scholarly journals The Three-Layered DNA Uptake Machinery at the Cell Pole in Competent Bacillus subtilis Cells Is a Stable Complex

2011 ◽  
Vol 193 (7) ◽  
pp. 1633-1642 ◽  
Author(s):  
M. Kaufenstein ◽  
M. van der Laan ◽  
P. L. Graumann
Keyword(s):  
Bacillus Subtilis ◽  
Stable Complex ◽  
Dna Uptake ◽  
Cell Pole

scholarly journals Single molecule dynamics of DNA receptor ComEA, membrane permease ComEC and taken up DNA in competent Bacillus subtilis cells

2020 ◽  
Author(s):  
Marie Burghard-Schrod ◽  
Alexandra Kilb ◽  
Kai Krämer ◽  
Peter L. Graumann
Keyword(s):  
Bacillus Subtilis ◽  
Cell Membrane ◽  
Single Molecule ◽  
Metal Binding ◽  
Outer Cell ◽  
Dna Uptake ◽  
Cell Pole ◽  
Catalytic Function ◽  
C Terminus ◽  
Capture Mechanism

AbstractIn competent gram-negative and gram-positive bacteria, double stranded DNA is taken up through the outer cell membrane and/or the cell wall, and is bound by ComEA, which in Bacillus subtilis is a membrane protein. DNA is converted to single stranded DNA, and transported through the cell membrane via ComEC. We show that in Bacillus subtilis, the C-terminus of ComEC, thought to act as a nuclease, is not only important for DNA uptake, as judged from a loss of transformability, but also for the localization of ComEC to the cell pole and its mobility within the cell membrane. Using single molecule tracking, we show that only 13% of ComEC molecules are statically localised at the pole, while 87% move throughout the cell membrane. These experiments suggest that recruitment of ComEC to the cell pole is mediated by a diffusion/capture mechanism. Mutation of a conserved aspartate residue in the C-terminus, likely affecting metal binding, strongly impairs transformation efficiency, suggesting that this periplasmic domain of ComEC could indeed serve a catalytic function as nuclease. By tracking fluorescently labeled DNA, we show that taken up DNA has a similar mobility within the periplasm as ComEA, suggesting that most taken up molecules are bound to ComEA. We show that DNA can be highly mobile within the periplasm, indicating that this subcellular space can act as reservoir for taken up DNA, before its entry into the cytosol.ImportanceBacteria can take up DNA from the environment and incorporate it into their chromosome in case similarity to the genome exists. This process of “natural competence” can result in the uptake of novel genetic information leading to horizontal gene transfer. We show that fluorescently labelled DNA moves within the periplasm of competent Bacillus subtilis cells with similar dynamics as DNA receptor ComEA, and thus takes a detour to get stored before uptake across the cell membrane into the cytosol by DNA permease ComEC. The latter assembles at a single cell pole, likely by a diffusion-capture mechanism, and requires its large C-terminus, including a conserved residue thought to confer nuclease function, for proper localization, function and mobility within the membrane.

scholarly journals Single molecule dynamics of DNA receptor ComEA, membrane permease ComEC and taken up DNA in competent Bacillus subtilis cells

2021 ◽  
Author(s):  
Marie Burghard-Schrod ◽  
Alexandra Kilb ◽  
Kai Krämer ◽  
Peter L. Graumann
Keyword(s):  
Bacillus Subtilis ◽  
Cell Membrane ◽  
Single Molecule ◽  
Metal Binding ◽  
Outer Cell ◽  
Dna Uptake ◽  
Dna Dynamics ◽  
Cell Pole ◽  
C Terminus ◽  
Capture Mechanism

In competent Gram-negative and Gram-positive bacteria, double stranded DNA is taken up through the outer cell membrane and/or the cell wall, and is bound by ComEA, which in Bacillus subtilis is a membrane protein. DNA is converted to single stranded DNA, and transported through the cell membrane via ComEC. We show that in Bacillus subtilis , the C-terminus of ComEC, thought to act as a nuclease, is not only important for DNA uptake, as judged from a loss of transformability, but also for the localization of ComEC to the cell pole and its mobility within the cell membrane. Using single molecule tracking, we show that only 13% of ComEC molecules are statically localised at the pole, while 87% move throughout the cell membrane. These experiments suggest that recruitment of ComEC to the cell pole is mediated by a diffusion/capture mechanism. Mutation of a conserved aspartate residue in the C-terminus, likely affecting metal binding, strongly impairs transformation efficiency, suggesting that this periplasmic domain of ComEC could indeed serve a catalytic function as nuclease. By tracking fluorescently labeled DNA, we show that taken up DNA has a similar mobility as a protein, in spite of being a large polymer. DNA dynamics are similar within the periplasm as those of ComEA, suggesting that most taken up molecules are bound to ComEA. We show that DNA can be highly mobile within the periplasm, indicating that this subcellular space can act as reservoir for taken up DNA, before its entry into the cytosol. Importance Bacteria can take up DNA from the environment and incorporate it into their chromosome, termed “natural competence” that can result in the uptake of novel genetic information. We show that fluorescently labelled DNA moves within the periplasm of competent Bacillus subtilis cells, with similar dynamics as DNA receptor ComEA. This indicates that DNA can accumulate in the periplasm, likely bound by ComEA, and thus can be stored before uptake at the cell pole, via integral membrane DNA permease ComEC. Assembly of the latter assembles at the cell pole likely occurs by a diffusion-capture mechanism. DNA uptake into cells thus takes a detour through the entire periplasm, and involves a high degree of free diffusion along and within the cell membrane.

