Nonequivalence of the nucleotide-binding subunits of an ABC transporter, the histidine permease, and conformational changes in the membrane complex.

The membrane-bound complex of the Salmonella typhimurium histidine permease, an ABC transporter (or traffic ATPase), is composed of two membrane proteins, HisQ and HisM, and two identical copies of an ATP-hydrolyzing protein, HisP. We have developed a technique that monitors quantitatively the sulfhydryl modification levels within the intact complex, and we have used it to investigate whether the HisP subunits behave identically within the complex. We show here that they interact differently with various thiol-specific reagents, thus indicating that, despite being identical, they are arranged asymmetrically. The possible basis of this asymmetry is discussed. We have also analyzed the occurrence of conformational changes during various stages of the activity cycle using thiol-specific reagents, fluorescence measurements, and circular dichroism spectroscopy. Cys-51, located close to the ATP-binding pocket, reflects conformational changes upon binding of ATP but does not participate in changes involved in signaling and translocation. The latter are shown to cause secondary structure alterations, as indicated by changes in alpha-helices; tertiary structure alterations also occur, as shown by fluorescence studies.

Single Specific Primer-Polymerase Chain Reaction (SSP-PCR) and Genome Walking.

The polymerase chain reaction (PCR) is used for selective amplification of DNA fragments from both prokaryotes and eukaryotes (1-3). The only requirement for amplification is that the sequence of the extremities of the DNA fragment to be amplified be known (4). This places a limitation on the use of PCR in the amplification of adjacent unknown regions. We have developed a method that allows the amplification of double-stranded DNA even when the sequence information is available at one end only (5). This method, the single specific primer-PCR (SSP-PCR), permits amplification of genes for which only a partial sequence information is available, and allows unidirectional genome walking from known into unknown regions of the chromosome.

Energy coupling in bacterial periplasmic transport systems. Studies in intact Escherichia coli cells.

Periplasmic permeases are composed of four proteins, one of which has an ATP-binding site that has been postulated to be involved in energy coupling. Previous data suggested that these permeases derive energy from substrate level phosphorylation (Berger, E. A. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1514-1518); however, conflicting results later cast doubt upon this hypothesis. Here, we make use of two well characterized periplasmic permeases and of a well characterized unc mutant (ATPase-) to examine this energetics problem in depth. We have utilized the histidine and maltose periplasmic permeases in Escherichia coli as model systems. Isogenic unc strains were used in order to study separately the effect of the proton-motive force and of ATP on transport. These parameters were analyzed concomitantly with transport assays. Starvation experiments indicate that both histidine and maltose transport require ATP generation and that a normal level of delta psi is not sufficient. Uncouplers such as carbonyl cyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol dissipated the delta psi without decreasing the ATP level and without significant effect on these permeases, showing that delta psi is not needed. Inhibition of ATP synthesis by arsenate eliminates transport through both permeases, confirming the need for ATP. In agreement with previous results with the glutamine permease (Plate, C. A. (1979) J. Bacteriol. 137, 221-225), valinomycin plus K+ dissipates delta psi without affecting ATP levels and inhibits histidine transport; however, maltose transport is not inhibited under these conditions. This result is discussed in terms of the artefactual side effects caused by valinomycin/K+ treatment on some periplasmic permeases. Histidine transport is also shown to be sensitive to changes in the cytoplasmic pH. It is concluded that periplasmic permeases indeed have an obligatory requirement for ATP (or a closely related molecule), whereas the proton-motive force is neither sufficient nor essential.

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.

Extensive homology between membrane-associated components of histidine and maltose transport systems of Salmonella typhimurium and Escherichia coli.

A strong homology was found between the amino acid sequences, deduced from DNA nucleotide sequences, of cytoplasmic membrane-associated components of the high affinity histidine transport system of Salmonella typhimurium (coded by the hisP gene) and the maltose-maltodextrin transport system of Escherichia coli (coded by the malK gene). When the HisP protein sequence was aligned with that of the NH2-terminal two-thirds of the MalK protein, 32% of the positions were identical, and an additional 35% were occupied by functionally similar amino acid residues. These results suggest that some, and possibly many, "periplasmic-binding protein-dependent" transport systems have evolved from a common ancestral system.

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.