A PEST-like sequence in listeriolysin O essential for Listeria monocytogenes pathogenicity.

Establishment and maintenance of an intracellular niche are critical to the success of an intracellular pathogen. Here, the pore-forming protein listeriolysin O (LLO), secreted by Listeria monocytogenes, was shown to contain a PEST-like sequence (P, Pro; E, Glu; S, Ser; T, Thr) that is essential for the virulence and intracellular compartmentalization of this pathogen. Mutants lacking the PEST-like sequence entered the host cytosol but subsequently permeabilized and killed the host cell. LLO lacking the PEST-like sequence accumulated in the host-cell cytosol, suggesting that this sequence targets LLO for degradation. Transfer of the sequence to perfringolysin O transformed this toxic cytolysin into a nontoxic derivative that facilitated intracellular growth.

Three sites of contact between the Bacillus subtilis transcription factor sigmaF and its antisigma factor SpoIIAB.

The developmental regulatory protein sigmaF of Bacillus subtilis, a member of the sigma70-family of RNA polymerase sigma factors, is regulated negatively by the antisigma factor SpoIIAB, which binds to sigmaF to form an inactive complex. Complex formation between SpoIIAB, which contains an inferred adenosine nucleotide binding pocket, and sigmaF is stimulated strongly by the presence of ATP. Here we report that SpoIIAB contacts sigmaF at three widely spaced binding surfaces corresponding to conserved regions 2.1, 3.1, and 4.1 of sigma70-like sigma factors. This conclusion is based on binding studies between SpoIIAB and truncated portions of sigmaF, the isolation of mutants of sigmaF that were partially resistant to inhibition by SpoIIAB in vivo and were defective in binding to the antisigma factor in vitro, and the creation of alanine substitution mutants of regions 2.1, 3.1, or 4.1 of sigmaF that were impaired in complex formation. Because the interaction of SpoIIAB with all three binding surfaces was stimulated by ATP, we infer that ATP induces a conformational change in SpoIIAB that is needed for tight binding to sigmaF. Finally, we discuss the possibility that another antisigma factor, unrelated to SpoIIAB, may interact with its respective sigma factor in a similar topological pattern of widely spaced binding surfaces located in or near conserved regions 2.1, 3.1, and 4.1.

Bacillus subtilis lon protease prevents inappropriate transcription of genes under the control of the sporulation transcription factor sigma G.

The Bacillus subtilis RNA polymerase sigma factor sigma G is a cell-type-specific regulatory protein that governs the transcription of genes that are expressed at an intermediate to late stage of sporulation in the forespore compartment of the sporangium. Here we report the identification of a mutation (lon-1) that causes inappropriate transcription of genes under the control of sigma G under nutritional and genetic conditions in which sporulation is prevented. The mutation is located at 245 degrees on the genetic map and lies within a newly identified open reading frame that is predicted to encode a homolog to Lon protease. Inappropriate transcription of sigma G-controlled genes in the lon-1 mutant is not prevented by mutations in genes that are normally required for the appearance of sigma G during sporulation but is prevented by a mutation in the structural gene (spoIIIG) for sigma G itself. In light of previous work showing that spoIIIG is subject to positive autoregulation, we propose that Lon protease is responsible (possibly by causing degradation of sigma G) for preventing sigma G-directed transcription of spoIIIG and hence the accumulation of sigma G in cells that are not undergoing sporulation. An integrated physical and genetic map is presented that encompasses 36 kb of uninterrupted DNA sequence from the lon pheA region of the chromosome, corresponding to 245 degrees to 239 degrees on the genetic map.