scholarly journals Methyltransferase DnmA is responsible for genome-wide N6-methyladenosine modifications at non-palindromic recognition sites in Bacillus subtilis

2020 ◽  
Vol 48 (10) ◽  
pp. 5332-5348
Author(s):  
Taylor M Nye ◽  
Lieke A van Gijtenbeek ◽  
Amanda G Stevens ◽  
Jeremy W Schroeder ◽  
Justin R Randall ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bacillus Subtilis ◽  
Consensus Sequence ◽  
Dna Uptake ◽  
Cellular Functions ◽  
Promoter Regions ◽  
Gram Positive Bacteria ◽  
Genome Wide ◽  
Domains Of Life ◽  
Reporter Assays

Abstract The genomes of organisms from all three domains of life harbor endogenous base modifications in the form of DNA methylation. In bacterial genomes, methylation occurs on adenosine and cytidine residues to include N6-methyladenine (m6A), 5-methylcytosine (m5C), and N4-methylcytosine (m4C). Bacterial DNA methylation has been well characterized in the context of restriction-modification (RM) systems, where methylation regulates DNA incision by the cognate restriction endonuclease. Relative to RM systems less is known about how m6A contributes to the epigenetic regulation of cellular functions in Gram-positive bacteria. Here, we characterize site-specific m6A modifications in the non-palindromic sequence GACGmAG within the genomes of Bacillus subtilis strains. We demonstrate that the yeeA gene is a methyltransferase responsible for the presence of m6A modifications. We show that methylation from YeeA does not function to limit DNA uptake during natural transformation. Instead, we identify a subset of promoters that contain the methylation consensus sequence and show that loss of methylation within promoter regions causes a decrease in reporter expression. Further, we identify a transcriptional repressor that preferentially binds an unmethylated promoter used in the reporter assays. With these results we suggest that m6A modifications in B. subtilis function to promote gene expression.

scholarly journals The Bacillus Subtilis K-State Promotes Stationary-Phase Mutagenesis via Oxidative Damage

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 190
Author(s):  
Holly A. Martin ◽  
Amanda A. Kidman ◽  
Jillian Socea ◽  
Carmen Vallin ◽  
Mario Pedraza-Reyes ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Oxidative Damage ◽  
Stationary Phase ◽  
Bacterial Cells ◽  
Dna Uptake ◽  
Cellular Division ◽  
Exogenous Dna ◽  
K Cells ◽  
Induced Mutagenesis ◽  
Underlying Mechanisms

Bacterial cells develop mutations in the absence of cellular division through a process known as stationary-phase or stress-induced mutagenesis. This phenomenon has been studied in a few bacterial models, including Escherichia coli and Bacillus subtilis; however, the underlying mechanisms between these systems differ. For instance, RecA is not required for stationary-phase mutagenesis in B. subtilis like it is in E. coli. In B. subtilis, RecA is essential to the process of genetic transformation in the subpopulation of cells that become naturally competent in conditions of stress. Interestingly, the transcriptional regulator ComK, which controls the development of competence, does influence the accumulation of mutations in stationary phase in B. subtilis. Since recombination is not involved in this process even though ComK is, we investigated if the development of a subpopulation (K-cells) could be involved in stationary-phase mutagenesis. Using genetic knockout strains and a point-mutation reversion system, we investigated the effects of ComK, ComEA (a protein involved in DNA transport during transformation), and oxidative damage on stationary-phase mutagenesis. We found that stationary-phase revertants were more likely to have undergone the development of competence than the background of non-revertant cells, mutations accumulated independently of DNA uptake, and the presence of exogenous oxidants potentiated mutagenesis in K-cells. Therefore, the development of the K-state creates conditions favorable to an increase in the genetic diversity of the population not only through exogenous DNA uptake but also through stationary-phase mutagenesis.

