Faculty Opinions recommendation of Transformation proteins and DNA uptake localize to the cell poles in Bacillus subtilis.

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.

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

Genes ◽

10.3390/genes11020190 ◽

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.

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

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2011.02.006 ◽

2011 ◽

Vol 111 (6) ◽

pp. 618-623 ◽

Cited By ~ 15

Author(s):

Masaomi Takeno ◽

Hisataka Taguchi ◽

Takashi Akamatsu

Keyword(s):

Bacillus Subtilis ◽

Uptake Rate ◽

Dna Uptake

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

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2012.02.004 ◽

2012 ◽

Vol 113 (6) ◽

pp. 689-693 ◽

Cited By ~ 6

Author(s):

Masaomi Takeno ◽

Hisataka Taguchi ◽

Takashi Akamatsu

Keyword(s):

Bacillus Subtilis ◽

Dna Uptake

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

Journal of Bacteriology ◽

10.1128/jb.01128-10 ◽

2011 ◽

Vol 193 (7) ◽

pp. 1633-1642 ◽

Cited By ~ 32

Author(s):

M. Kaufenstein ◽

M. van der Laan ◽

P. L. Graumann

Keyword(s):

Bacillus Subtilis ◽

Stable Complex ◽

Dna Uptake ◽

Cell Pole

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

10.1101/2020.09.29.319830 ◽

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.

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

Journal of Bacteriology ◽

10.1128/jb.00213-16 ◽

2016 ◽

Vol 198 (21) ◽

pp. 2975-2984 ◽

Cited By ~ 6

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.

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Mechanisms of transforming DNA uptake to the periplasm of Bacillus subtilis

10.1101/2021.03.09.434615 ◽

2021 ◽

Author(s):

Jeanette Hahn ◽

Micaela DeSantis ◽

David Dubnau

Keyword(s):

Bacillus Subtilis ◽

Virulence Genes ◽

Bacterial Species ◽

Epifluorescence Microscopy ◽

Cellular Distribution ◽

Dna Uptake ◽

Early Step ◽

Ratchet Mechanism ◽

New Evidence

ABSTRACTWe demonstrate here that the acquisition of DNAase resistance by transforming DNA, often assumed to indicate transport to the cytoplasm, actually reflects uptake to the periplasm, requiring a re-evaluation of conclusions about the roles of several proteins in transformation. The new evidence suggests that the transformation pilus is needed for DNA binding to the cell surface near the cell poles and for the initiation of uptake. The cellular distribution of the membrane-anchored ComEA of B. subtilis does not noticeably change during DNA uptake as does the unanchored ComEA of Vibrio and Neisseria. Instead, our evidence suggests that ComEA stabilizes the attachment of transforming DNA at localized regions in the periplasm and then mediates uptake, probably by a Brownian ratchet mechanism. Following that, the DNA is transferred to periplasmic portions of the channel protein ComEC, which plays a previously unsuspected role in uptake to the periplasm. We show that the transformation endonuclease NucA also facilitates uptake to the periplasm and that the previously demonstrated role of ComFA in the acquisition of DNAase resistance actually derives from the instability of ComGA when ComFA is deleted. These results prompt a new understanding of the early stages of DNA uptake for transformation.IMPORTANCETransformation is a widely distributed mechanism of bacterial horizontal gene transfer that plays a role in the spread of antibiotic resistance and virulence genes and more generally in evolution. Although transformation was discovered nearly a century ago and most, if not all of the proteins required have been identified in several bacterial species, much remains poorly understood about the molecular mechanism of DNA uptake. This study uses epifluorescence microscopy to investigate the passage of labeled DNA into the compartment between the cell wall and the cell membrane of Bacillus subtilis, a necessary early step in transformation. The roles of individual proteins in this process are identified, and their modes of action are clarified.

