Stephen C. Winans

Structure of a bacterial quorum-sensing transcription factor complexed with pheromone and DNA

Many proteobacteria are able to monitor their population densities through the release of pheromones known as N-acylhomoserine lactones. At high population densities, these pheromones elicit diverse responses that include bioluminescence, biofilm formation, production of antimicrobials, DNA exchange, pathogenesis and symbiosis. Many of these regulatory systems require a pheromone-dependent transcription factor similar to the LuxR protein of Vibrio fischeri. Here we present the structure of a LuxR-type protein. TraR of Agrobacterium tumefaciens was solved at 1.66 Å as a complex with the pheromone N-3-oxooctanoyl-l-homoserine lactone (OOHL) and its TraR DNA-binding site. The amino-terminal domain of TraR is an α/β/α sandwich that binds OOHL, whereas the carboxy-terminal domain contains a helix–turn–helix DNA-binding motif. The TraR dimer displays a two-fold symmetry axis in each domain; however, these two axes of symmetry are at an approximately 90° angle, resulting in a pronounced overall asymmetry of the complex. The pheromone lies fully embedded within the protein with virtually no solvent contact, and makes numerous hydrophobic contacts with the protein as well as four hydrogen bonds: three direct and one water-mediated.

The Bases of Crown Gall Tumorigenesis

The nine decades since Smith and Townsend demonstrated that Agrobacterium tumefaciens causes plant tumors (95) have been marked by a series of surprises. Among the most important of these was the report in 1958 that these tumors could be excised and propagated in vitro without exogenous plant hormones (7). Equally important were a series of reports beginning about the same time that tumors released compounds that agrobacteria could use as nutrients (24). Perhaps the most exciting discoveries, reported in the 1970s and 1980s, were that tumorigenesis required the transfer of fragments of oncogenic DNA to infected plant cells (10), that this process evolved from a conjugal transfer system (99), and that the genes that direct this process are expressed in response to host-released chemical signals (47). This DNA transfer process has become a cornerstone of plant molecular genetics. The genus Agrobacterium also has provided excellent models for several aspects of host-pathogen interactions, including intercellular transport of macromolecules (11), bacterial detection of host organisms (47), targeting of proteins to plant cell nuclei (3), and interbacterial chemical signaling via autoinducer-type pheromones (120).

Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates.

Many bacteria, including several pathogens of plants and humans, use a pheromone called an autoinducer to regulate gene expression in a cell density-dependent manner. Agrobacterium autoinducer [AAI, N-(3-oxo-octanoyl)-L-homoserine lactone] of A. tumefaciens is synthesized by the TraI protein, which is encoded by the tumor-inducing plasmid. Purified hexahistidinyl-TraI (H6-TraI) used S-adenosylmethionine to make the homoserine lactone moiety of AAI, but did not use related compounds. H6-TraI used 3-oxo-octanoyl-acyl carrier protein to make the 3-oxo-octanoyl moiety of AAI, but did not use 3-oxo-octanoyl-coenzyme A. These results demonstrate the enzymatic synthesis of an autoinducer through the use of purified substrates.

Detection of and Response to Signals Involved in Host-Microbe Interactions by Plant-Associated Bacteria

SUMMARY Diverse interactions between hosts and microbes are initiated by the detection of host-released chemical signals. Detection of these signals leads to altered patterns of gene expression that culminate in specific and adaptive changes in bacterial physiology that are required for these associations. This concept was first demonstrated for the members of the family Rhizobiaceae and was later found to apply to many other plant-associated bacteria as well as to microbes that colonize human and animal hosts. The family Rhizobiaceae includes various genera of rhizobia as well as species of Agrobacterium. Rhizobia are symbionts of legumes, which fix nitrogen within root nodules, while Agrobacterium tumefaciens is a pathogen that causes crown gall tumors on a wide variety of plants. The plant-released signals that are recognized by these bacteria are low-molecular-weight, diffusible molecules and are detected by the bacteria through specific receptor proteins. Similar phenomena are observed with other plant pathogens, including Pseudomonas syringae, Ralstonia solanacearum, and Erwinia spp., although here the signals and signal receptors are not as well defined. In some cases, nutritional conditions such as iron limitation or the lack of nitrogen sources seem to provide a significant cue. While much has been learned about the process of host detection over the past 20 years, our knowledge is far from being complete. The complex nature of the plant-microbe interactions makes it extremely challenging to gain a comprehensive picture of host detection in natural environments, and thus many signals and signal recognition systems remain to be described.

Adaptation of a conjugal transfer system for the export of pathogenic macromolecules

Conjugal transfer of bacterial plasmids requires a pore through which DNA can traverse the envelopes of the donor and recipient cells. Recent studies indicate that these pores, which are composed of approximately ten proteins, are evolutionarily related to the transport systems required for the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to plant cells and for toxin secretion from Bordetella pertussis.

