RESUMO
In addition to defense against foreign DNA, the CRISPR-Cas9 system of Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 controls a highly specific regulon of four genes that must be repressed for bacterial virulence. Regulation occurs through a protospacer adjacent motif (PAM)-dependent interaction of Cas9 with its endogenous DNA targets, dependent on a non-canonical small RNA (scaRNA) and tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage, highlighting the evolution of scaRNA to repress transcription without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also direct DNA cleavage. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Francisella/genética , Virulência/genética , DNA/genética , Clivagem do DNA , Regulação Bacteriana da Expressão Gênica/genética , Lipoproteínas/biossíntese , Lipoproteínas/genética , RNA/genética , Transcrição GênicaRESUMO
The discovery and characterization of the prokaryotic CRISPR-Cas immune system has led to a revolution in genome editing and engineering technologies. Despite the fact that most applications emerged after the discovery of the type II-A CRISPR-Cas9 system of Streptococcus pyogenes, its biological importance in this organism has received little attention. Here, we provide a comprehensive overview of the current knowledge about CRISPR-Cas systems from S. pyogenes. We discuss how the interplay between CRISPR-mediated immunity and horizontal gene transfer might have modeled the evolution of this pathogen. We review the current literature about the CRISPR-Cas systems present in S. pyogenes (types I-C and II-A), and describe their distinctive biochemical and functional features. Finally, we summarize the main biotechnological applications that have arisen from the discovery of the CRISPR-Cas9 system in S. pyogenes.
Assuntos
Sistemas CRISPR-Cas/genética , Streptococcus pyogenes/genética , Adaptação Fisiológica/genética , Loci Gênicos , RNA/biossíntese , Interferência de RNARESUMO
Most DNA-binding bacterial transcription factors contact DNA through a recognition α-helix in their DNA-binding domains. An emerging class of DNA-binding transcription factors, predominantly found in pathogenic bacteria interact with the DNA via a relatively novel type of DNA-binding domain, called the LytTR domain, which mainly comprises ß strands. Even though the crystal structure of the LytTR domain of the virulence gene transcription factor AgrA from Staphylococcus aureus bound to its cognate DNA sequence is available, the contribution of specific amino acid residues in the LytTR domain of AgrA to transcription activation remains elusive. Here, for the first time, we have systematically investigated the role of amino acid residues in transcription activation in a LytTR domain-containing transcription factor. Our analysis, which involves in vivo and in vitro analyses and molecular dynamics simulations of S. aureus AgrA identifies a highly conserved tyrosine residue, Y229, as a major amino acid determinant for maximal activation of transcription by AgrA and provides novel insights into structure-function relationships in S. aureus AgrA.
Assuntos
Proteínas de Bactérias/química , Staphylococcus aureus/genética , Transativadores/química , Ativação Transcricional , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mutagênese , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Relação Estrutura-Atividade , Transativadores/genética , Transativadores/metabolismo , Fatores de Virulência/genéticaRESUMO
Polypeptide aggregation into amyloid is linked with several debilitating human diseases. Despite the inherent risk of aggregation-induced cytotoxicity, bacteria control the export of amyloid-prone subunits and assemble adhesive amyloid fibres during biofilm formation. An Escherichia protein, CsgC potently inhibits amyloid formation of curli amyloid proteins. Here we unlock its mechanism of action, and show that CsgC strongly inhibits primary nucleation via electrostatically-guided molecular encounters, which expands the conformational distribution of disordered curli subunits. This delays the formation of higher order intermediates and maintains amyloidogenic subunits in a secretion-competent form. New structural insight also reveal that CsgC is part of diverse family of bacterial amyloid inhibitors. Curli assembly is therefore not only arrested in the periplasm, but the preservation of conformational flexibility also enables efficient secretion to the cell surface. Understanding how bacteria safely handle amyloidogenic polypeptides contribute towards efforts to control aggregation in disease-causing amyloids and amyloid-based biotechnological applications.