The cbaAB genes for bo3-type cytochrome c oxidase in Bacillus stearothermophilus.

Structural genes were cloned for cytochrome bo3-type cytochrome c oxidase recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. Sequencing and Northern blotting analyses indicated that the two genes cbaA and cbaB composed an operon encoding for subunits I and II, respectively, and that the oxidase was SoxB-type. They are the first genes for a SoxB-type cytochrome c oxidase whose natural substrate is known.

Over-expression of cbaAB genes of Bacillus stearothermophilus produces a two-subunit SoxB-type cytochrome c oxidase with proton pumping activity.

We constructed expression plasmids containing cbaAB, the structural genes for the two-subunit cytochrome bo(3)-type cytochrome c oxidase (SoxB type) recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. B. stearothermophilus cells transformed with the plasmids over-expressed an enzymatically active bo(3)-type cytochrome c oxidase protein composed of the two subunits, while the transformed Escherichia coli cells produced an inactive protein composed of subunit I without subunit II. The oxidase over-expressed in B. stearothermophilus was solubilized and purified. The oxidase contained protoheme IX and heme O, as the main low-spin heme and the high-spin heme, respectively. Analysis of the substrate specificity indicated that the high-affinity site is very specific for cytochrome c-551, a cytochrome c that is a membrane-bound lipoprotein of thermophilic Bacillus. The purified enzyme reconstituted into liposomal vesicles with cytochrome c-551 showed H(+) pumping activity, although the efficiency was lower than those of cytochrome aa(3)-type oxidases belonging to the SoxM-type.

Crystallization and preliminary X-ray studies of HisJ and LAO periplasmic proteins from Salmonella typhimurium.

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.

Protein globularization during folding. A study by synchrotron small-angle X-ray scattering.

Various conformational states of polypeptide chains were investigated by synchrotron small-angle X-ray scattering (SAXS). SAXS patterns of proteins and model polypeptides in globular states (native and "molten globule") and in non-globular states (unfolded protein as well as randomly coiled, partially alpha-helical and partially beta-structural synthetic polypeptides) were analyzed in terms of Guinier and Kratky plots. Large differences in the SAXS pattern have been found between globular and non-globular conformations of the polypeptide chains, and they have been interpreted in terms of differences in the shape and size of the globular and non-globular scatterers with the same molecular mass. The equilibrium and time-resolved unfolding curves of bovine carbonic anhydrase and yeast phosphoglycerate kinase were monitored by integrated SAXS intensity, and were found to be coincident with the curves measured by other physicochemical techniques, such as tryptophan fluorescence and peptide circular dichroism spectra. The intermolecular association of the protein "molten globule"-like intermediates accumulated during the guanidine hydrochloride-induced unfolding of bovine carbonic anhydrase has been investigated by various SAXS parameters. It has been shown that the integrated SAXS intensity is much less sensitive to the protein intermolecular association than the zero angle intensity and the radius of gyration. We propose the integrated SAXS intensity as a global parameter which is particularly appropriate for fast kinetic studies of protein coil to globule transitions. Time-resolved refolding curves of the above proteins were monitored by the integrated SAXS intensity to investigate the globularization process in protein folding. Two fast kinetic processes for bovine carbonic anhydrase and two fast (each within two seconds) as well as two slow (within 500 seconds) kinetic processes for yeast phosphoglycerate kinase have been recorded. The kinetic processes reflect both protein intramolecular globularization and its intermolecular association.

Genetic variation in proteins: comparison of one-dimensional and two-dimensional gel electrophoresis.

Two proteins with known characteristics on one-dimensional gels were studied by two-dimensional electrophoresis to compare the sensitivities of the two methods in detecting genetic variation. Two-dimensional electrophoresis was found to be less sensitive than several types of one-dimensional gels in distinguishing variants of both proteins. Denaturation of proteins in urea in the two-dimensional method makes it possible to distinguish closely related proteins that differ from each other by units of charge. Many more types of variation in protein sequences can be distinguished on one-dimensional gels in the absence of denaturants. The estimates of heterozygosity based on two-dimensional gels are lower than those based on other methods, at least in part, because of the limited types of sequence differences that can be detected on two-dimensional gels. The application of two-dimensional electrophoresis to the measurement of genetic variation and to the detection of new mutations should be made carefully, in view of the limited sensitivity of the method in finding differences in sequence.

Overproduction of the membrane-bound components of the histidine permease from Salmonella typhimurium: identification of the M protein.

