RESUMO
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.
Assuntos
Proteínas de Bactérias/química , Colífagos/genética , Enzimas de Restrição-Modificação do DNA/química , Escherichia coli/genética , Escherichia/genética , Plasmídeos/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Colífagos/metabolismo , Cristalografia por Raios X , Enzimas de Restrição-Modificação do DNA/genética , Enzimas de Restrição-Modificação do DNA/metabolismo , DNA Viral/química , DNA Viral/genética , DNA Viral/metabolismo , Escherichia/metabolismo , Escherichia/virologia , Escherichia coli/metabolismo , Escherichia coli/virologia , Expressão Gênica , Ilhas Genômicas , Genômica/métodos , Modelos Moleculares , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
GcvB is one of the most highly conserved Hfq-associated small RNAs in Gram-negative bacteria and was previously reported to repress several ABC transporters for amino acids. To determine the full extent of GcvB-mediated regulation in Salmonella, we combined a genome-wide experimental approach with biocomputational target prediction. Comparative pulse expression of wild-type versus mutant sRNA variants revealed that GcvB governs a large post-transcriptional regulon, impacting ~1% of all Salmonella genes via its conserved G/U-rich domain R1. Complementary predictions of C/A-rich binding sites in mRNAs and gfp reporter fusion experiments increased the number of validated GcvB targets to more than 20, and doubled the number of regulated amino acid transporters. Unlike the previously described targeting via the single R1 domain, GcvB represses the glycine transporter CycA by exceptionally redundant base-pairing. This novel ability of GcvB is focused upon the one target that could feedback-regulate the glycine-responsive synthesis of GcvB. Several newly discovered mRNA targets involved in amino acid metabolism, including the global regulator Lrp, question the previous assumption that GcvB simply acts to limit unnecessary amino acid uptake. Rather, GcvB rewires primary transcriptional control circuits and seems to act as a distinct regulatory node in amino acid metabolism.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Pequeno RNA não Traduzido/metabolismo , Salmonella/metabolismo , Fatores de Transcrição/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Proteína Reguladora de Resposta a Leucina/metabolismo , Mutação , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , RNA Citoplasmático Pequeno/biossíntese , RNA Citoplasmático Pequeno/genética , Pequeno RNA não Traduzido/genética , Salmonella/genética , Fatores de Transcrição/genéticaRESUMO
To date, the majority of studies of bacterial gene expression have been carried out on large communities, as techniques for analysis of expression in individual cells have not been available. Recent developments now allow us to use reporter genes to monitor gene expression in individual bacterial cells. Conventional reporters are not suitable for studies of living single cells. However, variants of GFP have proved to be ideal for the study of development, cell biology, and pathogenesis and are now the reporters of choice for microbial studies. In combination with techniques such as DFI and IVET and the use of flow cytometry and advanced fluorescence microscopy, the latest generation of GFP reporters allows the investigation of gene expression in individual bacterial cells within particular environments. These studies promise to bring a new level of understanding to the fields of bacterial pathogenesis and environmental microbiology.