TraG-like proteins are potential NTP hydrolases (NTPases) that are essential for DNA transfer in bacterial conjugation. They are thought to mediate interactions between the DNA-processing (Dtr) and the mating pair formation (Mpf) systems. TraG-like proteins also function as essential components of type IV secretion systems of several bacterial pathogens such as Helicobacter pylori. Here we present the biochemical characterization of three members of the family of TraG-like proteins, TraG (RP4), TraD (F), and HP0524 (H. pylori). These proteins were found to have a pronounced tendency to form oligomers and were shown to bind DNA without sequence specificity. Standard NTPase assays indicated that these TraG-like proteins do not possess postulated NTP-hydrolyzing activity. Surface plasmon resonance was used to demonstrate an interaction between TraG and relaxase TraI of RP4. Topology analysis of TraG revealed that TraG is a transmembrane protein with cytosolic N and C termini and a short periplasmic domain close to the N terminus. We predict that multimeric inner membrane protein TraG forms a pore. A model suggesting that the relaxosome binds to the TraG pore via TraG-DNA and TraG-TraI interactions is presented.Bacterial conjugation is responsible for the spread of genetic traits among a broad range of bacterial species. It is the primary mechanism for dissemination of antibiotic resistance among human pathogens (60). Nearly all functions required to mediate bacterial conjugation are encoded by conjugative plasmids, which are usually further endowed with antibiotic resistance genes (64). In general, transfer (Tra) proteins are grouped into functional classes, defined as those involved in mating pair formation (Mpf) and DNA processing (Dtr). Secretion systems used by some pathogens, such as Agrobacterium tumefaciens, Bordetella pertussis, Helicobacter pylori, and Legionella pneumophila, for delivering effector molecules to eukaryotic cells are composed of protein components evolutionarily related to those of Mpf complexes (12). Such macromolecular transfer systems ancestrally related to the conjugative Mpf complexes are called type IV secretion systems, as originally proposed by Salmond (46). Each of these systems secretes distinct DNA and/or protein substrates. TraG-like proteins (named for the protein of IncP plasmid RP4 [31]) are present in all conjugative DNA transfer systems and in several type IV secretion systems. Although TraG-like proteins are essential components in these secretion systems (12), their function remains poorly understood.The Dtr systems of conjugative plasmids are best characterized at the initiation stage of DNA processing. The relaxosome (20) is a complex of several Dtr proteins (relaxosomal proteins) bound to a specific DNA sequence, the origin of transfer (oriT) of the conjugative plasmid. This complex initiates DNA transfer by producing a single-stranded scission at the nic cleavage site within oriT. In this reaction, the catalytic key component, called the relaxase, becomes transiently ...

The perception and response to cellular death is an important aspect of multicellular eukaryotic life. For example, damage-associated molecular patterns activate an inflammatory cascade that leads to removal of cellular debris and promotion of healing. We demonstrate that lysis of Pseudomonas aeruginosa cells triggers a program in the remaining population that confers fitness in interspecies co-culture. We find that this program, termed P. aeruginosa response to antagonism (PARA), involves rapid deployment of antibacterial factors and is mediated by the Gac/Rsm global regulatory pathway. Type VI secretion, and, unexpectedly, conjugative type IV secretion within competing bacteria, induce P. aeruginosa lysis and activate PARA, thus providing a mechanism for the enhanced capacity of P. aeruginosa to target bacteria that elaborate these factors. Our finding that bacteria sense damaged kin and respond via a widely distributed pathway to mount a complex response raises the possibility that danger sensing is an evolutionarily conserved process.DOI: http://dx.doi.org/10.7554/eLife.05701.001

Quorum-sensing (QS) systems allow organisms, such as the pathogen Pseudomonas aeruginosa, to link gene expression with their population density and the diffusion and flow characteristics of their environment. The leading hypotheses about QS systems' biological functions necessitate that QS-controlled gene expression be suppressed until a threshold culture density (the quorum) is reached. Despite a detailed understanding of QS in P. aeruginosa, known regulatory elements do not fully explain how the quorum threshold for gene activation is produced. Here we investigated the mechanism with a screening approach that used random gene activation. These experiments uncovered a regulator without close homologs in other species that produces the quorum expression threshold. Expression of this regulator (named QteE) reduces LasR protein stability without affecting LasR transcription or translation. QteE also independently reduces RhlR levels. Because QteE can block QS when signal levels are high, it could provide a mechanism for individual cells to exert autonomous control over their QS regulons. This unique regulator governs two central QS control points in P. aeruginosa and shapes the expression pattern thought fundamental to the biological functions of QS.cell to cell signaling | quorum sensing | LasR | gene regulation

We examined the dependence of protein export and membrane protein insertion on SecE and SecA, two components of the secretion (Sec) apparatus of Escherichia coli. The magnitude of the secretion defect observed for signal sequence-containing proteins in cells depleted of SecE is larger and more general than that in many temperature-or cold-sensitive Sec mutants. In addition, we show that the proper insertion of the polytopic MalF protein (synthesized without a signal sequence) into the cytoplasmic membrane is also SecE-dependent. In contrast to an earlier study (McGovern, K., and Beckwith, J. (1991) J. Biol. Chem. 266, 20870 -20876), the membrane insertion of MalF also is inhibited by treatment of cells with sodium azide, a potent inhibitor of SecA. Therefore, our data strongly suggest that the cytoplasmic membrane insertion of MalF is dependent on the same cellular machinery as is involved in the export of signal sequence-containing proteins. We propose that the mechanism of export from the cytoplasm is related for both signal sequence-containing and cytoplasmic membrane proteins, but hydrophobic membrane proteins such as MalF may have a higher affinity for the Sec apparatus.