scholarly journals CRISPR-Cas immunity repressed by a biofilm-activating pathway inPseudomonas aeruginosa

2019 ◽  
Author(s):  
Adair L. Borges ◽  
Bardo Castro ◽  
Sutharsan Govindarajan ◽  
Tina Solvik ◽  
Veronica Escalante ◽  
...  
Keyword(s):  
Genetic Screen ◽  
Type I ◽  
Two Component System ◽  
Component System ◽  
Forward Genetic Screen ◽  
Immune Systems ◽  
Adaptive Immune ◽  
Biofilm Production ◽  
Two Component ◽  
Adaptive Immune Systems

CRISPR-Cas systems are adaptive immune systems that protect bacteria from bacteriophage (phage) infection. To provide immunity, RNA-guided protein surveillance complexes recognize foreign nucleic acids, triggering their destruction by Cas nucleases. While the essential requirements for immune activity are well understood, the physiological cues that regulate CRISPR-Cas expression are not. Here, a forward genetic screen identifies a two-component system (KinB/AlgB), previously characterized in regulatingPseudomonas aeruginosavirulence and biofilm establishment, as a regulator of the biogenesis and activity of the Type I-F CRISPR-Cas system. Downstream of the KinB/AlgB system, activators of biofilm production AlgU (a σEorthologue) and AlgR, act as repressors of CRISPR-Cas activity during planktonic and surface-associated growth. AmrZ, another biofilm activator, functions as a surface-specific repressor of CRISPR-Cas immunity.Pseudomonasphages and plasmids have taken advantage of this regulatory scheme, and carry hijacked homologs of AmrZ, which are functional CRISPR-Cas repressors. This suggests that while CRISPR-Cas regulation may be important to limit self-toxicity, endogenous repressive pathways represent a vulnerability for parasite manipulation.

scholarly journals Structural basis for inhibition of the type I-F CRISPR–Cas surveillance complex by AcrIF4, AcrIF7 and AcrIF14

2020 ◽  
Author(s):  
Clinton Gabel ◽  
Zhuang Li ◽  
Heng Zhang ◽  
Leifu Chang
Keyword(s):  
Genome Editing ◽  
Conformational Changes ◽  
Structural Basis ◽  
Type I ◽  
Structural Detail ◽  
Immune Systems ◽  
Helical Bundle ◽  
Adaptive Immune ◽  
Target Dna ◽  
Adaptive Immune Systems

Abstract CRISPR–Cas systems are adaptive immune systems in bacteria and archaea to defend against mobile genetic elements (MGEs) and have been repurposed as genome editing tools. Anti-CRISPR (Acr) proteins are produced by MGEs to counteract CRISPR–Cas systems and can be used to regulate genome editing by CRISPR techniques. Here, we report the cryo-EM structures of three type I-F Acr proteins, AcrIF4, AcrIF7 and AcrIF14, bound to the type I-F CRISPR–Cas surveillance complex (the Csy complex) from Pseudomonas aeruginosa. AcrIF4 binds to an unprecedented site on the C-terminal helical bundle of Cas8f subunit, precluding conformational changes required for activation of the Csy complex. AcrIF7 mimics the PAM duplex of target DNA and is bound to the N-terminal DNA vise of Cas8f. Two copies of AcrIF14 bind to the thumb domains of Cas7.4f and Cas7.6f, preventing hybridization between target DNA and the crRNA. Our results reveal structural detail of three AcrIF proteins, each binding to a different site on the Csy complex for inhibiting degradation of MGEs.

scholarly journals Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: insights from a natural arms race

2019 ◽  
Vol 374 (1772) ◽  
pp. 20180089 ◽  
Author(s):  
Amelia C. McKitterick ◽  
Kristen N. LeGault ◽  
Angus Angermeyer ◽  
Munirul Alam ◽  
Kimberley D. Seed
Keyword(s):  
High Throughput Sequencing ◽  
Mobile Genetic Element ◽  
Arms Race ◽  
Type I ◽  
Helicase Activity ◽  
Wild Populations ◽  
Immune Systems ◽  
Adaptive Immune ◽  
Adaptive Immune Systems

CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae . Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.

scholarly journals Distinct subcellular localization of a Type I CRISPR complex and the Cas3 nuclease in bacteria

2020 ◽  
Author(s):  
Sutharsan Govindarajan ◽  
Adair Borges ◽  
Joseph Bondy-Denomy
Keyword(s):  
Fusion Proteins ◽  
Cell Biology ◽  
Type I ◽  
Immune Systems ◽  
Adaptive Immune ◽  
Spatiotemporal Regulation ◽  
Cas9 Nuclease ◽  
Adaptive Immune Systems ◽  
Single Focus