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A Conserved Metal Binding Motif in the Bacillus subtilis Competence Protein ComFA Enhances Transformation

Journal of Bacteriology ◽

10.1128/jb.00272-17 ◽

2017 ◽

Vol 199 (15) ◽

Cited By ~ 3

Author(s):

Scott S. Chilton ◽

Tanya G. Falbel ◽

Susan Hromada ◽

Briana M. Burton

Keyword(s):

Bacillus Subtilis ◽

Amino Acid ◽

Gene Transfer ◽

Horizontal Gene Transfer ◽

Metal Binding ◽

Dna Uptake ◽

Content Type ◽

Efficient Transformation ◽

Dead Box ◽

The Dead

ABSTRACT Genetic competence is a process in which cells are able to take up DNA from their environment, resulting in horizontal gene transfer, a major mechanism for generating diversity in bacteria. Many bacteria carry homologs of the central DNA uptake machinery that has been well characterized in Bacillus subtilis. It has been postulated that the B. subtilis competence helicase ComFA belongs to the DEAD box family of helicases/translocases. Here, we made a series of mutants to analyze conserved amino acid motifs in several regions of B. subtilis ComFA. First, we confirmed that ComFA activity requires amino acid residues conserved among the DEAD box helicases, and second, we show that a zinc finger-like motif consisting of four cysteines is required for efficient transformation. Each cysteine in the motif is important, and mutation of at least two of the cysteines dramatically reduces transformation efficiency. Further, combining multiple cysteine mutations with the helicase mutations shows an additive phenotype. Our results suggest that the helicase and metal binding functions are two distinct activities important for ComFA function during transformation. IMPORTANCE ComFA is a highly conserved protein that has a role in DNA uptake during natural competence, a mechanism for horizontal gene transfer observed in many bacteria. Investigation of the details of the DNA uptake mechanism is important for understanding the ways in which bacteria gain new traits from their environment, such as drug resistance. To dissect the role of ComFA in the DNA uptake machinery, we introduced point mutations into several motifs in the protein sequence. We demonstrate that several amino acid motifs conserved among ComFA proteins are important for efficient transformation. This report is the first to demonstrate the functional requirement of an amino-terminal cysteine motif in ComFA.

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The YtrBCDEF ABC transporter is involved in the control of social activities in Bacillus subtilis

10.1101/2020.07.24.219923 ◽

2020 ◽

Author(s):

Martin Benda ◽

Lisa Schulz ◽

Jeanine Rismondo ◽

Jörg Stülke

Keyword(s):

Cell Wall ◽

Transcription Factor ◽

Bacillus Subtilis ◽

Abc Transporter ◽

Biofilm Formation ◽

Translational Regulation ◽

Constitutive Expression ◽

Competence Development ◽

Dna Uptake ◽

Genetic Competence

AbstractBacillus subtilis develops genetic competence for the uptake of foreign DNA when cells enter the stationary phase and a high cell density is reached. These signals are integrated by the competence transcription factor ComK which is subject to transcriptional, post-transcriptional and post-translational regulation. Many proteins are involved in the development of competence, both to control ComK activity and to mediate DNA uptake. However, the precise function they play in competence development is often unknown. In this study, we have tested whether proteins required for genetic transformation play a role in the activation of ComK or rather downstream of competence gene expression. While these possibilities could be distinguished for most of the tested factors, two proteins (PNPase and the transcription factor YtrA) are required both for full ComK activity and for the downstream processes of DNA uptake and integration. Further analyses of the role of the transcription factor YtrA for the competence development revealed that the constitutive expression of the YtrBCDEF ABC transporter in the ytrA mutant causes the loss of genetic competence. Moreover, constitutive expression of this ABC transporter also interferes with biofilm formation. Since the ytrGABCDEF operon is induced by cell wall-targeting antibiotics, we tested the cell wall properties upon overexpression of the ABC transporter and observed an increased thickness of the cell wall. The composition and properties of the cell wall are important for competence development and biofilm formation, suggesting that the increased cell wall thickness as a result of YtrBCDEF overexpression causes the observed phenotypes.

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