Intracistronic mapping of the defective site and the biochemical properties of beta subunit mutants of Escherichia coli H+-ATPase: correlation of structural domains with functions of the beta subunit

Sixteen mutants of Escherichia coli defective in H+-ATPase (proton-translocating ATPase) were tested for their ability to recombine with hybrid plasmids carrying various portions of the beta subunit cistron. Twelve mutations were mapped within the carboxyl half of the cistron corresponding to amino acid residues 279 to 459 (domain II), while four mutations were mapped within residues 17 to 278 (domain I). The biochemical properties of these mutants were analyzed in terms of the proton permeability of their membranes and the assembly properties of their F1F0 complex. The mutants were classified according to the properties into three types, I, II, and III. In 12 mutants of type I, proton conduction in membrane vesicles was blocked and little F1 was released from the membranes under conditions in which F1 could be released from wild-type membranes, suggesting that assembly of the F1F0 complex is structurally and functionally defective. F1 was partially purified with very low recovery from one of the type I mutants, KF16. ATPase activity was reconstituted from this F1 with the beta subunit of the wild type, confirming the genetic results. Only one mutant, KF38, was classified as type II. Its membranes were partially leaky to protons and its F1 was releasable, suggesting that the interaction of its F1 and F0 was unstable. Type III mutants, KF11 and KF43, had an F1F0 complex with very low activity, in which the structure of F1 was relatively similar to that of the wild type. F1 was purified as a single complex from KF43 in this study and from KF11 previously (H. Kanazawa, Y. Horiuchi, M. Takagi, Y. Ishino, and M. Futai (1980) J. Biochem. 88, 695-703). Reconstitution experiments in vitro showed that the F1's of both mutants were defective in the beta subunit. The properties of the altered F1 of KF43 differed from those of F1 of KF11, suggesting that the mutation sites of KF43 and KF11 were different. From the results of mapping mutation sites and the biochemical properties of the mutants, the correlation of structural domains with function of the beta subunit is discussed. Most type I and type II mutations except that of KF39 were mapped in domain II, while the type III mutations were mapped in domain I, suggesting that domain II is more important than domain I for the function of the beta subunit, especially in terms of proper assembly of the F1F0 complex.     

The secondary attachment site for bacteriophage lambda in the proA/B gene of Escherichia coli

We have determined the nucleotide sequence of a secondary λ attachment site in $\text{pro}\text{A}\text{B}$, a site that accounts for 3% of lysogens isolated from Escherichia coli strains deleted for the primary site. Direct sequence analysis of the transducing bacteriophages carrying the left and right att junctions, as well as the recombinant pro⁺ phage reveals that the $\text{pro}\text{A}\text{B}$ site shares an 11-nucleotide interrupted homology with the core sequence of the primary site. We have compared the $\text{pro}\text{A}\text{B}$att site with other secondary attachment sites to gain insights into the structural features important for λ integration.     

Further sequence analysis of the phage lambda receptor site. Possible implications for the organization of the lamB protein in Escherichia coli K12

We present the DNA sequence alterations due to seven lamB missense mutations yielding resistance to phages lambda and K10. They reveal five different amino acid positions in the LamB protein. Three positions (245, 247 and 249) define a new region required for phage adsorption. The two other positions (148 and 152) belong to a region where mutations to phage resistance has already been detected. These two regions are hydrophilic and could belong to turns of the protein located at the surface of the cell. All the missense mutational alterations to phage resistance sequenced in the LamB protein correspond to 10 sites located in four different segments of the polypeptide chain. We discuss their location in terms of the notion of phage receptor site and of a working model for the organization of this protein in the outer membrane of Escherichia coli.     

EcoA and EcoE: alternatives to the EcoK family of type I restriction and modification systems of Escherichia coli

The genes (hsd A) encoding EcoA, a restriction and modification system first identified in Escherichia coli 15T-, behave in genetic crosses as alleles of the genes (hsd K) encoding the archetypal type I restriction and modification system of E. coli K12. Nevertheless, molecular experiments have failed to detect relatedness between the A and K systems. We have cloned the hsd A genes and have identified, on the basis of DNA homology, related genes (hsd E) conferring a new specificity to a natural isolate of E. coli. We show that the overall organization of the genes encoding EcoA and EcoE closely parallels that for EcoK. Each enzyme is encoded by three genes, of which only one, hsdS, confers the specificity of DNA interaction. The three genes are in the same order as those encoding EcoK, i.e. hsdR, hsdM and hsdS and, similarly, they include a promoter between hsdR and hsdM from which the M and S genes can be transcribed. The evidence indicates that EcoA and EcoE are type I restriction and modification enzymes, but they appear to identify an alternative family to EcoK. For both families, the hsdR polypeptide is by far the largest, but the sizes of the other two polypeptides are reversed, with the smallest polypeptide of EcoK being the product of hsd S, and the smallest for the EcoA family being the product of hsdM. Physiologically, the A restriction and modification system differs from that of K and its relatives, in that A-specific methylation of unmodified DNA is particularly effective.     