AbstractCRISPR-Cas systems are prokaryotic adaptive immune systems that have been well characterized biochemically, but in vivo spatiotemporal regulation and cell biology remains largely unaddressed. Here, we used fluorescent fusion proteins to study the localization of the Type I-F CRISPR-Cas system native to Pseudomonas aeruginosa. When targeted to an integrated prophage, the crRNA-guided (Csy) complex and a majority of Cas3 molecules in the cell are recruited to a single focus. When lacking a target in the cell, however, the Csy complex is broadly nucleoid bound, while Cas3 is diffuse in the cytoplasm. Nucleoid association for the Csy proteins is crRNA-dependent, and inhibited by expression of anti-CRISPR AcrIF2, which blocks PAM binding. The Cas9 nuclease is also nucleoid localized, only when gRNA-bound, which is abolished by PAM mimic, AcrIIA4. Our findings reveal PAM-dependent nucleoid surveillance and spatiotemporal regulation in Type I CRISPR-Cas that separates the nuclease-helicase Cas3 from the crRNA-guided surveillance complex.

BIFURCATIONS IN A HUMAN MIGRATION MODEL OF SCHEURLE–SEYDEL TYPE-I: TURING BIFURCATION

2003 ◽  
Vol 13 (05) ◽  
pp. 1303-1308 ◽  
Author(s):  
SÁNDOR KOVÁCS
Keyword(s):  
Graduate Programs ◽  
Critical Value ◽  
Human Migration ◽  
Fickian Diffusion ◽  
Type I ◽  
Two Component System ◽  
Component System ◽  
Turing Bifurcation ◽  
Cross Diffusion ◽  
Two Component

In this paper we consider a model for the behavior of students in graduate programs at neighboring universities which is a modified form of the model proposed by [Scheurle & Seydel, 2000], and observe that the stationary solution of this two-component system becomes unstable in the presence of diffusion. We assume that both types of individuals are continuously distributed throughout a bounded two-dimensional spatial domain of two types (regular hexagon and rhombus), across whose boundaries there is no migration, and which simultaneously undergo simple (Fickian) diffusion but the spatial flow is influenced not only by its own but also by the other's density (cross diffusion). We will show that at a critical value of a parameter a Turing bifurcation takes place: a spatially nonhomogenous solution (pattern) arises.

scholarly journals The ColRS Two-Component System Regulates Membrane Functions and Protects Pseudomonas putida against Phenol

2006 ◽  
Vol 188 (23) ◽  
pp. 8109-8117 ◽  
Author(s):  
Paula Ann Kivistik ◽  
Marta Putrinš ◽  
Külliki Püvi ◽  
Heili Ilves ◽  
Maia Kivisaar ◽  
...  
Keyword(s):  
Pseudomonas Putida ◽  
Target Genes ◽  
Type I ◽  
Two Component System ◽  
Component System ◽  
Two Component ◽  
Encoding Gene ◽  
Phenol Tolerance

ABSTRACT As reported, the two-component system ColRS is involved in two completely different processes. It facilitates the root colonization ability of Pseudomonas fluorescens and is necessary for the Tn4652 transposition-dependent accumulation of phenol-utilizing mutants in Pseudomonas putida. To determine the role of the ColRS system in P. putida, we searched for target genes of response regulator ColR by use of a promoter library. Promoter screening was performed on phenol plates to mimic the conditions under which the effect of ColR on transposition was detected. The library screen revealed the porin-encoding gene oprQ and the alginate biosynthesis gene algD occurring under negative control of ColR. Binding of ColR to the promoter regions of oprQ and algD in vitro confirmed its direct involvement in regulation of these genes. Additionally, the porin-encoding gene ompA PP0773 and the type I pilus gene csuB were also identified in the promoter screen. However, it turned out that ompA PP0773 and csuB were actually affected by phenol and that the influence of ColR on these promoters was indirect. Namely, our results show that ColR is involved in phenol tolerance of P. putida. Phenol MIC measurement demonstrated that a colR mutant strain did not tolerate elevated phenol concentrations. Our data suggest that increased phenol susceptibility is also the reason for inhibition of transposition of Tn4652 in phenol-starving colR mutant bacteria. Thus, the current study revealed the role of the ColRS two-component system in regulation of membrane functionality, particularly in phenol tolerance of P. putida.

