Biomedical subjects
Publications and source records attributed to G F Ames.
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Periplasmic transport systems consist of a membrane-bound complex and a periplasmic substrate-binding protein and are postulated to function by translocating the substrate either through a nonspecific pore or through specific binding sites located in the membrane complex. We have isolated mutants carrying mutations in one of the membrane-bound components of the histidine permease of Salmonella typhimurium that allow transport in the absence of both histidine-binding proteins HisJ and LAO (lysine-, arginine-, ornithine-binding protein). All of the mutations are located in a limited region of the nucleotide-binding component of the histidine permease, HisP. The mutants transported substrate in the absence of binding proteins only when the membrane-bound complex was produced in large amounts. At low (chromosomal) levels, the mutant complex was unable to transport substrate in the absence of binding proteins but transported it efficiently in the presence of HisJ. The alterations responsible for the mutations were identified by DNA sequencing; they are closely related to a group of hisP mutations isolated as suppressors of HisJ interaction mutations (G. F.-L. Ames and E. N. Spudich, Proc. Natl. Acad. Sci. USA 73:1877-1881, 1976). The hisP suppressor mutations behaved similarly to these newly isolated mutations despite the entirely different selection procedure. The results are consistent with the HisP protein carrying or contributing to the existence of a substrate-binding site that can be mutated to function in the absence of a binding protein.
We have demonstrated that efficient polymerase chain reaction amplifications from chromosomal DNA can be carried out using whole bacterial cells as the starting material. Cells from the liquid or solid cultures can be used directly, without any pre-treatment, thus eliminating the need for DNA isolation.
The periplasmic histidine transport system (permease) of Escherichia coli and Salmonella typhimurium is composed of a soluble, histidine-binding receptor located in the periplasm and a complex of three membrane-bound proteins of which one, HisP, was shown previously to bind ATP. These permeases are energized by ATP. HisP is a member of a family of membrane transport proteins which is conserved in all periplasmic permeases and is presumed to be involved in coupling the energy of ATP to periplasmic transport. In this paper the nature of the ATP-binding site of HisP has been explored by identification of some of the residues that come into contact with ATP. HisP was derivatized with 8-azido-ATP (N3ATP). Both the underivatized and the derivatized forms of HisP were solubilized, purified, and digested with trypsin. The resulting tryptic peptides were resolved by high pressure liquid chromatography, and peptides modified by N3ATP were isolated and sequenced. Two peptides, X and Z, spanning amino acid residues 16-23 and 31-45, were found to contain sites of N3ATP attachment at His19 and Ser41, respectively. Both peptides are close to the amino-terminal end of HisP; peptide Z is located in one of the well conserved regions comprising the nucleotide-binding consensus motifs of the energy-coupling components of these permeases. These consensus motifs are found in many purine nucleotide-binding proteins. The relationship between the location of these residues and the overall structure of the ATP-binding site is discussed.
We show that a family of prokaryotic repetitive sequences, called REP (repetitive extragenic palindromic), (Stern et al., 1984) is involved in the formation of chromosomal rearrangements such as duplications. The join-points of seven RecA+ tandem duplications previously characterized in Salmonella typhimurium, that fuse the hisD gene to distant foreign promoters, were cloned and sequenced. In all seven cases they are shown to have originated by recombination between distant REP sequences. Importantly, several join-points had also occurred at REP sequences even in a RecA-background. Thus, REPs can recombine with each other by a RecA(-)-independent mechanism involved in the generation of chromosomal rearrangements. While all RecA+ duplications analysed resulted from recombination between REP sequences, some RecA-duplications did occur also outside of REP sequences, in one case by recombination within a 7 bp homology. Possible roles for the known interaction between DNA gyrase and REP in chromosomal rearrangements are discussed.
Bacterial periplasmic transport systems are complex permeases composed of a soluble substrate-binding receptor and a membrane-bound complex containing 2-4 proteins. Recent developments have clearly demonstrated that these permeases are energized by the hydrolysis of ATP. Several in vitro systems have allowed a detailed study of the essential parameters functioning in these permeases. Several of the component proteins have been shown to interact with each other and the actual substrate for the transport process has been shown to be the liganded soluble receptor. The affinity of this substrate for the membrane complex is approximately 10 microM. The involvement of ATP in energy coupling is mediated by one of the proteins in the membrane complex. For each specific permease, this protein is a member of a family of conserved proteins which bind ATP. The similarity between the members of this family is high and extends itself beyond the consensus motifs for ATP binding. Interestingly, over the last few years, several eukaryotic membrane-bound proteins have been discovered which bear a high level of homology to the family of the conserved components of bacterial periplasmic permeases. Most of these proteins are known to, or can be inferred to participate in a transport process, such as in the case of the multidrug resistance protein (MDR), the STE6 gene product of yeast, and possibly the cystic fibrosis protein. This homology suggests a similarity in the mechanism of action and possibly a common evolutionary origin. This exciting development will stimulate progress in both the prokaryotic and eukaryotic areas of research by the use of overlapping procedures and model building. We propose that this universal class of permeases be called 'Traffic ATPases' to distinguish them from other types of transport systems, and to highlight their involvement in the transport of a vast variety of substrates in either direction relative to the cell interior and their use of ATP as energy source.
