Transcriptome analysis of Sulfolobus solfataricus infected with two related fuselloviruses reveals novel insights into the regulation of CRISPR-Cas system

Fusco, Salvatore; Liguori, Rossana; Limauro, Danila; Bartolucci, Simonetta; She, Qunxin; Contursi, Patrizia

doi:10.1016/j.biochi.2015.04.006

Cited by 40 publications

(44 citation statements)

References 45 publications

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“…In contrast, in SSV1 lysogens, the provirus coexists with some episomal copies, and a constitutive extrusion of the viral particles occurs. Therefore, the lysogeny of SSV1 could be better defined as a carrier state (44). In this case, the UV irradiation only enhances the rate of SSV1 replication and progeny extrusion without causing cell lysis.…”

Section: Discussionmentioning

confidence: 99%

Unravelling the Role of the F55 Regulator in the Transition from Lysogeny to UV Induction of Sulfolobus Spindle-Shaped Virus 1

Fusco

She

Fiorentino

et al. 2015

J Virol

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Sulfolobus spindle-shaped virus 1 represents a model for studying virus-host interaction in harsh environments, and it is so far the only member of the family Fuselloviridae that shows a UV-inducible life cycle. Although the virus has been extensively studied, mechanisms underpinning the maintenance of lysogeny as well as those regulating the UV induction have received little attention. Recently, a novel SSV1 transcription factor, F55, was identified. This factor was able to bind in vitro to several sequences derived from the early and UV-inducible promoters of the SSV1 genome. The location of these binding sites together with the differential affinity of F55 for these sequences led to the hypothesis that this protein might be involved in the maintenance of the SSV1 lysogeny. Here, we report an in vivo survey of the molecular events occurring at the UV-inducible region of the SSV1 genome, with a focus on the binding profile of F55 before and after the UV irradiation. The binding of F55 to the target promoters correlates with transcription repression, whereas its dissociation is paralleled by transcription activation. Therefore, we propose that F55 acts as a molecular switch for the transcriptional regulation of the early viral genes. IMPORTANCEFunctional genomic studies of SSV1 proteins have been hindered by the lack of similarity with other characterized proteins. As a result, few insights into their in vivo roles have been gained throughout the last 3 decades. Here, we report the first in vivo investigation of an SSV1 transcription regulator, F55, that plays a key role in the transition from the lysogenic to the induced state of SSV1. We show that F55 regulates the expression of the UV-inducible as well as the early genes. Moreover, the differential affinity of this transcription factor for these targets allows a fine-tuned and temporal coordinated regulation of transcription of viral genes.T he majority of viruses isolated from Crenarchaea shows virion morphotypes that have not been observed for viruses infecting Bacteria and Eukarya. Consequently, eight novel viral families have been introduced in order to classify these novel viruses (Fuselloviridae, Lipothrixviridae, Rudiviridae, Guttaviridae, Globuloviridae, Bicaudaviridae, Ampullaviridae, and Clavaviridae), but so far, there are still several unique archaeal viruses that remain to be classified (1-3). Many analyses of environmental samples have shown that spindle-shaped viruses are abundant and occupy several niches, including deep sea hydrothermal vents (4, 5), hypersaline environments (6, 7), anoxic freshwaters (8), cold Antarctic lakes (9), terrestrial hot springs (10-12), and acidic mines (13, 14), where they frequently outnumber head-tailed viruses. Notably, this unique virion morphotype seems to be a hallmark of viruses infecting Archaea, since it has never been observed for bacteriophages or eukaryal viruses (15). To date, the family Fuselloviridae comprises nine members (SSV1, SSV2, SSV4, SSV5, SSV6, SSV7, SSV8, SSV9, and ASV1) isolated f...

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Section: Discussionmentioning

confidence: 99%

Unravelling the Role of the F55 Regulator in the Transition from Lysogeny to UV Induction of Sulfolobus Spindle-Shaped Virus 1

Fusco

She

Fiorentino

et al. 2015

J Virol

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“…Moreover, GC and purine skew analyses also indicated that the origin is within this area and appears to be well conserved in other fuselloviruses (49). Alternatively, this region could carry essential noncoding RNAs; however, none have been identified to date (10,50).…”

Section: Discussionmentioning

confidence: 99%

Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene vp3

Iverson

Goodman

Gorchels

et al. 2017

J Virol

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Viruses infecting the Archaea harbor a tremendous amount of genetic diversity. This is especially true for the spindle-shaped viruses of the family Fuselloviridae, where Ͼ90% of the viral genes do not have detectable homologs in public databases. This significantly limits our ability to elucidate the role of viral proteins in the infection cycle. To address this, we have developed genetic techniques to study the well-characterized fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1), which infects Sulfolobus solfataricus in volcanic hot springs at 80°C and pH 3. Here, we present a new comparative genome analysis and a thorough genetic analysis of SSV1 using both specific and random mutagenesis and thereby generate mutations in all open reading frames. We demonstrate that almost half of the SSV1 genes are not essential for infectivity, and the requirement for a particular gene correlates well with its degree of conservation within the Fuselloviridae. The major capsid gene vp1 is essential for SSV1 infectivity. However, the universally conserved minor capsid gene vp3 could be deleted without a loss in infectivity and results in virions with abnormal morphology. IMPORTANCE Most of the putative genes in the spindle-shaped archaeal hyperthermophile fuselloviruses have no sequences that are clearly similar to characterized genes. In order to determine which of these SSV genes are important for function, we disrupted all of the putative genes in the prototypical fusellovirus, SSV1. Surprisingly, about half of the genes could be disrupted without destroying virus function. Even deletions of one of the known structural protein genes that is present in all known fuselloviruses, vp3, allows the production of infectious viruses. However, viruses lacking vp3 have abnormal shapes, indicating that the vp3 gene is important for virus structure. Identification of essential genes will allow focused research on minimal SSV genomes and further understanding of the structure of these unique, ubiquitous, and extremely stable archaeal viruses.

