Mutations that alter the transmembrane signalling pathway in an ATP binding cassette (ABC) transporter.

The maltose transport system of Escherichia coli is a well-characterized member of the ATP binding cassette transporter superfamily. Members of this family share sequence similarity surrounding two short sequences (the Walker A and B sequences) which constitute a nucleotide binding pocket. It is likely that the energy from binding and hydrolysis of ATP is used to accomplish the translocation of substrate from one location to another. Periplasmic binding protein-dependent transport systems, like the maltose transport system of E.coli, possess a water-soluble ligand binding protein that is essential for transport activity. In addition to delivering ligand to the membrane-bound components of the system on the external face of the membrane, the interaction of the binding protein with the membrane complex initiates a signal that is transmitted to the ATP binding subunit on the cytosolic side and stimulates its hydrolytic activity. Mutations that alter the membrane complex so that it transports independently of the periplasmic binding protein also result in constitutive activation of the ATPase. Genetic analysis indicates that, in general, two mutations are required for binding protein-independent transport and constitutive ATPase. The mutations alter residues that cluster to specific regions within the membrane spanning segments of the integral membrane components MalF and MalG. Individually, the mutations perturb the ability of MBP to interact productively with the membrane complex. Genetic alteration of this signalling pathway suggests that other agents might have similar effects. These could be potentially useful for modulating the activities of ABC transporters such as P-glycoprotein or CFTR, that are implicated in disease.

Partition of nonreplicating DNA by the par system of bacteriophage P1.

P1 plasmid encodes a cis-acting centromere analog, parS, and two Par proteins that together stabilize plasmids by partitioning them to daughter bacteria. We infected immune bacteria with bacteriophage lambda into which parS had been inserted. The presence of P1 Par proteins in the infected cells was found to delay the appearance of cells cured of the nonreplicating, extrachromosomal lambda-parS DNA. This stabilization of lambda-parS, approximated in a computer simulation, demonstrates that active partition by the P1 par system does not require the act of plasmid replication and can be studied in its absence.

Allele-specific malE mutations that restore interactions between maltose-binding protein and the inner-membrane components of the maltose transport system.

Active accumulation of maltose and maltodextrins by Escherichia coli depends on an outer-membrane protein. LamB, a periplasmic maltose-binding protein (MalE, MBP) and three inner-membrane proteins, MalF, MalG and MalK. MalF and MalG are integral transmembrane proteins, while MalK is associated with the inner aspect of the cytoplasmic membrane via an interaction with MalG. Previously we have shown that MBP is essential for movement of maltose across the inner membrane. We have taken advantage of malF and malG mutants in which MBP interacts improperly with the membrane proteins. We describe the properties of malE mutations in which a proper interaction between MBP and defective MalF and MalG proteins has been restored. We found that these malE suppressor mutations are able to restore transport activity in an allele-specific manner. That is, a given malE mutation restores transport activity to different extents in different malF and malG mutants. Since both malF and malG mutations could be suppressed by allele-specific malE suppressors, we propose that, in wild-type bacteria, MBP interacts with sites on both MalF and MalG during active transport. The locations of different malE suppressor mutations indicate specific regions on MBP that are important for interacting with MalF and MalG.

Transport of p-nitrophenyl-alpha-maltoside by the maltose transport system of Escherichia coli and its subsequent hydrolysis by a cytoplasmic alpha-maltosidase.

In wild-type Escherichia coli the activity of the maltose transport system is dependent on a periplasmic maltose-binding protein. It has been possible, however, to isolate mutants in which transport activity is mediated by the membrane components of the system and is no longer dependent on the periplasmic binding protein. In this manuscript we show that in these binding protein-independent strains, p-nitrophenyl-alpha-maltoside is a potent inhibitor of maltose transport. In contrast, p-nitrophenyl-alpha-maltoside is only a weak inhibitor of maltose transport in wild-type bacteria. In addition, we show that p-nitrophenyl-alpha-maltoside is transported by the binding protein-independent strains but not by wild-type bacteria. We were able to detect transport of this compound because there is a cytoplasmic enzyme that cleaves p-nitrophenyl-alpha-maltoside. This enzyme has not previously been described. We show that although the synthesis of this enzyme is subject to the same regulation as the components of the maltose regulon, and is MalT dependent, it is not coded for by a known mal gene. We refer to this enzyme as alpha-maltosidase. These results strengthen our proposal that the membrane components of the maltose transport system comprise a recognition site for maltose and related substrates.

Genetic evidence for substrate and periplasmic-binding-protein recognition by the MalF and MalG proteins, cytoplasmic membrane components of the Escherichia coli maltose transport system.

We isolated mutants of Escherichia coli in which the maltose-binding protein (MBP) is no longer required for growth on maltose as the sole source of carbon and energy. These mutants were selected as Mal+ revertants of a strain which carries a deletion of the MBP structural gene, malE. In one class of these mutants, maltose is transported into the cell independently of MBP by the remaining components of the maltose system. The mutations in these strains map in either malF or malG. These genes code for two of the cytoplasmic membrane components of the maltose transport system. In some of the mutants, MBP actually inhibits maltose transport. We demonstrate that these mutants still transport maltose actively and in a stereospecific manner. These results suggest that the malF and malG mutations result in exposure of a substrate recognition site that is usually available only to substrates bound to MBP.

Anti-idiotypic route to anti-acetylcholine receptor antibodies and experimental myasthenia gravis.

trans-3,3'-Bis[alpha-(trimethylammonio)methyl]azobenzene bromide (BisQ) is a potent agonist of the acetylcholine receptor (AcChoR) of Electrophorus electricus. BisQ is highly constrained, suggesting that its structure is complementary to the combining site of the AcChoR when the latter is in its activated state. Antibodies produced in rabbits to a conjugate of bovine serum albumin and a derivative of BisQ mimicked the binding characteristics of the AcChoR with respect to the order of binding of a variety of agonists and to the preferred recognition of decamethonium ion (an agonist) over hexamethonium ion (an antagonist). Immunization of three rabbits with purified anti-BisQ yielded antisera having binding characteristics of anti-AcChoR in that, by complement fixation and enzyme immunoassay, crossreactions with receptor preparations from rat, Torpedo, and eel could be demonstrated in sera of all three rabbits immunized. Two of the three rabbits showed signs of muscle weakness similar to that seen after immunization with the AcChoR. One of the rabbits was injected intramuscularly with neostigmine and showed temporary improvement. Another showed post-tetanic exhaustion of hind-limb muscles after stimulation of the sciatic nerve at 50 Hz. Antibodies reactive with the AcChoR, therefore, were elicited by immunization with an antibody to a potent ligand of the AcChoR without the necessity of isolating the receptor itself. A similar mechanism may play a part in the etiology of at least some autoimmune diseases in which antibodies to various other receptors are involved.