During sporulation, Bacillus subtilis forms an asymmetric septum, dividing the cell into two compartments, a mother cell and a forespore. The site of asymmetric septation is linked to the membrane where FtsZ and SpoIIE initiate the formation of the Z-ring and the E-ring, respectively. These rings then serve as a scaffold for the other cell division and peptidoglycan synthesizing proteins needed to build the septum. However, despite decades of research, not enough is known about how the asymmetric septation site is determined. Here, we identified and characterized the interaction between SpoIIE and RefZ. We show that these two proteins transiently colocalize during the early stages of asymmetric septum formation when RefZ localizes primarily from the mother cell side of the septum. We propose that these proteins and their interplay with the spatial organization of the chromosome play a role in controlling asymmetric septum positioning.

Department of Microbial Genetics Institute of Molecular Biology Slovak Academy of Sciences Bratislava Slovakia

Laboratory of Microbial Genetics and Gene Expression Institute of Microbiology of the Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Higgins D., Dworkin J. Recent progress in Bacillus subtilis sporulation. FEMS Microbiol. Rev. 2012;36:131–148. PubMed PMC

Tan I.S., Ramamurthi K.S. Spore formation in Bacillus subtilis. Environ. Microbiol. Rep. 2014;6:212–225. PubMed PMC

Errington J., Wu L.J. Cell Cycle Machinery in Bacillus subtilis. Subcell Biochem. 2017;84:67–101. PubMed PMC

Bylund J.E., Haines M.A., Piggot P.J., Higgins M.L. Axial filament formation in Bacillus subtilis: induction of nucleoids of increasing length after addition of chloramphenicol to exponential-phase cultures approaching stationary phase. J. Bacteriol. 1993;175:1886–1890. PubMed PMC

Hilbert D.W., Piggot P.J. Compartmentalization of gene expression during Bacillus subtilis spore formation. Microbiol. Mol. Biol. Rev. 2004;68:234–262. PubMed PMC

Ben-Yehuda S., Fujita M., Liu X.S., Gorbatyuk B., Skoko D., Yan J., et al. Defining a centromere-like element in Bacillus subtilis by identifying the binding sites for the chromosome-anchoring protein RacA. Mol. Cell. 2005;17:773–782. PubMed

Ben-Yehuda S., Rudner D.Z., Losick R. RacA, a bacterial protein that anchors chromosomes to the cell poles. Science. 2003;299:532–536. PubMed

Wu L.J., Errington J. RacA and the Soj-Spo0J system combine to effect polar chromosome segregation in sporulating Bacillus subtilis. Mol. Microbiol. 2003;49:1463–1475. PubMed

Kloosterman T.G., Lenarcic R., Willis C.R., Roberts D.M., Hamoen L.W., Errington J., et al. Complex polar machinery required for proper chromosome segregation in vegetative and sporulating cells of Bacillus subtilis. Mol. Microbiol. 2016;101:333–350. PubMed PMC

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

Eichenberger P., Fawcett P., Losick R. A three-protein inhibitor of polar septation during sporulation in Bacillus subtilis. Mol. Microbiol. 2001;42:1147–1162. PubMed

Pogliano J., Osborne N., Sharp M.D., Mello A. A. De, Perez A., Sun Y.L., et al. A vital stain for studying membrane dynamics in bacteria: a novel mechanism controlling septation during Bacillus subtilis sporulation. Mol. Microbiol. 1999;31:1149–1159. PubMed PMC

Arigoni F., Guérout-Fleury A.M., Barák I., Stragier P. The SpoIIE phosphatase, the sporulation septum and the establishment of forespore-specific transcription in Bacillus subtilis: a reassessment. Mol. Microbiol. 1999;31:1407–1415. PubMed

Rawlings A.E., Levdikov V.M., Blagova E., Colledge V.L., Mas P.J., Tunaley J., et al. Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen. Protein Eng. Des. Sel. 2010;23:817–825. PubMed PMC

Levin P.A., Losick R., Stragier P., Arigoni F. Localization of the sporulation protein SpoIIE in Bacillus subtilis is dependent upon the cell division protein FtsZ. Mol. Microbiol. 1997;25:839–846. PubMed

