A piston model for transmembrane signaling of the aspartate receptor.

To characterize the mechanism by which receptors propagate conformational changes across membranes, nitroxide spin labels were attached at strategic positions in the bacterial aspartate receptor. By collecting the electron paramagnetic resonance spectra of these labeled receptors in the presence and absence of the ligand aspartate, ligand binding was shown to generate an approximately 1 angstrom intrasubunit piston-type movement of one transmembrane helix downward relative to the other transmembrane helix. The receptor-associated phosphorylation cascade proteins CheA and CheW did not alter the ligand-induced movement. Because the piston movement is very small, the ability of receptors to produce large outcomes in response to stimuli is caused by the ability of the receptor-coupled enzymes to detect small changes in the conformation of the receptor.

The ToxR protein of Vibrio cholerae forms homodimers and heterodimers.

The ToxR protein of Vibrio cholerae regulates the expression of several virulence factors that play important roles in the pathogenesis of cholera. Previous experiments with ToxR-alkaline phosphatase (ToxR-PhoA) fusion proteins suggested a model for gene regulation in which the inactive form of ToxR was a monomer and the active form of ToxR was a dimer (V. L. Miller, R. K. Taylor, and J. J. Mekalanos, Cell 48:271-279, 1987). In order to examine whether ToxR exists in a dimeric form in vivo, biochemical cross-linking analyses were carried out. Different dimeric cross-linked species were detected depending on the expression level of ToxR: when overexpressed, ToxR+ToxR homodimers and ToxR+ToxS heterodimers were detected, and when ToxR was expressed at normal levels, exclusively ToxR+ToxS heterodimers were detected. The amount of overexpression was quantitated by using ToxR-PhoA fusion proteins and was found to correspond to 2.7-fold the normal level of ToxR. The formation of both homodimeric ToxR species and heterodimeric ToxR+ToxS species is consistent with previously reported genetic data that suggested that both types of ToxR oligomeric interactions occur. However, variation in the amount of either the homodimeric or heterodimeric form detectable by this cross-linking analysis was not observed to correlate with laboratory culture conditions known to modulate ToxR activity. Thus, genetic and biochemical data indicate that ToxR is able to interact with both itself and ToxS but that these interactions may not explain mechanistically the observed changes in ToxR activity that occur in response to environmental conditions.

Roles for motility in bacterial-host interactions.

The ability to move in a directed manner may confer distinct advantages upon host-adapted prokaryotes. Potential benefits of motility include increased efficiency of nutrient acquisition, avoidance of toxic substances, the ability to translocate to preferred hosts and access optimal colonization sites within them, and dispersal in the environment during the course of transmission. The costs of motility also may be significant. These include the metabolic burden of synthesizing flagellar components, the energetic expense of fuelling flagellar motors and the presentation of polymeric and highly antigenic targets to the immune system. It is therefore not surprising that synthesis of the motility apparatus is usually subject to strict control. Studies of a variety of bacterial-host interactions demonstrate roles for motility, and its regulation, at points throughout the infectious cycle.

Analysis of Vibrio cholerae ToxR function by construction of novel fusion proteins.

The ToxR protein is a transmembrane protein that regulates the expression of several virulence factors of Vibrio cholerae. Previous analysis of fusion proteins between ToxR and alkaline phosphatase (ToxR-PhoA) suggested that ToxR was active as a dimer. In order to determine whether dimerization of the ToxR periplasmic domain was essential for activity, this domain was replaced by monomeric and dimeric protein domains. Surprisingly, PhoA (dimeric), beta-lactamase (monomeric, ToxR-Bla), or the leucine zipper of GCN4 (dimeric, ToxR-GCN4-M) could substitute functionally for the ToxR periplasmic domain. ToxR-GCN4 fusion proteins, in which the ToxR transmembrane domain was eliminated (ToxR-GCN4-C), were inactive, but an additional fusion protein that contained a heterologous membrane-spanning domain retained activity. Strains containing each of these ToxR fusion proteins were analysed for in vivo colonization properties and response to in vitro growth conditions that are known to affect expression of the ToxR regulon. Strains containing ToxR-GCN4-M and ToxR-Bla responded like wild-type strains to in vitro growth conditions. In the infant-mouse colonization model, strains containing ToxR fusion proteins were all deficient in colonization relative to strains containing wild-type ToxR, and strains containing monomeric ToxR-Bla were most severely outcompeted. These results suggest that, under in vitro conditions, ToxR does not require a dimerized periplasmic domain, but that, under in vivo conditions, the correct conformation of the ToxR periplasmic domain may be more important for function.

