Rhizobia are agriculturally important bacteria that can form nitrogen-fixing nodules on the roots of leguminous plants. Agricultural application of rhizobial inoculants can play an important role in increasing leguminous crop yields. In temperate rhizobia, genes involved in nodulation and nitrogen fixation are usually located on one or more large plasmids (pSyms) or on symbiotic islands. In addition, other large plasmids of rhizobia carry genes that are beneficial for survival and competition of rhizobia in the rhizosphere. Conjugative transfer of these large plasmids thus plays an important role in the evolution of rhizobia. Therefore, understanding the mechanism of conjugative transfer of large rhizobial plasmids provides foundations for maintaining, monitoring, and predicting the behaviour of these plasmids during field release events. In this minireview, we summarize two types of known rhizobial conjugative plasmids, including quorum sensing regulated plasmids and RctA-repressed plasmids. We provide evidence for the existence of a third type of conjugative plasmid, including pRleVF39c in Rhizobium leguminosarum bv. viciae strain VF39SM, and we provide a comparison of the different types of conjugation genes found in members of the rhizobia that have had their genomes sequenced so far.

Rhizobium leguminosarum strain VF39SM contains two plasmids that have previously been shown to be self-transmissible by conjugation. One of these plasmids, pRleVF39b, is shown in this study to carry a set of plasmid transfer genes that differs significantly from conjugation systems previously studied in the rhizobia but is similar to an uncharacterized set of genes found in R. leguminosarum bv. trifolii strain WSM2304. The entire sequence of the transfer region on pRleVF39b was determined as part of a genome sequencing project, and the roles of the various genes were examined by mutagenesis. The transfer region contains a complete set of mating pair formation (Mpf) genes, a traG gene, and a relaxase gene, traA, all of which appear to be necessary for plasmid transfer. Experimental evidence suggested the presence of two putative origins of transfer within the gene cluster. A regulatory gene, trbR, was identified in the region between traA and traG and was mutated. TrbR was shown to function as a repressor of both trb gene expression and plasmid transfer. Rhizobia are agriculturally important bacteria that are capable of forming nitrogen-fixing nodules on the roots of legumes, such as peas, lentils, and beans. In fast-growing rhizobia, such as the genera Rhizobium and Ensifer (Sinorhizobium), the genes required for establishment of this symbiosis (nod, nif, and fix genes) are usually located on one of the large plasmids, known as pSyms. In addition, other large rhizobial plasmids carry genes beneficial for bacterial fitness and competitiveness in the rhizosphere, such as genes encoding bacteriocin production, lipopolysaccharide production, exopolysaccharide production, utilization of different carbon sources, and synthesis of specific vitamins (1-9).Although plasmid conjugation has not been shown to enhance bacterial competitiveness directly, it is one of the most important methods for bacteria to acquire genetic information and adapt to changing environmental conditions. There is considerable evidence for rhizobial plasmid transfer both under laboratory conditions (10-13), and in natural environments (14-16). In addition, genome sequences of many rhizobial strains and rhizobial plasmids have revealed potential conjugation genes encoding DNA transfer and replication (Dtr) components and mating pair formation (Mpf) components, as well as putative origins of transfer (oriT).Two types of rhizobial conjugation systems have been characterized, including the quorum sensing (QS)-regulated conjugation system (type I) and the RctA-repressed conjugation system (type II). In a recent review, we demonstrated that these two conjugation systems are phylogenetically separate, consistent with their characterized transfer regulation (8). In addition, we proposed the presence of the type III conjugation system on plasmids pRL10JI, pRL11JI, and pRL12JI of Rhizobium leguminosarum bv. viciae 3841 (17), and pRleVF39d, pRleVF39e, pRleVF39f of R. leguminosarum bv. viciae VF39SM (18) based on sequence information (8). None of the type III...

Plasmid curing has shown that the ability to use glycerol as a carbon source is plasmid-encoded in Rhizobium leguminosarum. We isolated the locus responsible for glycerol utilization from plasmid pRleVF39c in R. leguminosarum bv. viciae VF39. This region was analyzed by DNA sequencing and mutagenesis. The locus encompasses a gene encoding GlpR (a DeoR regulator), genes encoding an ABC transporter, and genes glpK and glpD, encoding a kinase and dehydrogenase, respectively. All the genes except the regulatory gene glpR were organized into a single operon, and were required for growth on glycerol. The glp operon was strongly induced by both glycerol and glycerol 3-phosphate, as well as by pea seed exudate. GlpR repressed the operon in the absence of inducer. Mutation of genes encoding the ABC transporter abolished all transport of glycerol in transport assays using radiolabelled glycerol. This confirms that, unlike in other organisms such as Escherichia coli and Pseudomonas aeruginosa, which use facilitated diffusion, glycerol uptake occurs by an active process in R. leguminosarum. Since the glp locus is highly conserved in all sequenced R. leguminosarum and Rhizobium etli strains, as well as in Sinorhizobium spp. and Agrobacterium spp. and other alphaproteobacteria, this process for glycerol uptake is probably widespread. Mutants unable to use glycerol were deficient in competitiveness for nodulation of peas compared with the wild-type, suggesting that glycerol catabolism confers an advantage upon the bacterium in the rhizosphere or in the infection thread.

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.