Nucleotide sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli

The pstA(=phoT), pstB and phoU genes are situated at 84 minutes on the Escherichia coli genetic map. All of them are involved in the negative regulation of the phosphate regulon, and all of them except for phoU are required for the binding-protein-mediated, highly specific phosphate transport. We have determined the DNA sequence of about 4 X 10(3) bases of chromosomal segment containing these genes. Four translational reading frames (TRFs) were detected in the region. We attempted to assign the TRFs to the mutant alleles. Plasmids were constructed so that each contained only one of the TRFs, downstream from the lac promoter, to be used for the complementation tests. By this test, TRF-2, TRF-3 and TRF-4 were identified with the pstA(=phoT), pstB and phoU genes, respectively. Alkaline phosphatase-constitutive mutations of the two strains in our collection were complemented by the plasmid with the TRF-1 region. Therefore, we propose to designate the allele phoW. The order of the genes in this region has been established to be phoS-phoW-pstA(=phoT)-pstB-phoU counterclockwise on the E. coli genetic map.     

Inhibitory properties of endogenous subunit epsilon in the Escherichia coli F1 ATPase.

Homogeneous ? bound tightly to the purified Escherichia coli ATPase (ECF₁ from which ? had been removed and strongly inhibited its ATPase activity. ECF₁ containing ? had a lower specific activity than ECF₁ missing ?, provided that the ATPase assay was carried out at relatively high concentrations of enzyme. Antiserum specific for the ? subunit stimulated the ATPase, as did diluting the enzyme, apparently by dissociating ?. When the ATPase reaction was started by the addition of enzyme, the rate of ATP hydrolysis increased progressively during the first 3 min until a linear steady-state rate was reached. A prior incubation with ATP abolished the lag period and ADP prevented the ATP effect. ECF₁ missing ? gave a linear rate of ATP hydrolysis without a lag, unless ? was rebound to it before the assay. These results suggest that ECF₁ as purified is in an inhibited state due to the presence of the ? subunit, whose interaction with ECF₁ is governed by an equilibrium binding. ATP appears to convert ECF₁ to a form which more readily binds and releases ?.     

Non-random layout of the amino acid loci on the genome of Escherichia coli

A strong correlation exists between the relative frequencies of occurrence of the amino acids in bulk Escherichia coli protein and their genetic map positions when the latter are indexed against the position of the origin of DNA synthesis. The greater the production of the amino acid, the closer its operon is to the origin.     

Refined structure of alkaline phosphatase from Escherichia coli at 2.8 A resolution

The structure of alkaline phosphatase from Escherichia coli has been determined to 2.8 A resolution. The multiple isomorphous replacement electron density map of the dimer at 3.4 A was substantially improved by molecular symmetry averaging and solvent flattening. From these maps, polypeptide chains of the dimer were built using the published amino acid sequence. Stereochemically restrained least-squares refinement of this model against native data, starting with 3.4 A data and extending in steps to 2.8 A resolution, proceeded to a final overall crystallographic R factor of 0.256. Alkaline phosphatase-phosphomonoester hydrolase (EC 3.1.3.1) is a metalloenzyme that forms an isologous dimer with two reactive centers 32 A apart. The topology of the polypeptide fold of the subunit is of the alpha/beta class of proteins. Despite the similarities in the overall alpha/beta fold with other proteins, alkaline phosphatase does not have a characteristic binding cleft formed at the carboxyl end of the parallel sheet, but rather an active pocket that contains a cluster of three functional metal sites located off the plane of the central ten-stranded sheet. This active pocket is located near the carboxyl ends of four strands and the amino end of the antiparallel strand, between the plane of the sheet and two helices on the same side. Alkaline phosphatase is a non-specific phosphomonoesterase that hydrolyzes small phosphomonoesters as well as the phosphate termini of DNA. The accessibility calculations based on the refined co-ordinates of the enzyme show that the active pocket barely accommodates inorganic phosphate. Thus, the alcoholic or phenolic portion of the substrate would have to be exposed on the surface of the enzyme. Two metal sites, M1 and M2, 3.9 A apart, are occupied by zinc. The third site, M3, 5 A from site M2 and 7 A from site M1, is occupied by magnesium or, in the absence of magnesium, by zinc. As with other zinc-containing enzymes, histidine residues are ligands to zinc site M1 (three) and to zinc site M2 (one). Ligand assignment and metal preference indicate that the crystallographically found metal sites M1, M2 and M3 correspond to the spectroscopically deduced metal sites A, B and C, respectively. Arsenate, a product analog and enzyme inhibitor, binds between Ser102 and zinc sites M1 and M2. The position of the guanidinium group of Arg 166 is within hydrogen-bonding distance from the arsenate site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Essential interaction between lambdoid phage 21 terminase and the Escherichia coli integrative host factor

