55.2, a Phage T4 ORFan Gene, Encodes an Inhibitor of Escherichia coli Topoisomerase I and Increases Phage Fitness

Mattenberger, Yves Benoît; Silva, Filo; Belin, Dominique

doi:10.1371/journal.pone.0124309

Cited by 16 publications

(15 citation statements)

References 81 publications

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“…Conversely, mutations in the gyrB subunit of DNA gyrase exacerbate the lethality of dnaA46 (61). Consistent with those prior results, we demonstrated that a ppGpp-driven inhibition of replication initiation was effectively bypassed by expressing T4 gp55.2, an inhibitor of topo I (59).…”

Section: Discussionsupporting

confidence: 91%

“…Notably, topA mutants can suppress the temperature sensitivity of a dnaA allele, dnaA46 , which is deficient in replication initiation (58). To manipulate chromosome superhelicity, we took advantage of a recently described T4 phage protein, gp55.2, which specifically inhibits topo I activity (59). We introduced a low-copy-number plasmid with the T4 gene 55.2 under the control of an arabinose-inducible promoter into a wild-type strain along with the IPTG-inducible pRelA′ plasmid.…”

Section: Resultsmentioning

confidence: 99%

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The Stringent Response Inhibits DNA Replication Initiation in E. coli by Modulating Supercoiling of oriC

Kraemer

Sanderlin

Laub

2019

mBio

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The stringent response enables bacteria to respond to a variety of environmental stresses, especially various forms of nutrient limitation. During the stringent response, the cell produces large quantities of the nucleotide alarmone ppGpp, which modulates many aspects of cell physiology, including reprogramming transcription, blocking protein translation, and inhibiting new rounds of DNA replication. The mechanism by which ppGpp inhibits DNA replication initiation in Escherichia coli remains unclear. Prior work suggested that ppGpp blocks new rounds of replication by inhibiting transcription of the essential initiation factor dnaA, but we found that replication is still inhibited by ppGpp in cells ectopically producing DnaA. Instead, we provide evidence that a global reduction of transcription by ppGpp prevents replication initiation by modulating the supercoiling state of the origin of replication, oriC. Active transcription normally introduces negative supercoils into oriC to help promote replication initiation, so the accumulation of ppGpp reduces initiation potential at oriC by reducing transcription. We find that maintaining transcription near oriC, either by expressing a ppGpp-blind RNA polymerase mutant or by inducing transcription from a ppGpp-insensitive promoter, can strongly bypass the inhibition of replication by ppGpp. Additionally, we show that increasing global negative supercoiling by inhibiting topoisomerase I or by deleting the nucleoid-associated protein gene seqA also relieves inhibition. We propose a model, potentially conserved across proteobacteria, in which ppGpp indirectly creates an unfavorable energy landscape for initiation by limiting the introduction of negative supercoils into oriC. IMPORTANCE To survive bouts of starvation, cells must inhibit DNA replication. In bacteria, starvation triggers production of a signaling molecule called ppGpp (guanosine tetraphosphate) that helps reprogram cellular physiology, including inhibiting new rounds of DNA replication. While ppGpp has been known to block replication initiation in Escherichia coli for decades, the mechanism responsible was unknown. Early work suggested that ppGpp drives a decrease in levels of the replication initiator protein DnaA. However, we found that this decrease is not necessary to block replication initiation. Instead, we demonstrate that ppGpp leads to a change in DNA topology that prevents initiation. ppGpp is known to inhibit bulk transcription, which normally introduces negative supercoils into the chromosome, and negative supercoils near the origin of replication help drive its unwinding, leading to replication initiation. Thus, the accumulation of ppGpp prevents replication initiation by blocking the introduction of initiation-promoting negative supercoils. This mechanism is likely conserved throughout proteobacteria.