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We have devised a strategy to extend the use of the polymerase chain reaction (PCR) to amplify double-stranded DNA when sequence information is available only at one extremity. The only information required is a short stretch of sequence used to design a gene-specific primer, which is then used in combination with a second generic vector primer at the unknown end. The primers are used in a PCR reaction after ligating the unknown end to a generic vector. Restriction, ligation, amplification and sequencing of the products can be achieved within three days. This method eliminates the laborious steps of shotgun cloning, colony screening and culturing of cells. We have used this method to take two contiguous steps beyond the histidine transport operon in Salmonella typhimurium. We also demonstrate the usefulness of this technique to do chromosome walking in the absence of any restriction data.
Two periplasmic binding proteins, HisJ and LAO, which are involved in histidine and arginine transport, respectively, have been crystallized. Preliminary X-ray diffraction studies of the HisJ and LAO crystals show that both belong to the orthorhombic space group P2(1)2(1)2(1) and have unit cell dimensions of a = 39.26 A, b = 66.17 A, c = 88.33 A and a = 36.08 A, b = 78.34 A, c = 102.02 A, respectively. Both HisJ and LAO crystals diffract beyond 2.0 A resolution.
The periplasmic histidine transport system of Salmonella typhimurium has been reconstituted in isolated right-side-out membrane vesicles. The reconstituted system is entirely dependent on both the periplasmic protein, HisJ, and the membrane-bound complex, composed of proteins HisQ, HisM, and HisP. Transport is also dependent on the presence of ascorbate and phenazine methosulfate, which provide the energy for transport. Ascorbate oxidation generates a proton-motive-force, which allows ATP synthesis. ATP (or a cogenerated molecule) appears to be the immediate energy donor. Dissipation of the proton-motive-force or reduction of the level of ATP by a variety of treatments results in inhibition of transport. Vanadate inhibits transport, indicating that ATP utilization is necessary to energize transport. The interaction between liganded HisJ and the membrane complex has been measured directly: it displays Michaelis-Menten type kinetics, with a K1/2 of approximately 65 microM. The significance of this finding in terms of transport properties of whole cells is discussed.
The periplasmic histidine permease of Salmonella typhimurium has been reconstituted in inside-out vesicles (IOV) of Escherichia coli by disrupting the cells with a French press in the presence of a high concentration of the periplasmic histidine-binding protein, HisJ. Efflux from IOV, which is equivalent to uptake in whole cells, is induced by ATP. The reconstituted system depends on the presence of the membrane-bound permease proteins, HisQ, HisM, and HisP, and does not function if reconstitution is performed in the presence of a mutant HisJ protein, HisJ5625, that can bind histidine normally but can't interact properly with the membrane complex. Efflux is not induced by the nonhydrolyzable ATP analog, adenyl-5'-yl imidodiphosphate, supporting the contention that ATP hydrolysis is necessary. 8-Azido ATP inactivates IOV, indicating that the ATP effect occurs through the HisP protein, which has previously been shown to be modified by 8-azido ATP (Hobson, A., Weatherwax, R., and Ames, G.F.-L. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 733-7337). The estimated Km of the vesicles for ATP is about 200 microM. Vanadate, an inhibitor of phosphohydrolase enzymes, inhibits ATP-induced efflux. We conclude that ATP is likely to be the proximal energy source for periplasmic permeases.
The histidine periplasmic permease of Salmonella typhimurium has been partially purified and reconstituted into proteoliposomes. In this in vitro preparation, transport activity is completely dependent on the presence of all four permease proteins (HisJ, HisQ, HisM, and HisP) and on internal ATP. The reconstituted system shows initial rates of transport that are comparable to those obtained with right-side-out membrane vesicles and it establishes a 100-fold concentration gradient for histidine. Proteoliposomes also transport histidine when GTP replaces ATP. Proteoliposomes do not catalyze significant ATP hydrolysis until histidine transport is initiated by addition of substrate along with HisJ, the water-soluble histidine-binding protein. Both initially and throughout the course of substrate transport there is a concomitant hydrolysis of ATP, with an apparent stoichiometry (ATP/histidine) of 5:1. These experiments demonstrate directly that ATP is the source of energy for periplasmic permeases, thus resolving previous controversies on this topic.
We have constructed a fine-structure genetic map of the maltose transport operon in Salmonella typhimurium. We have isolated mal mutants by using indicator plates, penicillin selection, or a proton suicide technique. Mutants were obtained as spontaneous events or were induced by chemical mutagenesis and transposon insertion. Tn10 and Mu d(lac Ap)1 insertion mutations were used to create deletions. Mutations were also obtained in a gene that is equivalent to lamB in Escherichia coli, which codes for the lambda bacteriophage receptor. The gene products in the mutants were characterized by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis and immunoblotting. Our data indicate that the location of this operon on the Salmonella chromosome as well as the gene order and its orientation are the same as those in E. coli. This map will be useful in studying the mechanism of periplasmic transport in S. typhimurium.
