Intracellular glycosyl hydrolase PslG shapes bacterial cell fate, signaling, and the biofilm development of Pseudomonas aeruginosa

Biofilm formation is one of most important causes leading to persistent infections. Exopolysaccharides are usually a main component of biofilm matrix. Genes encoding glycosyl hydrolases are often found in gene clusters that are involved in the exopolysaccharide synthesis. It remains elusive about the functions of intracellular glycosyl hydrolase and why a polysaccharide synthesis gene cluster requires a glycosyl hydrolase-encoding gene. Here, we systematically studied the physiologically relevant role of intracellular PslG, a glycosyl hydrolase whose encoding gene is co-transcribed with 15 psl genes, which is responsible for the synthesis of exopolysaccharide PSL, a key biofilm matrix polysaccharide in opportunistic pathogen Pseudomonas aeruginosa. We showed that lack of PslG or its hydrolytic activity in this opportunistic pathogen enhances the signaling function of PSL, changes the relative level of cyclic-di-GMP within daughter cells during cell division and shapes the localization of PSL on bacterial periphery, thus results in long chains of bacterial cells, fast-forming biofilm microcolonies. Our results reveal the important roles of intracellular PslG on the cell fate and biofilm development.

WFPA801ΔpslG strain (having wild type level of ePsl production, Figure 1A) showed 130 reduced swimming zone compared to that of either PAO1 or WFPA801 ( Figure 1B), 131 indicating that the induction of ePsl production in WFPA801ΔpslG attenuated its 132 swimming ability. However, T4P-mediated twitching motility was not affected ΔpslG impacts the bacterial distribution and maximum thicknesses of pellicles. 137 We then investigated the effect of ΔpslG on the air-liquid interface biofilms, 138 termed as pellicles, by using confocal laser scanning microscopy. The total pellicle 139 biomass of ΔpslG is similar to that of PAO1 after 24h growth, although ΔpslG 140 produced much less ePsl and had defect on initial attachment ( Figure 1 and Figure 2A, 141 C). However, ΔpslG has significant higher maximum thickness than that of PAO1 142 ( Figure 2B). In addition, there is less bacteria in each section image of ΔpslG pellicles 143 compared to that of PAO1 ( Figure 2D, left and middle panel). The ePsl matrix in 144 ΔpslG pellicles shows weaker fluorescent intensities than that of PAO1 ( Figure 2D, 145 middle and right panels), which is consistent with their corresponding ePsl production. 146 In spite of that, the fibre-like ePsl can be detected in the pellicles of ΔpslG, which 147 have a radial pattern as previously described for PAO1 pellicles ( Figure 2D, middle 148 panels) (Wang et al., 2013). These results suggest that the pslG deletion might impact bacterial distribution within biofilms.

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To further understand the effect of ΔpslG, by employing bacterial tracking 152 techniques, we observed ΔpslG cell behavior at the single-cell level. Figure 3A shows 153 the surface coverage obtained by all tracked bacterial trajectories for a specific time 154 period during microcolony formation. Red color indicates the surface area that has 155 been visited by bacteria, while black color indicates a "fresh" surface area that has 156 never been visited. Bacterial cells are shown in blue. When compared ΔpslG and Figure 3E). Taken together, these results indicate that loss of pslG promotes 172 microcolonies formation by changing the surface exploration of bacteria at early 173 stages of biofilm formation, a phenomenon that has been reported mostly for strains 174 producing high level ePsl (Zhao et al., 2013).  (two-bright). The results show that none-bright type is observed most frequently in 203 both PAO1 and WFPA801ΔpslG strains, which has an occurrence probability of ~60% 204 for PAO1 and ~52% for WFPA801ΔpslG. However, interestingly, WFPA801ΔpslG 205 shows a relatively higher probability of two-bright type (~17%) than that of PAO1  intracellular c-di-GMP production (Irie et al., 2012). To see whether ePsl produced from ΔpslG has similar signaling functions, we set up a co-culture system to test the 216 signaling functions of ePsl. The system contained an ePsl donor strain and a reporter 217 strain. PAO1 harboring pcdrA::gfp in a plasmid was utilized as an intracellular 218 c-di-GMP reporter strain while PAO1, WFPA800, and ΔpslG strains were ePsl donors 219 respectively, in which WFPA800 was used as a negative control because it does not 220 produce ePsl. As shown in Figure 5A, PAO1 that produces wild type level of ePsl can 221 induce a stronger GFP fluorescence signal in the reporter stain compared to WFPA800 222 ( Figure 5A and 5B). Surprisingly, ePsl of ΔpslG strain stimulated a higher 223 fluorescence signal than that of PAO1 ( Figure 5B) although ΔpslG mutant produces 224 much less ePsl (about 30% of PAO1 level, Figure 1A), suggesting that ePsl 225 synthesized from ΔpslG mutant has stronger signaling effect on stimulating the 226 Aintracellular c-di-GMP production than that of wild type. 227 We then applied Raman spectroscopy to detect exopolysaccharides in the biofilm 228 formed by PAO1 and ΔpslG mutant. The ΔpslG mutant shows a different Raman 229 spectra at 865 cm -1 compared to that of PAO1 and WFPA800, indicating a change at 230 C-C stretching and C-O-C 1,4-glycosidic link ( Figure 5C). This result suggests that 231 the structure of ePsl produced by ΔpslG mutant strain is different from that of PAO1,     The other aspect as a consequence of the change in ePsl due to the loss of pslG is, 315 at the molecular level, the probability of both daughter cells having a higher level of 316 c-di-GMP than that of their mother cell in a division event is increased. The increased 317 level of c-di-GMP then would result in reduced cell motility and promote cells to 318 transit to biofilm style (Romling et al., 2013). This may also help cells to form long 319 chains by reducing the breakage of chains due to reduced cell motility and increased 320 ePsl production.

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As it is widely known that ePsl plays a very important role in the biofilms of P.

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The c-di-GMP levels were determined using pcdrA::gfp as a reporter as between each daughter cell (I dau ) and its mother cell (I mot ) was calculated, g = I dau /I mot .

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The relative standard deviation was estimated by              long cell-chain. The movie was taken at a frame interval of 1 min for 0.5h and was 818 played back at 5 fps. Scale bar, 5 µm.