pubmed.ncbi.nlm.nih.gov

The master regulator for entry into sporulation in Bacillus subtilis becomes a cell-specific transcription factor after asymmetric division - PubMed

  • ️Wed Jan 01 2003

The master regulator for entry into sporulation in Bacillus subtilis becomes a cell-specific transcription factor after asymmetric division

Masaya Fujita et al. Genes Dev. 2003.

Abstract

Gene transcription at the onset of sporulation in Bacillus subtilis is governed by Spo0A, a member of the response regulator family of transcription factors. Spo0A is traditionally viewed as the master regulator for entry into development. We now report that Spo0A continues to function after the initiation phase of sporulation and that it becomes a cell-specific transcription factor when the sporangium is divided into a mother cell and forespore. We observed that (1) Spo0A and Spo0A-directed gene transcription reached high levels in the mother cell; (2) an activated form of Spo0A impaired sporulation when produced in the forespore but not when produced in the mother cell; and (3) an inhibitor of Spo0A called Spo0A-N impaired sporulation and Spo0A-directed transcription when produced in the mother cell but not when produced in the forespore. Spo0A-N, which corresponds to the NH(2)-terminal domain of Spo0A, was shown to compete with the full-length response regulator for phosphorylation by the phosphorelay protein Spo0B. We propose that Spo0A is the earliest-acting transcription factor in the mother-cell line of gene expression and that in terms of abundance and transcriptional activity Spo0A may function predominantly as a cell-specific regulatory protein.

PubMed Disclaimer

Figures

Figure 1
Figure 1

Subcellular localization of GFP produced under the control of Spo0A during sporulation. Strains carrying a PspoIIG–gfp fusion (MF237) or a PspoIIE–gfp fusion (MF277) were induced to sporulate, treated with the vital membrane stain FM4-64 at hour 3 of sporulation, and observed by fluorescence microscopy. A strain carrying a Pspacc–gfp fusion (MF339) was used as a control. Bar, 1 μm.

Figure 2
Figure 2

Subcellular localization of Spo0A. Sporulating cells of the wild-type strain PY79 were collected at the indicated times after the start of sporulation. Immunofluorescence microscopy was carried out using anti-Spo0A antibodies or, as a control, anti-σA antibodies and a secondary antibody conjugated to FITC (green). The cells were also stained with DAPI (red) to visualize DNA. Representative cells are highlighted by rectangles. Arrowheads point to the forespore. Bar, 1 μm.

Figure 3
Figure 3

Artificial activation of Spo0A in the forespore inhibits sporulation. The strains used were: MF825 (PspoIIQ–spo0A), MF826 (PspoIIQ–sad67), MF827 (PspoIID–spo0A), and MF828 (PspoIID–sad67). Whole-cell extracts from cells collected at hour 3 of sporulation were subjected to SDS-PAGE and immunoblot analysis using anti-Spo0A antibodies (top panels). Cells were examined by phase-contrast microscopy at hour 4 of sporulation (bottom panels). Also shown are relative sporulation efficiencies for the strains.

Figure 4
Figure 4

Inhibition of Spo0A activation in the mother cell inhibits sporulation. (A) Whole-cell extracts from cells of strain PY79 (wild type), MF972 (Pspo0A–spo0A-N), MF973 (PspoIID–spo0A-N), and MF974 (PspoIIQ–spo0A-N) that had been collected at 1-h intervals after the start of sporulation were subjected to SDS-PAGE and immunoblot analysis using anti-Spo0A antibodies (top panel). Fluorescence microscopic images of cells that had been treated with FM4-64 at hour 4 of sporulation and sporulation efficiencies are shown for each of the strains (bottom panel). (B) Samples of purified Spo0A–His6 and Spo0A-N–His6 were subjected to SDS-PAGE and stained with Coomassie brilliant blue (lanes 1,2) or were subjected to SDS-PAGE and immunoblot analysis using anti-Spo0A antibodies (lanes 3–5). Samples of total protein from cells of strain MF972 collected at hour 2 of sporulation were subjected to SDS-PAGE and immunoblot analysis using anti-Spo0A antibodies (lanes 6–8). (Lane 1) Spo0A-N–His6 (14 pmole). (Lane 2) Spo0A–His6 (14 pmole). (Lane 3) Spo0A–His6 + Spo0A-N–His6 (0.035 pmole each). (Lane 4) Spo0A–His6 + Spo0A-N–His6 (0.14 pmole each). (Lane 5) Spo0A–His6 + Spo0A-N–His6 (0.35 pmole each). (Lane 6) Total protein (0.5 μg). (Lane 7) Total protein (1 μg). (Lane 8) Total protein (2 μg). Note that the His-tagged proteins migrated slightly slower than the corresponding untagged proteins. Positions of molecular weight markers (kD) are indicated on the left.

