A role for Fin (YabK) in sigma factor switching during sporulation in B. subtilis. (A) Cartoon depicting the sigma factors directing compartment-specific gene expression in sporangia at early (top) and late (bottom) stages of development. At early times, the sigma factors σ^F and σ^E direct gene expression in the forespore and mother cell, respectively. At later times, after the forespore is engulfed by the mother cell, σ^F is replaced by σ^G and σ^E is replaced by σ^K. (B) Model for the switch from σ^F to σ^G. To begin, σ^F activates transcription of the gene (sigG) for σ^G (arrow 1); however, σ^G activation is delayed through poorly understood mechanisms that likely involve σ^F (barred line 2) (see Discussion). To trigger the switch to σ^G, σ^F also directs synthesis of its own inhibitor, Fin (arrow 3). Once σ^F is inactivated by Fin (barred line 4), σ^G becomes active. This transition is reinforced by two mechanisms. First, σ^G continues to direct fin synthesis, resulting in sustained σ^F inhibition (arrow 5). Second, σ^G autoregulates its own gene, leading to large amounts of the late sigma factor (arrow 6). σ^G also inhibits σ^F by an unknown, Fin-independent pathway (barred line 7) (see Discussion). In the absence of Fin, unchecked σ^F activity prevents σ^G activation, likely due to the same mechanisms represented by barred line 2. Dashed arrows indicate transcriptional regulation. Lines with barred ends indicate inhibition by currently unknown mechanisms.

fin (yabK) is a small gene that is expressed in the forespore under the control of σ^F and σ^G. (A) The fin gene (previously annotated yabK), depicted within its chromosomal context. To our knowledge, this gene synteny is conserved in all Bacillus genomes completed to date. The black bar indicates the fin-containing region that was inserted at the sacA locus to complement the mutation of fin. The drawing is to scale (as indicated), except that 2 kb of the mfd gene is not shown. (B) Sequence of the fin coding and upstream promoter regions. The putative −10 and −35 promoter sequences are boxed, with the consensus σ^F- and σ^G-recognized sequences shown above (40). Also boxed are the fin ATG start codon and TAA stop codon. The RBS for fin is underlined, while the dashed line indicates the 3′ end of the upstream pth gene, which overlaps the predicted fin promoter elements. (C) The fin promoter (P_fin) is activated by σ^F and σ^G during sporulation. The accumulation of β-galactosidase from a P_fin-lacZ reporter gene was measured during sporulation of wild-type cells (WT; ⋄), cells deleted for sigF (ΔsigF; ○), and cells deleted for sigG (ΔsigG; ▵) (strains AHB1933, AHB1956, and AHB1934, respectively). AU, arbitrary units. (D) GFP-Fin localizes diffusely throughout the forespore during early and late sporulation. Cells deleted for the native fin gene and harboring a functional gfp-fin gene fusion at the amyE locus (strain AHB2085) were observed by fluorescence microscopy at hour 3.5 of sporulation. GFP fluorescence is shown in grayscale (GFP-Fin) or false-colored green (merge). Membrane fluorescence from the dye FM4-64 is shown in grayscale (membrane) or false-colored red (merge). White arrowheads indicate a forespore at an early stage of sporulation (immediately following asymmetric division), while the white arrows indicate a forespore at a later stage of sporulation (after engulfment; note that membranes surrounding engulfed forespores are not stained due to membrane impermeability for the FM4-64 dye). Bar = 1 μm.

The Fin protein is conserved among Bacillus species and displays similarity to the anti-σ^G factor CsfB. Multiple sequence alignments are shown for CsfB orthologs (top) and Fin (YabK) orthologs (bottom) from various Bacillus and related species. The full amino acid sequence for each protein is shown. Identical or similar residues present in more than 50% of the sequences are shaded black or gray, respectively. Conserved Cys-X-X-Cys (CxxC) and Tyr-X-X-Tyr (YxxY) motifs present in Fin and CsfB ortholog families are indicated. Accession numbers for protein sequences are listed in Materials and Methods. Note that CsfB is also conserved more widely in endospore formers, including Clostridium species, and that multiple sequence alignments of the entire CsfB family have been published previously (19, 29).

fin is required for the efficient switch from σ^F to σ^G in the forespore. (A) σ^F remains active at late times during sporulation in the absence of fin. The σ^F-dependent activation of a P_spoIIQ-lacZ reporter gene was monitored during sporulation of wild-type cells (WT; ⋄), cells deleted for fin (Δfin; ▵), and cells deleted for fin and harboring a fin complementation construct at the sacA locus (Δfin + sacA::fin; ○) (strains AHB881, AHB1953, and AHB1985, respectively). (B) Fin and σ^G additively repress σ^F at late times during sporulation. σ^F-Directed β-galactosidase production from a P_spoIIQ-lacZ reporter gene was measured during sporulation of wild-type cells (WT; ⋄), cells deleted for fin (Δfin; ▵), cells deleted for sigG (ΔsigG; ⧫), and cells simultaneously deleted for fin and sigG (ΔsigG Δfin; ▴) (strains AHB881, AHB1953, AHB882, and AHB1954, respectively). (C) Fin is required for full σ^G activation. The σ^G-dependent activation of a P_sspB-lacZ reporter gene was monitored during sporulation of wild-type cells (WT; ⋄), cells deleted for fin (Δfin; ▵), and cells deleted for fin and harboring a fin complementation construct at the sacA locus (Δfin + sacA::fin; ○) (strains AHB324, AHB1952, and AHB1984, respectively). (D) Deletion of csfB partially restores σ^G activity to fin mutant cells. P_sspB-lacZ reporter gene activation by σ^G was monitored during sporulation of wild-type cells (WT; ⋄), cells deleted for fin (Δfin; ▵), cells deleted for csfB (ΔcsfB; ⧫), and cells simultaneously deleted for fin and csfB (Δfin ΔcsfB; ▴) (strains AHB881, AHB1953, AHB1879, and AHB2112, respectively).

Cells lacking fin are defective for spore formation and are arrested after engulfment. The status of sporulation at hour 6 of sporulation was determined for wild-type cells (WT), cells deleted for fin (Δfin), and cells deleted for fin and harboring a fin complementation construct at the sacA locus (Δfin + sacA::fin) (strains PY79, AHB1931, and AHB1983, respectively), using a combination of fluorescence and phase-contrast microscopy (see Materials and Methods) (n > 190 for each sample). The remaining, nonsporulating ∼25 to 30% of cells from each population are not shown. Also indicated (bottom) are the sporulation efficiencies for the indicated strains. Sporulation efficiency was calculated as the number of heat-resistant spores present after 24 h of growth/sporulation in DSM normalized to the number present in the wild-type sample.