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How bacteria respond to material stiffness during attachment: a role of Escherichia coli flagellar motility Fangchao Song, Megan E Brasch, Hao Wang, James H. Henderson, Karen Sauer, and Dacheng Ren ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b04757 • Publication Date (Web): 21 Jun 2017 Downloaded from http://pubs.acs.org on June 22, 2017

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How bacteria respond to material stiffness during attachment: a role of Escherichia coli flagellar motility

Fangchao Song1,2,†, Megan E. Brasch1,2, Hao Wang1,2, James H. Henderson1,2, Karin Sauer3, Dacheng Ren1,2,4,5,*

1

Department of Biomedical and Chemical Engineering, Syracuse, New York, USA.

2

Syracuse Biomaterials Institute, Syracuse, New York, USA. 3Department of Biological

Science, Binghamton University, Binghamton, New York, USA. 4Department of Civil and Environmental Engineering, Syracuse, New York, USA.5Department of Biology, Syracuse University, Syracuse, New York, USA.

*Corresponding author: Dacheng Ren: Phone 001-315-443-4409. Fax 001-315-443-9175. Email: [email protected]

Current address:

Lawrence Berkeley National Laboratory, 1 Cyclotron Rd, MS955-0542, Berkeley, CA 94720

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Abstract Material stiffness has been shown to have potent effects on bacterial attachment and biofilm formation, but the mechanism is still unknown. In this study, response to material stiffness by Escherichia coli during attachment was investigated with biofilm assays and cell tracking using the Automated Contour-base Tracking for In Vitro Environments (ACTIVE) computational algorithm. By comparing the movement of E. coli cells attached on poly(dimethylsiloxane) (PDMS) surfaces of different Young’s moduli (0.1 and 2.6 MPa, prepared by controlling the degree of crosslinking) using ACTIVE, attached cells on stiff surfaces were found more motile during early stage biofilm formation than those on soft surfaces. To investigate if motility is important to bacterial response to material stiffness, E. coli RP437 and its isogenic mutants of flagellar motor (motB), and synthesis of flagella (fliC) and type I fimbriae (fimA) were compared for attachment on 0.1 MPa and 2.6 MPa PDMS surfaces. The motB mutant exhibited defects in response to PDMS stiffness (based on cell counting and tracking with ACTIVE), which was recovered by complementing the motB gene. Unlike motB results, mutants of fliC and fimA did not show significant defects on both face-up and face-down surfaces. Collectively, these findings suggest that E. coli cells can actively respond to material stiffness during biofilm formation, and motB is involved in this response.

Keywords bacteria, attachment, motility, material stiffness, mechanosensing

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Introduction Over 90% of bacteria on earth live in biofilms with surface-attached cells embedded in an extracellular matrix comprised of self-produced polysaccharides, DNA, RNA, and proteins.1 Due to high-level tolerance to antimicrobials and host immune factors, biofilm associated infections result in nearly 100,000 deaths and 28-45 billion dollars of losses each year in the U.S. alone.2-5 Additionally, biofilms formed on water pipes and ship hulls hydro-dynamically increase energy costs, putting a heavy burden on our economy.1,6 Understanding the mechanism of biofilm formation and the physiology of biofilm cells is critical for solving these problems. There are multiple steps in the transition from planktonic growth to biofilm formation, including initial attachment, cell cluster formation, biofilm maturation, and dispersion.1,78

Among these steps, initial attachment (including reversible and irreversible attachment)

plays an important role in biofilm formation and is known to be influenced by many properties of the substratum surface such as surface chemistry,9-12 stiffness,13-16 hydrophobicity,17-18 roughness,19-20 topography,21-24 and charge.9,25 Recently, we reported that a decrease in stiffness (with a decrease in Young’s modulus from 2.6 to 0.1 MPa) of cross-linked poly(dimethylsiloxane) (PDMS) promotes the attachment and growth of Escherichia coli and Pseudomonas aeruginosa; and that the cells attached on soft surfaces (0.1MPa) are more elongated and more sensitive to antibiotics compared to those on stiff surfaces (2.6MPa).26 These and other findings13-16 indicate that bacteria can sense the mechanical properties of a surface when making the decision of attachment and biofilm formation vs. planktonic growth. However, the research on bacterial mechanosensing to date has been limited to the general contact of a surface and the

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effects of material stiffness have not been explored. For general contact with a surface, previous studies have shown that bacteria can detect a surface using flagella, fimbriae or other outer membrane appendages.27-29 For example, flagella and pili are used by bacteria as adhesins to facilitate the initial contact with a surface by overcoming the hydrodynamic boundary layer and repulsive forces.29 To better understand mechanosensing of bacteria, it is important to investigate how bacteria differentiate “hard” surfaces from “soft” surfaces by sensing material stiffness during initial attachment. Motivated by this knowledge gap, this study was conducted to investigate the role of motility in mechanosensing of material stiffness by E. coli. We hypothesized that the stiffness of surface material can be sensed by bacteria and thus affect bacterial movement over the surface and the decision of permanent attachment and subsequent biofilm formation. To test this hypothesis, we compared the movement of E. coli cells attached on PDMS surfaces of different Young’s moduli. To understand the underlying mechanisms, we further compared wild-type E. coli with its isogenic mutants of flagellar motor, and the synthesis of flagella and type I fimbriae.

