Genetic analysis of a bacterial genetic exchange element: the gene transfer agent of Rhodobacter capsulatus

Abstract

An unusual system of genetic exchange exists in the purple nonsulfur bacterium Rhodobacter capsulatus. DNA transmission is mediated by a small bacteriophage-like particle called the gene transfer agent (GTA) that transfers random 4.5-kb segments of the producing cell's genome to recipient cells, where allelic replacement occurs. This paper presents the results of gene cloning, analysis, and mutagenesis experiments that show that GTA resembles a defective prophage related to bacteriophages from diverse genera of bacteria, which has been adopted by R. capsulatus for genetic exchange. A pair of cellular proteins, CckA and CtrA, appear to constitute part of a sensor kinase/response regulator signaling pathway that is required for expression of GTA structural genes. This signaling pathway controls growth-phase-dependent regulation of GTA gene messages, yielding maximal gene expression in the stationary phase. We suggest that GTA is an ancient prophage remnant that has evolved in concert with the bacterial genome, resulting in a genetic exchange process controlled by the bacterial cell.

Figure 1

Map of a region of the R. capsulatus chromosome containing genes necessary for GTA production. Locations of Tn5 and KIXX cartridge insertion found to abolish GTA production are shown. All ORFs are predicted to be transcribed left to right except for trmU. ORFs with weak or no similarity to known genes are shown in red or pink; ORFs homologous to known phage genes are shown in light blue; ORFs highly homologous to known cellular genes are shown in yellow. All predicted ORFs match R. capsulatus codon usage well, as defined by the highly expressed photosynthesis genes, except for those shown in pink (orfg17 and orfg19). More detailed information about sequence similarities is given in Table 1. Information about the sources of the DNA sequences used to construct this figure is in the Results section.

Figure 2

Comparisons of CtrA proteins and DNA binding sites. (A) Alignment of the CtrA amino acid sequences from R. capsulatus and C. crescentus. Identical amino acids (71%) are boxed, and the putative sequence specific DNA recognition domains of the proteins are shaded. (B) The consensus CtrA-binding site of C. crescentus compared with potential CtrA-binding sites located 5′ of the R. capsulatus ctrA gene. Sites 1 and 2 are ≈70 bp and 30 bp 5′ of the ctrA start codon, respectively.

Figure 3

Alignment of predicted CckA protein sequences of R. capsulatus and C. crescentus. The proteins are 44% identical over the sequence shown. Residues 274–691 of the C. crescentus protein (accession no. AF133718) are aligned with the available 419 aa of the R. capsulatus sequence (accession no. AF181079).

Figure 4

Growth-phase and CtrA dependence of β-galactosidase activities of orfg2′∷′lacZ plasmid-borne gene fusions. (A) Maps of the orfg2′∷′lacZ fusions used to investigate GTA gene expression. (B) β-galactosidase specific activities. The cell samples indicated left of the graph designate: Y262, plasmid-free Y262; Y262(pYNP), Y262 containing plasmid pYNP; Y262(pYP), Y262 containing plasmid pYP; YCKF(pYP), YCKF containing plasmid pYP; and YCKF(pCTRA, pYP), YCKF containing plasmids pCTRA and pYP. Cells were harvested over the growth phase at a, mid-log phase; b, late-log phase; c, early stationary phase; and d, late stationary phase.

Figure 5

RNA blot analysis of GTA gene transcripts. RNA was isolated from the strains indicated above the lines at three time points over the growth cycle: a, b, and c represent mid-log, late-log, and early stationary phases, respectively. Equal amounts (10 μg) of RNA were loaded in each lane. Approximate locations of molecular weight RNA standards are shown on the left (in kb). (A) Results when the blot was probed with a fragment of ctrA. (B) Results when the blot was probed with a fragment of orfg2.