Barák I., Behari J., Olmedo G., Guzmán P., Brown D.P., Castro E., et al. Structure and function of the Bacillus SpollE protein and its localization to sites of sporulation septum assembly. Mol. Microbiol. 1996;19:1047–1060. PubMed

Feucht A., Magnin T., Yudkin M.D., Errington J. Bifunctional protein requried for asymmetric cell division and cell-specific transcription in Bacillus subtilis. Genes Dev. 1996;10:794–803. PubMed

Duncan L., Alper S., Arigoni F., Losick R., Stragier P. Activation of cell-specific transcription by a serine phosphatase at the site of asymmetric division. Science. 1995;270:641–644. PubMed

Arigoni F., Duncan L., Alper S., Losick R., Stragier P. SpoIIE governs the phosphorylation state of a protein regulating transcription factor sigma F during sporulation in Bacillus subtilis. Proc. Natl. Acad. Sci. U. S. A. 1996;93:3238–3242. PubMed PMC

Iber D., Clarkson J., Yudkin M.D., Campbell I.D. The mechanism of cell differentiation in Bacillus subtilis. Nature. 2006;441:371–374. PubMed

Bradshaw N., Losick R. Asymmetric division triggers cell-specific gene expression through coupled capture and stabilization of a phosphatase. Elife. 2015;4:1–18. PubMed PMC

Campo N., Marquis K.A., Rudner D.Z. SpoIIQ anchors membrane proteins on both sides of the sporulation septum in Bacillus subtilis. J. Biol. Chem. 2008;283:4975–4982. PubMed

Barák I., Youngman P. SpoIIE mutants of Bacillus subtilis comprise two distinct phenotypic classes consistent with a dual functional role for the SpoIIE protein. J. Bacteriol. 1996;178:4984–4989. PubMed PMC

Khanna K., Garrido J.L., Sugie J., Pogliano K., Villa E. Asymmetric localization of the cell division machinery during Bacillus subtilis sporulation. Elife. 2021;10:1–24. PubMed PMC

Barák I., Muchová K. The positioning of the asymmetric septum during sporulation in Bacillus subtilis. PLoS One. 2018;13:1–15. PubMed PMC

Barák I., Wilkinson A.J. Division site recognition in Escherichia coli and Bacillus subtilis. FEMS Microbiol. Rev. 2007;31:311–326. PubMed

Eswaramoorthy P., Winter P.W., Wawrzusin P., York A.G., Shroff H., Ramamurthi K.S. Asymmetric division and differential gene expression during a bacterial developmental program requires DivIVA. PLoS Genet. 2014;10 PubMed PMC

Cha J.H., Stewart G.C. The divIVA minicell locus of Bacillus subtilis. J. Bacteriol. 1997;179:1671–1683. PubMed PMC

Wagner-Herman J.K., Bernard R., Dunne R., Bisson-Filho A.W., Kumar K., Nguyen T., et al. RefZ facilitates the switch from medial to polar division during spore formation in Bacillus subtilis. J. Bacteriol. 2012;194:4608–4618. PubMed PMC

Miller A.K., Brown E.E., Mercado B.T., Herman J.K. A DNA-binding protein defines the precise region of chromosome capture during Bacillus sporulation. Mol. Microbiol. 2016;99:111–122. PubMed

Karimova G., Pidoux J., Ullmann a, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc. Natl. Acad. Sci. U. S. A. 1998;95:5752–5756. PubMed PMC

Muchová K., Chromiková Z., Barák I. Linking the peptidoglycan synthesis protein complex with asymmetric cell division during bacillus subtilis sporulation. Int. J. Mol. Sci. 2020;21:1–13. PubMed PMC

Mehla J., Caufield J.H., Sakhawalkar N., Uetz P. A comparison of two-hybrid approaches for detecting protein–protein interactions. Methods Enzymol. 2017;586:333–358. PubMed PMC

Muchová K., Chromiková Z., Bradshaw N., Wilkinson A.J., Barák I. Morphogenic protein RodZ interacts with sporulation specific SpoIIE in Bacillus subtilis. PLoS One. 2016;11 PubMed PMC

