Contact-dependent traits in Pseudomonas syringae B728a

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 new environmental conditions. Such responses could help cells survive transitions from aquatic habitats fostering planktonic traits to attachment on surfaces, conditions that alternatively occur on leaves.

159 distribution were then used to determine significance [20]. A gene was considered significantly 160 differentially regulated if the p-value for the difference in relative expression between the filter 161 treatment and the liquid treatment was less than 0.001.

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163 Statistical analysis 164 The  181 Planktonic cells that remained in broth cultures served as a control. We compared gene 182 expression of cells on filters placed on KB agar and KB broth to test whether chemical 183 components of the agar, rather than simply the physical change it elicited, contributed to 184 induction of syfA expression as seen by Burch et al. [7]. By two hours after transfer to each of 185 the solid surfaces, cells exhibited significantly greater GFP fluorescence than those remaining in 186 the broth cultures (Fig 1). Similar levels of syfA induction occurred on all surfaces (Fig 1). Thus, 187 syfA induction clearly occurs rapidly upon encountering a surface and elevated expression on 188 agar appears to be due to the exposure of cells to a surface rather than any chemical components 189 of the agar. 192 Gene expression is estimated as the mean GFP fluorescence of individual cells harboring a 193 plasmid in which the syfA promoter was fused to a gfp reporter gene when harvested from these 194 surfaces at the various times shown on the abscissa. 7,038 cells were evaluated in total for 195 control cells that remained in a broth culture (blue); 3,068 cells were evaluated after application 196 to agar (green); 10,184 cells were evaluated after application to filters on an agar surface 197 (yellow); and 3,791 cells were evaluated after application to a filter placed on plastic (orange).
198 The vertical bars represent the standard error of the mean GFP fluorescence for a given cell.  S1). As expected, a wide range of 232 reads were recovered for a given gene (Fig S2). A heatmap revealed that while patterns of gene 233 regulation were similar among replicates in which cells were either planktonic or immobilized on 234 a filter, gene expression differed strongly between cells in these two conditions (Fig 3). Much of 235 the variation in relative gene expression levels of the average gene was thus associated with the 236 environment of the cells before RNA was harvested rather than other factors. 246 that in broth (Fig 4). Of the 1,390 genes that were induced on filter surfaces, 881 were induced 247 more than 2-fold while 509 exhibited lesser induction. Of the 1,766 genes that were repressed on 248 filter surfaces, 1,138 were repressed more than 2-fold while 628 were repressed less than 2-fold.
249 In total, 3,156 genes (60.46% of the P. syringae B728a genome) were found to be differentially 250 expressed in cells on the filter surface compared to those in broth culture (Fig 4). There was no 251 apparent relationship between the absolute level of expression of a gene and the likelihood that it 252 would exhibit differential expression in these two settings ( Fig S3).  Translation. Many genes encoding the 30S and 50S ribosomal protein subunits were induced 287 on the filter surface, as were genes encoding the elongation factor proteins Ts, P, Tu, and G.
288 Many genes encoding t-RNA synthetases were also induced. It thus appeared that translation as a 289 whole may have accelerated upon transition of cells from a planktonic to an immobilized state.

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Siderophore synthesis and iron metabolism. Many genes involved in siderophore 291 synthesis and transport were induced on the filter surface. Many genes such as pvdS, pvdG, pvdL, 292 pvdI, pvdJ, pvdK, pvdD, pvdE, pbdO, pvdN, pvdT, and pvdR were involved in regulation of 293 pyoverdine production and its transport. Many genes involved in achromobactin regulation, 294 synthesis, and transport including acsG, acsD, acsE, yhcA, acsC, acsB, acsA, carA-2, cbrB-2, 295 and cbrC-2 were also induced. Many genes involved in iron metabolism and transport were also 296 induced on the filter surface. This included the genes fecE, fecD, fecC, fecB, fecA, fecR, and the 297 RNA polymerase ECF sigma factor fecI. These results suggest that iron became less available on 298 the filter surfaces than in broth, perhaps due to diffusional limitations associated with the lack of 299 mixing of cells as would occur in a liquid medium. Alternatively, iron might commonly be less 300 available on the natural surfaces on which P. syringae typically inhabits, such as leaf surfaces, 301 and the filter mimicked physical cues that the bacteria might use to anticipate transition into such 302 low iron environments.

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Nucleotide metabolism and transport. Numerous genes involved in nucleotide 304 metabolism and transport were induced in immobilized cells. This included many genes involved 305 in purine and pyrimidine metabolism such as purA, purT, purC, purF, purB, purM, purN, purU-3, 306 purH, purD, purK, purE, and pyrB, pyrR, pyrH, pyrG, pyrF, pyrD, pyrC-2 respectively. As with 308 surfaces, increased transcription might be expected to also be linked to such increases, requiring 309 higher rates of nucleotide synthesis. Almost all genes with significant differential expression 310 involved in cell division were also induced on the filter surface. This included the cell division 311 proteins FtsK, FtsQ, and FtsL as well as the rod-shape determining proteins MreD and MreC.
312 MrdB, a cell cycle protein, was also induced. 509 Surprisingly, genes involved in phytotoxin synthesis and transport as well as many other genes 510 enabling production of other secondary metabolites were repressed on the filter surface ( . This study revealed that changes in gene expression upon 529 immobilization of cells were both very rapid and often transient as many genes that had become 530 induced by one hour of surface attachment exhibited subsequent decreases in expression by four 531 hours of attachment. By eight hours of attachment many of these genes exhibited continued 532 decreases in expression while others only then became induced. Interestingly, we found similar 533 functional genes in P. syringae to be up-regulated soon after cells attached to a surface, including 534 emrB encoding a drug resistance transporter as well as other genes in this operon, and glnH, 535 involved in the transport of glutamine (Fig S4). The gene marA, involved in resistance to a 536 variety of antimicrobial compounds and marR encoding its regulator as well as ahpF, grxA, and 537 katG and their regulator oxyR, collectively mediating oxidative stress tolerance were also up-538 regulated in both systems (Fig S4). Genes involved in attachment and DNA repair were up-539 regulated in response to attachment in both systems as well.