Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles. Here, we present the peptidisc for their facile capture into water-soluble particles. Unlike the nanodisc, which requires scaffold proteins of different lengths and precise amounts of matching lipids, reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide (NSP_r) and no extra lipids. Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein. We demonstrate the effectiveness of this ‘one size fits all’ method using five different membrane protein assemblies (MalFGK₂, FhuA, SecYEG, OmpF, BRC) during ‘on-column’, ‘in-gel’, and ‘on-bead’ reconstitution embedded within the membrane protein purification protocol. The peptidisc method is rapid and cost-effective, and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies.

Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.

Summary: The gene transfer agent produced by Rhodobacter capsulatus (RcGTA) resembles a small tailed bacteriophage that packages almost random genomic DNA segments that may be transferred to other R. capsulatus 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 polysaccharide is a receptor for the gene transfer agent of R. capsulatus, RcGTA.

The gene transfer agent (RcGTA) of Rhodobacter capsulatus is the model for a family of novel bacteriophage-related genetic elements that carry out lateral transfer of essentially random host DNA. Genuine and putative gene transfer agents have been discovered in diverse genera and are becoming recognized as potentially an important source of genetic exchange and microbial evolution in the oceans. Despite being discovered over 30 years ago, little is known about many essential aspects of RcGTA biology. Here, we validate the use of direct fluorescence reporter constructs, which express the red fluorescent protein mCherry in R. capsulatus. A construct containing the RcGTA promoter fused to mCherry was used to examine the single-cell expression profiles of wild type and RcGTA overproducer R. capsulatus populations, under different growth conditions and growth phases. The majority of RcGTA production clearly arises from a small, distinct sub-set of the population in the wild type strain and a larger sub-set in the overproducer. The most likely RcGTA release mechanism concomitant with this expression pattern is host cell lysis and we present direct evidence for the release of an intracellular enzyme accompanying RcGTA release. RcGTA ORF s is annotated as a ‘cell wall peptidase’ but we rule out a role in host lysis and propose an alternative function as a key contributor to RcGTA invasion of a target cell during infection.

Summary: The gtaI gene of Rhodobacter capsulatus encodes an N‐acyl‐homoserine lactone (acyl‐HSL) synthase. Immediately 5′ 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 RcGTA promoter region, and therefore modulation of RcGTA production appears to require at least one additional factor. We found that RcGTA production was stimulated by spent media from other species, and identified exogenous acyl‐HSLs that induce RcGTA.

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. IMPORTANCE Horizontal 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.