The periplasmic histidine permease of Salmonella typhimurium is composed of a soluble histidine-binding protein and three membrane-bound components. These latter are produced in very small amounts and only two, the Q and the P protein, have been previously identified. This paper describes the construction of a plasmid carrying the hisQ, hisM, and hisP genes under the control of the lambda PL promoter, thus allowing great overproduction of those gene products. The M protein has been identified in such overproducing strains and its nature confirmed by constructing in vitro hisM deletions within the plasmid. With these results the identification of all components of the histidine permease has been completed.

Structure of DNA polymerase alpha-primase complexes from mammalian cells analyzed by using monoclonal antibodies.

The molecular masses of two of the four DNA polymerase alpha-primase complex subunit peptides from various mammalian cells have been compared through the use of specific monoclonal antibodies. One monoclonal antibody (E4) binds to 77-kDa peptide from HeLa cells and cognate peptides from other mammalian cells (monkey, mouse, bovine, Indian muntjac, and hamster). Another monoclonal antibody (A5) binds the 180-kDa type peptide and its degradation product (160-kDa peptide) of the mammalian DNA polymerase alpha-primase complexes. Neither of these antibodies reacts with DNA polymerase alpha-primase complex from chicken cells. Comparative immunoblot analysis indicates that the molecular masses of the two main peptides of DNA polymerase alpha-primase complex isolated from the various mammalian sources are in excellent agreement with each other, except for the 77-kDa type peptide from bovine and Indian muntjac cells which was found to be significantly smaller (68 kDa) in these cases. The small molecular mass of bovine 77-kDa type peptide is not attributable to the action of a protease which may be present in the extract of bovine cells.

Identification and characterization of porins in Pseudomonas aeruginosa.

Earlier studies have shown that the major porin species in Pseudomonas aeruginosa outer membrane is protein F (OprF), which produces channels wider than those produced by Escherichia coli porins. In contrast, Yoshihara and Nakae ((1989) J. Biol. Chem. 264, 6297-6301) reported that protein F has no pore-forming activity as measured by the flux of L-arabinose, and that the channels in P. aeruginosa outer membrane, being produced by proteins C, "D," and "E," are much narrower than E. coli porin channels. In this study, we followed the protein purification scheme of Yoshihara and Nakae as closely as possible, and found that protein F had a specific activity for pore formation similar to that of proteins D1, D2, and E2. Furthermore, proteoliposome reconstitution assays showed conclusively that the channels formed by protein F, as well as by unfractionated outer membranes, allowed the diffusion of a tetrasaccharide, stachyose, at a significant rate, indicating that these channels are much larger than E. coli porin channels. It appears likely that in the study of Yoshihara and Nakae protein F was inadvertently inactivated during purification. We further suggest a hypothesis that resolves the apparent conflict between the presence of large diameter channels and the low permeability of the outer membrane in P. aeruginosa.

Phosphate-containing proteins of Salmonella typhimurium and Escherichia coli. Analysis by a new two-dimensional gel system.

The pattern of post-translational protein modifications involving a phosphate group was determined in the prokaryotes Salmonella typhimurium and Escherichia coli. A special two-dimensional gel electrophoretic separation was developed which utilizes acidic urea in the first dimension and neutral sodium dodecyl sulfate in the second dimension. This system allows survival and visualization of a number of proteins which are otherwise lost in systems employing basic conditions. The total number of phosphate-containing proteins thus obtained is approximately twenty. Among them are included proteins containing nucleotidylyl groups; two of these have been identified: glutamine synthetase (adenylylated) and regulatory protein PII (uridylylated). Two phosphate-containing proteins are shown to be regulated by the level of K+. The pattern of phosphorylation is shown to change with changing growth conditions and with specific mutations.

Nitrogen regulation in Salmonella typhimurium. Identification of an ntrC protein-binding site and definition of a consensus binding sequence.

We have investigated the DNA-binding ability of a nitrogen regulatory protein, the product of the ntrC gene, to several nitrogen-regulated promoters in Salmonella typhimurium. The ntrC protein is able to bind to the regulatory region (dhuA) of an operon coding for genes involved in the active transport of histidine, but not to another transport-related regulatory region, argTr. It bound to two different sites within the regulatory region of glnA (glutamine synthetase) and to one site in the regulatory region for the ntrBC operon. A consensus sequence has been derived from these four binding sites. The binding sequence displays dyad symmetry, as expected for the dimeric ntrC protein. The relationship of the binding sites to regulation of transcription initiation and termination, and to published homologies within the sequences of regulatory sites for nif genes is discussed.