The polymerase chain reaction (PCR) has been used to amplify DNA fragments by using eucaryotic genomic DNA as a template. We show that bacterial genomic DNA can be used as a template for PCR amplification. We demonstrate that DNA fragments at least as large as 4,400 base pairs can be amplified with fidelity and that the amplified DNA can be used as a substrate for most operations involving DNA. We discuss problems inherent in the direct sequencing of the amplified product, one of the important exploitations of this methodology. We have solved the problems by developing an "asymmetric amplification" method in which one of the oligonucleotide primers is used in limiting amounts, thus allowing the accumulation of single-stranded copies of only one of the DNA strands. As an illustration of the use of PCR in bacteria, we have amplified, sequenced, and subcloned several DNA fragments carrying mutations in genes of the histidine permease operon. These mutations are part of a preliminary approach to studying protein-protein interactions in transport, and their nature is discussed.
The histidine permease of Salmonella typhimurium consists of four protein components, one located in the periplasm and three in the cytoplasmic membrane. Genetic evidence indicated that the periplasmic protein interacts with the membrane proteins during transport. We have utilized two different methods to demonstrate that the periplasmic protein cross-links specifically to one of the membrane components, the Q protein. Formaldehyde, a water-soluble permeant molecule was used in vivo. Sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate, a photoactivatable cross-linking reagent, was used in vitro in a reconstituted membrane vesicle system. Furthermore, we show that a mutant periplasmic protein, capable of binding substrate but not transporting it, is defective in cross-linking to the membrane protein, indicating this interaction to be a crucial step in the mechanism of transport.
The promoter of nitrogen-regulated transport, argtr, has been mutationally altered in order to determine the features that are essential for its response to nitrogen availability. Deletions of all sequences upstream of position -44 or downstream of position +2 had no effect on nitrogen regulation of argTr. These deletions define a small region of 44 bp where all necessary features for nitrogen regulation are located. This region includes sequences highly homologous to the nif consensus promoter. Alteration of this particular sequence caused drastic changes in the response to changes of nitrogen availability, thus indicating that they are directly involved in regulation. This implies that the NtrC protein must also act within this small region of the promoter. The data are discussed in terms of current-hypotheses concerning nitrogen regulation. In addition, we have shown 1. that carbon regulation at this promoter must occur at a site upstream from the nitrogen promoter; 2. that nifA can replace ntrC in the regulation of argTr.
Bacterial periplasmic transport systems are complex, multicomponent permeases, present in Gram-negative bacteria. Many such permeases have been analyzed to various levels of detail. A generalized picture has emerged indicating that their overall structure consists of four proteins, one of which is a soluble periplasmic protein that binds the substrate and the other three are membrane bound. The liganded periplasmic protein interacts with the membrane components, which presumably form a complex, and which by a series of conformational changes allow the formation of an entry pathway for the substrate. The two extreme alternatives for such pathway involve either the formation of a nonspecific hydrophilic pore or the development of a ligand-binding site(s) on the membrane-bound complex. One of the membrane-bound components from each system constitutes a family of highly homologous proteins containing sequence domains characteristic of nucleotide-binding sites. Indeed, in several cases, they have been shown to bind ATP, which is thus postulated to be involved in the energy-coupling mechanism. Interestingly, eukaryotic proteins homologous to this family of proteins have been identified (mammalian mdr genes and Drosophila white locus), thus indicating that they perform a universal function, presumably related to energy coupling in membrane-related processes. The mechanism of energy coupling in periplasmic permeases is discussed.
A family of repetitive extragenic palindromic (REP) sequences is composed of hundreds of copies distributed throughout the chromosome. Their palindromic nature and conservation suggested that they are specifically recognized by a protein(s). We have identified DNA gyrase [DNA topoisomerase (ATP-hydrolysing), EC 5.99.1.3] as one of the REP-binding proteins. Gyrase has at least a 10-fold higher affinity for DNA containing REP sequences than for DNA not containing REP sequences. Binding effectiveness correlates directly with the number of REP sequences in the DNA. DNase I footprinting shows that gyrase protects 205 base pairs on a REP-containing DNA fragment enclosing the REP sequences. In agreement with the above results, a comparison of the REP consensus sequence with the sequence of previously identified pBR322 "strong" gyrase cleavage sites reveals a high degree of homology. Because REP sequences are numerous and found throughout the genome, we suggest they have physiological functions mediated through their interaction with gyrase, such as being sites of action for the maintenance of DNA supercoiling. In addition, we speculate that these interactions may be of a structural nature, such as involvement in the higher-order structure of the bacterial chromosome.