Transcriptional responses of Pseudomonas syringae to factors inherent during plant host associations, with a focus on quorum sensing and its regulation.

Transcriptional profiles of P. syringae pv. syringae B728a (Pss) were obtained during both epiphytic and endophytic phases of colonization of its host plant Phaseolus vulgaris as well as the conditions of water stress, oxidative stress, iron limitation, and nitrogen limitation in culture, mimicking the challenges faced in an on plants. The transcript profiles of Pss B728a support a model in which leaf surface sites specifically favor flagellar motility, swarming motility requiring HAA surfactant production, and chemosensing and chemotaxis, indicating that cells actively relocate on leaves after immigration. Phenylalanine degradation is also induced in cells on leaves, which may help them counteract phenylpropanoid-based defenses prior to leaf entry. In contrast, intercellular sites favor the high-level expression of genes for gamma-amino butyric acid (GABA) metabolism, the degradation of which would attenuate GABA-dependent host defenses. In addition, the synthesis of phytotoxins, two novel secondary metabolites, and syringolin A is induced in Pss cells in intercellular spaces, apparently contributing to its virulence. Syringolin A may also suppress host plant defenses as well as stomatal closure. The prominent catabolism of various non-sugar compounds, and the elaborations of secondary metabolites by Pss underscores the complexity of the chemical interface between it and host plants. A comparison of the transcriptome of Pss recovered from plants to that of cells exposed to osmotic stress, oxidative stress, and iron and nitrogen limitation in culture revealed that gene expression of cells in and on leaves was most similar to that of cells experiencing water stress in culture. Surprisingly, water was apparently more limiting in the apoplast than on the leaf surface. Global gene expression of mutants of Pss in which the global regulators gacS, retS, salA, rpoN, rpoE, rpoS, hrpL, ahlR and aefR were disrupted was also assessed in these same seven conditions to determine the roles of these regulators in mediating transcriptional responses to such stimuli. Complex patterns of gene expression were observed that revealed that the regulons differed greatly in size, and had variable overlap between other regulons and with the various stimulons. Genes enabling responses to water stress such as those for compatible solute synthesis and other osmoprotection mechanisms, which are strongly expressed both on the surface of leaves and within leaves, are rpoE -dependent. This reveals that rpoE is a key regulator mediating water stress responses and fitness on plants./nBecause of our particular interest in quorum sensing, we focused our analyses upon the ahlR and the aefR regulons. AefR was found to be a repressor of a variety of efflux pumps. The most prominent feature of the aefR regulon is that it oppositely regulates two sets of genes; while the ahlR regulon is positively regulated by aefR , genes encoding orthologs of the MexEF-OprN Resistance Nodulation Division multidrug efflux pump are repressed by aefR . This juxtaposition is especially striking in light of finding that mexS is a positive regulator of ahlI and thus quorum sensing, as mexS mutants induce expression of mexEF-oprN via MexT in P. aeruginosa . MexT/MexS is responsive to disulphide stress, a condition distinct from that conferred by reactive oxygen intermediates. Another regulator (Psyr_1729), likely a repressor of the adjacent genes encoding efflux pumps, positively regulates the expression of ahlI . The probable role of such efflux pumps is to export toxic, electrophilic oxidized products from cells. Such a scenario leads to the model that expression of multidrug efflux pumps, under the control of aefR , modulates quorum sensing indirectly through a change in cellular redox status. aefR is unique from other Pss regulators studied, in that its regulon overlaps little with that of global regulators such as gacS, salA , or rpoN . A distinct stimulus for the aefR regulon remains elusive, while we expect such a stimulus would be present during host interactions since an aefR mutant was impaired in apoplastic growth in plants./nThe ahlR (Psyr_1622) regulon was small, with only eight genes that are positively regulated, and all were in two adjacent, divergent operons. One operon is initiated by the ahlI promoter, to form a transcript sense to ahlI that extends into genes encoding a truncated pyruvated dehydrogenase E1 subunit, pdhT (Psyr_1623) as well as one