Many proteobacteria use acyl-homoserine lactones as quorum-sensing signals. Traditionally, biological detection systems have been used to identify bacteria that produce acyl-homoserine lactones, although the specificities of these detection systems can limit discovery. We used a sensitive approach that did not require a bioassay to detect production of long-acyl-chain homoserine lactone production by Rhodobacter capsulatus and Paracoccus denitrificans. These long-chain acyl-homoserine lactones are not readily detected by standard bioassays. The most abundant acyl-homoserine lactone was N-hexadecanoyl-homoserine lactone. The longchain acyl-homoserine lactones were concentrated in cells but were also found in the culture fluid. An R. capsulatus gene responsible for long-chain acyl-homoserine lactone synthesis was identified. A mutation in this gene, which we named gtaI, resulted in decreased production of the R. capsulatus gene transfer agent, and gene transfer agent production was restored by exogenous addition of N-hexadecanoyl-homoserine lactone. Thus, long-chain acyl-homoserine lactones serve as quorum-sensing signals to enhance genetic exchange in R. capsulatus.Many proteobacteria use acyl-homoserine lactone (acyl-HSL) signals in cell density-dependent gene regulation (9,11,45). Acyl-HSLs act as intercellular signals that allow bacterial species to monitor their population density and activate specific sets of genes at high cell densities. This type of cell density-dependent gene regulation, also called quorum sensing, was first described in the marine bacterium Vibrio fischeri (6, 21), which uses an acyl-HSL to activate luminescence gene expression. The V. fischeri quorum-sensing regulatory elements are LuxI and LuxR (8). The LuxI protein is the acyl-HSL synthase responsible for production of the N-3-oxohexanoyl-HSL. LuxR is a transcription factor that activates luminescence gene expression when bound by the acyl-HSL signal (8,9,11,12,23).Acyl-HSL signaling controls a number of bacterial processes, including virulence factor production, secondary metabolite production, and biofilm development in Pseudomonas aeruginosa (25,27,46) and conjugal transfer in Agrobacterium tumefaciens (10,29,49). Generally, LuxR and LuxI homologs serve as signal receptors and signal generators, respectively. Depending on the system, the signal varies in acyl group length and substitution (11), and these acyl side chain differences confer signal specificity (7, 35). Differences in acyl chain lengths are also a factor in signal permeability. Short-chain acyl-HSLs, like N-3-oxohexanoyl-HSL (3OC6-HSL) and butanoyl HSL, can diffuse freely through the cell membrane (14, 26). While still diffusible, long-chain acyl-HSLs like the P. aeruginosa signal N-3-oxododecanoyl-HSL (3OC12-HSL) appear to partition to the cell membrane. The MexAB-OprD efflux pump and perhaps other efflux pumps can aid in 3OC12-HSL export (26).Rhodobacter capsulatus and Paracoccus denitrificans are members of the ␣ group of the Proteobacteria. These free-living organi...

The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a bacteriophage-like genetic element with the sole known function of horizontal gene transfer. Homologues of RcGTA genes are present in many members of the alphaproteobacteria and may serve an important role in microbial evolution. Transcription of RcGTA genes is induced as cultures enter the stationary phase; however, little is known about cis-active sequences. In this work, we identify the promoter of the first gene in the RcGTA structural gene cluster. Additionally, gene transduction frequency depends on the growth medium, and the reason for this is not known. We report that millimolar concentrations of phosphate posttranslationally inhibit the lysis-dependent release of RcGTA from cells in both a complex medium and a defined medium. Furthermore, we found that cell lysis requires the genes rcc00555 and rcc00556, which were expressed and studied in Escherichia coli to determine their predicted functions as an endolysin and holin, respectively. Production of RcGTA is regulated by host systems, including a putative histidine kinase, CckA, and we found that CckA is required for maximal expression of rcc00555 and for maturation of RcGTA to yield gene transduction-functional particles.

The gene transfer agent (GTA) of the a-proteobacterium Rhodobacter capsulatus is a cell-controlled genetic exchange vector. Genes that encode the GTA structure are clustered in a 15-kb region of the R. capsulatus chromosome, and some of these genes show sequence similarity to known bacteriophage head and tail genes. However, the production of GTA is controlled at the level of transcription by a cellular two-component signal transduction system. This paper describes homologues of both the GTA structural gene cluster and the GTA regulatory genes in the a-proteobacteria Rhodopseudomonas palustris, Rhodobacter sphaeroides, Caulobacter crescentus, Agrobacterium tumefaciens and Brucella melitensis. These sequences were used in a phylogenetic tree approach to examine the evolutionary relationships of selected GTA proteins to these homologues and (pro)phage proteins, which was compared to a 16S rRNA tree. The data indicate that a GTA-like element was present in a single progenitor of the extant species that contain both GTA structural cluster and regulatory gene homologues. The evolutionary relationships of GTA structural proteins to (pro)phage proteins indicated by the phylogenetic tree patterns suggest a predominantly vertical descent of GTA-like sequences in the a-proteobacteria and little past gene exchange with (pro)phages.

We report the discovery of photoresponsive, flagellum-independent motility of the ␣-proteobacterium Rhodobacter capsulatus, a nonsulfur purple phototrophic bacterium. This motility takes place in the 1.5% agar-glass interface of petri plates but not in soft agar, and cells move toward a light source. The appearances of motility assay plates inoculated with wild-type or flagellum-deficient mutants indicate differential contributions from flagellar and flagellum-independent mechanisms. Electron microscopy confirmed the absence of flagella in flagellar mutants and revealed the presence of pilus-like structures at one pole of wild-type and mutant cells. We suggest that R. capsulatus utilizes a flagellum-independent, photoresponsive mechanism that resembles twitching motility to move in a line away from the point of inoculation toward a light source.

When Rhodobacter capsulatus cultures enter the stationary phase of growth, particles of the gene transfer agent (RcGTA) are released from cells. The morphology of RcGTA resembles that of a small, tailed bacteriophage, with a protein capsid surrounding a ~4 kb linear, double-stranded fragment of DNA. However, the DNA present consists of random segments of the R. capsulatus genome, which may be transferred to another strain of R. capsulatus. The recipient in RcGTA-mediated gene transduction may acquire new alleles and thus express a new phenotype. The genes encoding the structural proteins of the RcGTA are clustered on the R. capsulatus chromosome, whereas genes that encode proteins that regulate the production of RcGTA are scattered around the chromosome. These regulatory proteins include a homoserine lactone synthase (GtaI) that produces a quorum-sensing signal, a two-component sensor-kinase protein (CckA), and a two-component response regulator protein (CtrA). We review the proposed evolutionary origin of RcGTA, as well as environmental and cellular factors involved in the induction of this unusual process of genetic exchange.