Heterogeneity in surface sensing produces a division of labor in Pseudomonas aeruginosa populations

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition from planktonic to biofilm growth in many bacterial species. Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence. The current thinking in the field is that once cells attach to a surface, they uniformly respond with elevated c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment, P. aeruginosa engages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration.


7
demonstrating that the subpopulation of cells with high c-di-GMP is producing more 122 exopolysaccharide than their low c-di-GMP counterparts. As a complementary approach, 123 we separated 4 h surface-grown cells of the reporter strain into reporter "on" and "off" 124 subpopulations using flow-assisted cell sorting (FACS; Figure 1 -Supplement 6). We 125 then applied qRT-PCR to compare Pel and Psl transcript levels in these two populations. 126 Both the pel and psl operon transcripts were elevated in the reporter "on" subpopulation, 127 relative to the reporter "off" subpopulation ( Figure 1E). These data support that, with 128 respect to c-di-GMP signaling, there are at least two distinct subpopulations that arise 129 shortly after surface attachment. 130 The Wsp system is required for surface sensing 131 We next evaluated the relative contributions of the Wsp and Pil-Chp surface 132 sensing systems to surface-induced c-di-GMP production. Strains with mutations in the 133 Pil-Chp chemosensory system were not significantly defective in surface sensing activity.  <0.05 by T-test). We then mutated the c-di-GMP cyclase gene, wspR, to inactivate the 141 Wsp system. In addition, we deleted the gene encoding the methylesterase wspF, which 142 locks the system into the active state, regardless of whether cells are surface-associated. 143 We found that PAO1 ΔwspR strain exhibited extremely low levels of reporter activity  (Figure 2A). We repeated these experiments in the lab strain PA14 and saw a 148 similar trend for Wsp and Pil-Chp mutants (Figure 2 -Supplement 4). 149 Since the Pil-Chp surface sensing apparatus is polarly localized and the Wsp 150 system is localized laterally along the length of the cell body, we examined whether 151 reporter activity correlated with polar versus lateral attachment to the surface. We found 152 that reporter activity was very low in polarly attached cells, while cells attached along the 153 entire length of the cell body displayed a higher proportion of activated cells ( Figure 2B). 154 This finding is also consistent with the localization of the Wsp system and its role for 155 early c-di-GMP signaling during surface sensing. 156 Heterogeneity in c-di-GMP levels among cells correlates with Wsp system activity 157 The specific activity of purified WspR increases as a function of WspR 158 concentration when the protein is treated with beryllium fluoride to mimic 159 phosphorylation, supporting the idea that formation of subcellular clusters of WspR-P 160 potentiates its diguanylate cyclase activity and leads to elevated c-di-GMP(12). Fewer 161 than 1% of wild-type cells grown in broth have a visible WspR-YFP cluster. However, 162 after a short period of growth on an agar surface, WspR-YFP clusters were visible in 30-163 40% of wild type PAO1 cells, and this is dependent on sensing by the membrane-bound 164 protein WspA, which is laterally distributed in cells (9). To directly link WspR cluster 165 formation with diguanylate cyclase activity at the cellular level and with surface sensing, 166 we constructed a version of the c-di-GMP reporter that expresses mTFP1 instead of GFP 167 9 (pP cdrA ::mTFP1) to avoid the issue of spectral overlap with WspR-YFP. We monitored 168 reporter activity in two point mutants of WspR (L170D and E253A) that are driven by an 169 inducible promoter, translationally fused to eYFP and have been previously shown to 170 form large subcellular WspR clusters in a higher percentage of cells than wild-type 171 WspR. The WspR[L170D] protein is highly active for c-di-GMP production, and it forms of 345 vs. 320 RFU respectively, Mann-Whitney test, p < 0.001). These data indicate that 182 the heterogeneity observed in c-di-GMP signaling after surface attachment is due to the 183 heterogeneity in the activity of the Wsp system, as reflected by subcellular clustering of 184 active WspR-P. 185 We next asked whether the observed heterogeneity in c-di-GMP signaling in 186 response to a surface has a meaningful influence on biofilm formation. This was 187 particularly important since previous published results indicated that a wspR mutation 188 had only a small impact on biofilm production (22). However, these studies assessed 189 biofilm formation at later stages of biofilm growth that were well beyond initial surface 190 10 attachment. Therefore, we chose to compare a wspR mutant to wild type at earlier 191 biofilm stages. We performed in vitro biofilm assays and observed that a PAO1 ΔwspR 192 mutant was defective for biofilm formation relative to wild type PAO1 at 2, 4, and 6 193 hours post-attachment ( Figure 4A). However, at later stages of development (~24 h), the 194 wspR mutant caught up and produced similar amounts of biofilm biomass relative to wild 195 type levels. Complementation of the ΔwspR strain in trans restored wild type levels of 196 biofilm formation at all time points. These data suggest that the Wsp system rapidly 197 responds to surface contact to generate elevated levels of c-di-GMP, which accelerates 198 biofilm production. Given the importance of c-di-GMP signaling in biofilm production, 199 the fact that the ΔwspR strain can ultimately attain wild-stype levels of biofilm biomass 200 suggests that one of the many other known c-di-GMP cyclases present in P. aeruginosa 201 may ultimately compensate for c-di-GMP production in the absence of WspR.

