Salvatore Fusco

Tlys, a Newly Identified Sulfolobus Spindle-Shaped Virus 1 Transcript Expressed in the Lysogenic State, Encodes a DNA-Binding Protein Interacting at the Promoters of the Early Genes

ABSTRACT While studying the gene expression of the Sulfolobus spindle-shaped virus 1 (SSV1) in Sulfolobus solfataricus lysogenic cells, a novel viral transcript (Tlys) was identified. Transcriptional analysis revealed that Tlys is expressed only in the absence of UV irradiation and is downregulated during the growth of the lysogenic host. The correponding gene f55 lies between two transcriptional units (T6 and Tind) that are upregulated upon UV irradiation. The open reading frame f55 encodes a 6.3-kDa protein which shows sequence identity with negative regulators that fold into the ribbon-helix-helix DNA-binding motif. DNA-binding assays demonstrated that the recombinant F55, purified from Escherichia coli, is indeed a putative transcription factor able to recognize site specifically target sequences in the promoters of the early induced T5, T6, and Tind transcripts, as well as of its own promoter. Binding sites of F55 are included within a tandem-repeated sequence overlapping the transcription start sites and/or the B recognition element of the pertinent genes. The strongest binding was observed with the promoters of T5 and T6, and an apparent cooperativity in binding was observed with the Tind promoter. Taking together the transcriptional analysis data and the biochemical evidences, we surmise that the protein F55 is involved in the regulation of the lysogenic state of SSV1.

Molecular biology of fuselloviruses and their satellites

Fuselloviruses, also known as Sulfolobus Spindle-shaped viruses (SSVs), are “lemon”- or “spindle”-shaped double-stranded DNA viruses. Among them, SSV1, SSV2 and the satellite viruses pSSVx and pSSVi have been investigated at the structural, genetic, transcriptomic, proteomic and biochemical levels, thus becoming models for dissecting DNA replication/gene expression in Archaea. Important progress has been made including elucidation of temporal genome expression during virus infection and induction of replication, SSV1 lysogeny maintenance as well as differentially expression of pSSVx replicase. Future researches focusing on these model systems would yield insightful knowledge of life cycle and DNA replication of fuselloviruses.

Host and viral transcriptional regulators in Sulfolobus : an overview

The genus Sulfolobus includes microorganisms belonging to the domain Archaea, sub-kingdom Crenarchaeota, living in geographically distant acidic hot springs. Their adaptation to such particular habitats requires finely regulated mechanisms of gene expression, among which, those modulated by sequence-specific transcription factors (TFs) play a key role. In this review, we summarize the current knowledge on the repertoires of TFs found in Sulfolobus spp. and their viruses, focusing on the description of their DNA-binding domains and their structure–function relationship.

Structural and functional studies of Stf76 from the Sulfolobus islandicus plasmid–virus pSSVx: a novel peculiar member of the winged helix–turn–helix transcription factor family

The hybrid plasmid–virus pSSVx from Sulfolobus islandicus presents an open reading frame encoding a 76 amino acid protein, namely Stf76, that does not show significant sequence homology with any protein with known 3D structure. The recombinant protein recognizes specifically two DNA-binding sites located in its own promoter, thus suggesting an auto-regulated role of its expression. Circular dichroism, spectrofluorimetric, light scattering and isothermal titration calorimetry experiments indicated a 2:1 molar ratio (protein:DNA) upon binding to the DNA target containing a single site. Furthermore, the solution structure of Stf76, determined by nuclear magnetic resonance (NMR) using chemical shift Rosetta software, has shown that the protein assumes a winged helix–turn–helix fold. NMR chemical shift perturbation analysis has been performed for the identification of the residues responsible for DNA interaction. In addition, a model of the Stf76–DNA complex has been built using as template a structurally related homolog.

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

Fuselloviruses SSV1 and SSV2 are model systems to investigate virus-host relationships in stably infected cells thanks to their temperate nature. Although they are very similar in morphology, genome organization and gene synteny, their replication is induced by different stimuli, i.e.: by UV-light exposure (for SSV1) and by the growth progression of the host (for SSV2). In this study, we have analysed global gene expression in SSV1- and SSV2-lysogens of Sulfolobus solfataricus P2 in the absence of any stimuli. Additionally, the interplay among SSV1, SSV2 and the host has been investigated in a double-infected strain to explore both virus-host and virus-virus interactions. Whereas SSV1 did not induce major changes of the host gene expression, SSV2 elicited a strong host response, which includes the transcriptional activation of CRISPR loci and cas genes. As a consequence, a significant decrease of the SSV2 copy number has been observed, which in turn led to provirus-capture into the host chromosome. Results of this study have revealed novel aspects of the host-viral interaction in the frame of the CRISPR-response.

