Optogenetics reprogramming of planktonic cells for biofilm formation

Single-cell behaviors play essential roles during early-stage biofilms formation. In this study, we evaluated whether biofilm formation could be guided by precisely manipulating single cells behaviors. Thus, we established an illumination method to precisely manipulate the type IV pili (TFP) mediated motility and microcolony formation of Pseudomonas aeruginosa by using a combination of a high-throughput bacterial tracking algorithm, optogenetic manipulation and adaptive microscopy. We termed this method as Adaptive Tracking Illumination (ATI). We reported that ATI enables the precise manipulation of TFP mediated motility and microcolony formation during biofilm formation by manipulating bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) levels in single cells. Moreover, we showed that the spatial organization of single cells in mature biofilms can be controlled using ATI. Thus, the established method (i.e., ATI) can markedly promote ongoing studies of biofilms.


Introduction
This real-time information was further analyzed to identify mobile and immobile cells. Thereafter, 74 information on the regions containing selected cells with the motility phenotype of interest was 75 input into a digital micromirror device to generate a mask. The mask was finally projected on these 76 selected cells through an additional objective. We termed this illumination as ATI (Figure 1a 88 ATI enables precise manipulation of TFP-meditated motility and microcolony formation of 89 single cells. 90 To directly manipulate the TFP-meditated motility of the selected single cells, we incorporated an 91 optogenetic part into the chromosome of P. aeruginosa by using the mini-CTX system (Hoang,92 Kutchma, Becher, & Schweizer, 2000). This part encodes a heme oxygenase (bphO) and c-di-  The c-di-GMP reporter encodes two fluorescent proteins. A green fluorescent protein (GFP) is 101 expressed using a c-di-GMP regulatory promoter (PcdrA), and a red fluorescent protein (mCherry) 102 is expressed using a constitutive promoter (PA1O4O3; Figure 2a). Thus, the ratio of fluorescent 103 intensities derived from GFP (FG) and mCherry (FR) was used to determine the c-di-GMP levels 7 / 29 in single cells. We observed that c-di-GMP levels, as indicated by the FG/FR ratio, markedly increa-  were carried out at least three times and one representative example is shown. 121 We first selected 33-66% of single cells with mobile motility and illuminated them (0.05 122 mW·cm −2 ) along with their offspring for 7 hours by using ATI. Treatment with ATI for 3 hours  surfaces, whereas another cell preferred to detach from surfaces.

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Simultaneously, we observed that 7 hours of ATI treatment considerably increased the c-di-GMP 136 levels in these illuminated cells and their offspring, as indicated by the FG/FR ratio, which increased  indicates higher c-di-GMP level. Scale bar were set at 5μm. The c-di-GMP level from one mother 12 / 29 1). Thereafter, we continuously cultured these young biofilms (up to 72 hours) with a distinct 175 organization of cells in dark to allow their maturation. These distinct young biofilms developed to 176 mature biofilms possessing a distinct spatial organization of GFP-or RFP-labeled cells ( Figure   177 4b). These results revealed that cell manipulation by using ATI during early-stage biofilm were carried out at least three times and one representative image is depicted. 190 We developed the ATI method that could be used to precisely manipulate TFP-mediated motility 191 of single P. aeruginosa cells during early-stage biofilm formation. In this method, the bacteria 192 were modified using an optogenetic part that enabled illumination with near-infrared light to 193 directly regulate intracellular c-di-GMP levels. We showed that ATI could manipulate single cells 194 with a mobile phenotype to switch to an immobile phenotype. Consequently, these manipulated 195 cells could stall in their place to form microcolonies in advance, whereas unmanipulated cells with 196 a mobile phenotype were more likely to move and spread on surfaces, facilitating the control of 197 the location and time of early-stage biofilm formation. Accordingly, we showed that the spatial 198 organization of single cells could be precisely controlled in a young biofilm of P. aeruginosa cells.

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Notably, our results indicated that the organization of single cells in young biofilms affected 200 subsequent cell organization in mature biofilms, which enabled the further control of the structure 201 and spatial organization of cells in biofilms. It should be emphasized that the strategy used herein 202 to control the spatial organization of bacterial colonies is different than that using micro-three-203 dimensional printing (Connell, Kim, Shear, Bard, & Whiteley, 2014).

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In addition to using ATI to guide biofilm formation, we expect that our method can be used to 205 answer various questions or resolve problems in microbiology. This is because: 1) The hardware 206 used to build ATI, mainly an air objective, a commercial projector, and an LED controller, are  Notably, the time required for data processing limits the application of ATI for investigating a 217 quickly evolving bio-systems or cellular processes. For example, data processing of a live image 218 (processed in a commercial desktop equipped with an intel i7 CPU) in the present study typically 219 took 3 seconds, limiting the use of ATI for manipulating rapidly swimming bacteria, whose 220 velocity can typically reach tens of microns per second. In addition to the data processing speed, 221 the accuracy of the tracking algorithm limits the application of ATI for investigating microbes 222 with high cell densities. For example, the current ATI cannot be used to manipulate the phenotypes track single cells in a dense microcolony. Developing new tracking algorithms or using powerful 225 computers can address these limitations, thus considerably expanding ATI applications.

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Strains and Growth conditions 228 Bacterial strains and plasmids used in this study are listed in Table 1. Strains were grown on LB (1:100) in fresh FAB medium to OD600 = 0.4~0.6 before used.

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Insertion mutant bphS was constructed by mini-CTX system using a modified procedure for  Table 1.

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We schematically show the setup of the Adaptive Tracking Illuminations (ATI) (Figure 1a). More 255 specifically, an inverted fluorescent microscope (Olympus, IX71) was modified to build the ATI.

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The modification includes that: 1) A commercial DMD-based LED projector (Gimi Z3) was used sCMOS camera was used to collect bright-field or fluorescent images with 0.2 or 1/1800 frame 300 rate respectively. The cells with green or red fluorescence were selected respectively to be 301 manipulated using ATI with a power density of 0.05 mW·cm -2 during the first 10 hours.

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Afterwards, the flow cell contains the distinctive young biofilms were continuously cultured up to 303 3 days in the dark to allow these young biofilms to mature. Finally, a laser-scan confocal 304 microscope (Olympus FV1000) equipped with a 100×oil objective was used to image the cells 305 organizations as well as the three-dimensional (3D) structures of the mature biofilms using z-axis 306 scanning (0.5 µm per step). The confocal images acquired in different z-positions were used to 307 reconstruct the structure of mature biofilms using software ImageJ. Experiments of biofilm 308 cultivation were carried out at least three times.      increased after using ATI for 7 hours, which is sharply contrast to that the fluorescence intensity 419 of GFP mut3* in those unilluminated mobile cells remain in low.