In vitro assembly of a prohead-like structure of the Rhodobacter capsulatus gene transfer agent.

Department of Biology, University of Virginia, Charlottesville, VA 22904, USA. <>
Virology (Impact Factor: 3.28). 08/2007; 364(1):95-102. DOI: 10.1016/j.virol.2007.02.027
Source: PubMed

ABSTRACT 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.

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    ABSTRACT: The gene transfer agent of Rhodobacter capsulatus (GTA) is a unique phage-like particle that exchanges genetic information between members of this same species of bacterium. Besides being an excellent tool for genetic mapping, the GTA has a number of advantages for biotechnological and nanoengineering purposes. To facilitate the GTA purification and identify the proteins involved in GTA expression, assembly and regulation, in the present work we construct and transform into R. capsulatus Y262 a gene coding for a C-terminally His-tagged capsid protein. The constructed protein was expressed in the cells, assembled into chimeric GTA particles inside the cells and excreted from the cells into surrounding medium. Transmission electron micrographs of phosphotungstate-stained, NiNTA-purified chimeric GTA confirm that its structure is similar to normal GTA particles, with many particles composed both of a head and a tail. The mass spectrometric proteomic analysis of polypeptides present in the GTA recovered outside the cells shows that GTA is composed of at least 9 proteins represented in the GTA gene cluster including proteins coded for by Orf's 3, 5, 6-9, 11, 13, and 15.
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    ABSTRACT: 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 α-proteobacteria, 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 was not known. We report that millimolar concentrations of phosphate post-translationally inhibit the lysis-dependent release of RcGTA from cells in both a complex and a defined medium. Furthermore, we found that cell lysis requires the genes rcc00555 and rcc00556, which were expressed and studied in E. 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.
    Journal of bacteriology 08/2013; DOI:10.1128/JB.00669-13 · 2.69 Impact Factor

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