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 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.

A nucleotide insertion between two adjacent methyltransferases in Helicobacter pylori results in a bifunctional DNA methyltransferase

2011 ◽  
Vol 433 (3) ◽  
pp. 487-495 ◽  
Author(s):  
Ritesh Kumar ◽  
Desirazu N. Rao
Keyword(s):  
Helicobacter Pylori ◽  
Dna Methyltransferase ◽  
Stop Codon ◽  
Binding Motif ◽  
Modification System ◽  
Restriction Modification System ◽  
Adenine Methylation ◽  
Nucleotide Insertion ◽  
H Pylori ◽  
Catalytic Motif

Helicobacter pylori has a dynamic R-M (restriction–modification) system. It is capable of acquiring new R-M systems from the environment in the form of DNA released from other bacteria or other H. pylori strains. Random mutations in R-M genes can result in non-functional R-M systems or R-M systems with new properties. hpyAVIAM and hpyAVIBM are two solitary DNA MTase (methyltransferase) genes adjacent to each other and lacking a cognate restriction enzyme gene in H. pylori strain 26695. Interestingly, in an Indian strain D27, hpyAVIAM–hpyAVIBM encodes a single bifunctional polypeptide due to insertion of a nucleotide just before the stop codon of hpyAVIBM and, when a similar mutation was made in hpyAVIAM–hpyAVIBM from strain 26695, a functional MTase with an N-terminal C5-cytosine MTase domain and a C-terminal N6-adenine MTase domain was constructed. Mutations in the AdoMet (S-adenosylmethionine)-binding motif or in the catalytic motif of M.HpyAVIA or M.HpyAVIB selectively abrogated the C5-cytosine or N6-adenine methylation activity of M.HpyAVIA–M.HpyAVIB fusion protein. The present study highlights the ability of H. pylori to evolve genes with unique functions and thus generate variability. For organisms such as H. pylori, which have a small genome, these adaptations could be important for their survival in the hostile host environment.

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 Low-level expression of the Type II restriction–modification system confers potent bacteriophage resistance in Escherichia coli

DNA Research ◽  
2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Karolina Wilkowska ◽  
Iwona Mruk ◽  
Beata Furmanek-Blaszk ◽  
Marian Sektas
Keyword(s):  
Dna Methyltransferase ◽  
Host Bacterium ◽  
Type Ii ◽  
Potential Conflict ◽  
Modification System ◽  
Biological Assays ◽  
Restriction Modification System ◽  
Highly Sensitive ◽  
System Maintenance ◽  
Restriction Modification

Abstract Restriction–modification systems (R–M) are one of the antiviral defense tools used by bacteria, and those of the Type II family are composed of a restriction endonuclease (REase) and a DNA methyltransferase (MTase). Most entering DNA molecules are usually cleaved by the REase before they can be methylated by MTase, although the observed level of fragmented DNA may vary significantly. Using a model EcoRI R–M system, we report that the balance between DNA methylation and cleavage may be severely affected by transcriptional signals coming from outside the R–M operon. By modulating the activity of the promoter, we obtained a broad range of restriction phenotypes for the EcoRI R–M system that differed by up to 4 orders of magnitude in our biological assays. Surprisingly, we found that high expression levels of the R–M proteins were associated with reduced restriction of invading bacteriophage DNA. Our results suggested that the regulatory balance of cleavage and methylation was highly sensitive to fluctuations in transcriptional signals both up- and downstream of the R–M operon. Our data provided further insights into Type II R–M system maintenance and the potential conflict within the host bacterium.

scholarly journals Community-wide epigenetics provides novel perspectives on the ecology and evolution of marine microbiome

2021 ◽  
Author(s):  
Hoon Je Seong ◽  
Simon Roux ◽  
Chung Yeon Hwang ◽  
Woo Jun Sul
Keyword(s):  
Cell Cycle ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Distribution Patterns ◽  
Cellular Processes ◽  
Genome Wide ◽  
Methylation Mark ◽  
Domains Of Life ◽  
Epigenetic Analysis ◽  
Cycle Regulation

DNA methylation in prokaryotes is involved in many different cellular processes including cell cycle regulation and defense against viruses. To date, most prokaryotic methylation systems have been studied in culturable microorganisms, resulting in a limited understanding of DNA methylation from a microbial ecology perspective. Here, we analyze the distribution patterns of several microbial epigenetics marks in the ocean microbiome through genome-centric metagenomics across all domains of life. We show that overall, DNA methylation can readily be detected across dominant oceanic bacterial, archaeal, and viral populations, and microbial epigenetic changes correlate with population differentiation. Furthermore, our genome-wide epigenetic analysis of Pelagibacter suggests that GANTC, a DNA methyltransferase target motif, is related to the cell cycle and is affected by environmental conditions. Yet, the presence of this motif also partitions the phylogeny of the Pelagibacter phages, possibly hinting at a competitive co-evolutionary history and multiple effects of a single methylation mark.

