Escherichia coli to host tissue is mediated by pili, which are hair-like protein structures extending from the outer cell membrane of the bacterium. The chaperones FimC and PapD are key components in pilus assembly since they catalyse folding of subunits that are incorporated in type 1 and P pili, respectively, and also transport the subunits across the periplasmic space. Recently, compounds that inhibit pilus biogenesis and interfere with chaperone-subunit interactions have been discovered and termed pilicides. In this paper NMR spectroscopy was used to study the interaction of different pilicides with PapD and FimC in order to gain structural knowledge that would explain the effect that some pilicides have on pilus assembly. First relaxation-edited NMR experiments revealed that the pilicides bound to the PapD chaperone with mM affinity. Then the pilicide-chaperone interaction surface was investigated through chemical shift mapping using 15 N-labelled FimC. Principal component analysis performed on the chemical shift perturbation data revealed the presence of three binding sites on the surface of FimC, which interacted with three different classes of pilicides. Analysis of structure-activity relationships suggested that pilicides reduce pilus assembly in E. coli either by binding in the cleft of the chaperone, or by influencing the orientation of the flexible F1-G1 loop, both of which are part of the surface by which the chaperone forms complexes with pilus subunits. It is suggested that binding to either of these sites interferes with folding of the pilus subunits, which occurs during formation of the chaperone-subunit complexes. In addition, pilicides that influence the F1-G1 loop also appear to reduce pilus formation by their ability to dissociate chaperone-subunit complexes.

A monospecific antibody recognizing two membrane proteins in Acholeplasma laidlawii identified a plasmid clone from a genomic library. The nucleotide sequence of the 4.6-kbp insert contained four sequential genes coding for proteins of 39 kDa (Elax; N terminus not cloned), 36 kDa (E13), 57 kDa (E2), and 36 kDa (E3; C terminus not cloned). The N termini of the cloned E2, E1,B, and native A. laidlawii E2 proteins were verified by amino acid sequencing. Computer-aided searches showed that the translated DNA sequences were homologous to the four subenzymes of the pyruvate dehydrogenase complexes from gram-positive bacteria and humans. The plasmid-encoded 57-kDa (E2) protein was recognized by antibodies against the E2 subenzymes of the pyruvate and oxoglutarate dehydrogenase complexes from Bacillus subtilis. A substantial fraction of the E2 protein as well as part of the pyruvate dehydrogenase enzymatic activity was associated with the cytoplasmic membrane in A. Iaidlawii. In vivo complementation with three different Escherichia coli pyruvate dehydrogenase-defective mutants showed that the four plasmid-encoded proteins were able to restore pyruvate dehydrogenase enzyme activity in E. coli. Since A. laidlawii lacks oxoglutarate dehydrogenase and most likely branched-chain dehydrogenase enzyme complex activities, these results strongly suggest that the sequenced genes code for the pyruvate dehydrogenase complex.