Lambdoid phage 21 requires the Escherichia coli integrative host factor (IHF) for growth. lambda-21 hybrids that have 21 DNA packaging specificity also require IHF. IHF-independent (her) mutants have been isolated. her mutations map in the amino-terminal half of the 21 1 gene. The 1 gene encodes the small subunit of the 21 terminase, and the amino-terminal half of the 1 polypeptide is a functional domain for specifically binding 21 DNA. Hence changes in the DNA-binding domain of terminase, her mutations, render 21 terminase able to function in the absence of IHF. Three of four her mutations studied are trans-dominant. An in vitro system was used to show that packaging of 21 DNA is IHF-dependent. IHF is directly required during the early, terminase-dependent steps of assembly. It is concluded that IHF is a host factor required for function of the 21 terminase. It is proposed, in analogy to the role of IHF in lambda integration, that IHF facilitates proper binding of 21 terminase to phage DNA. Consistent with this proposal, possible IHF-binding sites are present in the 21 cohesive end site.     

Conservation and DNA sequence arrangement of the DNA polymerase I gene region from Klebsiella aerogenes, Klebsiella pneumoniae and Escherichia coli

Linked multiple mutation is observed after treatment of Escherichia coli with methyl methanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and N-ethyl-N-nitro-N-nitrosoguanidine but not ultraviolet light. Induction of linked multiple mutations requires the uvrE⁺ gene product indicating the involvement of the mismatch repair system. The observation of linked multiple mutations is not due to mutagenesis occurring in a subpopulation of cells. Growing point mutagenesis also occurs after treatment with these mutagens but not with ultraviolet light. It is likely that the excess of mutations observed with these mutagens at growing points is at least partly a relative effect, rather than one due to an absolute increase of reactivity at the DNA growing point region. This relative effect may result from the operation of an inducible repair mechanism which removes O⁶-alkylguanine residues from the DNA distal to the bacterial growing point. The adaptive response, first described by Robins &; Cairns (1979) prefers O⁶-methylguanine over O⁶-ethylguanine.     

Immunochemical analysis of molecular forms of protein synthesis initiation factors in crude cell lysates of Escherichia coli

Three initiation factors (IF1, IF2, and IF3) have been highly purified from Escherichia coli and extensively characterized, but little is known about the molecular forms of these proteins as they occur in vivo. We have analyzed molecular-weight and charge forms in crude cell lysates by polyacrylamide gel electrophoresis followed by immunoblotting with antibodies specific for the initiation factors. Freshly grown bacterial cells were lysed by sonication in buffer containing sodium dodecyl sulfate, and the lysate was fractionated by gel electrophoresis. Proteins from the gel were electrotransferred to a nitrocellulose sheet which was treated with a specific rabbit antiserum followed by radiolabeled Staphylococcus aureus protein A. Autoradiography showed only one major band each for IF1 and IF3, exactly corresponding to the isolated factors. For IF2, two molecular-weight forms were detected which were identical with purified IF2a and IF2b. No evidence for precursor forms was found. Two-dimensional gel analysis showed no charge heterogeneity for IF1, IF2a, and IF3, but multiple forms were seen for IF2b. Analysis of phosphoproteins from cells grown in radioactive phosphate medium ruled out the possibility that phosphorylation occurs on the initiation factors, elongation factors, or ribosomal proteins.     