Materials and Methods Bacterial strains and growth media The strains used in this study are summarized in Table 1. E. coli RP437, a model strain for studying motility and biofilm formation,30 and its isogenic mutants of motB, fliC, and fimA were used to investigate the roles of flagella and type I fimbriae in bacterial response to material stiffness during biofilm formation. To follow cells with fluorescence imaging, the plasmid pRSH10324 was cloned into the above strains to label the cells with

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constitutively expressed red fluorescent protein. E. coli RP437 and its mutants were routinely grown at 37oC with shaking at 200 rpm in Lysogeny Broth (henceforth LB medium) containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl in deionized water.24,

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The wild-type strain E. coli RP437/pRSH103, E. coli RP3087/pRSH103

(motB mutant), E. coli RP437 fliC/pRSH103, and E. coli RP437 fimA/pRSH103 were routinely grown at 37oC with shaking at 200 rpm in LB medium supplemented with 30 µg/mL tetracycline to keep the plasmid.

Preparation of PDMS surfaces PDMS surfaces were prepared using SYLGARD184 Silicone Elastomer Kit (Dow Corning Corporation, Midland, MI). The stiffness was adjusted by varying the mass ratio of base to curing agent (5:1 and 40:1 tested), as described previously.32-34 For each given ratio, the elastomer base and curing agent were thoroughly mixed and degased under vacuum for 30 min. Then, the mixture was poured into a petri-dish, cured at 60oC for 24 h, and incubated at room temperature for another 24 h to fully polymerize. The PDMS surface was then peeled off the petri-dish and cut into 1.0 cm by 0.6 cm pieces (~1.5 mm thick), which were sterilized by soaking in 200 proof ethanol for 20 min and dried with sterile air. All of the sterilized PDMS substrates were stored at room temperature until use. As we reported previously, the Young’s moduli of 5:1 and 40:1 PDMS are 2.6 MPa (stiff) and 0.1 MPa (soft), respectively, measured by dynamic mechanical analysis (DMA) (Q800, TA Instruments, DE, USA).26

Bacterial attachment on PDMS

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E. coli cells from overnight cultures were harvested by centrifugation at 8,000 rpm for 3 min at 4oC, washed with phosphate buffered saline (PBS) (pH 7.3) three times, and then used to inoculate PBS to a desired cell density. The cell density of inoculum was adjusted based on optical density at 600 nm (OD600). Each cell suspension (30 mL) was transferred to a petri-dish containing sterilized PDMS surfaces to form biofilms. Meanwhile, the cell suspension was dropped (10 µL in each drop) on a LB agar plate after a series of 10× dilution to determine the cell density of the inoculum. After cell attachment at 37oC for 2 h without shaking, the PDMS surfaces were gently washed by dipping in fresh PBS three times. The viability of cells was determined by counting colony forming units (CFU) using the drop plate assay as described previously.35 All conditions were tested with at least 3 biological replicates.

Cell tracking and data analysis Bacterial cells from overnight cultures were harvested by centrifugation at 8,000 rpm for 3 min at 4oC, washed with PBS (pH 7.3), and then used to inoculate 15 mL PBS to desired cell density in a petri-dish containing sterilized PDMS surfaces. The initial attachment was monitored using an Axio Observer Z1 fluorescence microscope (Carl Zeiss Inc., Berlin, Germany). Images were then taken every 5 s for 20 min for each sample to generate a live-cell stack, and the results were analyzed using the Automated Contour-base Tracking for In Vitro Environments (ACTIVE) computational algorithm.36 The representative results are shown in Movie S1-4. The cells were identified and contoured by optimizing the parameters in ACTIVE. Cells were then grouped into three categories: “still” (the mass center did not move more than a quarter of the body length

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during any 5 s interval), “rotating” (the mass center moved more than a quarter but less than one body length during any 5 s interval), and “moving” (the mass center moved more than one body length during any 5 s interval). The average moving speed was calculated for each individual cell.

Results Tracking bacterial motility using the Automated Contour-based Tracking for In Vitro Environments (ACTIVE) computational algorithm. Time-lapse images were analyzed using ACTIVE, a MATLAB based automated cell tracking package.36 This package has been shown to have high accuracy in tracking the motility of mouse fibroblasts in a complex in vitro model36 and we recently used it to track individual E. coli cells attached on PDMS surfaces.37 To validate if ACTIVE could accurately detect cells in early stage multicellular biofilms including motile cells, E. coli RP437/pGLO38 expressing strong green fluorescence upon induction with arabinose was used to test the tracking software. Representative images during initial attachment on a glass surface are shown in Figure 1A. By adjusting the parameter of the ACTIVE package (Figure S1), all E. coli cells were successfully detected (Figure 1B), and the cell contours were automatically identified with good accuracy (Figure 1C). ACTIVE was also validated for tracking bacterial cells over time as shown in Figure 1D. These setting were adopted for the rest of this study.