Role of ComFA in controlling the DNA uptake rate during transformation of competent Bacillus subtilis

2011 ◽  
Vol 111 (6) ◽  
pp. 618-623 ◽  
Author(s):  
Masaomi Takeno ◽  
Hisataka Taguchi ◽  
Takashi Akamatsu
Keyword(s):  
Bacillus Subtilis ◽  
Uptake Rate ◽  
Dna Uptake

Role of ComEA in DNA uptake during transformation of competent Bacillus subtilis

2012 ◽  
Vol 113 (6) ◽  
pp. 689-693 ◽  
Author(s):  
Masaomi Takeno ◽  
Hisataka Taguchi ◽  
Takashi Akamatsu
Keyword(s):  
Bacillus Subtilis ◽  
Dna Uptake

scholarly journals ZpdN, a Plasmid-Encoded Sigma Factor Homolog, Induces pBS32-Dependent Cell Death in Bacillus subtilis

2016 ◽  
Vol 198 (21) ◽  
pp. 2975-2984 ◽  
Author(s):  
B.-E. Myagmarjav ◽  
M. A. Konkol ◽  
J. Ramsey ◽  
S. Mukhopadhyay ◽  
D. B. Kearns
Keyword(s):  
Cell Death ◽  
Bacillus Subtilis ◽  
Mitomycin C ◽  
Sigma Factor ◽  
Natural Transformation ◽  
Potent Inhibitor ◽  
Dna Uptake ◽  
Content Type ◽  
Dependent Cell ◽  
Necessary And Sufficient

ABSTRACTThe ancestralBacillus subtilisstrain 3610 contains an 84-kb plasmid called pBS32 that was lost during domestication of commonly used laboratory derivatives. Here we demonstrate that pBS32, normally present at 1 or 2 copies per cell, increases in copy number nearly 100-fold when cells are treated with the DNA-damaging agent mitomycin C. Mitomycin C treatment also caused cell lysis dependent on pBS32-borne prophage genes. ZpdN, a sigma factor homolog encoded by pBS32, was required for the plasmid response to DNA damage, and artificial expression of ZpdN was sufficient to induce pBS32 hyperreplication and cell death. Plasmid DNA released by cell death was protected by the capsid protein ZpbH, suggesting that the plasmid was packaged into a phagelike particle. The putative particles were further indicated by CsCl sedimentation but were not observed by electron microscopy and were incapable of killingB. subtiliscells extracellularly. We hypothesize that pBS32-mediated cell death releases a phagelike particle that is defective and unstable.IMPORTANCEProphages are phage genomes stably integrated into the host bacterium's chromosome and less frequently are maintained as extrachromosomal plasmids. Here we report that the extrachromosomal plasmid pBS32 ofBacillus subtilisencodes a prophage that, when activated, kills the host. pBS32 also encodes both the sigma factor homolog ZpdN that is necessary and sufficient for prophage induction and the protein ComI, which is a potent inhibitor of DNA uptake by natural transformation. We provide evidence that the entire pBS32 sequence may be part of the prophage and thus that competence inhibition may be linked to lysogeny.

scholarly journals Polar Positioning of a Conjugation Protein from the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis

2009 ◽  
Vol 192 (1) ◽  
pp. 38-45 ◽  
Author(s):  
Melanie B. Berkmen ◽  
Catherine A. Lee ◽  
Emma-Kate Loveday ◽  
Alan D. Grossman
Keyword(s):  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Donor Cell ◽  
Subcellular Location ◽  
Conjugative Transfer ◽  
Protein Fusion ◽  
Cell Pole ◽  
A Cell ◽  
Green Fluorescent ◽  
Integrative And Conjugative Element

ABSTRACT ICEBs1 is an integrative and conjugative element found in the chromosome of Bacillus subtilis. ICEBs1 encodes functions needed for its excision and transfer to recipient cells. We found that the ICEBs1 gene conE (formerly yddE) is required for conjugation and that conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE. ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4. We found that a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells. At least one ICEBs1 product likely interacts with ConE to target it to the membrane and cell poles, as ConE-GFP was dispersed throughout the cytoplasm in a strain lacking ICEBs1. We also visualized the subcellular location of ICEBs1. When integrated in the chromosome, ICEBs1 was located near midcell along the length of the cell, a position characteristic of that chromosomal region. Following excision, ICEBs1 was more frequently found near a cell pole. Excision of ICEBs1 also caused altered positioning of at least one component of the replisome. Taken together, our findings indicate that ConE is a critical component of the ICEBs1 conjugation machinery, that conjugative transfer of ICEBs1 from B. subtilis likely initiates at a donor cell pole, and that ICEBs1 affects the subcellular position of the replisome.

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