Converting a transmembrane receptor to a soluble receptor: recognition domain to effector domain signaling after excision of the transmembrane domain.

The bacterial aspartate receptor was reconstructed to eliminate the transmembrane domain, thus connecting the recognition domain directly to the effector domain. The resulting soluble receptor folded correctly and was no longer an integral membrane protein. Upon aspartate binding, this soluble receptor was stabilized to a similar extent as that of the native receptor. Of interest, this soluble receptor retained the ability to signal from the recognition to the effector domain. This result defines more clearly the role of the membrane and transmembrane domains in signal transduction and suggests that some ligand-induced motions in receptor proteins do not require the membrane or transmembrane domain for information transmission.

ToxR proteins with substitutions in residues conserved with OmpR fail to activate transcription from the cholera toxin promoter.

The ToxR protein of Vibrio cholerae is an integral membrane protein that coordinately regulates the expression of virulence genes required for successful infection. ToxR has been shown to bind directly to and activate transcription of the cholera toxin (ctx) promoter. Within the amino-terminal cytoplasmic region of ToxR, several amino acids are strictly conserved among ToxR, OmpR, and the other members of a family of bacterial regulatory proteins. To better understand the function of this region, two approaches were taken: conserved residues were changed by site-directed mutagenesis, and random mutations that eliminated ToxR-mediated transcriptional activation were isolated. Several classes of mutations were identified: those that abolish promoter DNA binding and transcriptional activation (toxR R96K, toxR R68K, and toxR R68L), those that abolish transcriptional activation but retain the ability to bind promoter DNA (toxR R96L), and those that have an intermediate phenotype (toxR R77L, toxR E51K, and toxR E51D). The toxR E51K allele had reduced activity in both Escherichia coli and V. cholerae but also exerted a dominant-negative effect over wild-type ToxR when assayed in V. cholerae. This result provides additional evidence that ToxR acts as an oligomer in the transcriptional activation process. From this mutational analysis of conserved amino acid residues within the OmpR-homologous region of ToxR, we conclude that this region is essential for transcriptional activation at the level of DNA binding and other steps that lead to activation of the ctx promoter.

One of three transmembrane stretches is sufficient for the functioning of the SecE protein, a membrane component of the E. coli secretion machinery.

The E. coli secE (prlG) gene codes for an integral cytoplasmic membrane protein which is part of the cell's secretory machinery. A deletion of nearly the entire gene renders the cell dependent on the presence of a complementing secE+ plasmid, indicating that the SecE protein is essential for growth. Deletions which remove carboxy-terminal sequences or substantial amounts near the amino-terminus of SecE can still complement the lethal deletion. This deletion analysis suggests that the essential domain of the SecE protein includes only a single one of its three hydrophobic membrane-spanning segments. Two of three dominant prlG signal sequence suppressors map to this segment. Consistent with the insensitivity of SecE to major structural changes, several cold-sensitive mutations cause lethality not because of any change in the protein, but because of a reduction in its level of expression. Our results suggest that higher levels of the protein are needed at the lower temperature. These findings are discussed in terms of the interactions between various components of the secretory machinery.

Direct measurement of small ligand-induced conformational changes in the aspartate chemoreceptor using EPR.

Ligand-binding-induced conformational changes in the Salmonella typhimurium aspartate receptor were studied using spin-labeling electron paramagnetic resonance. Cysteine residues, introduced by site-directed mutagenesis at several positions in the aspartate receptor periplasmic domain, were used to attach covalently a thiol-specific spin label. The electron paramagnetic resonance spectra of these labeled proteins were obtained in the presence and absence of the ligand aspartate, and used to calculate the distance change between spin labels. The results support a model in which transmembrane signaling is executed by a combined movement of alpha helix 4 (which leads into transmembrane domain 2) relative to alpha helix 1 (connected to transmembrane domain 1), as well as a coming together of the two subunits. Ligand binding causes spin labels at position 39 and 179 (within one subunit) to move further from each other and spin labels at position 39 and 39' (between two subunits) to move closer to each other. Both of these changes are very small-less than 2.5 A. No similar changes were detected in any aspartate receptor samples solubilized in detergent, suggesting that the membrane is required for these conformational changes. This is the first case of physically measured ligand-induced changes in a full-length 1-2 transmembrane domain receptor, and the results suggest that very small ligand-induced movements can result in large effects on the activity of downstream proteins.