New evidence supports the prophage origin of RcGTA.

Gene transfer agents (GTAs) are phage-like entities that package and transfer random host genome fragments between prokaryotes. RcGTA, produced by Rhodobacter capsulatus, is hypothesized to originate from a prophage ancestor. Most of the evidence supporting this hypothesis came from the finding of RcGTA-like genes in phages. More than 75% of the RcGTA genes have a phage homolog. However, only a few RcGTA homologs have been identified in a (pro)phage genome, leaving the hypothesis that GTAs evolved from prophages through gene loss with only weak evidence. We herein report the discovery of an inducible prophage (vB_MseS-P1) from a Mesorhizobium sediminum strain that contains the largest number (12) of RcGTA homologs found in a phage genome to date. We also identified three putative prophages and two prophage remnants harboring 12-14 RcGTA homologs in a Methylobacterium nodulans strain. The protein remote homology detection also revealed more RcGTA homologs from other phages than we previously thought. Moreover, the head-tail gene architecture of these newly discovered prophage-related elements closely resembles that of RcGTA. Furthermore, vB_MseS-P1 virions have structural proteins similar to RcGTA particles. Close phylogenetic relationships between certain prophage genes and RcGTA-like genes in Alphaproteobacteria further support the shared ancestry between RcGTA and prophages. Our findings provide new relatively direct evidence of the origin of RcGTA from a prophage progenitor.IMPORTANCEGTAs are important genetic elements in certain groups of bacteria and contribute to the genetic diversification, evolution, and ecological adaptation of bacteria. RcGTA, a common type of GTA, is known to package and transfer random fragments of the bacterial genome to recipient cells. However, the origin of RcGTA is still elusive. It has been hypothesized that RcGTA evolved from a prophage ancestor through gene loss. However, the few RcGTA homologs identified in a (pro)phage genome leave the hypothesis lacking direct evidence. This study uncovers the presence of a large number of RcGTA homologs in an inducible prophage and several putative prophages. The similar head-tail gene architecture and structural protein compositions of these newly discovered prophage-related elements and RcGTA further demonstrate an unprecedentedly observed close evolutionary relationship between prophages and RcGTA. Together, our findings provide more direct evidence supporting the origin of RcGTA from prophage.

Extracellular Polysaccharide Receptor and Receptor-Binding Proteins of the Rhodobacter capsulatus Bacteriophage-like Gene Transfer Agent RcGTA.

A variety of prokaryotes produce a bacteriophage-like gene transfer agent (GTA), and the alphaproteobacterial Rhodobacter capsulatus RcGTA is a model GTA. Some environmental isolates of R. capsulatus lack the ability to acquire genes transferred by the RcGTA (recipient capability). In this work, we investigated the reason why R. capsulatus strain 37b4 lacks recipient capability. The RcGTA head spike fiber and tail fiber proteins have been proposed to bind extracellular oligosaccharide receptors, and strain 37b4 lacks a capsular polysaccharide (CPS). The reason why strain 37b4 lacks a CPS was unknown, as was whether the provision of a CPS to 37b4 would result in recipient capability. To address these questions, we sequenced and annotated the strain 37b4 genome and used BLAST interrogations of this genome sequence to search for homologs of genes known to be needed for R. capsulatus recipient capability. We also created a cosmid-borne genome library from a wild-type strain, mobilized the library into 37b4, and used the cosmid-complemented strain 37b4 to identify genes needed for a gain of function, allowing for the acquisition of RcGTA-borne genes. The relative presence of CPS around a wild-type strain, 37b4, and cosmid-complemented 37b4 cells was visualized using light microscopy of stained cells. Fluorescently tagged head spike fiber and tail fiber proteins of the RcGTA particle were created and used to measure the relative binding to wild-type and 37b4 cells. We found that strain 37b4 lacks recipient capability because of an inability to bind RcGTA; the reason it is incapable of binding is that it lacks CPS, and the absence of CPS is due to the absence of genes previously shown to be needed for CPS production in another strain. In addition to the head spike fiber, we found that the tail fiber protein also binds to the CPS.

DNA Gyrase Inhibitors Increase the Frequency of Bacteriophage-like RcGTA-Mediated Gene Transfer in Rhodobacter capsulatus.

Rhodobacter capsulatus produces a bacteriophage-like particle called the gene transfer agent (RcGTA) that mediates horizontal gene transfer. RcGTA particles transfer random ~4.5-kb fragments of genomic DNA that integrate into recipient genomes by allelic replacement. This work addresses the effect of sub-inhibitory concentrations of antibiotics on gene transfer by RcGTA. A transduction assay was developed to test the effects of various substances on gene transfer. Using this assay, low concentrations of DNA gyrase inhibitors were found to increase the frequency of gene transfer. Novobiocin was studied in more detail, and it was found that this antibiotic did not influence the production or release of RcGTA but instead appeared to act on the recipient cells. The target of novobiocin in other species has been shown to be the GyrB subunit of DNA gyrase (a heterotetramer of 2GyrA and 2GyrB). R. capsulatus encodes GyrA and GyrB homologues, and a GyrB overexpression plasmid was created and found to confer resistance to novobiocin. The presence of the overexpression plasmid in recipient cells greatly diminished the novobiocin-mediated increase in gene transfer, confirming that this effect is due to the binding of novobiocin by GyrB. The results of this work show that antibiotics affect gene transfer in R. capsulatus and may be relevant to microbial genetic exchange in natural ecosystems.

