The gene transfer agent produced by Rhodobacter capsulatus (RcGTA) resembles a small tailed bacteri-ophage that packages almost random genomic DNA segments that may be transferred to other R. capsula-tus cells. Gene transfer agents are produced by a number of prokaryotes; however, no receptors have been identified. We investigated the RcGTA recipient capability of wild-type R. capsulatus cells at different culture growth phases, and found that the frequency of RcGTA-dependent acquisition of an allele increases as cultures enter the stationary phase. We also found that RcGTA adsorption to cells follows a similar trend. RcGTA recipient capability and adsorption were found to be dependent on the GtaR/I quorum-sensing (QS) system. Production of an extracellular polysaccharide was found to be regulated by GtaR/I QS, as was production of the cell capsule. A number of QS-regulated putative polysaccharide biosynthesis genes were identified, and mutagenesis of two of these genes, rcc01081 and rcc01932, yielded strains that lack a capsule. Furthermore, these mutants were impaired in RcGTA recipient capability and adsorption, as was a non-encapsulated wild-type isolate of R. capsulatus. Overall, our results indicate that capsular polysaccha-ride is a receptor for the gene transfer agent of R. cap-sulatus, RcGTA.

SummaryThe gtaI gene of Rhodobacter capsulatus encodes an N-acyl-homoserine lactone (acyl-HSL) synthase. Immediately 5Ј of the gtaI gene is ORF rcc00328 that encodes a potential acyl-HSL receptor protein. A combination of genetic and biochemical approaches showed that rcc00328 (renamed gtaR) modulates the production of a genetic exchange element called the gene transfer agent (RcGTA), and regulates the transcription of gtaI. Although gtaI mutants exhibited decreased levels of RcGTA production, mutagenesis of gtaR did not, whereas a gtaR/gtaI double mutant produced wild-type levels of RcGTA. Because mutagenesis of gtaR suppressed the effect of the gtaI mutation, we suggest that the GtaR protein is a negative transcriptional regulator of RcGTA gene expression. We discovered that the gtaR and gtaI genes are co-transcribed, and also negatively regulated by the GtaR protein in the absence of acyl-HSL. A His-tagged GtaR protein was purified, and DNA-binding experiments revealed a binding site in the promoter region of the gtaRI operon. This GtaR protein did not bind to the

Gene transfer agents (GTAs) morphologically resemble small, double-stranded DNA (dsDNA) bacteriophages; however, their only known role is to package and transfer random pieces of the producing cell genome to recipient cells. The best understood GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that homologues of three genes involved in natural transformation in other bacteria, comEC, comF, and comM, are essential for RcGTA-mediated gene acquisition. This paper gives genetic and biochemical evidence that RcGTA-borne DNA entry into cells requires the ComEC and ComF putative DNA transport proteins and genetic evidence that putative cytoplasmic ComM protein of unknown function is required for recipient capability. Furthermore, the master regulator of RcGTA production in <1% of a cell population, CtrA, which is also required for gene acquisition in recipient cells, is expressed in the vast majority of the population. Our results indicate that RcGTA-mediated gene transfer combines key aspects of two bacterial horizontal gene transfer mechanisms, where donor DNA is packaged in transducing phage-like particles and recipient cells take up DNA using natural transformation-related machinery. Both of these differentiated subsets of a culture population, donors and recipients, are dependent on the same response regulator, CtrA. IMPORTANCEHorizontal gene transfer (HGT) is a major driver of bacterial evolution and adaptation to environmental stresses. Traits such as antibiotic resistance or metabolic properties can be transferred between bacteria via HGT; thus, HGT can have a tremendous effect on the fitness of a bacterial population. The three classically described HGT mechanisms are conjugation, transformation, and phage-mediated transduction. More recently, the HGT factor GTA was described, where random pieces of producing cell genome are packaged into phage-like particles that deliver DNA to recipient cells. In this report, we show that transport of DNA borne by the R. capsulatus RcGTA into recipient cells requires key genes previously thought to be specific to natural transformation pathways. These findings indicate that RcGTA combines central aspects of phage-mediated transduction and natural transformation in an efficient, regulated mode of HGT.T he first evidence of prokaryotic genetic exchange was transformation, a term coined in 1928 by Griffith (1). Subsequently, conjugation was observed in 1946 (2), followed by transduction, which was discovered in 1952 (3). More recently, another prokaryotic mode of horizontal gene transfer, dependent on an extracellular particle called a gene transfer agent (GTA), was described (4), and GTAs subsequently have been discovered in diverse prokaryotes (5).GTAs varying in morphology have been reported, although most resemble small, tailed double-stranded DNA (dsDNA) bacteriophages. The general criteria that define a GTA are (i) the DNA packaged within the head is insufficient to carry the GTA structural genes; (ii) all GTA particles package only random parts o...

SummaryGene transfer agents (GTAs) are genetic exchange elements that resemble small DNA bacteriophages that transfer random pieces of the producing cell's genome to recipient cells. The best-studied GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that the putative response regulator CtrA, which is essential for RcGTA production, is required for RcGTA-mediated gene acquisition, and confirmed that a RecA homologue is required. It was also discovered that a DprA (DNA-protecting protein A) homologue is essential for RcGTA-mediated gene acquisition, and that dprA expression is induced by gtaIdependent quorum-sensing and non-phosphorylated CtrA. Modelling of the R. capsulatus DprA structure indicated the presence of a C-terminal region that resembles a dsDNA-binding protein domain. Purified His-tagged R. capsulatus DprA protein bound to both single-stranded (ss)DNA and double-stranded (ds)DNA, but with a greater affinity for ssDNA. Additionally, DprA protected dsDNA from endonuclease digestion, and increased the rate of nucleation of Escherichia coli RecA onto ssDNA. Single-cell expression analyses revealed that dprA is expressed in the majority of cells throughout a population. Overall, the results suggest that incorporation of RcGTA DNA into the recipient cell genome proceeds through a homologous recombination pathway resembling DNA recombination in natural transformation.

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