Proteomic Analysis and Identification of the Structural and Regulatory Proteins of the Rhodobacter capsulatus Gene Transfer Agent

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.

The Caulobacter crescentus phage phiCbK: genomics of a canonical phage

“…While gp101 does not possess the conserved fibritin domain of the BcepNazgul protein, it does contain a pair of apparent ligand-binding domains (IPR000421, IPR008979), one of which is C-terminal, which may be involved in receptor binding. The band 3 protein gp99 contains an immunoglobulin-like (Ig) domain (IPR007110) and a phage tail-like domain (PF13550), and is weakly related (E = 2 x 10 -22 , 15.9% identity) to the Rhodobacter capsulatus gene transfer agent orf 15 (ABK27263), which is believed to be responsible for host specificity [73]. The N-proximal position of the gp99 Ig-like domain is not likely to play a role in receptor binding, as phage tail fibers typically have their receptor-binding domains in their C-termini.…”

Selection for reducing energy cost of protein production drives the GC content and amino acid composition bias in gene transfer agents

“…No unifying pattern of directional selection towards the lower carbon content was detected across all genes and all taxa ( Figure S5). This lack of an overall signal was not surprising because GTA genes can be horizontally transferred across taxa (8), have different evolutionary rates among and within taxa (8), and are likely to reach unequal translation levels during GTA production (27). These differences would make the carbon content optimization gene-and taxonspecific, blurring the net effect.…”

Insights into Origin and Evolution of α-proteobacterial Gene Transfer Agents

“…Seventeen of the genes are 10 found in a single locus that has a genomic architecture typical of a siphovirus (Lang & Beatty 11 2007). The majority of the genes in the locus encode structural components of the RcGTA 12 particle (Chen et al 2009) and are thus referred to as the structural cluster. The remaining four 13 loci contain seven genes shown to be important for RcGTA production, release, and DNA 14 transfer (Fogg et al 2012;Hynes et al 2012Hynes et al , 2016Westbye et al 2015).…”