E. coli cells were more motile on 5:1 (stiff) PDMS than 40:1 (soft) PDMS during attachment.

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ACTIVE was used to track the movement of E. coli RP437/pRSH103 during early stage biofilm formation on PDMS surfaces with two different levels of stiffness. To focus on attachment, moving planktonic cells were excluded from the analysis by setting a filter in ACTIVE which excludes all the cells that moved more than one cell body length in the first 5 s of the experiment. The detected cells were categorized into three groups as shown in Figure 2 including “still cells” that did not move, “rotating cells” that moved in a circular motion (either in a full or partial circle) or moved back and forth but the mass center moved less than one body length during any 5 s interval of the experiment, and “moving cells” that had the mass center moved for more than one body length during any 5 s of the experiment. The cell types were automatically categorized by comparing images frame by frame using ACTIVE (5 s per frame). Previously, we showed that E. coli attaches more (based on biomass) on relatively soft (40:1) PDMS than harder (5:1, 10:1, or 20:1) PDMS.26 However, the mechanism of such difference in attachment is unknown. By tracking the cells during attachment in the present study, we found that PDMS stiffness also affected the movement of E. coli cells. As shown in Figure 3A and Movies S1 and S2, most of the wild-type E. coli RP437 cells carrying pRSH103 for red fluorescence (henceforth wild-type cells) were “still” on 40:1 (soft) PDMS surfaces, while the fractions of “rotating cells” and “moving cells” were significantly higher on stiff surfaces (p > 0.001 for both cases, t test). Specifically, on 40:1 (soft) PDMS surfaces, 69 ± 5 % of the attached wild-type cells were not actively moving (“still”), and the rotating and moving cells represented only 21 ± 4 % and 9 ± 3 % of the population, respectively (Figure 7A). On 5:1 (stiff) PDMS surfaces, however, the fraction of still cells was only 48 ± 4 % of the whole population, while the fraction of

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rotating cells increased to 34 ± 3 % and that of moving cells increased to 18 ± 3 % (Figure 7A). In addition to the differences in the type of cell movement, the speed of movement was also analyzed for each cell using ACTIVE. A representative set of results is shown in Figure 3B, in which each dot represents the velocity of a single cell at a particular time point, and each black line shows the average velocity of all cells at a given time point. The average velocity calculated from all cells over all time points is shown on the top of each plot. Consistent with the results of cell movement, the wild-type cells were found to be more motile on 5:1 (stiff ) surfaces than 40:1 (soft) surfaces; e.g., the average speed on 40:1 (soft) and 5:1 (stiff) PDMS surfaces was 2.7 ± 0.3 µm/min and 6.1 ± 0.4 µm/min, respectively (Figure 7B).

motB is important to the response of E. coli to PDMS stiffness during initial attachment. The above findings support our hypothesis that the stiffness of surface material can affect bacterial response and thus the decision of attachment and subsequent biofilm formation. The observed response appeared to be motility related; thus, we compared the wild-type strain with its mutants of motB, fimA, and fliC for attachment on soft (40:1; 0.1 MPa) and stiff (5:1; 2.6 MPa) PDMS (Table 1). These mutants were chosen because motility (motB), flagella (fliC), and pili (fimA) are known to be involved in initial attachment.39 The attachment (for 2 h) was carried out in PBS without any carbon source so that attachment could be studied in the absence of cell growth. The cell density of inoculum was controlled to be between 3 × 107 cells/mL and 6 × 107 cells/mL. After 2 h attachment,

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a total of (1.1 ± 0.4) × 106 cells/cm2 and (5.3 ± 2.4) × 103 cells/cm2 of the wild-type strain were detected on soft (0.1 MPa) and stiff (2.6 MPa) surfaces, respectively. Thus, the increase in stiffness from 0.1 MPa to 2.6 MPa led to a decrease in the number of attached wild-type cells by more than two orders of magnitude (Figure 4A), which is consistent with our earlier report26. The fliC and fimA mutants also exhibited marked reduction in attachment when the stiffness of PDMS increased from 0.1 MPa to 2.6 MPa. Specifically, the number of attached E. coli RP437 fliC mutant cells was (1.6 ± 0.2) × 105 cells/cm2 and (2.3 ± 0.7) × 103 cells/cm2 on 40:1 (soft) and 5:1 (stiff) surfaces, respectively, corresponding to a decrease of two orders of mangnitude (Figure 4A). The attachment of E. coli RP437 fimA mutant decreased from (2.0 ± 0.3) × 105 cells/cm2 on 40:1 PDMS (soft) surfaces to (4.4 ± 0.5) × 102 cells/cm2 on 5:1 PDMS (stiff) surfaces, corresponding to a decrease of more than two orders of magnitude as well (Figure 4A). It is not surprising that these mutants have lower numbers of attached cells compared to the wildtype strain on the same surfaces because both flagella and type I fimbriae are known to be important to initial attachment.29, 40 Despite showing decreased attachment relative to the wild-type strain (~ two orders of magnitude) on both soft and stiff surfaces, these mutants still exhibited significant difference in attachment between 5:1 (stiff) and 40:1 (soft) PDMS, especially on inverted surfaces (below). Thus, these two genes were not included in our further study. In comparison, E. coli RP437 motB mutant showed a drastically reduced difference in attachment between soft and stiff surfaces on both face-up and face-down surfaces. On face-up surfaces, while the wild-type strain and its motB mutant had similar numbers of cells attached on 40:1 PDMS (0.1 MPa; soft) (p = 0.57, t test), the motB mutant had about

