RESUMEN
The diversity of bacteriophages is likely unparalleled in the biome due to the immense variety of hosts and the multitude of viruses that infect them. Recent efforts have led to description at the genomic level of numerous bacteriophages that infect the Actinobacteria, but relatively little is known about those infecting other prokaryotic phyla, such as the purple non-sulfur photosynthetic α-proteobacterium Rhodobacter capsulatus. This species is a common inhabitant of freshwater ecosystems and has been an important model system for the study of photosynthesis. Additionally, it is notable for its utilization of a unique form of horizontal gene transfer via a bacteriophage-like element known as the gene transfer agent (RcGTA). Only three bacteriophages of R. capsulatus had been sequenced prior to this report. Isolation and characterization at the genomic level of 26 new bacteriophages infecting this host advances the understanding of bacteriophage diversity and the origins of RcGTA. These newly discovered isolates can be grouped along with three that were previously sequenced to form six clusters with four remaining as single representatives. These bacteriophages share genes with RcGTA that seem to be related to host recognition. One isolate was found to cause lysis of a marine bacterium when exposed to high-titer lysate. Although some clusters are more highly represented in the sequenced genomes, it is evident that many more bacteriophage types that infect R. capsulatus are likely to be found in the future.
Asunto(s)
Proteínas Bacterianas/genética , Bacteriófagos/genética , Regulación Bacteriana de la Expresión Génica , Variación Genética , Rhodobacter capsulatus/virología , Técnicas de Transferencia de GenRESUMEN
Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.
Asunto(s)
Bacteriófagos/fisiología , Técnicas de Transferencia de Gen , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/virología , Siphoviridae/fisiología , Bacteriófagos/genética , Bacteriófagos/ultraestructura , Microscopía por Crioelectrón , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Transferencia de Gen Horizontal , Siphoviridae/genética , Siphoviridae/ultraestructuraRESUMEN
Genetic exchange mediated by viruses of bacteria (bacteriophages) is the primary driver of rapid bacterial evolution. The priority of viruses is usually to propagate themselves. Most bacteriophages use the small terminase protein to identify their own genome and direct its inclusion into phage capsids. Gene transfer agents (GTAs) are descended from bacteriophages, but they instead package fragments of the entire bacterial genome without preference for their own genes. GTAs do not selectively target specific DNA, and no GTA small terminases are known. Here, we identified the small terminase from the model Rhodobacter capsulatus GTA, which then allowed prediction of analogues in other species. We examined the role of the small terminase in GTA production and propose a structural basis for random DNA packaging.IMPORTANCE Random transfer of any and all genes between bacteria could be influential in the spread of virulence or antimicrobial resistance genes. Discovery of the true prevalence of GTAs in sequenced genomes is hampered by their apparent similarity to bacteriophages. Our data allowed the prediction of small terminases in diverse GTA producer species, and defining the characteristics of a "GTA-type" terminase could be an important step toward novel GTA identification. Importantly, the GTA small terminase shares many features with its phage counterpart. We propose that the GTA terminase complex could become a streamlined model system to answer fundamental questions about double-stranded DNA (dsDNA) packaging by viruses that have not been forthcoming to date.
Asunto(s)
Empaquetamiento del ADN , Endodesoxirribonucleasas/genética , Transferencia de Gen Horizontal , Rhodobacter capsulatus/genética , Bacteriófagos/genética , Proteínas de la Cápside/genética , ADN , Proteínas de Unión al ADN , Farmacorresistencia Bacteriana , Endodesoxirribonucleasas/metabolismo , Evolución Molecular , Familia de Multigenes , Rhodobacter capsulatus/virología , Alineación de Secuencia , Análisis de Secuencia de Proteína , Transducción Genética , Ensamble de VirusRESUMEN
Viruses and bacteriophages recognize cell surface proteins using receptor-binding proteins. In most tailed bacteriophages, receptor-binding proteins are located on the bacteriophage tail. The gene transfer agent of Rhodobacter capsulatus, RcGTA, morphologically resembles a tailed bacteriophage and binds to a capsular polysaccharide covering R. capsulatus cells. Here, we report that the RcGTA capsid (head) is decorated by spikes that are needed for binding to the capsule. The triangular spikes measured ~12nm and appeared to be attached at the capsid vertices. Head spike production required the putative carbohydrate-binding protein ghsB (rcc01080) previously thought to encode a side tail fiber protein. We found that ghsB is likely co-transcribed with ghsA (rcc01079) and that ghsA/ghsB is regulated by the CckA-ChpT-CtrA phosphorelay homologues and a quorum-sensing system. GhsA and GhsB were found to be CckA-dependent RcGTA maturation factors, as GhsA- and GhsB-deficient particles were found to have altered native-gel electrophoresis migration. Additionally, we provide electron microscopy images showing that RcGTA contains side tail fibers and a baseplate-like structure near the tip of the tail, which are independent of ghsB.
