Occurrence and expression of gene transfer agent genes in marine bacterioplankton

Abstract

Genes with homology to the transduction-like gene transfer agent (GTA) were observed in genome sequences of three cultured members of the marine Roseobacter clade. A broader search for homologs for this host-controlled virus-like gene transfer system identified likely GTA systems in cultured Alphaproteobacteria, and particularly in marine bacterioplankton representatives. Expression of GTA genes and extracellular release of GTA particles ( approximately 50 to 70 nm) was demonstrated experimentally for the Roseobacter clade member Silicibacter pomeroyi DSS-3, and intraspecific gene transfer was documented. GTA homologs are surprisingly infrequent in marine metagenomic sequence data, however, and the role of this lateral gene transfer mechanism in ocean bacterioplankton communities remains unclear.

FIG. 1.

Phylogenetic tree of concatenated amino acid sequences of orfg3 (portal protein), orfg5 (capsid protein), and orfg12 (unknown protein) homologs constructed with the neighbor-joining algorithm and percent accepted mutation correction for genomic data available as of 1 December 2007. Accession numbers for each concatenated gene are listed in parentheses; the numbers within squares indicate the numbers of R. capsulatus-like GTA genes contained within the genome (15 possible); the filled circles indicate marine bacteria, open stars indicate that gene neighborhoods are shown in Fig. 2, checkmarks indicate organisms for which GTA activity has been experimentally verified, and boldface text indicates members of the Roseobacter clade. Bootstrap values of >50% are indicated on the nodes. Two nonmarine Rhizobiales, Brucella melitensis 16 M and Brucella suis 1330, contain GTA-like genes but are omitted from this tree because they lack one of the three concatenated homologs (orfg5 and orfg3, respectively).

FIG. 2.

Gene neighborhoods of R. capsulatus-like GTA genes in R. capsulatus and one representative bacterium from each clade shown in Fig. 1. The checkmarks indicate organisms for which GTA activity has been experimentally verified. The numbers within the arrows indicate R. capsulatus-like GTA gene homologs as defined by BLASTp matches having E values of 10⁻⁴ or lower. The R. capsulatus gene orientation and order were taken from Lang et al. (20). Complete genome sequences are available at NCBI (http://www.ncbi.nlm.nih.gov).

FIG. 3.

Transmission electron micrograph of GTA-like particles filtered from S. pomeroyi DSS-3 cultures. The long filamentous structures are most likely flagellar filaments (8). Bars = 50 nm.

FIG. 4.

Production of GTA particles in S. pomeroyi DSS-3 cultures. (A) Detection of GTA production by epifluorescence microscopy. (B) Expression of an S. pomeroyi DSS-3 GTA gene (orfg3; portal) (n = 4 ± standard deviation [SD]) as a function of the cell number (n = 2 ± SD). The dotted line indicates the transition between log growth phase and stationary phase.

FIG. 5.

Transfer of genetic markers between mutant strains of S. pomeroyi DSS-3. (A) Cultures of DSS-3 spontaneous mutants (Rif^r or Strep^r) were grown together or individually and plated on double-antibiotic plates. CFU of double mutants when grown together (Rif^r + Strep^r; replicates 1 and 2) provide an estimate of GTA-mediated gene transfer, since spontaneous double mutants in individually grown cultures (Rif^r or Strep^r) occur at very low numbers. (B) CFU from Kan^r₁₅₀ S. pomeroyi DSS-3 filtrate incubated with WT cells provide evidence for GTA activity. The values are averages (n = 2 plus standard deviation).

FIG. 6.

Presence of GTA genes in the GOS metagenomic library (29) calculated as percentages of cells carrying homologs for each of the 15 GTAs. The dark-gray bars represent “diagnostic genes” (i.e., genes present in >90% of GTA-containing organisms as listed in Fig. 1). The light-gray bars represent “nondiagnostic genes” (i.e., those less common in GTA-containing organisms). The mean abundances across all 15 homologs are given in parentheses.