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an order of magnitude more cells on the 5:1 PDMS (2.6 MPa; stiff) than the wild-type strain (Figure 4A). This finding indicates that motB may be involved in mechanosensing of/response to PDMS stiffness. To comfirm if this change was indeed caused by inactivation of motB, the motB mutant was complemented using a plasmid, pRGH03, which carries the motB gene under a constitutive promoter. As controls, the empty vector pRSH103 (the plasmid used to construct pRGH03) was also eletroporated into the wilde-type E. coli RP437 and its motB mutant. Attachement was carried out for all the strains for 2 h. As shown in Figure 4A, the defects of motB mutant were fully recovered by motB complementation with pRGH03.

Attachment on inverted surfaces. It was interesting to observe that mutation of motB caused defects in the response of E. coli to PDMS stiffness. The experiments above were based on attachment on face-up surfaces. To verify if the observed results are intrinsic rather than being caused by gravity driven settlement, we repeated the attachment experiments using face-down surfaces with similar inocululm densities (between 2 × 107 cells/mL and 4 × 107 cells/mL). The numbers of attached cells of all tested strains were at least one order of magnitude lower than those on face-up surfaces, suggesting that gravity does facilitate cell settlement and attachment. Nevertheless, E. coli still showed preference in attachment on 40:1 (soft) PDMS surfaces compared to 5:1 (stiff) PDMS surfaces (p > 0.001, t test). For example, the number of wild-type cells attached on 40:1 (soft) PDMS surfaces was (5.2 ± 1.8) × 103 cells/cm2, which is 1.5 orders of magnitude higher than that on 5:1 (stiff) PDMS surfaces [(2.2 ± 0.6) × 102 cells/cm2] (Figure 4B). Similar to the results of face-up

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surfaces, the motB mutant also exhibited defects in response to PDMS stiffness; e.g., the difference in the number of attached cells was reduced to 0.8 log for the motB mutant (Figure 4B). This defect was recovered in the complemented strain (Figure 4B), as observed for attachment on face-up surfaces. Several other mutants related to bacterial motility (fimA, fliC, luxS, fdlDA, trg, tar, and cheY) were also tested; however, none of them showed statisically signficant effects (p > 0.1 for all the cases; t test; Figure S2).

Movement of motB mutant on 40:1 (soft) and 5:1 (stiff) PDMS. It has been shown that motB is involved in the general sensing of surface contact by E. coli.27-28 To further understand the role of motB in mechanosensing of material stiffness, the CFU results of motB mutant were corroborated with imaging analysis using ACTIVE. Unlike the wild-type strain, the majority of motB mutant cells were found to be “still” on both soft and stiff surfaces (face-up) (Figure 5A; Movies S3 and S4). The defects of motB mutant in movement on PDMS surface during attachment was fully recovered by the complementation of motB gene (Figure 6A; Movies S5 and S6). The results of movement type and speed of the wild-type strain, its motB mutant, and motB complemented strain are summarized from at least 3 movies (~20 min each) with more than 200 cells each in Figure 7. The fraction of “still”, “rotating”, and “moving” cells of motB mutant was 84 ± 3 %, 11 ± 2 %, 5 ± 1 % on 40:1 (soft) PDMS surfaces, and 71 ± 2 %, 23 ± 2 %, 6 ± 1 % on 5:1 (stiff) PDMS surfaces, respectively. The fraction of “still”, “rotating” and “moving” cells of wild-type strain was 69 ± 5 %, 21 ± 4 %, 9 ± 3 % on 40:1 (soft) PDMS surfaces, and 48 ± 4 %, 34 ± 3 %, 18 ± 3 % on 5:1 (stiff) PDMS surfaces, respectively. These results showed that the distribution of motB mutant cells in

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these three categories is more distinct than the wild-type strain, indicating that the absence of a functional motB gene may cause defects in mechanosensing of material stiffness and the subsequent attachment. Complementation of the motB gene rescued these defects. For example, the fraction of “still”, “rotating” and “moving” cells of motB complemented strain was 68 ± 3 %, 20 ± 1 %, 12 ± 2 % on 40:1 (soft) PDMS surfaces, and 49 ± 5 %, 30 ± 4 %, 22 ± 3 % on 5:1 (stiff) PDMS surfaces. These results are similar to those of the wild-type strain (p > 0.1 for all the cases; t test). In addition to the type of cell movement, the average velocity of cell movement was found to be higher on 5:1 (stiff) PDMS than 40:1 (soft) PDMS (Figure 7B). Specifically, velocities of the wild-type strain and its motB mutant were 6.1 ± 0.4 µm/min and 3.5 ± 0.3 µm/min on stiff surfaces, and 2.7 ± 0.3 µm/min and 2.0 ± 0.4 µm/min on soft surfaces, respectively. This confirms that both the wild-type and its motB mutant are more motile on stiff surfaces. However, the difference in movement speed between soft and stiff surfaces is more profound for the wild-type strain (3.4 µm/min) than the motB mutant (1.5 µm/min). The distribution of the velocity on stiff and soft surfaces is consistent with these results (Figure 7C). The average movement speed of motB complement strain on 40:1 (soft) and 5:1 (stiff) PDMS was 5.6 ± 0.9 µm/min and 3.2 ± 0.4 µm/min on stiff and soft surfaces, respectively, which is not significantly different from the wild-type strain (p = 0.64 for soft surfaces; p = 0.36 for stiff surfaces; t test).