Asunto(s)
Cápsulas Bacterianas/metabolismo , Bacteriófagos/fisiología , Cápside/metabolismo , Rhodobacter capsulatus/metabolismo , Rhodobacter capsulatus/virología , Acoplamiento Viral , Bacteriófagos/genética , Bacteriófagos/ultraestructura , Cápside/ultraestructura , Genes Virales , Microscopía Electrónica de TransmisiónRESUMEN
The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a bacteriophage-like genetic element with the sole known function of horizontal gene transfer. Homologues of RcGTA genes are present in many members of the alphaproteobacteria and may serve an important role in microbial evolution. Transcription of RcGTA genes is induced as cultures enter the stationary phase; however, little is known about cis-active sequences. In this work, we identify the promoter of the first gene in the RcGTA structural gene cluster. Additionally, gene transduction frequency depends on the growth medium, and the reason for this is not known. We report that millimolar concentrations of phosphate posttranslationally inhibit the lysis-dependent release of RcGTA from cells in both a complex medium and a defined medium. Furthermore, we found that cell lysis requires the genes rcc00555 and rcc00556, which were expressed and studied in Escherichia coli to determine their predicted functions as an endolysin and holin, respectively. Production of RcGTA is regulated by host systems, including a putative histidine kinase, CckA, and we found that CckA is required for maximal expression of rcc00555 and for maturation of RcGTA to yield gene transduction-functional particles.
Asunto(s)
Bacteriófagos/fisiología , Fosfatos/metabolismo , Proteínas Quinasas/metabolismo , Rhodobacter capsulatus/virología , Transducción Genética , Liberación del Virus , Bacteriófagos/genética , Medios de Cultivo/química , Regulación de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica , Histidina Quinasa , Interacciones Huésped-Parásitos , Rhodobacter capsulatus/metabolismoRESUMEN
The α-proteobacterium Rhodobacter capsulatus is a model organism for the study of bacterial photosynthesis and the bacteriophage-like gene transfer agent. Characterization of phages that infect Rhodobacter is extremely rare, and scarce for the α-proteobacteria in general. Here, we describe the discovery of the only functional Mu-like transposing phage to have been identified in the α-proteobacteria, RcapMu, resident in the genome-sequenced R. capsulatus SB1003 strain. RcapMu packages ~42kb of total DNA, including <3kb of host DNA with no conserved motifs, indicative of replicative transposition with little insertion site preference. The phage genome contains 58 ORFs with comparable organization to known transposable phages. Shotgun proteomics of purified RcapMu particles detected all proteins with predicted structural functions as well as seven hypothetical proteins. Overall, comparison of RcapMu to enterobacteria phage Mu and other Mu-like phages revealed only regional homology to these phages, providing further evidence for the promiscuous, modular nature of bacteriophage evolution.
Asunto(s)
Bacteriófagos/clasificación , Bacteriófagos/genética , Genoma Viral , Rhodobacter capsulatus/virología , Proteínas Virales , Secuencia de Aminoácidos , Secuencia de Bases , Elementos Transponibles de ADN , ADN Bacteriano/genética , ADN Viral/genética , Sistemas de Lectura Abierta , Rhodobacter capsulatus/genética , Análisis de Secuencia de ADN , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The gene transfer agent (GTA) is a phage-like particle capable of exchanging double-stranded DNA fragments between cells of the photosynthetic bacterium Rhodobacter capsulatus. Here we show that the major capsid protein of GTA, expressed in E. coli, can be assembled into prohead-like structures in the presence of calcium ions in vitro. Transmission electron microscopy (TEM) of uranyl acetate staining material and thin sections of glutaraldehyde-fixed material demonstrates that these associates have spherical structures with diameters in the range of 27-35 nm. The analysis of scanning TEM images revealed particles of mass approximately 4.3 MDa, representing 101+/-11 copies of the monomeric subunit. The establishment of this simple and rapid method to form prohead-like particles permits the GTA system to be used for genome manipulation within the photosynthetic bacterium, for specific targeted drug delivery, and for the construction of biologically based distributed autonomous sensors for environmental monitoring.
Asunto(s)
Transferencia de Gen Horizontal , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/virología , Bacteriófagos/genética , Bacteriófagos/ultraestructura , Secuencia de Bases , ADN Bacteriano/genética , ADN Viral/genética , Genes Bacterianos , Genes Virales , Microscopía Electrónica , Familia de Multigenes , Sistemas de Lectura Abierta , Rhodobacter capsulatus/ultraestructura , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación , Ensamble de VirusRESUMEN
The gene transfer agent produced by Rhodobacter capsulatus (RcGTA) is a model for several virus-like elements that seem to function solely for mediating gene exchange. Several genes that encode RcGTA are clearly related to bacteriophage genes but the cellular regulatory mechanisms that control RcGTA production indicate that RcGTA is more than just a defective prophage. Genome sequencing projects show that seemingly functional RcGTA-like structural gene clusters are present in many other species of alpha-proteobacteria, which might also produce RcGTA-like particles. Here, we use the genomic sequence data that are currently available to identify candidate GTA-producing species and propose an evolutionary scheme for RcGTA-like elements in the alpha-proteobacteria.