Fig 1.
Expression of the sigG gene and the switch from σF to σG during B. subtilis sporulation.
(A) Cartoon depicting the sigma factors that govern gene expression during early (top) and late (bottom) stages of B. subtilis sporulation. At early times, the sigma factors σF and σE control gene expression in the forespore and mother cell, respectively. At later times, after forespore engulfment by the mother cell, σF is replaced by σG and σE is replaced by σK. The switch from σF to σG is indicated by a dashed arrow. (B) The switch from σF to σG. The σF-dependent expression of a PspoIIQ-lacZ reporter (open diamonds) and σG-dependent expression of a PsspB-lacZ reporter (closed squares) were monitored during sporulation of wild type cells. (Strains AHB881 and AHB317, respectively.) All lacZ reporters used in this study, unless otherwise noted, were at the non-essential amyE locus. β-Galactosidase activity is reported as the percent maximum for each. The timing of the σF-to-σG switch, between sporulation hours 2.5 and 3, is indicated by a dashed gray line. (C) Cartoon depicting the sigG gene in its native chromosomal location downstream of the spoIIGA-sigE operon, and our new, simplified model for its transcriptional regulation by σF at early times and σG itself at late times. Dashed box/arrow indicates region depicted in (D). (D) Sequence of the sigG upstream regulatory region and 5’ coding region (codons 1–28). The -35 and -10 sigG promoter elements [9] are boxed, with the consensus σF and σG-recognized sequences shown above [38]. Also boxed are the sigG +1 transcription start site, RBS, and GTG start codon. Gray dashed arrows indicate complementary sequences predicted to form a hairpin structure that blocks translation of read-through transcripts originating from PspoIIG [52]; note that this hairpin cannot form in transcripts originating from PsigG. Green, blue, and purple solid arrows indicate complementary sequences predicted to form hairpin secondary structures in mRNA transcripts originating from PsigG.
Fig 2.
PsigG activity is detected both before and after the switch from σF to σG.
(A, B) The activities of PsigG reporters harboring the native GTG start codon, PsigG-sigG1-28-lacZ (PsigG-GTG; open circles) or engineered to harbor an ATG start codon, PsigG-ATG-sigG2-28-lacZ (PsigG-ATG; closed circles), were measured during a time course of sporulation. (Strains EBM177 and EBM175, respectively.) Background β-galactosidase activity was measured in a strain without a lacZ reporter, PY79 (no lacZ; asterisks). Note that (B) provides a zoomed view and statistical significance of early sporulation data points from the boxed area of (A). (C, D) Strains harboring PsigG reporters with altered sigG 5’ coding sequence, one engineered to reduce secondary structure (RSS) in sigG codons 2–28, PsigG-ATG-RSSsigG2-28-lacZ (PsigG-ATG-RSS; open triangles), and another harboring comGA codons 2–8 in place of sigG codons 2–28, PsigG-ATG-comGA2-8-lacZ (PsigG-ATG-comGA; open diamonds), were monitored for β-galactosidase production during sporulation. (Strains EBM237 and JJB31, respectively.) Data for the PsigG-ATG-sigG2-28-lacZ (PsigG-ATG; closed circles) strain (EBM175) and PY79 (no lacZ, asterisks), the same as shown in (A) and (B), are also included for reference. Note that (D) provides a zoomed view and statistical significance of early sporulation data points from the boxed area of (C). The timing of the σF-to-σG switch, between sporulation hours 2.5 and 3, is indicated in each panel by a dashed gray line. For all panels, error bars indicate ± standard deviations based on three independent experiments. *p < 0.05, **p < 0.01, NS not significant, Student’s t-test.
Fig 3.
Late σF-dependent expression of both PsigG and PspoIIQ requires SpoIIQ, σE, and SpoIIIAA-AH.
The activity of PsigG-lacZ (A, C, D) or PspoIIQ-lacZ (B, E) was monitored during sporulation of strains with the following genotypes: wild type (WT; open circles), ΔsigG (closed circles), ΔsigG ΔsigF (open squares), ΔsigG ΔspoIIQ (open diamonds), ΔsigG ΔsigE (open triangles), and ΔsigG ΔspoIIIAA-AH (closed triangles). (PsigG-lacZ strains were JJB31, JJB73, JJB75, JJB79, JJB85, and JJB77, respectively. PspoIIQ-lacZ strains were AHB881, AHB882, AHB915, AHB916, AHB917, and AHB1017, respectively.) For clarity, only data from a subset of these strains are presented in each graph (as labeled) and, also for clarity, the data for the ΔsigG strain of each (closed circles) is presented in all graphs. Note that (D) provides a zoomed view of the data from the boxed area of (C). The timing of the σF-to-σG switch, between sporulation hours 2.5 and 3, is indicated in each panel by a dashed gray line. For all panels, error bars indicate ± standard deviations based on three independent experiments.
Fig 4.
Suboptimal spacing of the PsigG -10 and -35 elements diminishes sigG expression.