Evolution of DNA packaging in gene transfer agents.

Gene transfer agents (GTAs) are virus-like particles encoded and produced by many bacteria and archaea. Unlike viruses, GTAs package fragments of the host genome instead of the genes that encode the components of the GTA itself. As a result of this non-specific DNA packaging, GTAs can transfer genes within bacterial and archaeal communities. GTAs clearly evolved from viruses and are thought to have been maintained in prokaryotic genomes due to the advantages associated with their DNA transfer capacity. The most-studied GTA is produced by the alphaproteobacterium Rhodobacter capsulatus (RcGTA), which packages random portions of the host genome at a lower DNA density than usually observed in tailed bacterial viruses. How the DNA packaging properties of RcGTA evolved from those of the ancestral virus remains unknown. To address this question, we reconstructed the evolutionary history of the large subunit of the terminase (TerL), a highly conserved enzyme used by viruses and GTAs to package DNA. We found that RcGTA-like TerLs grouped within viruses that employ the headful packaging strategy. Because distinct mechanisms of viral DNA packaging correspond to differences in the TerL amino acid sequence, our finding suggests that RcGTA evolved from a headful packaging virus. Headful packaging is the least sequence-specific mode of DNA packaging, which would facilitate the switch from packaging of the viral genome to packaging random pieces of the host genome during GTA evolution.

The CckA-ChpT-CtrA Phosphorelay Controlling Rhodobacter capsulatus Gene Transfer Agent Production Is Bidirectional and Regulated by Cyclic di-GMP.

Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphotransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus, these proteins are key regulators of the gene transfer agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphotransfer to ChpT and thence to CtrA, to demonstrate biochemically that they form a phosphorelay. The secondary messenger cyclic di-GMP changed CckA from a kinase to a phosphatase, resulting in reversal of the phosphotransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for the lytic release of RcGTA. However, phosphatase activity was needed to produce functional RcGTA particles. The binding of cyclic di-GMP to the wild-type and mutant CckA proteins was evaluated directly using a pulldown assay based on biotinylated cyclic di-GMP and streptavidin-linked beads.IMPORTANCE The CckA, ChpT, and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus This work demonstrates biochemically that CckA, ChpT, and CtrA also form a functional phosphorelay in the latter group and that the direction of phosphotransfer is reversed by cyclic di-GMP. It is important to improve understanding of more representatives of this pathway in order to obtain deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

The Protease ClpXP and the PAS Domain Protein DivL Regulate CtrA and Gene Transfer Agent Production in Rhodobacter capsulatus.

Several members of the Rhodobacterales (Alphaproteobacteria) produce a conserved horizontal gene transfer vector, called the gene transfer agent (GTA), that appears to have evolved from a bacteriophage. The model system used to study GTA biology is the Rhodobacter capsulatus GTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in the Alphaproteobacteria and is an essential regulator of RcGTA production: it controls the production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that dysregulation of the CckA-ChpT-CtrA phosphorelay either by the loss of the PAS domain protein DivL or by substitution of the autophosphorylation residue of the hybrid histidine kinase CckA decreased CtrA phosphorylation and greatly increased RcGTA protein production in R. capsulatus We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels in R. capsulatus and identify ClpX(P) to be essential for the maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in the RhodobacteralesIMPORTANCE Members of the Rhodobacterales are abundant in ocean and freshwater environments. The conserved GTA produced by many Rhodobacterales may have an important role in horizontal gene transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay; however, several questions about GTA regulation remain. Our identification that a short DivL homologue and ClpXP regulate CtrA in R. capsulatus extends the model of CtrA regulation from Caulobacter crescentus to a member of the Rhodobacterales We found that the magnitude of RcGTA production greatly depends on DivL and CckA kinase activity, adding yet another layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles.

Functional and Evolutionary Characterization of a Gene Transfer Agent's Multilocus "Genome".

Gene transfer agents (GTAs) are phage-like particles that can package and transfer a random piece of the producing cell's genome, but are unable to transfer all the genes required for their own production. As such, GTAs represent an evolutionary conundrum: are they selfish genetic elements propagating through an unknown mechanism, defective viruses, or viral structures "repurposed" by cells for gene exchange, as their name implies? In Rhodobacter capsulatus, production of the R. capsulatus GTA (RcGTA) particles is associated with a cluster of genes resembling a small prophage. Utilizing transcriptomic, genetic and biochemical approaches, we report that the RcGTA "genome" consists of at least 24 genes distributed across five distinct loci. We demonstrate that, of these additional loci, two are involved in cell recognition and binding and one in the production and maturation of RcGTA particles. The five RcGTA "genome" loci are widespread within Rhodobacterales, but not all loci have the same evolutionary histories. Specifically, two of the loci have been subject to frequent, probably virus-mediated, gene transfer events. We argue that it is unlikely that RcGTA is a selfish genetic element. Instead, our findings are compatible with the scenario that RcGTA is a virus-derived element maintained by the producing organism due to a selective advantage of within-population gene exchange. The modularity of the RcGTA "genome" is presumably a result of selection on the host organism to retain GTA functionality.