encoding a full length pyruvate dehydrogenase E1 subunit, pdhQ (Psyr_1625), thus producing a transcript of over 5 kb that is antisense to both ahlR and qrpR . Pyruvate accumulated in a pdhQ mutant and pyoverdine-dependent fluorescence was altered or quenched. The other ahlR -regulated operon originates from the same intergenic region as ahlI (Psyr_1621), and extends, on the opposite strand, through five genes (Psyr_1620-Psyr_1616). This second operon was termed the pao operon (paoABCDE ), as it was necessary for the accumulation of pyruvate that occurs when pdhQ is deleted. GC-MS and HPLC-MS profiling revealed that at least five unidentified compounds, as well as pyruvate accumulated in cells in the absence of pdhQ , but only when the pao operon was expressed. High accuracy mass spectrometry reveals that at least one of these compounds with the chemical formula C 10 H 8 O 2 is probably an oxidized phenolic. Elevated levels of oxidized glutathione, methionine sulfoxide, and methanethiol in an ahlR ahlI double mutant in stationary phase cultures are consistent with the AhlR regulon contributing to cellular redox equilibrium./n/nBoth ahlR and qrpR have their own active promoters and their expression is not responsive to AHL. AhlR can positively regulate its target promoter(s) even in the absence of its ligand 3oxoC6-AHL, but its activity is greatly enhanced upon ligand interaction. Given the position of AhlR in the phylogeny of LuxR homologs, at a clade branch equidistant between apo-repressors and ligand-dependent activators, it is noteworthy that it has ligand-independent, yet ligand-responsive activator activity. QrpR is a MarR-family repressor of the promoter of pdhQ . The ligand needed to de-repress QrpP is not known, but it is not 3oxoC6-AHL. In the absence of AhlR and AhlI, pdhQ expression is strongly de-repressed in the presence of a paoABCDE transcript. We posit that a compound originating from these enzymes is the ligand that elicits QrpR-dependent de-repression of pdhQ . Exogenous application of pyruvate did not de-repress pdhQ . Phylogenetic assessment indicates that QrpR orthologs form a clade distinct from other well-described MarR homologs./nThe second ortholog of the pyruvade dehydrogenase complex E1 subunit in Pss B728a, Psyr_0518, exhibits constitutive expression in a variety of genetic backgrounds and growth conditions. The pH of the medium is greatly decreased in a Psyr_0518 mutant but this phenotype is reversed by expression of pdhQ , in trans. Mutants blocked in expression of ahlR -dependent genes did not exhibit altered growth on the surface of host plants, or in the interior of host or non-host plants, while mutants in the housekeeping pyruvate dehydrogenase E1 ortholog (Psyr_0518) had greatly impaired apoplastic growth which could be restored by overexpression of the quorum-controlled ortholog pdhQ ./nThese data taken together are consistent with a model whereby a redox stress, a potential inducing stimulus of the ahlR regulon, can be eliminated by expression of efflux pumps under the control of MexEF-OprN at low cell densities, whereas at high cell densities efflux is inadequate for this process since cellular activity needed for the process is low and the products exported by the pumps will accumulate in the vicinity of the cells. Under such circumstances, the AhlR regulon mediates metabolism of these toxic compounds. The presumed toxic effects of certain breakdown products on the pyruvate dehydrogenase E1 subunit are circumvented by expression of a toxin-resistant quorum-controlled enzyme PdhQ. The regulation of this metabolism by AHL signaling, QrpR, and transcriptional read-through events is exquisitely complex, as may be required to ensure cell density- or environmental porosity-dependent expression of this metabolism in anticipation of cellular stress, while avoiding endogenously generated cytotoxicity when more effective strategies are appropriate./nWe also dissected the effects of iron on ahlI expression using both promoter-probe fusions and transcript abundance, to ascertain independently, the effects of iron on AHL production and AHL perception, and to resolve the effects of iron on these processes over time. Initial iron limitation does not alter ahlI expression, but another event linked with iron starvation that occurs after the central iron homeostasis limitation response is initiated, influences ahlI expression. Site-directed mutagenesis of loci encoding central iron homeostasis regulators Fur, PrrF1, and PrrF2, reveals that they are not required for the dependence of ahlI on iron availability.

Advisor: Lindow, Steven E