Figure 5
Figure 5

Spo0A-N competes with Spo0A for phosphorylation by phosphorelay proteins in vitro. Kinase reactions were performed as described in Materials and Methods, and the proteins were analyzed by SDS-PAGE. (A) Reaction mixtures (20 μL) contained 0.2 μM KinA (lanes 1–8), 0.2 μM Spo0F (lanes 2–8), 0.2 μM Spo0B (lanes 3–6), 2 μM Spo0A (lanes 4,6,7), and 2 μM Spo0A-N (lanes 5,6,8). Reaction mixtures were incubated at 25°C for 1 h and then stopped by adding SDS-loading buffer. Samples were subjected to electrophoresis through a 16% SDS–polyacrylamide gel, and radioactive proteins were visualized by autoradiography (right panel). Fourteen picomoles of each purified protein was subjected to electrophoresis and stained with Coomassie as a reference for electrophoretic mobility (left panel). (B) Reaction mixtures contained 0.2 μM KinA, 0.2 μM Spo0F, 0.2 μM Spo0B, and 2 μM Spo0A. Increasing amounts of Spo0A-N (0, 2, 4, and 6 μM, from left to right) were added to the reactions. The relative levels of Spo0A∼P are indicated.

Figure 6
Figure 6

Inhibition of Spo0A activation inhibits expression of the spoIIG operon. Culture samples from strains MF290 (♦, amyE∷PspoIIG–lacZ), MF1161 (▵, amyE∷PspoIIG–lacZ, zej82∷amyE∷PspoIIQ–spo0A-N), MF1151 (▴, amyE∷PspoIIG–lacZ, zej82∷amyE∷PspoIID–spo0A-N), and MF1140 (▪, amyE∷PspoIIG–lacZ, zej82∷amyE∷Pspo0A–spo0A-N), were collected at 1-h intervals after the start of sporulation and analyzed for β-galactosidase activity.

Figure 7
Figure 7

Subcellular localization of GFP synthesized under the control of Spo0A, σE, or σF in the cells producing Spo0A-N. The strains used were MF237 (PspoIIG-gfp), MF1146 (PspoIIG–gfp, Pspo0A–spo0A-N), MF1156 (PspoIIG–gfp, PspoIID–spo0A-N), MF1166 (PspoIIG–gfp, PspoIIQ–spo0A-N), MF248 (PspoIID–gfp), MF1144 (PspoIID–gfp, Pspo0A–spo0A-N), MF1157 (PspoIID–gfp, PspoIID–spo0A-N), MF1167 (PspoIID–gfp, PspoIIQ–spo0A-N), PE128 (PspoIIQ–gfp), MF1145 (PspoIIQ–gfp, Pspo0A–spo0A-N), MF1158 (PspoIIQ–gfp, PspoIID–spo0A-N), and MF1168 (PspoIIQ–gfp, PspoIIQ–spo0A-N). Cells were collected at hour 3 of sporulation, treated with the membrane strain FM4-64, and visualized by fluorescence microscopy. Bar, 1 μm.

Similar articles

Cited by

References

    1. Ben-Yehuda S, Losick R. Asymmetric cell division in B. subtilis involves a spiral-like intermediate of the cytokinetic protein FtsZ. Cell. 2002;109:257–266. - PubMed
    1. Ben-Yehuda S, Rudner DZ, Losick R. RacA, a bacterial protein that anchors chromosomes to the cell poles. Science. 2003;299:532–536. - PubMed
    1. Burbulys D, Trach KA, Hoch JA. Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell. 1991;64:545–552. - PubMed
    1. Burkholder WF, Kurtser I, Grossman AD. Replication initiation proteins regulate a developmental checkpoint in Bacillus subtilis. Cell. 2001;104:269–279. - PubMed
    1. Chung JD, Stephanopoulos G, Ireton K, Grossman AD. Gene expression in single cells of Bacillus subtilis: Evidence that a threshold mechanism controls the initiation of sporulation. J Bacteriol. 1994;176:1977–1984. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources