Caulobacter crescentus Cell Cycle-Regulated DNA Methyltransferase Uses a Novel Mechanism for Substrate Recognition

Biochemistry ◽  
2017 ◽  
Vol 56 (30) ◽  
pp. 3913-3922 ◽  
Author(s):  
Clayton B. Woodcock ◽  
Aziz B. Yakubov ◽  
Norbert O. Reich
Keyword(s):  
Cell Cycle ◽  
Dna Methyltransferase ◽  
Caulobacter Crescentus ◽  
Substrate Recognition

scholarly journals Beta class amino methyltransferases from bacteria to humans: evolution and structural consequences

2020 ◽  
Vol 48 (18) ◽  
pp. 10034-10044 ◽  
Author(s):  
Clayton B Woodcock ◽  
John R Horton ◽  
Xing Zhang ◽  
Robert M Blumenthal ◽  
Xiaodong Cheng
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Dna Methyltransferase ◽  
Caulobacter Crescentus ◽  
Substrate Recognition ◽  
Its Sequence ◽  
Amino Groups ◽  
Dna And Rna ◽  
Acid Type ◽  
Target Molecules

Abstract S-adenosyl-l-methionine dependent methyltransferases catalyze methyl transfers onto a wide variety of target molecules, including DNA and RNA. We discuss a family of methyltransferases, those that act on the amino groups of adenine or cytosine in DNA, have conserved motifs in a particular order in their amino acid sequence, and are referred to as class beta MTases. Members of this class include M.EcoGII and M.EcoP15I from Escherichia coli, Caulobacter crescentus cell cycle–regulated DNA methyltransferase (CcrM), the MTA1-MTA9 complex from the ciliate Oxytricha, and the mammalian MettL3-MettL14 complex. These methyltransferases all generate N6-methyladenine in DNA, with some members having activity on single-stranded DNA as well as RNA. The beta class of methyltransferases has a unique multimeric feature, forming either homo- or hetero-dimers, allowing the enzyme to use division of labor between two subunits in terms of substrate recognition and methylation. We suggest that M.EcoGII may represent an ancestral form of these enzymes, as its activity is independent of the nucleic acid type (RNA or DNA), its strandedness (single or double), and its sequence (aside from the target adenine).

scholarly journals Cell cycle regulated DNA methyltransferase: fluorescent tracking of a DNA strand-separation mechanism and identification of the responsible protein motif

2020 ◽  
Vol 48 (20) ◽  
pp. 11589-11601
Author(s):  
Olivia Konttinen ◽  
Jason Carmody ◽  
Sarath Pathuri ◽  
Kyle Anderson ◽  
Xiaofeng Zhou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Methyltransferase ◽  
Caulobacter Crescentus ◽  
Dna Recognition ◽  
Dna Methyltransferases ◽  
Terminal Segment ◽  
Recognition Site ◽  
Separation Mechanism ◽  
Human Pathogens ◽  
Strand Separation

Abstract DNA adenine methylation by Caulobacter crescentus Cell Cycle Regulated Methyltransferase (CcrM) is an important epigenetic regulator of gene expression. The recent CcrM-DNA cocrystal structure shows the CcrM dimer disrupts four of the five base pairs of the (5′-GANTC-3′) recognition site. We developed a fluorescence-based assay by which Pyrrolo-dC tracks the strand separation event. Placement of Pyrrolo-dC within the DNA recognition site results in a fluorescence increase when CcrM binds. Non-cognate sequences display little to no fluorescence changes, showing that strand separation is a specificity determinant. Conserved residues in the C-terminal segment interact with the phospho-sugar backbone of the non-target strand. Replacement of these residues with alanine results in decreased methylation activity and changes in strand separation. The DNA recognition mechanism appears to occur with the Type II M.HinfI DNA methyltransferase and an ortholog of CcrM, BabI, but not with DNA methyltransferases that lack the conserved C-terminal segment. The C-terminal segment is found broadly in N4/N6-adenine DNA methyltransferases, some of which are human pathogens, across three Proteobacteria classes, three other phyla and in Thermoplasma acidophilum, an Archaea. This Pyrrolo-dC strand separation assay should be useful for the study of other enzymes which likely rely on a strand separation mechanism.

scholarly journals Genomic Adaptations to the Loss of a Conserved Bacterial DNA Methyltransferase

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Diego Gonzalez ◽  
Justine Collier
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Experimental Evolution ◽  
Dna Methyltransferase ◽  
Metabolic Regulation ◽  
Caulobacter Crescentus ◽  
Point Mutations ◽  
Rich Medium ◽  
Content Type ◽  
Evolution Approach

ABSTRACTCcrM is an orphan DNA methyltransferase nearly universally conserved in a vast group ofAlphaproteobacteria.InCaulobacter crescentus, it controls the expression of key genes involved in the regulation of the cell cycle and cell division. Here, we demonstrate, using an experimental evolution approach, thatC. crescentuscan significantly compensate, through easily accessible genetic changes like point mutations, the severe loss in fitness due to the absence of CcrM, quickly improving its growth rate and cell morphology in rich medium. By analyzing the compensatory mutations genome-wide in 12 clones sampled from independent ΔccrMpopulations evolved for ~300 generations, we demonstrated that each of the twelve clones carried at least one mutation that potentially stimulatedftsZexpression, suggesting that the low intracellular levels of FtsZ are the major burden of ΔccrMmutants. In addition, we demonstrate that the phosphoenolpyruvate-carbohydrate phosphotransfer system (PTS) actually modulatesftsZandmipZtranscription, uncovering a previously unsuspected link between metabolic regulation and cell division inAlphaproteobacteria. We present evidence that point mutations found in genes encoding proteins of the PTS provide the strongest fitness advantage to ΔccrMcells cultivated in rich medium despite being disadvantageous in minimal medium. This environmental sign epistasis might prevent such mutations from getting fixed under changing natural conditions, adding a plausible explanation for the broad conservation of CcrM.IMPORTANCEIn bacteria, DNA methylation has a variety of functions, including the control of DNA replication and/or gene expression. The cell cycle-regulated DNA methyltransferase CcrM modulates the transcription of many genes and is critical for fitness inCaulobacter crescentus. Here, we used an original experimental evolution approach to determine which of its many targets make CcrM so important physiologically. We show that populations lacking CcrM evolve quickly, accumulating an excess of mutations affecting, directly or indirectly, the expression of theftsZcell division gene. This finding suggests that the most critical function of CcrM inC. crescentusis to promote cell division by enhancing FtsZ intracellular levels. During this work, we also discovered an unexpected link between metabolic regulation and cell division that might extend to otherAlphaproteobacteria.

scholarly journals The CtrA Response Regulator Mediates Temporal Control of Gene Expression during the Caulobacter Cell Cycle

1999 ◽  
Vol 181 (8) ◽  
pp. 2430-2439 ◽  
Author(s):  
Ann Reisenauer ◽  
Kim Quon ◽  
Lucy Shapiro
Keyword(s):  
Cell Cycle ◽  
Inverted Repeat ◽  
Transcription Initiation ◽  
Dna Methyltransferase ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Temporal Control ◽  
Temporal Expression ◽  
Recognition Sequence ◽  
Control Of Gene Expression

ABSTRACT In its role as a global response regulator, CtrA controls the transcription of a diverse group of genes at different times in theCaulobacter crescentus cell cycle. To understand the differential regulation of CtrA-controlled genes, we compared the expression of two of these genes, the fliQ flagellar gene and the ccrM DNA methyltransferase gene. Despite their similar promoter architecture, these genes are transcribed at different times in the cell cycle. PfliQ is activated earlier than PccrM. Phosphorylated CtrA (CtrA∼P) bound to the CtrA recognition sequence in both promoters but had a 10- to 20-fold greater affinity for PfliQ. This difference in affinity correlates with temporal changes in the cellular levels of CtrA. Disrupting a unique inverted repeat element in PccrMsignificantly reduced promoter activity but not the timing of transcription initiation, suggesting that the inverted repeat does not play a major role in the temporal control of ccrMexpression. Our data indicate that differences in the affinity of CtrA∼P for PfliQ and PccrM regulate, in part, the temporal expression of these genes. However, the timing offliQ transcription but not of ccrMtranscription was altered in cells expressing a stable CtrA derivative, indicating that changes in CtrA∼P levels alone cannot govern the cell cycle transcription of these genes. We propose that changes in the cellular concentration of CtrA∼P and its interaction with accessory proteins influence the temporal expression offliQ, ccrM, and other key cell cycle genes and ultimately the regulation of the cell cycle.

scholarly journals The cell cycle-regulated DNA adenine methyltransferase CcrM opens a bubble at its DNA recognition site

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
John R. Horton ◽  
Clayton B. Woodcock ◽  
Sifa B. Opot ◽  
Norbert O. Reich ◽  
Xing Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Methyltransferase ◽  
Caulobacter Crescentus ◽  
Recognition Site ◽  
Van Der Waals Interactions ◽  
Target Sequence ◽  
Recognition Sequence ◽  
Methyltransferase Domain ◽  
Target Strand ◽  
Dna Strands

Abstract The Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM) methylates the adenine of hemimethylated GANTC after replication. Here we present the structure of CcrM in complex with double-stranded DNA containing the recognition sequence. CcrM contains an N-terminal methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the 5-base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson–Crick polar hydrogen bonds to ensure high enzymatic specificity.

scholarly journals A cell cycle-regulated adenine DNA methyltransferase from Caulobacter crescentus processively methylates GANTC sites on hemimethylated DNA

1998 ◽  
Vol 95 (6) ◽  
pp. 2874-2879 ◽  
Author(s):  
A. J. Berdis ◽  
I. Lee ◽  
J. K. Coward ◽  
C. Stephens ◽  
R. Wright ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Methyltransferase ◽  
Caulobacter Crescentus ◽  
A Cell

scholarly journals The CcrM DNA Methyltransferase of Agrobacterium tumefaciens Is Essential, and Its Activity Is Cell Cycle Regulated

2001 ◽  
Vol 183 (10) ◽  
pp. 3065-3075 ◽  
Author(s):  
Lyn Sue Kahng ◽  
Lucy Shapiro
Keyword(s):  
Cell Cycle ◽  
Agrobacterium Tumefaciens ◽  
Dna Methyltransferase ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Transcriptional Start Site ◽  
Binding Motif ◽  
Modification System ◽  
Cellular Processes ◽  
Restriction Modification System

ABSTRACT DNA methylation is now recognized as a regulator of multiple bacterial cellular processes. CcrM is a DNA adenine methyltransferase found in the alpha subdivision of the proteobacteria. Like the Dam enzyme, which is found primarily in Escherichia coli and other gamma proteobacteria, it does not appear to be part of a DNA restriction-modification system. The CcrM homolog ofAgrobacterium tumefaciens was found to be essential for viability. Overexpression of CcrM is associated with significant abnormalities of cell morphology and DNA ploidy. Mapping of the transcriptional start site revealed a conserved binding motif for the global response regulator CtrA at the −35 position; this motif was footprinted by purified Caulobacter crescentus CtrA protein in its phosphorylated state. We have succeeded in isolating synchronized populations of Agrobacterium cells and analyzing their progression through the cell cycle. We demonstrate that DNA replication and cell division can be followed in an orderly manner and that flagellin expression is cyclic, consistent with our observation that motility varies during the cell cycle. Using these synchronized populations, we show that CcrM methylation of the chromosome is restricted to the late S phase of the cell cycle. Thus, within the alpha subdivision, there is a conserved cell cycle dependence and regulatory mechanism controlling ccrMexpression.

scholarly journals Bacterial cell cycle control by citrate synthase independent of enzymatic activity

2019 ◽  
Author(s):  
Matthieu Bergé ◽  
Julian Pezzatti ◽  
Víctor González-Ruiz ◽  
Laurence Degeorges ◽  
Serge Rudaz ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Citrate Synthase ◽  
Caulobacter Crescentus ◽  
Tricarboxylic Acid Cycle ◽  
Cell Cycle Control ◽  
Tca Cycle ◽  
Cell Cycle Regulators ◽  
Central Metabolism ◽  
Cycle Enzyme

ABSTRACTCoordination of cell cycle progression with central metabolism is fundamental to all cell types and likely underlies differentiation into dispersal cells in bacteria. How central metabolism is monitored to regulate cell cycle functions is poorly understood. A forward genetic selection for cell cycle regulators in the polarized alpha-proteobacterium Caulobacter crescentus unearthed the uncharacterized CitA citrate synthase, a TCA (tricarboxylic acid) cycle enzyme, as unprecedented checkpoint regulator of the G1→S transition. We show that loss of the CitA protein provokes a (p)ppGpp alarmone-dependent G1-phase arrest without apparent metabolic or energy insufficiency. While S-phase entry is still conferred when CitA is rendered catalytically inactive, the paralogous CitB citrate synthase has no overt role other than sustaining TCA cycle activity when CitA is absent. With eukaryotic citrate synthase paralogs known to fulfill regulatory functions, our work extends the moonlighting paradigm to citrate synthase coordinating central (TCA) metabolism with development and perhaps antibiotic tolerance in bacteria.

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