RESUMEN
Bacteria have evolved a multitude of systems to prevent invasion by bacteriophages and other mobile genetic elements. Comparative genomics suggests that genes encoding bacterial defence mechanisms are often clustered in 'defence islands', providing a concerted level of protection against a wider range of attackers. However, there is a comparative paucity of information on functional interplay between multiple defence systems. Here, we have functionally characterised a defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a suite of thirty environmentally-isolated coliphages, we demonstrate multi-layered and robust phage protection provided by a plasmid-encoded defence island that expresses both a type I BREX system and the novel GmrSD-family type IV DNA modification-dependent restriction enzyme, BrxU. We present the structure of BrxU to 2.12 Å, the first structure of the GmrSD family of enzymes, and show that BrxU can utilise all common nucleotides and a wide selection of metals to cleave a range of modified DNAs. Additionally, BrxU undergoes a multi-step reaction cycle instigated by an unexpected ATP-dependent shift from an intertwined dimer to monomers. This direct evidence that bacterial defence islands can mediate complementary layers of phage protection enhances our understanding of the ever-expanding nature of phage-bacterial interactions.
Asunto(s)
Proteínas Bacterianas/química , Colifagos/genética , Enzimas de Restricción-Modificación del ADN/química , Escherichia coli/genética , Escherichia/genética , Plásmidos/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Colifagos/metabolismo , Cristalografía por Rayos X , Enzimas de Restricción-Modificación del ADN/genética , Enzimas de Restricción-Modificación del ADN/metabolismo , ADN Viral/química , ADN Viral/genética , ADN Viral/metabolismo , Escherichia/metabolismo , Escherichia/virología , Escherichia coli/metabolismo , Escherichia coli/virología , Expresión Génica , Islas Genómicas , Genómica/métodos , Modelos Moleculares , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
A type II restriction-modification system was found in a native plasmid of Pseudomonas savastanoi pv. savastanoi MLLI2. Functional analysis of the methyltransferase showed that the enzyme acts by protecting the DNA sequence CTGCAG from cleavage. Restriction endonuclease expression in recombinant Escherichia coli cells resulted in mutations in the REase sequence or transposition of insertion sequence 1A in the coding sequence, preventing lethal gene expression. Population screening detected homologous RM systems in other P. savastanoi strains and in the Pseudomonas syringae complex. An epidemiological survey carried out by sampling olive and oleander knots in two Italian regions showed an uneven diffusion of carrier strains, whose presence could be related to a selective advantage in maintaining the RM system in particular environments or subpopulations. Moreover, carrier strains can coexist in the same orchards, plants, and knot tissues with non-carriers, revealing unexpected genetic variability on a very small spatial scale. Phylogenetic analysis of the RM system and housekeeping gene sequences in the P. syringae complex demonstrated the ancient acquisition of the RM systems. However, the evolutionary history of the gene complex also showed the involvement of horizontal gene transfer between related strains and recombination events.
Asunto(s)
Proteínas Bacterianas/genética , Evolución Biológica , Enzimas de Restricción-Modificación del ADN/genética , Pseudomonas/enzimología , Pseudomonas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Enzimas de Restricción-Modificación del ADN/química , Enzimas de Restricción-Modificación del ADN/metabolismo , Elementos Transponibles de ADN , Escherichia coli/genética , Transferencia de Gen Horizontal , Datos de Secuencia Molecular , Nerium/microbiología , Olea/microbiología , Organismos Modificados Genéticamente/genética , FilogeniaRESUMEN
The product of gene 0.3 of bacteriophage T7, ocr, is a potent inhibitor of type I DNA restriction and modification enzymes. We have used biophysical methods to examine the mass, stability, shape and surface charge distribution of ocr. Ocr is a dimeric protein with hydrodynamic behaviour equivalent to a prolate ellipsoid of axial ratio 4.3 +/- 0.7:1 and mass of 27 kDa. The protein is resistant to denaturation but removal of the C-terminal region reduces stability substantially. Six amino acids, N4, D25, N43, D62, S68 and W94, are all located on the surface of the protein and N4 and S68 are also located at the interface between the two 116 amino acid monomers. Negatively charged amino acid side chains surround W94 but these side chains are not part of the highly acidic C-terminus after W94. Ocr is able to displace a short DNA duplex from the binding site of a type I enzyme with a dissociation constant of the order of 100 pM or better. These results suggest that ocr is of a suitable size and shape to effectively block the DNA binding site of a type I enzyme and has a large negatively charged patch on its surface. This charge distribution may be complementary to the charge distribution within the DNA binding site of type I DNA restriction and modification enzymes.