Discussion In this study, we compared E. coli RP437 and its isogenic mutants of motB, fliC, and fimA genes for attachment on PDMS surfaces with varying level of stiffness. The results

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show that motB mutation negatively impacted the capability of E. coli cells to differentiate soft and stiff surfaces during attachment, which was recovered by complementing the motB gene. The defects in attachment primarily occured on stiff surfaces, on which there were 10 times more attached motB mutant cells than the wildtype cells. Cell tracking during the initial attachment showed that motB mutation also reduced the difference in moving speed of E. coli cells between 40:1 (soft) and 5:1 (stiff) PDMS surfaces. This finding was corroborated by the CFU results. Collectively, the findings of this study demonstrate that motB is involved in the response of E. coli to PDMS stiffness during early-stage biofilm formation. The observed differences were caused by surface stiffness, rather than surface chemistry or hydrophobicity. Using X ray photoelectron spectroscopy (XPS), Jiang et al.41 showed that the changes in the composition of main elements C, O, and Si are negligible for PDMS made with the same base: curing agent ratios of this study. We recently confirmed that these PDMS surfaces do not affect the growth of bacteria, and thus, the observed results are not due to toxicity.26 Based on the structure of base and crosslinking agent, there is a possibility that the C=C on soft surfaces may be slightly more than those on stiff surfaces. However, this should be a minor effect since the maximum difference is only 5% (5% of –CH3 group could be possibly replaced by C=C double bond on 40:1 surfaces.). Moreover, the negligible change in surface chemistry did not cause the difference in surface hydrophobicity. Using the same material and the ratio of base to crosslinking agent, Mata et al.42 reported that the contact angles of such PDMS are all the same. Thus, we believe that the differences in the movement and the number of

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attached E. coli cells between 40:1 (soft) and 5:1 (stiff) PDMS surfaces were caused by surface stiffness. Based on these results and those of our earlier study,26 we speculate that material stiffness may affect bacterial biofilm formation by influencing attachment and cell motility. During initial attachment, E. coli may use extracellular appendages (e.g., flagella) to sense the stiffness of substrate material. If attachment is favorable, the cells will reduce motility and start biofilm growth. In comparison, if the stiffness is not desired, the cells may move more before settling, as observed on 5:1 (stiff) PDMS surfaces. It is also possible that some cells may leave the surface and return to the planktonic stage if the surface is not favorable for attachment. Our results suggest that the E. coli cells do not prefer 5:1 (stiff) PDMS surfaces for attachment since the cells were more motile on these surfaces than 40:1 (soft) PDMS. And the mutant cells with paralyzed MotB partially lost the ability to sense and/or respond to the stiffness of PDMS. It is worth noticing that the motB mutant cells still exhibited a moving speed around 3 µm/min even with mutation of this critical gens. We speculate that such movement may be caused by Brownian motion since the displacement of 1 µm beads in water in 5 seconds was around 0.9 µm calculated by Einstein equation,43 corresponding to a velocity around 10 µm/min. The bigger sized E. coli motB mutants are expected to have weaker influence by Brownian motion, consistent with our observation here. Further studies of cell tracking in 3D with high resolution microscopy will help answer if there is any other kinds of motility involved in the movement and the difference between soft and stiff surfaces. The rotation of flagella seems to be more important in response to surface stiffness than the flagella itself, because only the motB mutant showed significant differences from

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the wild-type strain on both face-up and face-down surfaces. Although the results of faceup surfaces in Figure 4A showed difference between fliC mutant and the wild-type strain, such difference was not observed on face-down surfaces shown in Figure S2. Thus, the effects shown in Figure 4A may be partially caused by gravity related factors. This is interesting to investigate in future. MotB is part of the flagellar motor which generates the force of its rotation for swimming.44 Lele et al.28 reported that the force generated by MotB increases with the mechanical load on flagella, such as that caused by increase in medium viscosity, attachent of flagella to a particle, or tethering bacteria on a surface. We specculate that contacting with a stiff surface may cause a heavier mechanical load on bacterial flagella compared to the contact with a soft surface. The higher force generation by MotB protein may lead to faster movement on the surface or detachment, as we observed for 5:1 (stiff) PDMS in this study. Mutation of the motB gene could render the cells non-motile and thus less capable of surface attachment. Consistently, we found that E. coli motB mutant lost the ability of swimming, which was partially restored by motB complementation (image not shown). The difference in attachment and movement speed was markedly reduced by deleting the motB gene, consistent with the importance of motB in response to material stiffness. However, the motB mutant still exhibited some difference in attachment between soft and stiff surfaces. This suggests that MotB may not be an indispensable sensor of the surface stiffness, but does facilliate mechanosensing. To further understand how MotB is invovled in the mechanosensing/response and if other genes are also important, it will be important to study more mutants and create

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double mutants of important genes. This is part of our ongoing work. It will be also interesting to investigate the effects in a wider range of surface stiffness to understand if there is an optimum stiffness for adhesion or if it is a rather monotonic trend. How surface stiffness influences long-term biofilm formation is another unanswered question. In summary, the results of this study indicate that, in addition to sensing the general contact with a surface, bacteria have specific genes involved in response to material stiffness during attachment. Further mechanistic studies at the molecular level will reveal if other genes are involved and how the sensing of stiffness is translated to the changes in cell attachment and biofilm growth. This knowledge may help identify novel agonists or antagonists of these pathways to either promote or reduce biofilm formation on a material when a particular level of stiffness is desired.

