The RNA polymerase sigma factor F is a developmental regulatory protein that is activated in a cell-specific manner following the formation of the polar septum during the process of spore formation in the bacterium Bacillus subtilis. Activation of F depends on the membrane-bound phosphatase SpoIIE, which localizes to the septum, and on the formation of the polar septum itself. SpoIIE is responsible for dephosphorylating and thereby activating the phosphoprotein SpoIIAA, which, in turn, triggers the release of F from the anti-F factor SpoIIAB. Paradoxically, however, the presence of unphosphorylated SpoIIAA is insufficient to cause F activation as SpoIIAA reaches substantial levels in mutants blocked in polar septation. We now describe mutants of SpoIIE, SpoIIAA, and SpoIIAB that break the dependence of F activation on polar division. Analysis of these mutants indicates that unphosphorylated SpoIIAA must reach a threshold concentration in order to trigger the release of F from SpoIIAB. Evidence is presented that this threshold is created by the action of SpoIIAB, which can form an alternative, long lived complex with SpoIIAA. We propose that formation of the SpoIIAASpoIIAB complex serves as a sink that traps SpoIIAA in an inactive state and that only when unphosphorylated SpoIIAA is in excess to the sink does activation of F take place.

Cell-specific activation of the transcription factor F during the process of sporulation in Bacillus subtilis is governed by an antisigma factor SpoIIAB and an antiantisigma factor SpoIIAA. SpoIIAB, which exists as a dimer, binds to F in a complex of stoichiometry F ⅐SpoIIAB 2 . Escape from the complex is mediated by SpoIIAA, which reacts with the complex to cause the release of free F . Previous evidence indicated that Arg-20 in SpoIIAB is a contact site for both F and SpoIIAA and that contact with F is mediated by Arg-20 on only one of the two subunits in the F ⅐SpoIIAB 2 complex. Here we report the construction of heterodimers of SpoIIAB in which one subunit is wild type and the other subunit is a mutant for Arg-20. We show that the dissociation constant for the binding of F to the heterodimer was similar to that for the wild type, a finding consistent with the idea that F contacts Arg-20 on only one of the two subunits. Although SpoIIAA was highly effective in causing the release of F from the wild type homodimer, the anti-antisigma factor had little effect on the release of F from the heterodimer. This finding is consistent with a model in which SpoIIAA docks on the F ⅐SpoIIAB 2 complex, making contact with the subunit in which Arg-20 is not in contact with F . SpoIIAB is both an anti-F factor and a protein kinase that phosphorylates and thereby inactivates SpoIIAA. We show that SpoIIAA effectively displaces F from a complex of F with a mutant (SpoIIAB R105A ) that is impaired in the kinase function of SpoIIAB. This result shows that SpoIIAAmediated displacement of F from SpoIIAB does not require concomitant phosphorylation of SpoIIAA.

The enterococci are low-GC Gram-positive bacteria that have emerged as leading causes of hospital-acquired infection. They are also commensals of the gastrointestinal tract of healthy humans and most other animals with gastrointestinal flora and are important for food fermentations. Here we report the availability of draft genome sequences for 28 enterococcal strains of diverse origin, including the species Enterococcus faecalis, E. faecium, E. casseliflavus, and E. gallinarum.The enterococci are ecologically diverse, Gram-positive lactic acid bacteria that are found in the gastrointestinal consortia of humans, other mammals, reptiles, amphibians, birds, and insects and are utilized in production of fermented foods and probiotics (1, 2, 16). However, they have also emerged as leading causes of hospital-acquired infection at extraintestinal sites, including the heart, urinary tract, and surgical site wounds (9, 10). These infections have become a leading health care concern because of increasing antibiotic resistance mediated by mobile genetic elements (12). The ability of enterococci to colonize extraintestinal sites and cause infection involves both core and variable genetic traits (3, 15) and is not fully understood.Enterococci exhibit diversity in metabolic capabilities (such as the ability of Enterococcus faecalis, but not E. faecium, to respire [11]) and in clinically relevant phenotypes such as toxin production (12) and biofilm formation (13). Enterococci can also be motile or nonmotile, and pigmented or nonpigmented (4), yet little is known about the mechanism and significance of these traits. Insights into basic enterococcal physiology are limited by a paucity of genome data. The genome sequences of the vancomycin-resistant bloodstream isolate E. faecalis V583 (14), the human oral isolate E. faecalis OG1RF (3), and the commercial probiotic strain E. faecalis Symbioflor (6) have been determined, and a draft genome sequence for E. faecium DO has been available in GenBank since 2002. Here we announce the availability of draft genome sequences of 28 additional enterococcal strains, including 16 E. faecalis strains, 8 E. faecium strains, 3 E. casseliflavus strains, and 1 E. gallinarum strain. In addition to expanding the Enterococcus nucleotide sequence in GenBank by approximately an order of magnitude, this is the first report of genome sequencing of the motile E. gallinarum strain and the motile, pigmented E. casseliflavus strains.Most of the enterococcal strains utilized for our genome sequencing project are of clinical origin. The remaining strains are commensal isolates or were obtained from animals or insects. E. faecalis strains selected for this project were previously described in a study of E. faecalis strain diversity (12). To maximize the information yield from genome sequencing, we selected 16 E. faecalis strains that represent the deepest phylogenetic nodes in the E. faecalis multilocus sequence typing (MLST) dendrogram from the 106 strains previously examined (12). These strains include commens...

SpoIIE is a dual-function protein in Bacillus subtilis that contributes to the switch from medial to polar cell division during sporulation and is responsible for activating the cell-specific transcription factor F . SpoIIE consists of an N-terminal domain with 10 membrane-spanning segments (region I), a C-terminal phosphatase domain (region III), and a central domain (region II) of uncertain function. To investigate the role of SpoIIE in polar division, we took advantage of a system for efficiently producing polar septa during growth in a SpoIIE-dependent manner using cells engineered to produce the sporulation protein in response to an inducer. The results show that regions II and III play a critical role in polar septum formation and that specific amino acid substitutions in those regions affect the abilities of SpoIIE both to promote polar division and to localize to the division machinery. Additionally, we show that neither the phosphatase function of SpoIIE nor the N-terminal, membrane-spanning region is needed for the switch to asymmetric division.