Agrobacterium Bioassay Strain for Ultrasensitive Detection of N-Acylhomoserine Lactone-Type Quorum-Sensing Molecules: Detection of Autoinducers in Mesorhizobium huakuii

ABSTRACT An ultrasensitive bioassay system for the detection of N-acylhomoserine lactones (AHLs) was constructed in Agrobacterium tumefaciens by using the T7 expression system to overproduce the AHL receptor TraR. This strain detected many diverse AHLs, some at extremely low concentrations. We used this strain to detect for the first time AHLs made by Mesorhizobium huakuii, which symbiotically fixes nitrogen in association with the legume Astragalus sinicus, a source of green manure throughout eastern Asia.

Cell–cell communication in the plant pathogen Agrobacterium tumefaciens

The plant pathogen Agrobacterium tumefaciens induces the formation of crown gall tumours at wound sites on host plants by directly transforming plant cells. This disease strategy benefits the bacteria as the infected plant tissue produces novel nutrients, called opines, that the colonizing bacteria can use as nutrients. Almost all of the genes that are required for virulence, and all of the opine uptake and utilization genes, are carried on large tumour-inducing (Ti) plasmids. The observation more than 25 years ago that specific opines are required for Ti plasmid conjugal transfer led to the discovery of a cell–cell signalling system on these plasmids that is similar to the LuxR–LuxI system first described in Vibrio fischeri. All Ti plasmids that have been described to date carry a functional LuxI-type N-acylhomoserine lactone synthase (TraI), and a LuxR-type signal receptor and transcriptional regulator called TraR. The traR genes are expressed only in the presence of specific opines called conjugal opines. The TraR–TraI system provides an important model for LuxR–LuxI-type systems, especially those found in the agriculturally important Rhizobiaceae family. In this review, we discuss current advances in the biochemistry and structural biology of the TraR–TraI system.

A LuxR-type regulator from Agrobacterium tumefaciens elevates Ti plasmid copy number by activating transcription of plasmid replication genes

TraR, a LuxR‐type quorum‐sensing transcription factor in Agrobacterium tumefaciens, activates genes required for conjugal transfer of the Ti plasmid and also enhances the copy number of a nopaline‐type Ti plasmid. Here, we show that TraR increases the copy number of an octopine‐type Ti plasmid up to eightfold and that TraR activates transcription of the repABC operon up to 25‐fold. The ability of TraR to increase copy number was strictly dependent on several TraR‐activated promoters of this operon, indicating that TraR affects copy number solely at the level of transcription. Promoter resections and mRNA transcript analysis revealed the presence of three TraR‐dependent promoters. Two TraR‐dependent transcription start sites are located 45.5 and 65.5 nucleotides downstream of a site called tra box II, whereas the third start site lies 42.5 nucleotides downstream of a site called tra box III. Purified TraR bound to both tra boxes with comparable affinities, causing moderate DNA bending. TraR bound and bent these two sites independently rather than synergistically. Alteration of tra box III to match the consensus sequence dramatically increased TraR‐dependent expression of repABC and plasmid copy number. TraR‐dependent elevation of Ti plasmid copy number caused a three‐ to fourfold increase in plant tumorigenesis.

The A. tumefaciens transcriptional activator OccR causes a bend at a target promoter, which is partially relaxed by a plant tumor metabolite

Octopine is released from crown gall tumors as a nutrient source and a signal molecule for the plant pathogen Agrobacterium tumefaciens. Some or all octopine-inducible genes are regulated by a protein called OccR. Primer extension analysis showed that OccR protein represses the occR gene and both represses and activates the occQ operon, which is divergently transcribed from occR. These promoters initiate transcription 46 bp apart. This regulatory system was reconstituted in vitro using purified OccR protein and Escherichia coli RNA polymerase. OccR binds with high affinity to a single site overlapping these promoters. Octopine shortens the DNAase I footprint of OccR and increases the gel mobility of OccR-DNA complexes by relaxing an OccR-incited DNA bend.

Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens

The DNA sequence of a cluster of pKM101 conjugal transfer genes was determined and aligned with the genetic map of the plasmid. Eighteen genes were identified, at least eight and probably 11 of which are required for efficient conjugation. These tra genes are homologous to and colinear with genes found in the virB operon of Agrobacterium tumefaciens TI plasmids. Seven pKM101 tra genes are also homologous to ptl genes of Bordetella pertussis, which direct the export of pertussis toxin. We used TnphoA to construct translational fusions between pKM101 genes and the Escherichia coli phoA gene, which encodes alkaline phosphatase, and provide evidence that at least 11 of the 18 genes are either fully or partially exported from the cytoplasm.