Funding Sources: U.S. National Institute of Health (1R21EY025750-01A1) and U.S. National Science Foundation (CAREER-1055644 and CMMI-1334611)

Acknowledgements The authors thank the U.S. National Institute of Health (1R21EY025750-01A1) and U.S. National Science Foundation (CAREER-1055644 and CMMI-1334611) for partial support of this work. We are grateful to Dr. John S. Parkinson at the University of Utah for sharing the mutant strains.

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Figure S1: Parameters for tracking bacterial motility on surfaces using ACTIVE. Figure S2: Attachment of the wild-type strain and its mutants related to bacterial motility on face-down stiff (2.6 MPa) and soft (0.1 MPa) PDMS after 2 h attachment in PBS. The cell number of each strain on soft PDMS was normalized as 100%. Movie S1: Movement of the wild-type cells on soft PDMS surfaces (0.1 MPa). Movie S2: Movement of the wild-type cells on stiff PDMS surfaces (2.6 MPa). Movie S3: Movement of the motB mutant cells on soft PDMS surfaces (0.1 MPa). Movie S4: Movement of the motB mutant cells on stiff PDMS surfaces (2.6 MPa). Movie S5: Movement of the motB complemented cells on soft PDMS surfaces (0.1 MPa). Movie S6: Movement of the motB complemented cells on stiff PDMS surfaces (2.6 MPa).

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References (1) Hall-Stoodley, L.; Costerton, J.; Stoodley, P. Bacterial Biofilms: from the Natural Environment to Infectious Diseases. Nat. Rev. Microbiol. 2004, 2, 95-108. (2) Klevens, R.; Morrison, M.; Nadle, J.; Petit, S.; Gershman, K.; Ray, S.; Harrison, L. H.; Lynfield, R.; Dumyati, G.; Townes, J. Invasive Methicillin-Resistant Staphylococcus aureus Infections in the United States. JAMA, J. Am. Med. Assoc. 2007, 298, 1763-1771. (3) Klevens, R.; Edwards, J.; Richards, C.; Horan, T.; Gaynes, R.; Pollock, D.; Cardo, D. Estimating Health Care-Associated Infections and Deaths in US Hospitals, 2002. Public Health Rep. 2007, 122, 160-166. (4) Häussler, S.; Parsek, M. R. Biofilms 2009: New Perspectives at the Heart of SurfaceAssociated Microbial Communities. J. Bacteriol. 2010, 192, 2941-2949. (5) Costerton, J.; Stewart, P.; Greenberg, E. Bacterial Biofilms: a Common Cause of Persistent Infections. Science 1999, 284, 1318-1322. (6) Costerton, J.; Lewandowski, Z.; Caldwell, D.; Korber, D.; Lappin-Scott, H. Microbial Biofilms. Annu. Rev. Microbiol. 1995, 49, 711-745. (7) Stoodley, P.; Sauer, K.; Davies, D.; Costerton, J. Biofilms as Complex Differentiated Communities. Annu. Rev. Microbiol. 2002, 56, 187-209. (8) Sauer, K.; Camper, A.; Ehrlich, G.; Costerton, J.; Davies, D. Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm. J. Bacteriol. 2002, 184, 1140-1154. (9) Renner, L.; Weibel, D. Physicochemical Regulation of Biofilm Formation. MRS Bull. 2011, 36, 347-355.

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(10) Cheng, G.; Zhang, Z.; Chen, S.; Bryers, J.; Jiang, S. Inhibition of Bacterial Adhesion and Biofilm Formation on Zwitterionic Surfaces. Biomaterials 2007, 28, 4192-4199. (11) Nejadnik, M.; van der Mei, H.; Norde, W.; Busscher, H. Bacterial Adhesion and Growth on a Polymer Brush-Coating. Biomaterials 2008, 29, 4117-4121. (12) Hou, S.; Burton, E.; Wu, R.; Luk, Y.; Ren, D. Prolonged Control of Patterned Biofilm Formation by Bio-Inert Surface Chemistry. Chem. Commum. 2009, 10, 12071209. (13) Lichter, J; Thompson, M.; Delgadillo, M.; Nishikawa, T.; Rubner, M.; Van Vliet, K. Substrata

