Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation

Significance The temporal control of DNA methylation by the CcrM DNA methyltransferase mediates cell cycle progression in the asymmetrically dividing bacterium Caulobacter crescentus. Limiting the presence of CcrM to a specific time in the cell cycle is achieved through temporally controlled transcription and Lon-mediated proteolysis. We discovered that a chromosomal DNA-based platform stimulates CcrM proteolysis by Lon. Upon cell division, CcrM is completely degraded by Lon in progeny swarmer cells. In progeny stalked cells, which initiate DNA replication immediately upon division, CcrM proteolysis is incomplete and the remaining enzyme is sequestered away from DNA at the cell pole. Thus, the cell ensures that once DNA replication starts, chromosomal DNA cannot serve as a substrate for remethylation until chromosome replication is complete. The cell cycle-regulated methylation state of Caulobacter DNA mediates the temporal control of transcriptional activation of several key regulatory proteins. Temporally controlled synthesis of the CcrM DNA methyltransferase and Lon-mediated proteolysis restrict CcrM to a specific time in the cell cycle, thereby allowing the maintenance of the hemimethylated state of the chromosome during the progression of DNA replication. We determined that a chromosomal DNA-based platform stimulates CcrM degradation by Lon and that the CcrM C terminus both binds to its DNA substrate and is recognized by the Lon protease. Upon asymmetric cell division, swarmer and stalked progeny cells employ distinct mechanisms to control active CcrM. In progeny swarmer cells, CcrM is completely degraded by Lon before its differentiation into a replication-competent stalked cell later in the cell cycle. In progeny stalked cells, however, accumulated CcrM that has not been degraded before the immediate initiation of DNA replication is sequestered to the cell pole. Single-molecule imaging demonstrated physical anticorrelation between sequestered CcrM and chromosomal DNA, thus preventing DNA remethylation. The distinct control of available CcrM in progeny swarmer and stalked cells serves to protect the hemimethylated state of DNA during chromosome replication, enabling robustness of cell cycle progression.

[1]  L. Shapiro,et al.  Bacterial scaffold directs pole-specific centromere segregation , 2014, Proceedings of the National Academy of Sciences.

[2]  G. Laloux,et al.  How do bacteria localize proteins to the cell pole? , 2014, Journal of Cell Science.

[3]  Pamela J. B. Brown,et al.  Sequential evolution of bacterial morphology by co-option of a developmental regulator , 2014, Nature.

[4]  Tyson A. Clark,et al.  Global methylation state at base-pair resolution of the Caulobacter genome throughout the cell cycle , 2013, Proceedings of the National Academy of Sciences.

[5]  J. Casadesús,et al.  DNA methylation in bacteria: from the methyl group to the methylome. , 2015, Current opinion in microbiology.

[6]  M. Laub,et al.  Proteotoxic Stress Induces a Cell-Cycle Arrest by Stimulating Lon to Degrade the Replication Initiator DnaA , 2013, Cell.

[7]  G. Jensen,et al.  A Self-Associating Protein Critical for Chromosome Attachment, Division, and Polar Organization in Caulobacter , 2008, Cell.

[8]  Justine Collier.  Cell cycle control in Alphaproteobacteria. , 2016, Current opinion in microbiology.

[9]  E. Huitema,et al.  Bacterial Birth Scar Proteins Mark Future Flagellum Assembly Site , 2006, Cell.

[10]  L. Shapiro,et al.  A Caulobacter DNA methyltransferase that functions only in the predivisional cell. , 1994, Journal of molecular biology.

[11]  L. Shapiro,et al.  Phospho-signal flow from a pole-localized microdomain spatially patterns transcription factor activity , 2017, bioRxiv.

[12]  R. Losick,et al.  Protein subcellular localization in bacteria. , 2010, Cold Spring Harbor perspectives in biology.

[13]  D. Mazel,et al.  Genome Engineering in Vibrio cholerae: A Feasible Approach to Address Biological Issues , 2012, PLoS genetics.

[14]  Alison K. Hottes,et al.  DnaA coordinates replication initiation and cell cycle transcription in Caulobacter crescentus , 2005, Molecular microbiology.

[15]  Jing Liu,et al.  Adaptor-mediated Lon proteolysis restricts Bacillus subtilis hyperflagellation , 2014, Proceedings of the National Academy of Sciences.

[16]  A. Goldberg,et al.  DNA stimulates ATP-dependent proteolysis and protein-dependent ATPase activity of protease La from Escherichia coli. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[17]  J. Casadesús,et al.  Epigenetic Gene Regulation in the Bacterial World , 2006, Microbiology and Molecular Biology Reviews.

[18]  Lucy Shapiro,et al.  A DNA methylation ratchet governs progression through a bacterial cell cycle , 2007, Proceedings of the National Academy of Sciences.

[19]  Y. Kikuchi,et al.  Oral Immunization with ATP-Dependent Protease-Deficient Mutants Protects Mice against Subsequent Oral Challenge with Virulent Salmonella enterica Serovar Typhimurium , 2003, Infection and Immunity.

