Biochemical characterization of the C4-dicarboxylate transporter DctA from Bacillus subtilis.

Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C(4)-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed.

Na(+):aspartate coupling stoichiometry in the glutamate transporter homologue Glt(Ph).

The Na(+) aspartate symporter Glt(Ph) from Pyrococcus horikoshii is the only member of the glutamate transporter family for which crystal structures have been determined. The cation:aspartate coupling stoichiometry is unknown, thus hampering the elucidation of the ion coupling mechanism. Here we measure transport of (22)Na(+) and [(14)C]aspartate in proteoliposomes containing purified Glt(Ph) and demonstrate that three Na(+) ions are symported with aspartate.

Quality control of overexpressed membrane proteins.

Overexpression of membrane proteins in Escherichia coli frequently leads to the formation of aggregates or inclusion bodies, which is undesirable for most studies. Ideally, one would like to optimize the expression conditions by monitoring simultaneously and rapidly both the amounts of properly folded and aggregated membrane protein, a requirement not met by any of the currently available methods. Here, we describe a simple gel-based approach with green fluorescent protein as folding indicator to detect well folded and aggregated proteins simultaneously.

Rigidity of the subunit interfaces of the trimeric glutamate transporter GltT during translocation.

Glutamate transporters are trimeric membrane proteins in which each protomer contains a separate translocation path. To determine whether structural rearrangements take place at the subunit interfaces during transport, intersubunit disulfide bridges were introduced in the bacterial transporter GltT. None of the intersubunit cross-links, which had been designed across the entire interface, affected the glutamate transport activity, indicating that the subunit interfaces are rigid during turnover.

Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus.

Flagellation in archaea is widespread and is involved in swimming motility. Here, we demonstrate that the structural flagellin gene from the crenarchaeaon Sulfolobus solfataricus is highly expressed in stationary-phase-grown cells and under unfavorable nutritional conditions. A mutant in a flagellar auxiliary gene, flaJ, was found to be nonmotile.

Identification and functional characterization of the Lactococcus lactis CodY-regulated branched-chain amino acid permease BcaP (CtrA).

Transcriptome analyses have previously revealed that a gene encoding the putative amino acid transporter CtrA (YhdG) is one of the major targets of the pleiotropic regulator CodY in Lactococcus lactis and Bacillus subtilis. The role of ctrA in L. lactis was further investigated with respect to both transport activity as well as CodY-mediated regulation.