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Biomacromolecules 2008, 9, 1571-1578. (14) Saha, N.; Monge, C.; Dulong, V.; Picart, C.; Glinel, K. Influence of Polyelectrolyte Film Stiffness on Bacterial Growth. Biomacromolecules 2013, 14, 520-528. (15) Bakker, D.; Huijs, F.; de Vries, J.; Klijnstra, J.; Busscher, H.; van der Mei, H. Bacterial Deposition to Fluoridated and Non-Fluoridated Polyurethane Coatings with Different Elastic Modulus and Surface Tension in a Parallel Plate and a Stagnation Point Flow Chamber. Colloids Surf., B 2003, 32, 179-190. (16) Kolewe, K.; Peyton, S.; Schiffman, J. Fewer Bacteria Adhere to Softer Hydrogels. ACS Appl. Mater. Interfaces 2015, 7, 19562-19569. (17) Packham, D. Surface Energy, Surface Topography and Adhesion. Int. J. Adhes. Adhes. 2003, 23, 437-448. (18) Gilber, P. Biofilm Community Interactions: Chance or Necessity? Contributions Made at the Meeting of Biofilm Club, 5, Powys, UK, 2001. Cardiff: BioLine 2001, 1, 1122

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(19) Singh, A.; Vyas, V.; Patil, R.; Sharma, V.; Scopelliti, P.; Bongiorno, G.; Podestà, A.; Lenardi, C.; Gade, W.; Milani, P. Quantitative Characterization of the Influence of the Nanoscale Morphology of Nanostructured Surfaces on Bacterial Adhesion and Biofilm Formation. PLoS one 2011, 6, e25029. (20) Díaz, C.; Cortizo, M.; Schilardi, P.; Saravia, S.; Mele, M. Influence of the NanoMicro Structure of the Surface on Bacterial Adhesion. Mater. Res. 2007, 10, 11-14. (21) Scheuerman, T.; Camper, A.; Hamilton, M. Effects of Substratum Topography on Bacterial Adhesion. J. Colloid Interface Sci. 1998, 208, 23-33. (22) Perni, S.; Prokopovich, P. Micropatterning with Conical Features can Control Bacterial Adhesion on Silicone. Soft Matter 2013, 9, 1844-1851. (23) Crawford, R.; Webb, H.; Truong, V.; Hasan, J.; Ivanova, E. Surface Topographical Factors Influencing Bacterial Attachment. Adv. Colloid Interface Sci. 2012, 179, 142-149. (24) Hou, S.; Gu, H.; Smith, C.; Ren, D. Microtopographic Patterns Affect Escherichia coli Biofilm Formation on Poly (dimethylsiloxane) Surfaces. Langmuir 2011, 27, 26862691. (25) An, Y.; Friedman, R. Concise Review of Mechanisms of Bacterial Adhesion to Biomaterial Surfaces. J. Biomed. Mater. Res. 1998, 43, 338-348. (26) Song, F.; Ren, D. Stiffness of Cross-linked Poly (dimethylsiloxane) Affects Bacterial Adhesion and Antibiotic Susceptibility of Attached Cells. Langmuir 2014, 30, 10354-10362. (27) Belas, R. Biofilms, Flagella, and Mechanosensing of Surfaces by Bacteria. Trends Microbiol. 2014, 22, 517-527.

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(28) Lele, P.; Hosu, B.; Berg, H. Dynamics of Mechanosensing in the Bacterial Flagellar Motor. PNAS 2013, 110, 11839-11844. (29) Petrova, O.; Sauer, K. Sticky Situations: Key Components that Control Bacterial Surface Attachment. J. Bacteriol. 2012, 194, 2413-2425. (30) Parkinson, J.; Houts, S. Isolation and Behavior of Escherichia coli Deletion Mutants Lacking Chemotaxis Functions. J. Bacteriol. 1982, 151, 106-113. (31) Maniatis, T.; Fritsch, E.; Sambrook, J. Molecular Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, 1982. (32) Evans, N.; Minelli, C.; Gentleman, E.; LaPointe, V.; Patankar, S.; Kallivretaki, M.; Chen, X.; Roberts, C.; Stevens, M. Substrate Stiffness Affects Early Differentiation Events in Embryonic Stem Cells. Eur. Cells Mater. 2009, 18, 13-14. (33) Wang, Z. Polydimethylsiloxane Mechanical Properties Measured by Macroscopic Compression and Nanoindentation Techniques. Ph. D. Thesis, University of South Florida, May 2011. (34) Fuard, D.; Tzvetkova-Chevolleau, T.; Decossas, S.; Tracqui, P.; Schiavone, P. Optimization of Polydimethylsiloxane (PDMS) Substrates for Studying Cellular Adhesion and Motility. Microelectron. Eng. 2008, 85, 1289-1293. (35) Chen, C.; Nace, G.; Irwin, P. A 6× 6 Drop Plate Method for Simultaneous Colony Counting and MPN Enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli. J. Microbiol. Methods 2003, 55, 475-479. (36) Baker, R.; Brasch, M.; Manning, M.; Henderson, J. Automated, Contour-based Tracking and Analysis of Cell Behaviour over Long Time Scales in Environments of Varying Complexity and Cell Density. J. R. Soc., Interface 2014, 11, 20140386.