[20]  Robert T Sauer,et al.  AAA+ proteases: ATP-fueled machines of protein destruction. , 2011, Annual review of biochemistry.

[21]  Justine Collier.  Epigenetic regulation of the bacterial cell cycle. , 2009, Current opinion in microbiology.

[22]  Jared M. Schrader,et al.  Dynamic translation regulation in Caulobacter cell cycle control , 2016, Proceedings of the National Academy of Sciences.

[23]  H. Lam,et al.  A Landmark Protein Essential for Establishing and Perpetuating the Polarity of a Bacterial Cell , 2006, Cell.

[24]  L. Shapiro,et al.  A spindle-like apparatus guides bacterial chromosome segregation , 2010, Nature Cell Biology.

[25]  L. Shapiro,et al.  A Localized Complex of Two Protein Oligomers Controls the Orientation of Cell Polarity , 2017, mBio.

[26]  R. Sauer,et al.  Recognition of misfolded proteins by Lon, a AAA(+) protease. , 2008, Genes & development.

[27]  L. Shapiro,et al.  Caulobacter Lon protease has a critical role in cell-cycle control of DNA methylation. , 1996, Genes & development.

[28]  B. Zehnbauer,et al.  Identification and purification of the Lon+ (capR+) gene product, a DNA-binding protein. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[29]  R. Hancock,et al.  Involvement of the Lon Protease in the SOS Response Triggered by Ciprofloxacin in Pseudomonas aeruginosa PAO1 , 2012, Antimicrobial Agents and Chemotherapy.

[30]  Peter Chien,et al.  Regulated Proteolysis in Bacteria: Caulobacter. , 2016, Annual review of genetics.

[31]  Dieter Braun,et al.  Protein-binding assays in biological liquids using microscale thermophoresis. , 2010, Nature communications.

[32]  Zemer Gitai,et al.  Dynamic Polar Sequestration of Excess MurG May Regulate Enzymatic Function , 2010, Journal of bacteriology.

[33]  J. Elf,et al.  Oufti: an integrated software package for high‐accuracy, high‐throughput quantitative microscopy analysis , 2016, Molecular microbiology.

[34]  Justine Collier.  Regulation of chromosomal replication in Caulobacter crescentus. , 2012, Plasmid.

[35]  L. Shapiro,et al.  A Polymeric Protein Anchors the Chromosomal Origin/ParB Complex at a Bacterial Cell Pole , 2008, Cell.

[36]  Zachary D. Smith,et al.  DNA methylation: roles in mammalian development , 2013, Nature Reviews Genetics.

[37]  Lucy Shapiro,et al.  DNA methylation affects the cell cycle transcription of the CtrA global regulator in Caulobacter , 2002, The EMBO journal.

[38]  J. Bujnicki,et al.  Defining the crucial domain and amino acid residues in bacterial Lon protease for DNA binding and processing of DNA-interacting substrates , 2017, The Journal of Biological Chemistry.

[39]  F. Yildiz,et al.  The LonA Protease Regulates Biofilm Formation, Motility, Virulence, and the Type VI Secretion System in Vibrio cholerae , 2016, Journal of bacteriology.

[40]  P. Modrich,et al.  DNA mismatch repair: functions and mechanisms. , 2006, Chemical reviews.

[41]  Lucy Shapiro,et al.  Bacterial DNA Methylation: a Cell Cycle Regulator? , 1999, Journal of bacteriology.

[42]  M. Charette,et al.  DNA-stimulated ATPase activity on the lon (CapR) protein , 1984, Journal of bacteriology.

[43]  Jared M. Schrader,et al.  The Global Regulatory Architecture of Transcription during the Caulobacter Cell Cycle , 2015, PLoS genetics.

[44]  Peter L. Freddolino,et al.  A legacy role for DNA binding of Lon protects against genotoxic stress , 2018, bioRxiv.

[45]  Ellen M. Quardokus,et al.  MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis , 2016, Nature Microbiology.

[46]  T. Bickle,et al.  Biology of DNA restriction , 1993 .

[47]  Jian-Kang Zhu,et al.  Regulation and function of DNA methylation in plants and animals , 2011, Cell Research.

[48]  M. Laub,et al.  Regulated proteolysis of a transcription factor complex is critical to cell cycle progression in Caulobacter crescentus , 2013, Molecular microbiology.

[49]  Haibi Wang,et al.  Caulobacter PopZ forms an intrinsically disordered hub in organizing bacterial cell poles , 2016, Proceedings of the National Academy of Sciences.

[50]  H. McAdams,et al.  The functions of DNA methylation by CcrM in Caulobacter crescentus: a global approach , 2014, Nucleic acids research.

[51]  N. Reich,et al.  Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition. , 2017, Biochemistry.

[52]  P. Viollier,et al.  End-in-Sight: Cell Polarization by the Polygamic Organizer PopZ. , 2017, Trends in microbiology.