Streptococcus pyogenes TrxSR Two-Component System Regulates Biofilm Production in Acidic Environments

2021 ◽  
Author(s):  
Masanori Isaka ◽  
Akira Okamoto ◽  
Yutaka Miura ◽  
Ichiro Tatsuno ◽  
Jun-ich Maeyama ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Regulatory System ◽  
Mass Spectrometry Analysis ◽  
M Protein ◽  
Two Component System ◽  
Component System ◽  
Isogenic Mutant ◽  
Spectrometry Analysis ◽  
Biofilm Production ◽  
Two Component

Bacteria form biofilms for their protection against environmental stress and produce virulence factors within the biofilm. Biofilm formation in acidified environments is regulated by a two-component system, as shown by studies on isogenic mutants of the sensor protein of the two-component regulatory system in Streptococcus pyogenes . In this study, we found that the LiaS histidine kinase sensor mediates biofilm production and pilus expression in an acidified environment through glucose fermentation. The liaS isogenic mutant produced biofilms in a culture acidified by hydrochloric acid but not glucose, suggesting that the acidified environment is sensed by another protein. In addition, the trxS isogenic mutant could not produce biofilms or activate the mga promoter in an acidified environment. Mass spectrometry analysis showed that TrxS regulates M Protein, consistent with the transcriptional regulation of emm , which encodes M protein. Our results demonstrate that biofilm production during environmental acidification is directly under the control of TrxS.

scholarly journals Structure reveals mechanism of CRISPR RNA-guided nuclease recruitment and anti-CRISPR viral mimicry

2018 ◽  
Author(s):  
MaryClare F. Rollins ◽  
Saikat Chowdhury ◽  
Joshua Carter ◽  
Sarah M. Golden ◽  
Heini M. Miettinen ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type I ◽  
Structural Homology ◽  
Immune Systems ◽  
Helical Bundle ◽  
Adaptive Immune ◽  
Double Stranded Dna ◽  
Viral Mimicry ◽  
Cas Genes ◽  
Adaptive Immune Systems

AbstractBacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here we present the cryo-EM structure of the type I-F CRISPR RNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a Ȉ180-degree rotation of the C-terminal helical bundle on the “large” Cas8f subunit. We show that the dsDNA-induced conformational change in Cas8f exposes a Cas2/3 “nuclease recruitment helix” that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f “nuclease recruitment helix”, implying that cas genes may sometimes serve as genetic fodder for the evolution of anti-CRISPRs.

Structural plasticity and in vivo activity of Cas1 from the type I-F CRISPR–Cas system

2016 ◽  
Vol 473 (8) ◽  
pp. 1063-1072 ◽  
Author(s):  
Max E. Wilkinson ◽  
Yoshio Nakatani ◽  
Raymond H.J. Staals ◽  
Sebastian N. Kieper ◽  
Helen K. Opel-Reading ◽  
...  
Keyword(s):  
Structural Plasticity ◽  
Type I ◽  
Immune Systems ◽  
Adaptive Immune ◽  
Foreign Dna ◽  
Adaptive Immune Systems

CRISPR–Cas systems are adaptive immune systems that defend prokaryotes against foreign DNA. In a type I-F CRISPR–Cas system we show that Cas1 is required for adaptation to new threats, and that this protein is characterized by remarkable structural plasticity.

scholarly journals Regulation of the type I protein secretion system by the MisR/MisS two-component system in Neisseria meningitidis

Microbiology ◽  
2009 ◽  
Vol 155 (5) ◽  
pp. 1588-1601 ◽  
Author(s):  
Soma Sannigrahi ◽  
Xinjian Zhang ◽  
Yih-Ling Tzeng
Keyword(s):  
Neisseria Meningitidis ◽  
Growth Phase ◽  
Sequence Similarity ◽  
Secretion System ◽  
Type I ◽  
Two Component System ◽  
Promoter Elements ◽  
Component System ◽  
Rtx Toxin ◽  
Two Component

Neisseria meningitidis, an obligate human pathogen, remains a leading cause of meningitis and fatal sepsis. Meningococci are known to secrete a family of proteins, such as FrpC, with sequence similarity to the repeat-in-toxin (RTX) proteins via the type I secretion system. The meningococcal type I secretion proteins are encoded at two distant genetic loci, NMB1400 (hlyB) and NMB1738/1737 (hlyD/tolC), and are separated from the RTX toxin-like substrates. We have characterized the promoter elements of both hlyB and hlyD by primer extension and lacZ reporter fusions and revealed the growth phase-dependent upregulation of both genes. In addition, we showed that the MisR/MisS two-component system negatively regulates the expression of hlyB and hlyD/tolC. Direct binding of MisR to hlyB and hlyD promoters was demonstrated by electrophoretic mobility shift assay (EMSA), and DNase I protection assays identified MisR binding sites overlapping the promoter elements. Direct repression of hlyB transcription by MisR was supported by in vitro transcription assays. Mutations in the MisR/S system affected, but did not eliminate, the growth phase-dependent upregulation of hlyB, suggesting additional regulatory mechanisms. Increased secretion of RTX toxin-like proteins was detected in the cell-free media from misS mutant cultures, indicating that the amounts of extracellular RTX toxin-like proteins are, in part, controlled by the abundance of the type I secretion apparatus. This is, to our knowledge, the first example of a two-component system mediating secretion of cytotoxin family proteins by controlling expression of the type I secretion proteins.

Sign in / Sign up

Export Citation Format

Share Document