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Summary The type II secretion system is a multi-protein complex that spans the cell envelope of gramnegative bacteria and promotes the secretion of proteins, including several virulence factors. This system is homologous to the type IV pilus biogenesis machinery and contains five proteins, EpsGK, termed the pseudopilins that are structurally homologous to the type IV pilins. The major pseudopilin EpsG has been proposed to form a pilus-like structure in an energy-dependent process that requires the ATPase, EpsE. A key remaining question is how the membrane-bound EpsG interacts with the cytoplasmic ATPase, and if this is a direct or indirect interaction. Previous studies have established an interaction between the bitopic inner membrane protein EpsL and EpsE; therefore, in this study we used in vivo cross-linking to test the hypothesis that EpsG interacts with EpsL. Our findings suggest that EpsL may function as a scaffold to link EpsG and EpsE and thereby transduce the energy generated by ATP hydrolysis to support secretion. The recent discovery of structural homology between EpsL and a protein in the type IV pilus system implies that this interaction may be conserved and represent an important functional interaction for both the type II secretion and type IV pilus systems.

Deletions or substitutions of amino acids at the carboxyl-terminus of the heat-labile enterotoxin B subunit (EtxB) affect its assembly into pentamers in a temperature-dependent manner. At 42 degrees C, the mutations prevent the B subunits from achieving their final pentameric structure resulting in membrane association of the monomers. However, mutant B subunits produced at 30 degrees C assemble, in the periplasm, into pentamers that remain stable when transferred to 42 degrees C, indicating that the mutant pentamers are stable under conditions where their formation is inhibited. The mutant pentamers are, similarly to wild-type pentamers, SDS-resistant and stable, in vitro, at temperatures up to 65 degrees C. This suggests that although the C-terminal amino acids are part of the subunit interface, they appear not to contribute significantly to the stability of the final pentameric complex, but are instead essential for the formation or stabilization of an assembly intermediate in the pentamerization process. Single second site mutations suppress the assembly defect of mutant EtxB191.5, which carries substitutions at its C-terminus. The Thr-->Ile replacement at position 75 in the alpha 2-helix probably restores the van der Waals contact between residues 75 and 101, which had been greatly reduced by the Met-->Leu substitution at position 101 in the beta 6-strand of EtxB191.5. Interaction between the alpha 2-helix and beta 6-strand which contains the C-terminus probably stabilizes a conformation essential for assembly and is therefore required for the formation of pentamers.

Summary PsiB, an anti-SOS protein, shown previously to prevent activation of RecA protein, was purified from the crude extract of PsiB overproducing cells. PsiB is probably a tetrameric protein, whose subunit has a sequence-deduced molecular mass of 15741 daltons. Using an immuno-assay with anti-PsiB antibodies, we have monitored PsiB cell concentrations produced by F and R6-5 piasmids: the latter type produces a detectable level of PsiB protein while the former does not. The discrepancy can be assigned to a Tn 10 outgoing promoter located upstream of psiB. When we inserted a Tn 10 promoter upstream of F psiB, the F PsiB protein concentration reached the level of R6-5 PsiB. We describe here the physiological role that PsiB protein may have in the cell and how it causes an anti-SOS function. We observed that PsiB protein was transiently expressed by a wild-type F sex factor during its transmission to an Escherichia coli K-12 recipient, In an F+× F- cross, PsiB concentration increased at least 10-fold in F- recipient bacteria after 90 minutes and declined thereafter; the psiB gene may be repressed when F plasm id replicates vegetatively. PsiB protein may be induced zygotically so as to protect F single-stranded DNA transferred upon conjugation. PsiB protein, when overproduced, may interfere with RecA protein at chromosomal single-stranded DNA sites generated by discontinuous DNA replication, thus causing an SOS inhibitory pheno-type.