(A) Alignment of ten B. subtilis promoters activated by σF, including PsigG. Nucleotides comprising the -10 and -35 elements of each promoter are in bold and shaded gray. Transcription start sites, if known, are underlined [41]. The consensus promoter sequence for σF is shown above [38]. Red arrowheads indicate two notable features of PsigG that differ from the other σF-target promoters: shorter spacing of the -10 and -35 promoter elements (14 nt as opposed to the more common 15 nt; left arrowhead), and a T at position -7 (more typically an A or G; right arrowhead). (B, C) PsigG activation is significantly stimulated by increasing the spacing between the -10 and -35 elements to 15 nt. β-Galactosidase production was monitored during sporulation of strains harboring PsigG-lacZ (WT [14 nt]; open circles) and 15ntPsigG-lacZ (15 nt; closed circles), a variant in which a single nucleotide was inserted between the PsigG -10 and -35 elements to increase their spacing to 15 nt. (Strains JJB31 and JJB51, respectively.) Note that (C) provides a zoomed view of the data from the boxed area of (B). (D, E) The T at position -7 at most only modestly influences PsigG activation. The activity of PsigG-lacZ (WT; closed circles),T→APsigG-lacZ (T→A; closed diamonds), and T→GPsigG-lacZ (T→G; open diamonds) was measured during sporulation. (Strains JJB31, JJB87, and JJB89, respectively.) The T→A and T→G variants were engineered to harbor an A or G, respectively, at position -7 in place of T. Note that (E) provides a zoomed view of the data from the boxed area of (D). The timing of the σF-to-σG switch, between sporulation hours 2.5 and 3, is indicated in each panel by a dashed gray line. For all relevant panels, error bars indicate ± standard deviations based on three independent experiments. *p < 0.05, **p < 0.01 Student’s t-test.
Fig 5.
An mRNA hairpin formed between the sigG leader sequence and RBS significantly decreases PsigG-lacZ translation.
(A) Depiction of the mutations in the PsigG+10→+30-lacZ reporter variant, as well as variants designed to harbor only a subset of these mutations. The sigG transcription start site (+1), sigG ribosome-binding site (RBS), and ATG start codon are indicated. (*Note that the PsigG-lacZ reporter gene used here harbored the non-native ATG start codon in place of the native sigG GTG start codon.) Also indicated, with green arrows, are complementary sequences predicted to form the RBS hairpin structure shown in (B). (B) Depiction of the sigG RBS-hairpin structure predicted to form in the sigG mRNA leader sequence. The 5’ end of the sigG mRNA (+1), the sigG core RBS, and native GTG (GUG) translation start codon are labeled. Asterisks indicate the six nucleotide positions altered in the +10→+15 mutant. The prediction and graphic were generated by ViennaRNA, with bases color-coded according to their partition function probabilities [68]. (C) Alteration to nucleotides upstream of the sigG RBS stimulates expression from a PsigG-lacZ reporter gene. The activities of PsigG+10→+30-lacZ (+10→+30; closed circles), PsigG+24→+30-lacZ (+24→+30; open squares), PsigG+10→+18-lacZ (+10→+18; closed squares), PsigG+18-lacZ (+18; closed triangles), PsigG+10→+15-lacZ (+10→+15; open diamonds), and the corresponding wild type PsigG-lacZ (WT; open circles) were measured during sporulation. (Strains AHB883, AHB2124, AHB2126, JC68, JC70, and AHB1274, respectively.) (D) The +10→+15 alterations do not impact β-galactosidase production from a transcriptional lacZ reporter. β-Galactosidase production was monitored during sporulation of strains harboring PsigG-RBS-lacZ (WT [+RBS]; open squares) and PsigG+10→+15-RBS-lacZ (+10→+15 [+RBS]; closed squares). (Strains AM3 and AM4, respectively.) In these transcriptional reporters, lacZ is separated from the sigG RBS by a spacer and provided with an engineered RBS. (E) Mutations that weaken/eliminate or strengthen the sigG RBS-hairpin increase or decrease PsigG-lacZ expression, respectively. The activities of PsigGmut7-lacZ (mut7; closed squares), PsigGmut2-lacZ (mut2; open squares), and the corresponding wild type PsigG-lacZ (WT; open circles) were measured during sporulation. (Strains JJB55, JJB37, and JJB31, respectively.) The mRNA leader sequence for each PsigG-lacZ variant is indicated to the right, with the 5’ end of the mRNA (+1), core RBS, and the mut7 or mut2 mutations (in blue) labeled and underlined. Also depicted is the capacity for formation of a hairpin structure (green lines connecting complementary base-pairs). For all relevant panels, error bars indicate ± standard deviations based on three independent experiments.
Fig 6.
The identified transcriptional and translational regulation of sigG diminishes sigG expression by 4-6-fold and is required to prevent aberrant activity of σG.