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“…Given that many Cas proteins encode nucleases 6 the fine-tuned regulation of these systems to avoid toxicity is likely a key factor to enable safe retention of a CRISPR-Cas immune system 7 . Multiple signals have been shown to activate CRISPR-Cas function in diverse organisms, such as quorum sensing 8,9 , temperature 10 , membrane stress 11,12 , altered host metabolite levels 13,14 , and phage infection 15-18 . However, relatively little is known regarding the factors and/or signals that serve to temper CRISPR-Cas activity and mitigate the risk of acquiring and expressing a nucleolytic immune system.…”

Section: Figurementioning

confidence: 99%

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

Borges

Castro

Govindarajan

et al. 2019

Preprint

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10 11 CRISPR-Cas systems are adaptive immune systems that protect bacteria from 12 bacteriophage (phage) infection. To provide immunity, RNA-guided protein 13 surveillance complexes recognize foreign nucleic acids, triggering their 14 destruction by Cas nucleases. While the essential requirements for immune 15 activity are well understood, the physiological cues that regulate CRISPR-Cas 16 expression are not. Here, a forward genetic screen identifies a two-component 17 system (KinB/AlgB), previously characterized in regulating Pseudomonas 18 aeruginosa virulence and biofilm establishment, as a regulator of the biogenesis 19 and activity of the Type I-F CRISPR-Cas system. Downstream of the KinB/AlgB 20 system, activators of biofilm production AlgU (a σ E orthologue) and AlgR, act as 21 repressors of CRISPR-Cas activity during planktonic and surface-associated 22 growth. AmrZ, another biofilm activator, functions as a surface-specific repressor 23 of CRISPR-Cas immunity. Pseudomonas phages and plasmids have taken 24 advantage of this regulatory scheme, and carry hijacked homologs of AmrZ, 25 which are functional CRISPR-Cas repressors. This suggests that while CRISPR-26 Cas regulation may be important to limit self-toxicity, endogenous repressive 27 pathways represent a vulnerability for parasite manipulation. 29Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-30 associated (cas) genes are RNA-guided nucleases found in nearly half of all bacteria 1 . 31CRISPR-Cas systems are mechanistically diverse, with six distinct types (I-VI) identified, 32 based on signature genes, mechanisms of action, and the type of nucleic acid target 1 . 33Our strong understanding of the basic components to enable sequence specific DNA 34 and RNA cleavage have enabled functional transplantation into heterologous bacterial 2,3 35 and eukaryotic 4,5 hosts. Given that many Cas proteins encode nucleases 6 the fine-tuned 36 regulation of these systems to avoid toxicity is likely a key factor to enable safe retention 37 of a CRISPR-Cas immune system 7 . Multiple signals have been shown to activate 38 CRISPR-Cas function in diverse organisms, such as quorum sensing 8,9 , temperature 10 , 39 membrane stress 11,12 , altered host metabolite levels 13,14 , and phage infection [15][16][17][18] . 40However, relatively little is known regarding the factors and/or signals that serve to 41 temper CRISPR-Cas activity and mitigate the risk of acquiring and expressing a 42 nucleolytic immune system. 44Type I CRISPR-Cas systems are comprised of a multi-subunit RNA-guided surveillance 45 complex, a trans-acting nuclease (Cas3) [19][20][21] , and proteins dedicated to spacer 46 acquisition, Cas1 and Cas2 22,23 . Pseudomonas aeruginosa has become a powerful 47 model organism for studying Type I CRISPR-Cas mechanisms 24-29 , functions 3,23,30,31 , 48 evolution 32-34 , and interactions with phages utilizing anti-CRISPR proteins [35][36][37][38] . The P. 49aeruginosa strain PA14 possesses an active Type I-F CRISPR-Cas immune system ...

show abstract

Transcriptome analysis of Sulfolobus solfataricus infected with two related fuselloviruses reveals novel insights into the regulation of CRISPR-Cas system

Cited by 40 publications

References 45 publications

Unravelling the Role of the F55 Regulator in the Transition from Lysogeny to UV Induction of Sulfolobus Spindle-Shaped Virus 1

Unravelling the Role of the F55 Regulator in the Transition from Lysogeny to UV Induction of Sulfolobus Spindle-Shaped Virus 1

Extreme Mutation Tolerance: Nearly Half of the Archaeal Fusellovirus Sulfolobus Spindle-Shaped Virus 1 Genes Are Not Required for Virus Function, Including the Minor Capsid Protein Gene vp3

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

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