Contact-dependent traits in Pseudomonas syringae B728a

Abstract

Production of the biosurfactant syringafactin by the plant pathogen Pseudomonas syringae B728a is a surface contact-dependent trait. Expression of syfA, as measured using a gfp reporter gene fusion was low in planktonic cells in liquid cultures but over 4-fold higher in cells immobilized on surfaces as varied as glass, plastic, paper, parafilm, agar, membrane filters, and leaves. Induction of syfA as measured by GFP fluorescence was rapid, occurring within two hours after immobilization of cells on surfaces. Comparison of the global transcriptome by RNA sequencing of planktonic cells in a nutrient medium with that of cells immobilized for 2 hours on filters placed on this solidified medium revealed that, in addition to syfA, 3156 other genes were differentially expressed. Genes repressed in immobilized cells included those involved in quaternary ammonium compound (QAC) metabolism and transport, compatible solute production, carbohydrate metabolism and transport, organic acid metabolism and transport, phytotoxin synthesis and transport, amino acid metabolism and transport, and secondary metabolism. Genes induced in immobilized cells included syfA plus those involved in translation, siderophore synthesis and transport, nucleotide metabolism and transport, flagellar synthesis and motility, lipopolysaccharide (LPS) 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. That many genes are rapidly differentially expressed upon transfer of cells from a planktonic to an immobilized state suggests that cells experience the two environments differently. It seems possible that surface contact initiates anticipatory changes in P. syringae gene expression, which enables rapid and appropriate physiological responses to the different environmental conditions such as might occur in a biofilm. Such responses could help cells survive transitions from aquatic habitats fostering planktonic traits to attachment on surfaces, conditions that alternatively occur on leaves.

Fig 1. Induction of syfA expression in cells of Pseudomonas syringae B728a as a function of time.

Gene expression is estimated as the mean GFP fluorescence of individual cells harboring a plasmid in which the syfA promoter was fused to a gfp reporter gene when harvested from these conditions at the various times shown on the abscissa. 7,038 cells were evaluated in total for control cells that remained in a broth culture (blue); 3,068 cells were evaluated after application to an agar surface (green); 10,184 cells were evaluated after application to filters placed on an agar surface (yellow); and 3,791 cells were evaluated after application to a filter moistened with KB broth that was then placed on plastic (orange). The vertical bars represent the standard error of the mean GFP fluorescence for a given cell.

Fig 2. The expression of syfA increases rapidly after immobilization of cells of Pseudomonas syringae B728a.

Gene expression is estimated as the mean GFP fluorescence of individual cells harboring a plasmid in which the syfA promoter was fused to a gfp reporter gene when harvested 6 hours after application to glass (green), polycarbonate plastic (yellow), paper (orange), parafilm (red), bean leaves (purple), agar (grey), Isopore filters (pink), Durapore filters (aqua), Teflon filters (brown), or recovered from broth cultures at the time of application to surfaces or after 6 hours (blue). The vertical bars represent the standard error of the mean GFP fluorescence for a given cell. Bars sharing a letter are not significantly different at p<0.05 by Tukey HSD test.

Fig 3. Heat map illustrating different patterns of gene expression in cells of Pseudomonas syringae B728a.

Heat map illustrating different patterns of gene expression in cells of Pseudomonas syringae B728a 2 hours after application to a filter surface compared to that in broth culture. Red indicates high expression and yellow indicates low expression.

Fig 4. Volcano plot illustrating the differential expression of genes in Pseudomonas syringae B728a.

Volcano plot illustrating the differential expression of genes in Pseudomonas syringae B728a 2 hours after application to a filter surface. Genes that are significantly differentially expressed compared to that in broth culture are shown in red, and those that do not differ are shown in black.

Fig 5. Proportion of up-regulated and down-regulated genes of Pseudomonas syringae B728a.

Proportion of up-regulated and down-regulated genes of Pseudomonas syringae B728a in those functional gene categories that exhibited differential expression in immobilized cells as compared to planktonic cells 2 hours after application to a filter surface. (A) Proportion of up-regulated genes in each functional gene category and (B) proportion of down-regulated genes in each functional gene category.