Purification and characterization of the periplasmic lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium.

The lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium has been purified to homogeneity and characterized. The dissociation constants (KD) were determined by equilibrium dialysis assay to be 14, 15, and 29 nM for L-arginine, L-lysine, and L-ornithine respectively. L-Histidine was found to be a relatively good ligand (KD, 500 nM). Methods have been developed for the separation of liganded from unliganded LAO, for the estimation of bound ligand, and for unliganding LAO. Liganded and unliganded LAO are shown to have distinct UV spectra. The UV spectrum also varies with the nature of the substrate. Inhibition studies with substrate analogs yielded information useful for understanding the nature of the ligand-binding pocket.

One intact ATP-binding subunit is sufficient to support ATP hydrolysis and translocation in an ABC transporter, the histidine permease.

The membrane-bound complex of the Salmonella typhimurium histidine permease, a member of the ABC transporters (or traffic ATPases) superfamily, is composed of two integral membrane proteins, HisQ and HisM, and two copies of an ATP-binding subunit, HisP, which hydrolyze ATP, thus supplying the energy for translocation. The three-dimensional structure of HisP has been resolved. Extensive evidence indicates that the HisP subunits form a dimer. We investigated the mechanism of action of such a dimer, both within the complex and in soluble form, by creating heterodimers between the wild type and mutant HisP proteins. The data strongly suggest that within the complex both subunits hydrolyze ATP and that one subunit is activated by the other. In a heterodimer containing one wild type and one hydrolysis defective subunit both hydrolysis and ligand translocation occur at half the rate of the wild type. Soluble HisP also hydrolyzes ATP if one subunit is inactive; its specific activity is identical to that of the wild type, indicating that only one of the subunits in a soluble dimer is involved in hydrolysis. We show that the activating ability varies depending on the nature of the substitution of a well conserved residue, His-211.

Identification of a membrane protein as a histidine transport component in Salmonella typhimurium.

A component of high-affinity histidine transport in Salmonella typhimurium has been identified. It is a basic (pI about 9.0) membrane-bound protein, the P protein. It is shown to be coded for by the distal half of the previously described hisP gene by analysis of numerous hisP mutants, two of which exhibit P proteins with altered electrophoretic mobilities. Upon separation of the cytoplasmic (inner) from the outer membrane, it can be shown that the P protein is located in the cytoplasmic membrane. The P protein is under the same regulatory controls as histidine transport--i.e., transport operon promoter dhuA and nitrogen regulation. A wild-type cell contains about 200 molecules of P protein. As a result of this work we now divide the hisP gene into two genes: the hisP gene proper and the hisQ gene, which codes for another essential component of histidine transport, the Q protein. The P protein was shown previously by genetic analysis to interact with the periplasmic histidine-binding protein J, another essential component of histidine transport. Possible mechanism for the interaction of the J, P, and Q components in histidine transport, and of P and Q in lysine/arginine/ornithine transport, are discussed.

Two-dimensional gel electrophoresis of membrane proteins.

A high-resolution method for two-dimensional separation of membrane proteins is described. It involves a nondiscriminating solubilization of a membrane preparation with sodium dodecyl sulfate, followed by electrophoresis in the first dimension according to charge (by isoelectric focusing). The electrophoresis in the second dimension is in the presence of sodium dodecyl sulfate, thus separating proteins on the basis of molecular weight. Electrophoresis in the first dimension is either on a thin slab gel, or on a small-diameter tube; electrophoresis in the second dimension is on a thin slab gel. Up to 100 mug of protein can be analyzed. The two-dimensional system is a modification of the one recently described by O'Farrell (1975). About 150 different proteins can be visualized in Escherichia coli or Salmonella typhimurim cell envelopes; examples of differences between mutant and wild-type strains are presented. The method is applicable also to membrane preparations from other sources: a two-dimensional separation of plasma membrane proteins from HeLa cells is presented.

Reconstitution of periplasmic transport in inside-out membrane vesicles. Energization by ATP.

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.

Regulation of a transport operon promoter in Salmonella typhimurium: identification of sites essential for nitrogen regulation.

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.