scholarly journals Regulation of podJ Expression during theCaulobacter crescentus Cell Cycle

1999 ◽  
Vol 181 (13) ◽  
pp. 3967-3973 ◽  
Author(s):  
William B. Crymes ◽  
Daolong Zhang ◽  
Bert Ely
Keyword(s):  
Cell Cycle ◽  
Binding Site ◽  
Promoter Activity ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Negative Regulator ◽  
Nucleotide Sequence Analysis ◽  
Global Response ◽  
Temporal Regulation ◽  
Polar Organelle

ABSTRACT The polar organelle development gene, podJ, is expressed during the swarmer-to-stalked cell transition of theCaulobacter crescentus cell cycle. Mutants with insertions that inactivate the podJ gene are nonchemotactic, deficient in rosette formation, and resistant to polar bacteriophage, but they divide normally. In contrast, hyperexpression of podJresults in a lethal cell division defect. Nucleotide sequence analysis of the podJ promoter region revealed a binding site for the global response regulator, CtrA. Deletion of this site results in increased overall promoter activity, suggesting that CtrA is a negative regulator of the podJ promoter. Furthermore, synchronization studies have indicated that temporal regulation is not dependent on the presence of the CtrA binding site. Thus, although the level of podJ promoter activity is dependent on the CtrA binding site, the temporal control of podJ promoter expression is dependent on other factors.

scholarly journals Regulation of Bacterial Cell Cycle Progression by Redundant Phosphatases

2020 ◽  
Vol 202 (17) ◽  
Author(s):  
Jérôme Coppine ◽  
Andreas Kaczmarczyk ◽  
Kenny Petit ◽  
Thomas Brochier ◽  
Urs Jenal ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Environmental Changes ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Model Organism ◽  
Replication Initiation ◽  
Cycle Progression ◽  
Content Type ◽  
Two Component

ABSTRACT In the model organism Caulobacter crescentus, a network of two-component systems involving the response regulators CtrA, DivK, and PleD coordinates cell cycle progression with differentiation. Active phosphorylated CtrA prevents chromosome replication in G1 cells while simultaneously regulating expression of genes required for morphogenesis and development. At the G1-S transition, phosphorylated DivK (DivK∼P) and PleD (PleD∼P) accumulate to indirectly inactivate CtrA, which triggers DNA replication initiation and concomitant cellular differentiation. The phosphatase PleC plays a pivotal role in this developmental program by keeping DivK and PleD phosphorylation levels low during G1, thereby preventing premature CtrA inactivation. Here, we describe CckN as a second phosphatase akin to PleC that dephosphorylates DivK∼P and PleD∼P in G1 cells. However, in contrast to PleC, no kinase activity was detected with CckN. The effects of CckN inactivation are largely masked by PleC but become evident when PleC and DivJ, the major kinase for DivK and PleD, are absent. Accordingly, mild overexpression of cckN restores most phenotypic defects of a pleC null mutant. We also show that CckN and PleC are proteolytically degraded in a ClpXP-dependent way before the onset of the S phase. Surprisingly, known ClpX adaptors are dispensable for PleC and CckN proteolysis, raising the possibility that as yet unidentified proteolytic adaptors are required for the degradation of both phosphatases. Since cckN expression is induced in stationary phase, depending on the stress alarmone (p)ppGpp, we propose that CckN acts as an auxiliary factor responding to environmental stimuli to modulate CtrA activity under suboptimal conditions. IMPORTANCE Two-component signal transduction systems are widely used by bacteria to adequately respond to environmental changes by adjusting cellular parameters, including the cell cycle. In Caulobacter crescentus, PleC acts as a phosphatase that indirectly protects the response regulator CtrA from premature inactivation during the G1 phase of the cell cycle. Here, we provide genetic and biochemical evidence that PleC is seconded by another phosphatase, CckN. The activity of PleC and CckN phosphatases is restricted to the G1 phase since both proteins are degraded by ClpXP protease before the G1-S transition. Degradation is independent of any known proteolytic adaptors and relies, in the case of CckN, on an unsuspected N-terminal degron. Our work illustrates a typical example of redundant functions between two-component proteins.

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