StpA represses CRISPR-Cas immunity in H-NS deficient Escherichia coli

Mitić, Damjan; Radovčić, Marin; Markulin, Dora; Ivančić-Baće, Ivana

doi:10.1016/j.biochi.2020.04.020

Cited by 13 publications

(17 citation statements)

References 55 publications

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“…Alternatively, H-NS and StpA in the WT strain may form a heteromeric complex (92), which could have a stronger suppression effect on expression of crRNA than only H-NS in the stpA null mutant. Of note, a recent report documented that ectopic expression of StpA on a multicopy-number plasmid suppressed transcription of casA in an hns cas1 double mutant of E. coli, resulting in a decrease of DNA interference during phage infection (93). The result is in accordance with our observation that ectopic expression of StpA at a high level suppressed transcription of cas genes in the hns null mutant (Fig.…”

Section: Discussionsupporting

confidence: 93%

“…S13). It is also documented that although inactivation of stpA further increased transcription of casA in the hns cas1 mutant, stpA seems to have no additional role to further improve immunity against bacteriophage infection in that strain (93). This finding is different from our observation in that stpA deletion in an hns null mutant reduced transcription of cas genes (Fig.…”

Section: Discussioncontrasting

confidence: 89%

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Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

Sun

Mao

Fei

et al. 2020

Appl Environ Microbiol

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ABSTRACT Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (Pcas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli. Although the H-NS paralogue StpA also binds Pcas, its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of Pcas. Third, by comparing transcriptional activities of the intact Pcas and the Pcas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in Pcas. Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of Pcas. Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. IMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli. However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli. In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.

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Section: Discussionsupporting

confidence: 93%

Section: Discussioncontrasting

confidence: 89%

Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

Sun

Mao

Fei

et al. 2020

Appl Environ Microbiol

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“…Cells were lacking H-NS (∆hns) to de-repress transcription of Cascade and Cas3 encoding genes. At 30 • C, wild type Cas3 cells were fully protected from plaque formation when challenged by λvir, but this protection was lost at 37 • C (Figure 5), as expected from Figure 1 and [28]. Cas3 W406A cells showed λvir plaques at both temperatures, and, in addition, Cas3 W406A plaques were significantly larger than those of wild type Cas3 at 37 • C, further indicating that the Cas3 W406A mutation is ineffective for CRIPSR interference (Figure 5).…”

Section: Cas3 W406a Does Not Protect E Coli Cells From Phage Lysissupporting

confidence: 76%

“…Conformational Re-Arrangement of Cas3 in Transition from 30 • C to 37 • C In CRISPR systems Cas3 nuclease is targeted to destruction of MGEs, and potentially provides MGE DNA as protospacers for updating the CRISPR immunity locus. In E. coli, we previously reported that the efficacy of CRISPR defence against phage λ is restricted by temperature [26,28]. This can be seen as increasing frequencies of phage λ plaques at 35-37 • C on lawns of ∆hns E. coli cells expressing Cas3, Cascade and λ-targeting crRNA when compared with plaques at 30 • C (Figure 1A).…”

Section: Cas3 Nuclease Activity Is Modulated By Temperature Changes That Correspond Withmentioning

confidence: 84%

A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity

Liu

Matošević

Mitić

et al. 2021

IJMS

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Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a ‘gate’ for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells.

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“…However, both strong activation and no significant effect of CRP on the cas3 promoter have been reported, which has tentatively been related to the different growth phases of the E. coli cultures assayed in the two studies (Yang et al 2014 , 2020 ). Similarly, two recent publications (Mitić et al 2020 ; Sun et al 2020a ) documented contradictory results showing that when the gene encoding the H-NS paralog StpA was inactivated or deleted in distinct genetic backgrounds, the cas operon transcription was either increased (in an hns cas1 double mutant) or reduced ( hns null mutant). Nevertheless, overexpression of StpA suppressed transcription in both cases.…”

Section: Crispr-cas Controlmentioning

confidence: 98%

Digging into the lesser-known aspects of CRISPR biology

2021

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A long time has passed since regularly interspaced DNA repeats were discovered in prokaryotes. Today, those enigmatic repetitive elements termed clustered regularly interspaced short palindromic repeats (CRISPR) are acknowledged as an emblematic part of multicomponent CRISPR-Cas (CRISPR associated) systems. These systems are involved in a variety of roles in bacteria and archaea, notably, that of conferring protection against transmissible genetic elements through an adaptive immune-like response. This review summarises the present knowledge on the diversity, molecular mechanisms and biology of CRISPR-Cas. We pay special attention to the most recent findings related to the determinants and consequences of CRISPR-Cas activity. Research on the basic features of these systems illustrates how instrumental the study of prokaryotes is for understanding biology in general, ultimately providing valuable tools for diverse fields and fuelling research beyond the mainstream.

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StpA represses CRISPR-Cas immunity in H-NS deficient Escherichia coli

Cited by 13 publications

References 55 publications

Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity

Digging into the lesser-known aspects of CRISPR biology

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