202
Cyclic di-GMP heterogeneity leads to diversification in surface exploration at the lineage 203 level 204 We hypothesized that heterogeneity in c-di-GMP signaling dictated by the Wsp complex 205 could impact the surface behavior of the two observed subpopulations. We predicted that 206 the subpopulation of cells with high c-di-GMP after surface attachment would produce 207 biofilm matrix exopolysaccharides and contribute to initial microcolony formation, while 208 the cells with low c-di-GMP would exhibit increased surface motility and detachment, 209 which is known to be inhibited by exopolysaccharide production. To test this hypothesis, 210 we tracked both reporter activity and surface behavior for cells within a single field of 211 view for 40 h. From our single-cell tracking data, we generated family trees across at 212 least four generations of cells, using a previously described technique (23). We tracked 213 11 the time-averaged P cdrA ::gfp ASV reporter activity (I c-di-GMP ), surface motility behavior 214 (F motile , defined as the fraction of time that cells are motile), and detachment behavior 215 (tree asymmetry λ where λ = 0 represents both daughter cells remaining attached to the 216 surface and λ = 1 represents when one daughter cell detaches or travels outside the field 217 of view).

218
In P. aeruginosa, surface exploration is mainly accomplished by twitching  Figure 4D) and spatial trajectories ( Figure 4E).

229
Families with the highest I c-di-GMP had the lowest F motile and λ (Family 1, Figure 4B-E).

230
In these families, daughter cells remained attached following cell division, exhibited 231 continuously elevated c-di-GMP, did not move appreciable distances on the surface, and 232 ultimately produced small microcolonies. In contrast, families of cells with low I c-di-GMP 233 had the highest F motile and λ. For these families, daughter cells frequently detached or 234 traveled outside the field of view, had lower c-di-GMP levels, traveled larger distances 235 on the surface, and ultimately did not form microcolonies (Family 3, Figure 4B-E).  especially if their number is not large or if they are assymetrically partitioned, which may 290 be one mechanism that leads to the heterogeneity in c-di-GMP levels.

291
If we overwhelm WspR-generated c-di-GMP heterogeneity by using 292 optogentically-induced sustained c-di-GMP production, we find that phenotypic 293 heterogeneity is lost, and that illuminated cells deterministically become sessile and form 294 microcolonies. Interestingly, our optogenetic experiments show that sustained c-di-GMP 295 production for more than one generation is required before commitment to the sessile

332
To create MPAO1 attTn7::P(A1/04/03)::GFPmut, the miniTn7 from pBT270 was 333 integrated into the chromosome of P. aeruginosa PAO1 with the helper plasmid pTNS2,  were gated based on forward and side scatter to remove particles smaller than a single P. 477 aeruginosa cell and large aggregates. 478 We used PAO1 cells that did not express GFP (wild type PAO1; Figure 1   PelA or PslA expression in the P cdrA ::gfp ASV sorted "on" population (high GFP) relative to 517 the "off" population (low GFP) for the three biological replicates.     Approximately 56.6% of the population falls into the reporter "on" population. e)

625
Example of gating for "on" cells from wild type PA14 pP cdrA ::gfp ASV cells that had been 626 attached to glass in LB medium for 4 hours prior to FACS sorting. Approximately 12.8% 627 31 of the population falls into the reporter "on" population. f) Example of gating for "on" 628 cells from wild type PA14 pP cdrA ::gfp ASV cells that had been attached to glass in Jensen's 629 media (a condition in which Pel is more abundantly produced than in LB) for 4 hours.

630
Approximately 31.9% of the population falls into the reporter "on" population.