The identification of a novel Sulfolobus islandicus CAMP-like peptide points to archaeal microorganisms as cell factories for the production of antimicrobial molecules

AbstractBackgroundPathogenic bacteria easily develop resistance to c onventional antibiotics so that even relatively new molecules are quickly losing efficacy. This strongly encourages the quest of new antimicrobials especially for the treatment of chronic infections. Cationic antimicrobial peptides (CAMPs) are small positively charged peptides with an amphipathic structure, active against Gram-positive and Gram-negative bacteria, fungi, as well as protozoa.ResultsA novel (CAMP)-like peptide (VLL-28) was identified in the primary structure of a transcription factor, Stf76, encoded by pSSVx, a hybrid plasmid–virus from the archaeon Sulfolobus islandicus. VLL-28 displays chemical, physical and functional properties typical of CAMPs. Indeed, it has a broad-spectrum antibacterial activity and acquires a defined structure in the presence of membrane mimetics. Furthermore, it exhibits selective leakage and fusogenic capability on vesicles with a lipid composition similar to that of bacterial membranes. VLL-28 localizes not only on the cell membrane but also in the cytoplasm of Escherichia coli and retains the ability to bind nucleic acids. These findings suggest that this CAMP-like peptide could exert its antimicrobial activity both on membrane and intra cellular targets.ConclusionsVLL-28 is the first CAMP-like peptide identified in the archaeal kingdom, thus pointing to archaeal microorganisms as cell factories to produce antimicrobial molecules of biotechnological interest. Furthermore, results from this work show that DNA/RNA-binding proteins could be used as sources of CAMPs.

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

ABSTRACT 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. IMPORTANCE Functional 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.

A standardized protocol for the UV induction of Sulfolobus spindle‑shaped virus 1

The Fuselloviridae prototype member Sulfolobus spindle-shaped virus 1 is a model of UV-inducible viruses infecting Crenarchaeota. Previous works on SSV1 UV induction were bases on empirically determined parameters that have not yet been standardized. Thus, in many peer reviewed literature, it is not clear how the fluence and irradiance have been determined. Here, we describe a protocol for the UV induction of SSV1 replication, which is based on the combination of the following instrumentally monitored parameters: (1) the fluence; (2) the irradiance; (3) the exposure time, and (4) the exposure distance. With the aim of finding a good balance between the viral replication induction and the host cells viability, UV-irradiated cultures were monitored for their ability to recover in the aftermath of the UV exposure. This UV irradiation procedure has been set up using the well-characterized Sulfolobus solfataricus P2 strain as model system to study host–virus interaction.

Insights into the anticancer properties of the first antimicrobial peptide from Archaea

BACKGROUND The peptide VLL-28, identified in the sequence of an archaeal protein, the transcription factor Stf76 from Sulfolobus islandicus, was previously identified and characterized as an antimicrobial peptide, possessing a broad-spectrum antibacterial activity. METHODS Through a combined approach of NMR and Circular Dichroism spectroscopy, Dynamic Light Scattering, confocal microscopy and cell viability assays, the interaction of VLL-28 with the membranes of both parental and malignant cell lines has been characterized and peptide mechanism of action has been studied. RESULTS It is here demonstrated that VLL-28 selectively exerts cytotoxic activity against murine and human tumor cells. By means of structural methodologies, VLL-28 interaction with the membranes has been proven and the binding residues have been identified. Confocal microscopy data show that VLL-28 is internalized only into tumor cells. Finally, it is shown that cell death is mainly caused by a time-dependent activation of apoptotic pathways. CONCLUSIONS VLL-28, deriving from the archaeal kingdom, is here found to be endowed with selective cytotoxic activity towards both murine and human cancer cells and consequently can be classified as an ACP. GENERAL SIGNIFICANCE VLL-28 represents the first ACP identified in an archaeal microorganism, exerting a trans-kingdom activity.

Metagenomics of microbial and viral life in terrestrial geothermal environments

Geothermally heated regions of Earth, such as terrestrial volcanic areas (fumaroles, hot springs, and geysers) and deep-sea hydrothermal vents, represent a variety of different environments populated by extremophilic archaeal and bacterial microorganisms. Since most of these microbes thriving in such harsh biotopes, they are often recalcitrant to cultivation; therefore, ecological, physiological and phylogenetic studies of these microbial populations have been hampered for a long time. More recently, culture-independent methodologies coupled with the fast development of next generation sequencing technologies as well as with the continuous advances in computational biology, have allowed the production of large amounts of metagenomic data. Specifically, these approaches have assessed the phylogenetic composition and functional potential of microbial consortia thriving within these habitats, shedding light on how extreme physico-chemical conditions and biological interactions have shaped such microbial communities. Metagenomics allowed to better understand that the exposure to an extreme range of selective pressures in such severe environments, accounts for genomic flexibility and metabolic versatility of microbial and viral communities, and makes extreme- and hyper-thermophiles suitable for bioprospecting purposes, representing an interesting source for novel thermostable proteins that can be potentially used in several industrial processes.