Brown E.E., Miller A.K., Krieger I.V., Otto R.M., Sacchettini J.C., Herman J.K. A DNA-binding protein tunes septum placement during Bacillus subtilis sporulation. J. Bacteriol. 2019;201:1–22. PubMed PMC

Wollman A.J.M., Muchová K., Chromiková Z., Wilkinson A.J., Barák I., Leake M.C. Single-molecule optical microscopy of protein dynamics and computational analysis of images to determine cell structure development in differentiating Bacillus subtilis. Comput. Struct. Biotechnol. J. 2020;18:1474–1486. PubMed PMC

Wagner S., Baarst L., Ytterberg A.J., Klussmerer A., Wagner C.S., Nord O., et al. Consequences of membrane protein overexpression in Escherichia coli. Mol. Cell. Proteomics. 2007;6:1527–1550. PubMed

Lopez-Garrido J., Ojkic N., Khanna K., Wagner F.R., Villa E., Endres R.G., et al. Chromosome translocation inflates Bacillus forespores and impacts cellular morphology. Cell. 2018;172:758–770.e14. PubMed PMC

Wu L.J., Errington J. Bacillus subtilis spoIIIE protein required for DNA segregation during asymmetric cell division. Science. 1994;264:572–575. PubMed

Chareyre S., Li X., Anjuwon-Foster B.R., Updegrove T.B., Clifford S., Brogan A.P., et al. Cell division machinery drives cell-specific gene activation during differentiation in Bacillus subtilis. Proc. Natl. Acad. Sci. U. S. A. 2024;121:1–11. PubMed PMC

Meeske A.J., Rodrigues C.D.A., Brady J., Lim H.C., Bernhardt T.G., Rudner D.Z. High-throughput genetic screens identify a large and diverse collection of new sporulation genes in Bacillus subtilis. PLoS Biol. 2016;14:1–33. PubMed PMC

Cleverley R.M., Rutter Z.J., Rismondo J., Corona F., Tsui H.C.T., Alatawi F.A., et al. The cell cycle regulator GpsB functions as cytosolic adaptor for multiple cell wall enzymes. Nat. Commun. 2019;10:1–17. PubMed PMC

Galperin M.Y., Yutin N., Wolf Y.I., Alvarez R.V., Koonin E.V. Conservation and evolution of the sporulation gene set in diverse members of the firmicutes. J. Bacteriol. 2022;204:1–23. PubMed PMC

Makroczyová J., Jamroškovič J., Krascsenitsová E., Labajová N., Barák I. Oscillating behavior of Clostridium difficile Min proteins in Bacillus subtilis. Microbiologyopen. 2016;5:387–401. PubMed PMC

Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A., et al., editors. Current Protocols in Molecular Biology. John Wiley & Sons, Inc.; Hoboken, NJ: 2001.

Harwood C.R. Wiley; Chichester; New York: 1990. Molecular Biological Methods for Bacillus.

Youngman P., Perkins J.B., Losick R. Construction of a cloning site near one end of Tn917 into which foreign DNA may be inserted without affecting transposition in Bacillus subtilis or expression of the transposon-borne erm gene. Plasmid. 1984;12:1–9. PubMed

Backman K., Ptashne M., Gilbert W. Construction of plasmids carrying the cI gene of bacteriophage lambda. Proc. Natl. Acad. Sci. U. S. A. 1976;73:4174–4178. PubMed PMC

Lewis P.J., Marston A.L. GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis. Gene. 1999;227:101–109. PubMed

Miller J.H. Cold Spring Harbor Laboratory; New York: 1972. Experiments in Molecular Genetics.

Jamroskovic J., Pavlendová N., Muchová K., Wilkinson A.J., Barák I. An oscillating Min system in Bacillus subtilis influences asymmetrical septation during sporulation. Microbiology. 2012;158:1972–1981. PubMed PMC

Ju J., Luo T., Haldenwang W.G. Forespore expression and processing of the SigE transcription factor in wild-type and mutant Bacillus subtilis. J. Bacteriol. 1998;180:1673–1681. PubMed PMC

Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33:103–119. PubMed