribosyltransferases from Escherichia coli K-12, and its derivative purine-sensitive mutant

Orotate phosphoribosyltransferase (OPT) was purified from both Escherichia coli K-12 strain and its derivative, a purine-sensitive mutant. The wild-type OPT had a molecular weight (M.W.) of 47,000 and was composed of two identical subunits (M.W. 23,500). The wild-type OPT showed maximum activity at pH 9.5, and no activity was seen in the absence of Mg2+ or Mn2+ ion. It also catalyzed a reverse reaction, namely orotidine-5'-monophosphate (OMP) pyrophosphorolysis. In this reverse reaction, tripolyphosphate, tetrapolyphosphate, and trimetaphosphate were also effective as pyrophosphate donors. The apparent Km values of the wild-type OPT were 30 microM for orotate and 40 microM for 5-phosphoribosyl 1-pyrophosphate (PRib-PP), and also 3.6 microM for OMP and 13 microM for PPi. On the other hand, the mutant OPT showed increased apparent Km values for all four substrates, 440 microM for orotate, 360 microM for PRib-PP, 33 microM for OMP, and 250 microM for PPi. The mutant OPT required a higher concentration of Mg2+ ion for maximum activity than the wild-type OPT. The nature of the purine-sensitive phenotype of the mutant is discussed from the standpoint of the reactivity of the mutant OPT, which has an increased Km value for PRib-PP (about 9-fold).     

Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12

We have cloned genes responsible for enterobactin synthesis (entD) and transport (fepA,fes) from Escherichia coli K-12. Relevant recombinant plasmids enabled EntD- and transport-defective mutants to grow on iron-limiting medium. Subcloning and deletion analysis demonstrated that the gene order is entD-fepA-fes. Protein synthesis studies in minicells suggest that FepA is first translated as an Mr 84 000 precursor, which is subsequently cleaved to the active Mr 81 000 receptor; the fes gene product is an Mr 44 000 protein; no polypeptide has been identified as the entD gene product.     

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.     

Dynamics of IS-related genetic rearrangements in resting Escherichia coli K-12

An analysis of restriction fragment length polymorphism (RFLP) using eight residential insertion sequence (IS) elements as hybridization probes reveals that the genome of resting bacteria is more dynamic than it was long believed. Escherichia coli strains stored in agar stabs for up to 30 yr accumulate a genetic variation which is correlated to time of storage. This spontaneous mutagenesis is often IS-specific, with particularly high activity for IS5, and thus suggests that transpositional DNA rearrangements are a major cause for the observed genetic polymorphism. The RFLP patterns indicate a burst of IS30 transposition to occur occasionally. Mutation rate is estimated by two different methods to roughly 10(-5) IS-related DNA rearrangements per bacterial chromosome per hour of storage for the eight IS elements studied. A pedigree derived from the RFLP data reveals that populations had evolved independently in each stab and showed no signs of convergence. Relics of an assumed ancestral population were still present in the stab cultures, but the elder stabs provided mostly mutants. These results indicate that cells placed under nutritional deprivation might have a highly plastic genome and suggest that such plasticity might play an adaptive role.      

Optimization of the purification of mitochondrial F1-adenosine triphosphatase.

A simple technique of purification of the soluble pig heart mitochondrial F1-ATPase is described. It consists of removal of extrinsic proteins from mitochondrial membranes before extraction with chloroform and ammonium sulfate fractionation. A high degree of purity, an excellent stability and a good yield are attained after gel filtration through an Ultrogel ACA 34 column equilibrated in the presence of 50% glycerol. The tested properties of the F1-ATPase prepared by this method are similar to those of the same enzyme extracted by sonication. The enzyme is virtually devoid of tightly bound nucleotides. In addition, some characteristics of the behaviour of the beta subunit are shown.     

A genome rearrangement has orphaned the Escherichia coli K-12 AcpT phosphopantetheinyl transferase from its cognate Escherichia coli O157:H7 substrates

Phosphopantetheinyl transferases (PPTases) are enzymes that catalyse the transfer of a 4'-phosphopantetheine moiety from CoA to a conserved serine residue of a carrier protein. These carrier proteins use the 4'-phosphopantetheine thiol to shuttle intermediates between the active sites of biosynthetic enzymes involved in fatty acid, non-ribosomal peptide and polyketide synthesis. Three PPTases have been previously been identified in Escherichia coli K-12 and other E. coli strains by homology searches and are encoded by the genes acpS, entD and acpT. Both AcpS and EntD have been well studied whereas the function of AcpT has been an enigma because no carrier protein substrate could be found. We report genetic and biochemical evidence that AcpT modifies two carrier proteins encoded in O-island 138, a cluster of fatty acid biosynthesis-like genes located adjacent to acpT in the genome of the pathogenic E. coli strain O157:H7 (E. coli K-12 and several other sequenced E. coli and Shigella strains lack O-island 138). The two carrier proteins of O-island 138 of strain O157:H7 are not modified (or only very poorly modified) by AcpS, the PPTase responsible for 4'-phosphopantetheine attachment to the acyl carrier protein (AcpP) of fatty acid synthesis. We demonstrate that AcpT cannot functionally replace AcpS in E. coli K-12 either in its native chromosomal location or upon insertion of acpT into the acpS chromosomal location. However, in the absence of AcpS activity AcpT does allow very slow growth thus providing a rationale for its retention in the absence of its cognate substrates. These results together with phylogenetic analyses and comparisons of the E. coli and Shigella strains of known genome sequence strongly argue that AcpT has been orphaned from its cognate substrates by a deletion event that occurred in a common ancestor of these organisms. This seems one of the few cases where a chromosomal rearrangement has been functionally demonstrated to be a deletion event rather than an insertion event in the reference organism. We also show that the previously reported suppression of an acpS mutation by the deletion of Lon protease is an artifact of the increased capsular polysaccharide production of lon strains.