The gene transfer agent-like particle of the marine phototrophic bacterium Rhodovulum sulfidophilum

Abstract

Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum, we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5 kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40 nm and a tail length of about 60 nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum. However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.

Keywords: Artificial RNAs; Extracellular nucleic acids; Gene transfer; Genome sequence; Rhodobacter capsulatus.

Fig. 1

Detection of 4.5-kb DNA in the extracellular soluble DNA fraction of Rhodovulum sulfidophilum. Extracellular soluble DNA fractions from the stationary phase of Rdv. sulfidophilum cultures were electrophoresed on 0.5% agarose gels and stained by ethidium bromide. Lane 1, DNA from PYS medium; lane 2, DNA from glucose containing medium; and lane 3, DNA from α-cyclodextrin containing medium. M, size marker. DNA sizes (2–20 kb) are shown to the left of the panel. The 4.5-kb DNA bands are indicated with an arrow. See text for full description.

Fig. 2

Organization of the GTA structural gene cluster of Rhodobacter capsulatus and the GTA-like gene cluster of Rhodovulum sulfidophilum. The arrows show the approximate scale of the open reading frames (ORFs). A 1-kb scale bar is shown. The numbers, 2–15, in the genes of Rdv. sulfidophilum are homologs of the same numbered genes of Rba. capsulatus. The predicted functions of the ORFs are shown for Rba. capsulatus. GTA structural genes and other host-associated genes are shaded in gray and black, respectively. See text for full description.

Fig. 3

Transmission electron micrograph of the GTA-like particle of Rhodovulum. sulfidophilum. The preparation was negatively stained with 2% (wt/vol) phosphotungstic acid (pH 7.0). Bar=100 nm.

Fig. 4

Analysis of the DNA from the Rhodovulum sulfidophilum GTA-like particle. (A) The DNA preparation from the GTA-like particle or the particle itself was treated with DNase I and analyzed by 0.8% agarose gel electrophoresis. Lane 1, the DNA from the particle fraction purified by phenolization; lane 2, the same sample as in lane 1 but treated with DNase I; lane 3, the particle was first treated with DNase I, then the DNA sample was prepared by phenolization; lanes 4 and 5, DNase I treated and untreated plasmid DNA (pGEM-3Z), respectively, as a positive control for DNase I activity. (B) The particle fraction was directly subjected to 1% agarose gel electrophoresis (lane 6). M, size markers (phage lambda DNA treated with HindIII). Sizes of markers (in kb) are indicated on the left-hand side of the panel.

Fig. 5

The DNAs in the particle are random fragments from the host genome. Using primers specific for the genes nifH, soxA, pufQ, and regA (known to have widespread location on the genome of Rhodovulum sulfidophilum[18]), PCRs were performed with the GTA-like particle fraction as a template. The PCR products analyzed by 2% agarose gel electrophoresis are shown. “Genome” (for control) and “GTA” indicate the templates used for the PCRs. Exactly the same sized bands were amplified from “Genome” and “GTA” DNA by each primer set. M, size marker, 100-bp DNA ladder (Invitrogen). See text for full description.