UC Berkeley

Leaf surfaces are a harsh habitat for microbes since they are subjected to high fluxes of ultraviolet radiation, fluctuations in temperature, low nutrient availability, and frequent drying. Despite this, leaves can be heavily colonized by bacteria. I explored the ability of bacteria to modify their local environment to better tolerate or avoid the water stress they would experience on leaves by addressing the role of the biosurfactant syringafactin produced by the model epiphytic bacterium Pseudomonas syringae B728a. Syringafactin is very hygroscopic, binding nearly three times its weight in water, but only in atmospheres of high relative humidity. The water stress status of bacteria differing in syringafactin production was assessed in different settings by measuring the fluorescence of cells harboring a plasmid containing the promoter of proU, encoding the synthesis of the compatible solute proline, fused to a promoterless gfp reporter gene. The wild-type syringafactin-producing strain experienced less water stress than the mutant strain on membrane filters exposed to high relative humidity, but not on filters exposed to low relative humidity. Syringafactin production was also associated with lower water stress in bacteria on leaves, irrespective of the dryness of the air in which the plants were incubated. The high relative humidity-dependent water-binding capabilities of syringafactin are consistent with a model in which the laminar boundary layer surrounding leaves is nearly saturated with water vapor, irrespective of the dryness of air away from the leaf. Thus, by producing syringafactin, P. syringae hydrates its local microenvironment by sequestering liquid water, on an otherwise dry leaf, from the humid air trapped at the leaf surface.

The production of syringafactin requires syfA, encoding a synthase necessary for syringafactin production. Therefore, as a readout for syringafactin production, I used a P. syringae strain harboring a plasmid containing the syfA promoter fused to a promoterless gfp reporter gene. GFP fluorescence exhibited by P. syringae, was much higher in cells cultured on nutrient agar surfaces compared to in cells in the corresponding broth culture. These observations suggested that expression of this trait was dependent on contact of cells with a solid surface. Immobilization of this strain on various surfaces, including various membranes, plastic surfaces, and on leaves, resulted in rapid increases in apparent expression of syfA within two hours compared to that of cells that were maintained in a planktonic state in broth cultures. These findings indicated that immobilization of cells was sufficient to induce the production of this biosurfactant. These findings also suggested that P. syringae might either experience different environmental conditions upon immobilization or might use contact with the surface as a cue to express certain traits in anticipation of their necessity for optimum fitness upon change from a liquid habitat, in which cells might exist in a planktonic state, to a dry habitat, such as the leaf surface. To explore what additional traits besides syringafactin production might be expressed in a contact-dependent fashion in P. syringae, I compared the global transcriptome of planktonic cells grown in a broth medium with those immobilized for two hours on a filter surface placed on a similar medium. A large fraction of the genes exhibited either higher (26.63%) or lower expression (33.83%) upon immobilization on the filter surface. Genes in the functional categories of translation, siderophore synthesis and transport, nucleotide metabolism and transport, flagellar synthesis and motility, lipopolysaccharide synthesis and transport, energy generation, transcription, chemosensing and chemotaxis, replication and DNA repair, iron-sulfur proteins, peptidoglycan/cell wall polymers, terpenoid backbone synthesis, iron metabolism and transport, and cell division were significantly more likely to be up-regulated upon immobilization while those in the categories of quaternary ammonium compound metabolism and transport, compatible solute synthesis, carbohydrate metabolism and transport, organic acid metabolism and transport, phytotoxin synthesis and transport, amino acid metabolism and transport, and secondary metabolism were repressed on surfaces compared to in liquid.

While surface contact is increasingly being recognized as an important cue for the differential gene expression in various microorganisms, little is known of the mechanisms by which cells perceive that they are immobilized on such a surface. The mechanisms by which P. syringae B728a mediates surface contact-dependent production of syringafactin, and other-contact dependent processes are largely unknown. I identified genes that alter expression of syfA on agar surfaces using random transposon mutagenesis of P. syringae, harboring a syfA:gfp fusion. Putative regulatory genes contributing to activation of syfA expression identified by this method included efeO, tsr, and mexB while those contributing to its repression included psrA, cyoC, and algW. Some but not all of these putative regulatory genes also exhibited differential contact- dependent expression in immobilized cells compared to in planktonic cells. The KEGG database was used to analyze the metabolic pathways in which these genes might participate. Furthermore, I formulated possible pathways that could account for the surface-dependent regulation of syfA and potentially the regulation of other surface-regulated genes. One of these pathways is a stress sensing pathway that AlgW is part of which may play a role in activating genes involved in adhesion. Another pathway includes Tsr which may regulate genes involved in swarming, a type of bacterial motility that can only occur on a surface. These potential pathways reveal that a variety of mechanisms may be involved in regulating surface-induced genes in bacteria.