Sequence information within the lamB genes in required for proper routing of the bacteriophage lambda receptor protein to the outer membrane of Escherichia coli K-12

The structure of Satellite tobacco necrosis virus (STNV) has been determined to 3.0 Å resolution by X-ray crystallography. Electron density maps were obtained with phases based on one heavy-atom derivative and several cycles of phase refinement using the 60-fold non-crystallographic symmetry in the particle. A model for one protein subunit was built using a computer graphics display. The subunit is constructed mainly of a β-roll structure forming two β-sheets, each of four antiparallel strands. The N-termini of the subunits form bundles of three α-helices extending into the RNA region of the virus at the 3-fold axis. The topology of the polypeptide chain is the same as, and the conformation clearly similar to, that of the shell domains of the Tomato bushy stunt virus (TBSV) and Southern bean mosaic virus (SBMV) protein subunits. The subunit packing in the T = 1 STNV structure is, however, significantly different from the packing of these T = 3 viruses: parts of some of the structural elements facing the RNA in TBSV and SBMV are utilized for subunit-subunit contacts in STNV. No RNA structure is obvious in the present icosahedrally averaged electron density maps. The protein surface facing the RNA contains mainly hydrophilic residues, especially lysine and arginine.     

A high-affinity calcium-stimulated ATPase activity in the hen oviduct shell gland

The hen oviduct shell gland is a highly active calcium-transporting epithelial tissue which is responsible for the mineralization of the egg shell. We have identified a calcium-stimulated ATPase present at high specific activity in membrane preparations from shell gland mucosal shavings. In the presence of optimal MgCl₂ (5 mm) and a Ca²⁺ buffer, ATP hydrolysis was stimulated by addition of low concentrations of free Ca²⁺ (K_0.5 ~0.4 μm); but not by similar concentrations of Mn²⁺, Zn²⁺, Co²⁺, or La²⁺. This stimulation was specific for ATP; there was little or no effect of Ca²⁺ on hydrolysis of ADP, AMP, GTP, ITP, or p-nitrophenyl phosphate. Calcium-stimulated ATPase activity was inhibited by chlorpromazine, trifluoperazine, and quercetin, as well as by sulfhydryl-blocking agents, but not by oligomycin or ouabain. No significant effect of calmodulin was observed. Finally, low concentrations of free Ca²⁺ (10 to 100 μm) in the presence or absence of Mg²⁺ stimulated transfer of ³²P from [γ-³²P]ATP to a 105,000 molecular weight shell gland membrane protein. This phosphoprotein was sensitive to hydrolysis by heating or by hydroxylamine treatment at acidic pH, and its formation was not inhibited by addition of K⁺. The specific activity of Ca²⁺-ATPase in total membrane preparations from laying hen shell gland ranged from 80 to 150 nmol/min/ mg protein, similar to or greater than levels found in purified plasma membrane fractions from a variety of tissues. No significant activity was found in membrane preparations from the magnum or isthmus regions of the oviduct, which are not involved in egg shell calcification. The characteristics of the Ca²⁺-ATPase, its high specific activity, and its preferential localization in the shell gland region of the oviduct suggest a role for an ATP-dependent calcium transport system in egg shell mineralization.     

Structure-function relationship in Escherichia coli initiation factors. Environment of the Cys residue and evidence for a hydrophobic region in initiation factor IF3 by fluorescence and ESR spectroscopy