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(37) Gu, H.; Chen, A.; Song, X.; Brasch, M.; Henderson, J.; Ren, D. How Escherichia coli Lands and Forms Cell Clusters on a Surface: a New Role of Surface Topography. Sci. Rep. 2016, 6, 29516. (38) Hou, S.; Burton, E.; Simon, K.; Blodgett, D.; Luk, Y.; Ren, D. Inhibition of Escherichia coli Biofilm Formation by Self-assembled Monolayers of Functional Alkanethiols on Gold. Appl. Environ. Microbiol. 2007, 73, 4300-4307. (39) Pratt, L.; Kolter, R. Genetic Analysis of Escherichia coli Biofilm Formation: Roles of Flagella, Motility, Chemotaxis and Type I Pili. Mol. Microbiol. 1998, 30, 285-293. (40) Busscher, H.; van der Mei, H. How do Bacteria Know They are on a Surface and Regulate Their Response to an Adhering State? PLoS Pathog. 2012, 8, e1002440. (41) Jiang, F. Exploratory Study of Pre-osteoblastic Cell Behaviour on PDMS Substrates with Tunable Stiffness. Ph.D. Thesis, Université catholique de Louvain, May 2010. (42) Mata, A.; Fleischman, A.; Roy, S. Characterization of Polydimethylsiloxane (PDMS) Properties for Biomedical Micro/Nanosystems. Biomed. Microdevices 2005, 7, 281-293. (43) Catipovic, M.; Tyler, P.; Trapani, J.; Carter, A. Improving the Quantification of Brownian Motion. Am. J. Phys. 2013, 81, 485-491. (44) Chun, S.; Parkinson, J. Bacterial Motility: Membrane Topology of the Escherichia coli MotB Protein. Science 1988, 239, 276-278.

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Table 1. List of E. coli strains and plasmids used in this study. E. coli strains Genotype and plasmids E. coli Strains RP437 thr-1(Am) leuB6 his-4 metF159(Am) eda-50 rpsL1356 thi-1ara14mtl-1 xyl-5 tonA31 tsx-78 lacY1 FRP3087 RP437 motB ∆fliC

RP437 ∆fliC

∆fimA

RP437 ∆fliA

Plasmids pRSH103 pRHG03 pGLO

Tetr, constitutive rfp motB complementation Ampr, araC promoter

Characteristics

Source/ Reference

Wild-type strain for biofilm study

(30)

Motility mutant (point mutation in motB) Flagella mutant (deletion mutation of fliC) Type I fimbriae mutant (deletion mutation of fimA)

(30)

Red fluorescence motB complementation Green fluorescence

(24) (37) (38)

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(37)

(37)

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Figure 1. Validation of ACTIVE for tracking motile bacterial cells. (A) Image of E. coli RP437/pGLO (shown in gray scale). (B) E. coli RP437/pRSH103 cells identified by ACTIVE. (C) Contour profiles were obtained based on the fluorescence intensity. (D) Cell tracks over 12 min (each cell was assigned a separate color).

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Figure 2. Three types of movement of E. coli cells on surfaces categorized by ACTIVE (bar = 2 µm). “still” refers to the cells that the mass center did not move more than a quarter of the body length during any 5 s interval; “rotating” refers to the cells that the mass center moved more than a quarter but less than one body length during any 5 s interval; “moving” refers to the cells that the mass center moved more than one body length during any 5 s interval.

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Figure 3. Effect of material stiffness on the motility of attached E. coli cells. (A) Percentages of “still”, “rotating” and “moving” cells during attachment of the wild-type cells from a representative sample. (B) Movement speed of the wild-type cells on PDMS surfaces. Each dot represents a cell, and each black line shows the average velocity of all cells at a given time point.

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Figure 4. Attachment of the wild-type strain and its mutants of motB, fimA, and fliC on (A) face-up and (B) face-down 5:1 PDMS (2.6 MPa, stiff) and 40:1 PDMS (0.1 MPa, soft) after 2 h attachment in PBS. The cell number of each strain on 40:1 PDMS was normalized as 100%.

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Figure 5: Effect of material stiffness on the motility of attached motB mutant cells. (A) Percentages of “still”, “rotating” and “moving” motB mutant cells attachment from a representative sample. (B) Movement speed of the motB mutant on PDMS surfaces. Each dot represents a cell, and each black line shows the average velocity of all cells at a given time point.

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Figure 6: Effect of material stiffness on the motility of attached E. coli motB complemented cells. (A) Percentages of “still”, “rotating” and “moving” cells of the E. coli motB complemented strain from a representative sample. (B) Movement speed of the wild-type E. coli motB complemented cells on PDMS surfaces. Each dot represents a cell, and each black lines shows the average velocity of all cells at a given time point.

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Figure 7. Motility of the wild type strain, its motB mutant, and motB complemented strain on PDMS with different levels of stiffness. (A) Percentages of “still”, “rotating” and “moving” cells during attachment. The results were calculated by averaging the data from at least 320 frames in 3 movies. Error bars represent standard error. (B) Movement speed of the wild-type strain, its motB mutant and motB complemented strain on 40:1 and 5:1 PDMS surfaces. Each bar represents the average speed of all the cells under the given condition. The results were calculated from at least 200 cells in 3 movies (~20 min long for each) excluding the cells with zero velocity. Bars represent standard errors. (C) Distributions of movement speeds of the wild-type strain, its motB mutant, and motB 30 ACS Paragon Plus Environment

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complement strains on 40:1 and 5:1 PDMS. Both the wild-type and motB mutant were engineered to carry the empty vector for comparison with the complemented strain.

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