A single amino acid substitution in a histidine-transport protein drastically alters its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Mutation hisJ5625 has altered the histidine-binding protein J of Salmonella typhimurium such that histidine transport is impaired, even though binding of histidine by the J protein is unimpaired [Kustu, S.G., & Ames, G.F. (1974) J. Biol. Chem. 249, 6976--6983]. We have determined by protein analytical methods that the only effect of this mutation has been the substitution of a cysteine residue for an arginine at a site in the interior of the polypeptide chain. This arginine residue is therefore potentially essential for the transport function of the protein. The mutant protein migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis more slowly than the wild type protein, as if its molecular weight were greater by as much as 2000. Since this behavior is apparently due to a single amino acid replacement, a molecular weight difference even between two closely related proteins should not be inferred solely on the basis of sodium dodecyl sulfate gel electrophoresis.

The chaperonin GroEL does not recognize apo-alpha-lactalbumin in the molten globule state.

We investigate here the interaction between GroEL and two kinds of non-native alpha-lactalbumin. alpha-Lactalbumin is a Ca(2+)-binding protein which assumes a molten globule state in the absence of Ca2+ (apo-alpha-lactalbumin) at neutral pH. Our results, obtained by molecular-sieve chromatography and hydrogen-exchange measurements, show that apo-alpha-lactalbumin in this molten globule state is not bound to GroEL either in the absence or in the presence of KCl. On the other hand, we show by molecular-sieve chromatography that alpha-lactalbumin, in which the four disulphide bonds are fully reduced, is bound to GroEL when 50 mM KCl is present. The results demonstrate that the protein state recognized by GroEL is more unfolded and expanded than the typical molten globule state of alpha-lactalbumin.

Purification and characterization of HisP, the ATP-binding subunit of a traffic ATPase (ABC transporter), the histidine permease of Salmonella typhimurium. Solubility, dimerization, and ATPase activity.

The nucleotide-binding subunit, HisP, of the histidine permease, a traffic ATPase (ABC transporter), has been purified as a soluble protein and characterized. Addition of a 6-histidine extension (HisP(His6)) allows a rapid and effective metal affinity purification, giving a 30-fold purification with a yield of 50%. HisP(his6) is indistinguishable from underivatized HisP when incorporated into the permease membrane-bound complex, HisQMP2. Purified HisP(his6) has a strong tendency to precipitate; 5 mM ATP and 20% glycerol maintain it in solution at a high protein concentration. HisP(his6) is active as a dimer, binds ATP with a Kd value of 205 microM, and hydrolyzes it at a rate comparable to that of HisQMP2; in contrast to the latter, it does not display cooperativity for ATP. HisP(his6) has been characterized with respect to substrate and inhibitor specificity and various physico-chemical characteristics. Its pH optimum is 7 and it requires a cation for activity, with Co2+ and Mn2+ being more effective than Mg2+ at lower concentrations but inhibitory in the higher concentration range. In contrast to the intact complex, HisP(his6) is not inhibited by vanadate but is inhibited by N-ethylmaleimide. Neither the soluble receptor, HisJ, nor the transport substrate, histidine, has any effect on the activity.

Genetic determination of enzymes synthesizing O-specific sugars of Salmonella lipopolysaccharides.

Nikaido, Hiroshi (Massachusetts General Hospital, Boston, Mass.), Kishiko Nikaido, and P. Helena Mäkelä. Genetic determination of enzymes synthesizing O-specific sugars of Salmonella lipopolysaccharides. J. Bacteriol. 91:1126-1135. 1966.-Levels of enzymes involved in the biosynthesis of various nucleotide sugars were examined in parental strains and recombinants obtained in crosses between Salmonella of groups B, C(2), and C(1) with the O antigen specificities 4, 5, 12; 6, 8; and 6,7, respectively. The results showed that smooth strains of groups B and C(2) possessed the enzymes for the synthesis of guanosine diphosphate mannose, cytidine diphosphate abequose, and thymidine diphosphate rhamnose; these sugars are constituents of their lipopolysaccharides. Group C(1) lipopolysaccharide is devoid of both abequose and rhamnose, and the corresponding enzymes for cytidine diphosphate abequose synthesis, as well as the enzyme(s) catalyzing the last step(s) of thymidine diphosphate rhamnose synthesis, were undetectable in S. montevideo of this group. Two other enzymes also involved in the biosynthesis of thymidine diphosphate rhamnose were present at a low level of activity; their function in this strain is not known. The analysis of enzyme levels in recombinants indicated that genes determining at least eight of the enzymes involved in the biosynthesis of nucleotide-bound mannose, rhamnose, and abequose were located in the O locus known to determine the specificity of the O antigen. In three rough recombinant strains, enzyme levels indicated that crossing-over had presumably occurred within the O locus. The results also suggested a high degree of nonhomology in this region of the chromosome between groups B and C(1).