(A, B) Expression of a PsigG reporter is increased by 4-6-fold in the absence of the four regulatory features identified in this study. β-Galactosidase production was monitored during sporulation of strains harboring PsigG-sigG1-28-lacZ (WTPsigG-lacZ; open circles) or a variant in which all four features of sigG that dampen expression were simultaneously removed or repaired, 15ntPsigGmut7-ATG-RSSsigG2-28-lacZ (quadPsigG-lacZ; closed circles) (strains EBM177 and EBM262, respectively.) Note that (B) provides a zoomed view of the data from the boxed area of (A). (C, D) Expression of sigG from regulatory sequences lacking the four regulatory features identified in this study causes aberrant σG activity during a time course of sporulation. β-Galactosidase production from (C) the σF-dependent PspoIIQ-lacZ reporter or (D) the σG-dependent PsspB-lacZ reporter was monitored during sporulation of strains in which sigG was expressed from its wild type regulatory sequences (WTPsigG-sigG; open squares) or from regulatory sequences modified to remove or repair the four features identified in this study to dampen sigG expression (quadPsigG-sigG; closed squares). (PspoIIQ-lacZ strains were CFB429 and CFB431, respectively. PsspB-lacZ strains were CFB435 and CFB437, respectively.) The black arrow in (D) indicates aberrant σG activity at early times of sporulation. The timing of the σF-to-σG switch, between sporulation hours 2.5 and 3, is indicated in each panel by a dashed gray line. For all panels, error bars indicate ± standard deviations based on three independent experiments.
Fig 7.
Misexpression of sigG causes ectopic σG activity in a subset of vegetative cells.
(A, B) GFP production from a σG-dependent PsspB-gfp reporter was monitored by fluorescence microscopy of vegetatively growing cells in which sigG was expressed from its wild type regulatory sequences (WTPsigG) or from regulatory sequences modified to remove or repair the four features identified in this study to dampen sigG expression (quadPsigG). Additionally, cells either harbored wild type csfB, or were deleted for the gene (ΔcsfB). (Strains EBM192 [WTPsigG], EBM276 [quadPsigG], EBM282 [WTPsigG ΔcsfB], and EBM287 [quadPsigG-sigG ΔcsfB].) In (A), representative microscopy images are shown with GFP fluorescence (false-colored green) merged with membrane fluorescence from the dye FM 4–64 (false-colored red). Yellow arrowheads indicate vegetative cells with visible GFP fluorescence. Scale bar 5 μm. In (B), GFP fluorescence intensity (with background subtracted) for more than 500 cells of each strain, including a “no GFP” control strain (PY79) lacking the PsspB-gfp reporter, is shown in column scatter graph format, with each cell represented by a black dot. Cells exhibiting fluorescence intensity above the cut off value (three standard deviations above mean auto-fluorescence of the “no GFP” strain; gray dashed line) were determined to have detectable σG activity. The percentage of cells with this activity is indicated above the respective strain. Note that ~60000 fluorescence units is the upper limit of detection under our microscopy settings. (C) Simultaneous deletion of csfB and misexpression of sigG from quadPsigG causes synthetic toxicity to vegetatively growing B. subtilis. The same strains described in (A) were struck onto LB agar and were photographed after ~18 hours of growth at 37°C.
Fig 8.
Misexpression of sigG causes premature σG activity in the forespore during sporulation.
GFP production from a σG-dependent PsspB-gfp reporter was monitored by fluorescence microscopy of sporulating cells (hour 3.5) in which sigG was expressed from its wild type regulatory sequences (WTPsigG) or from regulatory sequences modified to remove or repair the four features identified in this study to dampen sigG expression (quadPsigG). Additionally, cells either harbored wild type csfB, or were deleted for the gene (ΔcsfB). (Strains EBM192 [WTPsigG], EBM276 [quadPsigG] and EBM282 [WTPsigG ΔcsfB].) (A) Representative microscopy images for each strain. GFP fluorescence is depicted in grayscale (PsspB-GFP) or false-colored green (Merge). Membrane fluorescence from the dye FM 4–64 is shown in grayscale (Membranes) or false-colored red (Merge). Following asymmetric division and during engulfment, the FM 4–64 dye labels all membranes including the double membranes separating the mother cell and forespore; after the completion of engulfment, the membranes surrounding the forespore are no longer accessible to the dye and therefore remain unlabeled. Yellow arrowheads indicate late engulfment forespores, identifiable by their FM 4-64-labeled engulfing membranes, with visible GFP fluorescence. Scale bar 5 μm. (B) GFP fluorescence intensity (with background subtracted) for more than 200 late-engulfment forespores or (C) their corresponding mother cells for the three strains described in (A) as well as a “no GFP” control strain (PY79) lacking the PsspB-gfp reporter are shown in column scatter graph format, with each cell represented by a black dot. Cells exhibiting fluorescence intensity above the cut off value (three standard deviations above mean auto-fluorescence of the “no GFP” strain; gray dashed line) were determined to have detectable σG activity. The percentage of cells with this activity is noted above the respective strain.