Summary: The transcriptional regulator MvfR is required for full Pseudomonas aeruginosa virulence, the function of multiple quorum sensing (QS)‐regulated virulence factors and the synthesis of 4‐hydroxy‐2‐alkylquinolines (HAQs), including the Pseudomonas quinolone signal (PQS). Here we investigate the role of MvfR in the QS circuitry and P. aeruginosa pathogenesis. We demonstrate using a combination of biochemical and molecular approaches, including transcription profiling, that MvfR is involved in the regulation of multiple P. aeruginosa QS‐controlled genes without altering the expression of lasRI/rhlRI or the production of N‐acyl‐ l‐homoserine lactone (AHL) signals. Dissection of how mvfR is interwoven into the P. aeruginosa QS circuitry reveals that the MvfR system, through the essential contribution of PqsE, positively regulates a subset of genes dependant on both LasR and RhlR. Animal studies show that MvfR contributes to P. aeruginosa virulence by controlling the transcription of genes not under RhlR regulation, and that reduced virulence of a mvfR mutant is caused by the loss of pqsE expression and not only a deficiency in HAQs/PQS production. This study provides novel insights into the unique role of the MvfR system in AHL‐mediated QS and further supports its importance in P. aeruginosa pathogenesis.

Summary: MvfR (PqsR), a Pseudomonas aeruginosa LysR‐type transcriptional regulator, plays a critical role in the virulence of this pathogen. MvfR modulates the expression of multiple quorum sensing (QS)‐regulated virulence factors; and the expression of the phnAB and pqsA‐E genes that encode functions mediating 4‐hydroxy‐2‐alkylquinolines (HAQs) signalling compounds biosynthesis, including 3,4‐dihydroxy‐2‐heptylquinoline (PQS) and its precursor 4‐hydroxy‐2‐heptylquinoline (HHQ). PQS enhances the in vitro DNA‐binding affinity of MvfR to the pqsA‐E promoter, to suggest it might function as the in vivo MvfR ligand. Here we identify a novel MvfR ligand, as we show that HHQ binds to the MvfR ligand‐binding‐domain and potentiates MvfR binding to the pqsA‐E promoter leading to transcriptional activation of pqsA‐E genes. We show that HHQ is highly produced in vivo, where it is not fully converted into PQS, and demonstrate that it is required for MvfR‐dependent gene expression and pathogenicity; PQS is fully dispensable, as pqsH^– mutant cells, which produce HHQ but completely lack PQS, display normal MvfR‐dependent gene expression and virulence. Conversely, PQS is required for full production of pyocyanin. These results uncover a novel biological role for HHQ; and provide novel insights on MvfR activation that may aid in the development of therapies that prevent or treat P. aeruginosa infections in humans.

With the advent of recombinant DNA techniques, the field of molecular plant pathology witnessed dramatic shifts in the 1970s and 1980s. The new and conventional methodologies of bacterial molecular genetics put bacteria center stage. The discovery in the mid-1980s of the hrp/hrc gene cluster and the subsequent demonstration that it encodes a type III secretion system (T3SS) common to Gram negative bacterial phytopathogens, animal pathogens, and plant symbionts was a landmark in molecular plant pathology. Today, T3SS has earned a central role in our understanding of many fundamental aspects of bacterium-plant interactions and has contributed the important concept of interkingdom transfer of effector proteins determining race-cultivar specificity in plant-bacterium pathosystems. Recent developments in genomics, proteomics, and structural biology enable detailed and comprehensive insights into the functional architecture, evolutionary origin, and distribution of T3SS among bacterial pathogens and support current research efforts to discover novel antivirulence drugs.

The hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and ability to elicit the hypersensitive reaction in resistant plants. Some hrp gene products constitute elements of the type III secretion system, by which effector proteins are exported and delivered into plant cells. Here, we show that the hrpZ gene product from the bean halo-blight pathogen, Pseudomonas syringae pv. phaseolicola (HrpZ_Psph), is secreted in an hrp-dependent manner in P. syringae pv. phaseolicola and exported by the type III secretion system in the mammalian pathogen Yersinia enterocolitica. HrpZ_Psph was found to associate stably with liposomes and synthetic bilayer membranes. Under symmetric ionic conditions, addition of 2 nM of purified recombinant HrpZ_Psph to the cis compartment of planar lipid bilayers provoked an ion current with a large unitary conductivity of 207 pS. HrpZ_Psph-related proteins from P. syringae pv. tomato or syringae triggered ion currents similar to those stimulated by HrpZ_Psph. The HrpZ_Psph-mediated ion-conducting pore was permeable for cations but did not mediate fluxes of Cl⁻. Such pore-forming activity may allow nutrient release and/or delivery of virulence factors during bacterial colonization of host plants.

Type III secretion systems (TTSSs) are essential mediators of the interaction of many Gram-negative bacteria with human, animal or plant hosts. Extensive sequence and functional similarities exist between components of TTSS from bacteria as diverse as animal and plant pathogens. Recent crystal structure determinations of TTSS proteins reveal extensive structural homologies and novel structural motifs and provide a basis on which protein interaction networks start to be drawn within the TTSSs, that are consistent with and help rationalize genetic and biochemical data. Such studies, along with electron microscopy, also established common architectural design and function among the TTSSs of plant and mammalian pathogens, as well as between the TTSS injectisome and the flagellum. Recent comparative genomic analysis, bioinformatic genome mining and genome-wide functional screening have revealed an unsuspected number of newly discovered effectors, especially in plant pathogens and uncovered a wider distribution of TTSS in pathogenic, symbiotic and commensal bacteria. Functional proteomics and analysis further reveals common themes in TTSS effector functions across phylogenetic host and pathogen boundaries. Based on advances in TTSS biology, new diagnostics, crop protection and drug development applications, as well as new cell biology research tools are beginning to emerge.

Plant pathogenic bacteria and rhizobia infect higher plants albeit the interactions with their hosts are principally distinct and lead to completely different phenotypic outcomes, either pathogenic or mutualistic, respectively. Bacterial protein delivery to plant host plays an essential role in determining the phenotypic outcome of plant-bacteria interactions. The involvement of type III secretion systems (T3SSs) in mediating animal- and plant-pathogen interactions was discovered in the mid-80’s and is now recognized as a multiprotein nanomachine dedicated to trans-kingdom movement of effector proteins. The discovery of T3SS in bacteria with symbiotic lifestyles broadened its role beyond virulence. In most T3SS-positive bacterial pathogens, virulence is largely dependent on functional T3SSs, while in rhizobia the system is dispensable for nodulation and can affect positively or negatively the mutualistic associations with their hosts. This review focuses on recent comparative genome analyses in plant pathogens and rhizobia that uncovered similarities and variations among T3SSs in their genetic organization, regulatory networks and type III secreted proteins and discusses the evolutionary adaptations of T3SSs and type III secreted proteins that might account for the distinguishable phenotypes and host range characteristics of plant pathogens and symbionts.