Histidine-binding Protein Research Articles

The Histidine-binding Protein J, a Histidine Transport Component, Has Two Different Functional Sites

Abstract Previous work from this laboratory has shown that the histidine-binding protein J is a component of the high affinity histidine transport system in Salmonella typhimurium and that it is the product of the hisJ gene. We now present evidence that the J protein has a second type of site, in addition to its histidine-binding site, and that the second site is essential for the J protein to function in transport. The existence of a second functional site had been inferred from the properties of a mutant strain, TA300. This strain has a single mutation in the hisJ gene and produces a J protein with altered electrophoretic mobility in the presence of sodium dodecyl sulfate. The J protein from this strain binds histidine with normal affinity but functions very poorly in histidine transport. We postulate that the J protein from TA300 has been altered at a site other than its histidinebinding site. Possible biochemical functions of this second site are discussed. Properties of TA300 are compared with those of other strains in which histidine-binding by the J protein has been altered.

83] Isolation of transport mutants in bacteria

This chapter describes the methods used for the isolation of several classes of mutants in the histidine transport system in Salmonella typhimurium. These methods are applicable to other organisms and to other transport systems. Three classes of mutants have been isolated and characterized carefully. (1) Mutants with an elevated level of histidine transport ( dhuA site). (2) Mutants with a complete loss of the histidine high affinity permease (hisP gene). (3) Mutants lacking a known component of the histidine high affinity permease, the histidine-binding protein J ( hisJ gene). All three sites are 40% cotransducible with the purF gene and 98% with each other. Any of the mutants can be obtained from cultures that have been mutagenized before starting the selection, in order to increase the frequency of mutation or to obtain special types of mutation. J. R. Roth has full descriptions of various mutagenic procedures.

Hydrophobic chromatography in the purification of the histidine-binding protein j from Salmonella typhimurium

Upon increasing the length of the side chains in a homologous series of ω-aminoalkyl agaroses (Seph—C n —NH 2), more and more proteins become adsorbed onto the column and it is thus possible to achieve purification by selective exclusion of a desired protein. This principle is illustrated in the purification of the histidine-binding protein j from Salmonella typhimurium, vising ω-aminodecyl or ω-aminododecyl agarose columns. The protein was purified 7- to 10-fold in one step from either the shock fluid or the crude extract of the bacteria. Since protein j has an isoelectric pH of 5.5 and yet does not bind to the positively charged columns at pH 7, it seems that this protein is not likely to have available hydrophobic pockets and may thus be remarkably hydrophilic, compared with the other proteins in the shock fluid.

The hisP protein, a known histidine transport component in Salmonella typhimurium, is also an arginine transport component.

The hisP protein is an essential component of the high-affinity histidine transport system in Salmonella typhimurium. Our present studies demonstrate that this protein is also an essential component of an arginine transport system. Strains with a mutation in the hisP gene are unable to transport arginine for use as a sole nitrogen source. However, such strains have normal high-affinity transport of arginine, indicating that the hisP protein is not required for all arginine transport systems. Histidine does not appear to compete with arginine for transport through the hisP system, suggesting that the hisP protein may not be a "carrier" for these amino acids. The hisJ protein, a periplasmic histidine-binding protein, is known to function in conjunction with the hisP protein in the transport of histidine. The hisJ protein does not function with the hisP protein in arginine transport.

The Histidine-binding Protein J Is a Component of Histidine Transport

Abstract The histidine-binding protein J, previously shown to be involved in histidine transport in Salmonella typhimurium (Ames, G. F., and Lever, J. (1970) Proc. Nat. Acad. Sci. U. S. A. 66, 1096), is shown unequivocally to be the product of the hisJ gene. A hisJ mutant with an altered J protein and a correspondingly altered histidine transport has been isolated and characterized. The J protein from this strain has an increased temperature sensitivity, besides having altered chromatographic and electrophoretic mobilities. The in vivo effect of the altered J protein is expressed as an increased temperature sensitivity of histidine transport. Our data indicate that the hisJ gene is the structural gene for the J protein and that the J protein is an obligatory component of histidine transport. As expected, there is an excellent correlation between the specificity of transport in the wild type strain and the specificity of binding of the wild type J protein for a variety of amino acids, amino acid analogues, and inhibitors.

Purification and Properties of a Component of Histidine Transport in Salmonella typhimurium

Abstract The purification of the J histidine-binding protein, a component of high affinity histidine transport in Salmonella typhimurium, is described. A mutant strain producing elevated levels of this protein is used as the optimal source of this protein. The purification involves a modified osmotic shock procedure, chromatography on DEAE-cellulose, and isoelectric focusing. The final preparation is homogeneous by several criteria. The J protein has a molecular weight of 25,000 by gel filtration and 26,000 by sedimentation equilibrium. Histidine binding is reversible, with a dissociation constant for the J protein-histidine complex of 0.15 µm. The J protein at saturating substrate (10-6 m histidine) maximally binds 1 molecule of histidine per molecule protein. Histidine binding decreases slightly at high ionic strength, and decreases with increase in pH. Binding activity is relatively stable to boiling. The protein has an isoelectric pH of 5.5 and an amino acid composition representative of S. typhimurium protein. The J protein is located near the cell surface, as indicated by its release by osmotic shock and during spheroplast formation. The J protein has no detectable enzymatic activity. In addition to the J protein, osmotic shock treatment releases three separable components which bind histidine.

Purification and Characterization of a Histidine-binding Protein from Salmonella typhimurium LT-2 and Its Relationship to the Histidine Permease System

Abstract A histidine-binding protein has been isolated from the shock fluid of osmotically shocked Salmonella typhimurium and has been purified by conventional techniques. It has a molecular weight of 25,000 and binds 1 histidine molecule per molecule of protein. The binding protein is stable to wide variations of temperature, ionic strength, and pH. It binds histidine with a Kd of 1.5 µm. The protein also binds arginine but with a much weaker affinity. The transport of histidine by Salmonella typhimurium was found to be an energy-dependent process. The Km for histidine was 0.07 µm when measured by the growing cells method but it was 1 µm when determined in the presence of an inhibitor of protein synthesis. Histidine transport is inhibited by large excesses of arginine. Osmotically shocked exhibit a reduction in the initial rate and the steady state level of histidine accumulation. The simultaneous release of the histidine-binding protein and the reduction of transport caused by osmotic shock, as well as the similarity of kinetic constants and specificity of the binding protein and the histidine permease system suggest that the histidine-binding protein is a functional component of the histidine permease system.

Components of histidine transport: histidine-binding proteins and hisP protein.

The high-affinity (K(m) = 3 x 10(-8) M) transport system for histidine in Salmonella typhimurium has been resolved into three components: J, K, and P. J, which is a histidine-binding protein released by osmotic shock, is specified by the hisJ gene: hisJ mutants lack the binding protein and are defective in histidine transport. Another class of mutants-dhuA, which is closely linked to hisJ-has five times the normal level of binding protein and has an increased rate of histidine transport. P, which is a protein specified by the hisP gene, is required for J binding protein to be operative in transport. hisP mutants, though defective in transport, have normal levels of J binding protein. K, a third transport component, works in parallel to J, and also requires the P protein in order to be operative in transport. A second histidine-binding protein has been found but its relation to K is unclear. hisJ, dhuA, and hisP have been mapped and are in a cluster (near purF) on the S. typhimurium chromosome.