The reactions of rabbit muscle pyruvate kinase with 5′-p-fluorosulfonylbenzoyl adenosine (5′-FSBA) and 5′-p-fluorosulfonylbenzoyl guanosine (5′-FSBG) from pH 7.0 to 8.0 exhibit biphasic inactivation kinetics. These reactions are characterized by three events: a fast reaction yielding partially active enzyme (with 67% of its original activity for the 5′-FSBA reaction and 45% for the 5′-FSBG reaction) which is reactivated by dithiothreitol, and two slower reactions yielding fully inactive enzymes; the product of only one of the two slower reactions is reactivated by dithiothreitol. These reactions are termed fast dithiothreitol-sensitive, slow dithiothreitol-sensitive, and dithiothreitol-insensitive inactivations. The rates of all three phases of the reactions with 5′-FSBA and 5′-FSBG increase as the pH is raised. The 5′-FSBG reaction can be described in terms of initial reaction with a single ionizable group of pK_a 7.80, 8.60, and 7.94 for the fast dithiothreitol-sensitive, slow dithiothreitol-sensitive, and dithiothreitol-insensitive reactions, respectively; pH-independent rate constants of 0.173, 0.133, and 0.0165 min^?1 are calculated for these three phases of the overall reaction. The pH dependence of the dithiothreitol-insensitive inactivation by 5′-FSBA coincides with that for 5′-FSBG, but the data for the dithiothreitol-sensitive reactions with 5′-FSBA indicate that the reaction in each phase occurs at more than one site over the pH range tested. Differential protection by ligands against inactivation by 5′-FSBA and 5′-FSBG at pH 7.4 and 8.0 indicates that, for the fast dithiothreitol-sensitive reactions, the cysteine residues participating in the two reactions are not identical, although in both cases modification has been attributed to formation of a disulfide. For 5′-FSBA, the partial inactivation appears to result from modification of cysteine residues at the noncatalytic nucleotide site, whereas for 5′-FSBG the inactivation is due to modification within the catalytic metal-nucleotide site. Reaction with 5′-FSBG seems to occur at the same locus for both the fast and slow dithiothreitol-sensitive phases, with the rate difference being ascribable to negative cooperativity among subunits. For the slow dithiothreitol-sensitive inactivation by 5′-FSBA, protection by Mg²⁺ and by Mg²⁺ plus ADP suggests that the targets of modification include the active-site cysteine that is modified by 5′-FSBG. The dithiothreitol-insensitive inactivation, shown to be due to reaction of 5′-FSBA with a tyrosine, may result from reaction of both nucleotide analogs with the same residue, although differential protection by the natural ligands suggests that 5′-FSBA and 5′-FSBG bind to two subsites within the active site.     

Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening

The kinetics of formation and of dissociation of open complexes (RPo) between Escherichia coli RNA polymerase (R) and the lambda PR promoter (P) have been studied as a function of temperature in the physiological range using the nitrocellulose filter binding assay. The kinetic data provide further evidence for the mechanism R + P in equilibrium I1 in equilibrium I2 in equilibrium RPo, where I1 and I2 are kinetically distinguishable intermediate complexes at this promoter which do not accumulate under the reaction conditions investigated. The overall second-order association rate constant (ka) increases dramatically with increasing temperature, yielding a temperature-dependent activation energy in the range 20 kcal (near 37 degrees C) to 40 kcal (near 13 degrees C) (1 kcal = 4.184 kJ). Both isomerization steps (I1----I2 and I2----RPo) appear to be highly temperature dependent. Except at low temperatures (less than 13 degrees C) the step I1----I2, which we attribute to a conformational change in the polymerase with a large negative delta Cp degrees value, is rate-limiting at the reactant concentrations investigated and hence makes the dominant contribution to the apparent activation energy of the pseudo first-order association reaction. The subsequent step I2----RPo, which we attribute to DNA melting, has a higher activation energy (in excess of 100 kcal) but only becomes rate-limiting at low temperature (less than 13 degrees C). The initial binding step R + P in equilibrium I1 appears to be in equilibrium on the time-scale of the isomerization reactions under all conditions investigated; the equilibrium constant for this step is not a strong function of temperature and is approximately 10(7) M-1 under the standard ionic conditions of the assay (40 mM-Tris . HCl (pH 8.0), 10 mM-MgCl2, 0.12 M-KC1). The activation energy of the dissociation reaction becomes increasingly negative at low temperatures, ranging from approximately -9 kcal near 37 degrees C to -30 kcal near 13 degrees C. Thermodynamic (van't Hoff) enthalpies delta H degrees of open complex formation consequently are large and temperature-dependent, increasing from approximately 29 to 70 kcal as the temperature is reduced from 37 to 13 degrees C. The corresponding delta Cp degrees value is approximately -2.4 kcal/deg. We propose that this large negative delta Cp degrees value arises primarily from the burial of hydrophobic surface in the conformational change (I1 in equilibrium I2) in RNA polymerase in the key second step of the mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cloning and deletion mapping of the recFdnaN region of the Escherichia coli chromosome

By cloning a 3.6-kb EcoRI fragment of the Escherichia coli chromosome with pBR322 we located more precisely recF relative to dnaN. By deletion mapping we localized functional recF to a 1.65-kb region of the cloned fragment and allowed rough mapping of the C terminus of dnaN. Cloned recF⁺, separated from functional flanking genes dnaN and gyrB, complemented chromosomal recF mutations presumably by coding for a cytodiffusible product. The protein encoded by dnaN was observed as a band on a polyacrylamide gel from minicells. Identification of a recF protein was not made.