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Section: Discussionsupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

The Stringent Response Inhibits DNA Replication Initiation in E. coli by Modulating Supercoiling of oriC

Kraemer

Sanderlin

Laub

2019

mBio

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“…Four of the mutants suppress the toxicity of phage T4 gene vs.1, an exported lysozyme whose toxicity is weaker than that of PAI2::PhoA (23). Finally, one mutant was isolated as a suppressor of phage T4 gene 55.2, a topoisomerase inhibitor that exhibit the strongest toxicity of the three proteins used (24).…”

Section: Resultsmentioning

confidence: 99%

AraC mutations that suppress inactivating mutations in the C-terminal domain of the RpoA subunit ofEscherichia coliRNA polymerase

Belin

Silva

2020

Preprint

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In E. coli, transcriptional activation is often mediated by the C-terminal domain of RpoA, the α subunit of RNA polymerase. Mutations that prevent activation of the arabinose PBAD promoter are clustered in a small region of the α–CTD domain around K271. To determine the target(s) of RpoA in the PBAD promoter, we have isolated suppressors of rpoA α–CTD mutations. The suppressors map to the N-terminal domain of AraC, the main transcriptional regulator of ara gene expression. No mutation was found in the large DNA regulatory region between araC and PBAD, suggesting that, in this system, RpoA does not activate transcription through its direct DNA binding. One class of araC mutations result in substitutions in the core of the N-terminal domain suggesting that they may affect its conformation. Another class of suppressors define genetically a domain that potentially interacts with the C-terminal domain of RpoA. Surprisingly, in rpoA+ strains lacking CRP, the araC mutations largely restore arabinose gene expression, suggesting that they somehow strengthen the AraC:α–CTD interaction. Thus, the N-terminal domain of AraC exhibits at least three activities: dimerization, arabinose binding and transcriptional activation via RpoA.ImportanceGene expression is most often controlled at the level of transcription by regulators that interact with RNA polymerase. The C-terminal domain of Escherichia coli RpoA is attached to the core enzyme by a flexible linker and serves as a hub that interacts with many regulators and even with DNA sites to activate transcription. Mutations in a RpoA subdomain interfere with activation of the main arabinose promoter by AraC, the regulator that either activates or represses expression of the arabinose operons. We define here genetically the target of RpoA in the main arabinose promoter. Suppressors of most RpoA mutations map to the N-terminal domain of AraC that promotes its dimerization and binds to arabinose, the inducer. Thus, our results identify a third function for this AraC domain. Some suppressors define a potential binding site for RpoA, while others, at internal residues, probably affect the conformation of the AraC domain.

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“…This means that topology-modifying factors already exist in diverse MGEs that could have secondary effects on CRISPR-Cas activity and thus prove useful in the context of a molecular arms race 30,31 . For instance, though not studied in the context of bacterial defense systems, the fitness of phage T4 is improved via the expression of an accessory protein that modifies DNA supercoiling and the propensity of R-loops to form 32 . Other phages, such as those in the T5-like family, incorporate regular nicks into their genome, the function of which has eluded description for over 40 years 33 .…”

Section: Discussionmentioning

confidence: 99%

The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity

Forsberg

Schmidtke

Werther

et al. 2020

Preprint

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To overcome CRISPR-Cas defense systems, many phages and mobile genetic elements encode CRISPR-Cas inhibitors called anti-CRISPRs (Acrs). Nearly all mechanistically characterized Acrs directly bind their cognate Cas protein to inactivate CRISPR immunity. Here, we describe AcrIIA22, an unconventional Acr found in hypervariable genomic regions of Clostridial bacteria and their prophages from the human gut microbiome. Uncovered in a functional metagenomic selection, AcrIIA22 does not bind strongly to SpyCas9 but nonetheless potently inhibits its activity against plasmids. To gain insight into its mechanism, we obtained an X-ray crystal structure of AcrIIA22, which revealed homology to PC4-like nucleic-acid binding proteins. This homology helped us deduce that acrIIA22 encodes a DNA nickase that relieves torsional stress in supercoiled plasmids, rendering them less susceptible to SpyCas9, which is highly dependent on negative supercoils to form stable R-loops. Modifying DNA topology may provide an additional route to CRISPR-Cas resistance in phages and mobile genetic elements.

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55.2, a Phage T4 ORFan Gene, Encodes an Inhibitor of Escherichia coli Topoisomerase I and Increases Phage Fitness

Cited by 16 publications

References 81 publications

The Stringent Response Inhibits DNA Replication Initiation in E. coli by Modulating Supercoiling of oriC

The Stringent Response Inhibits DNA Replication Initiation in E. coli by Modulating Supercoiling of oriC

AraC mutations that suppress inactivating mutations in the C-terminal domain of the RpoA subunit ofEscherichia coliRNA polymerase

The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity

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