An adaptive tracking illumination system for optogenetic control of single bacterial cells.

Single-cell behaviors are essential during early-stage biofilm formation. In this study, we aimed to evaluate whether single-cell behaviors could be precisely and continuously manipulated by optogenetics. We thus established adaptive tracking illumination (ATI), a novel illumination method to precisely manipulate the gene expression and bacterial behavior of Pseudomonas aeruginosa on the surface at the single-cell level by using the combination of a high-throughput bacterial tracking algorithm, optogenetic manipulation, and adaptive microscopy. ATI enables precise gene expression control by manipulating the optogenetic module gene expression and type IV pili (TFP)-mediated motility and microcolony formation during biofilm formation through bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) level modifications in single cells. Moreover, we showed that the spatial organization of single cells in mature biofilms could be controlled using ATI. Therefore, this novel method we established might markedly answer various questions or resolve problems in microbiology. KEY POINTS: • High-resolution spatial and continuous optogenetic control of individual bacteria. • Phenotype-specific optogenetic control of individual bacteria. • Capacity to control biologically relevant processes in engineered single cells.

Carbon Starvation Induces the Expression of PprB-Regulated Genes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa can cause severe infections in humans. This bacterium often adopts a biofilm lifestyle that is hard to treat. In several previous studies, the PprA-PprB two-component system (TCS), which controls the expression of type IVb pili, BapA adhesin, and CupE fimbriae, was shown to be involved in biofilm formation (M. Romero, H. Silistre, L. Lovelock, V. J. Wright, K.-G. Chan, et al., Nucleic Acids Res 46:6823-6840, 2018, https://doi.org/10.1093/nar/gky324; S. de Bentzmann, C. Giraud, C. S. Bernard, V. Calderon, F. Ewald F, et al., PLoS Pathog 8:e1003052, 2012, https://doi.org/10.1371/journal.ppat.1003052). However, signals or environmental conditions that can trigger the PprA-PprB TCS are still unknown, and the molecular mechanisms of PprB-mediated biofilm formation are poorly characterized. Here, we report that carbon starvation stress (CSS) can induce the expression of pprB and genes in the PprB regulon. CSS-induced pprB transcription is mediated by the stress response sigma factor RpoS rather than the two-component sensor PprA. We also observed a strong negative regulation of PprB on the transcription of itself. Further experiments showed that PprB overexpression greatly enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa Specifically, under the background of PprB overexpression, both the BapA adhesin and CupE fimbriae displayed positive effects on CCA and CSA, while the type IVb pili showed an unexpected negative effect on CCA and no effect on CSA. In addition, expression of the PprB regulon genes were significantly increased in 3-day colony biofilms, indicating a possible carbon limitation state. The CSS-RpoS-PprB-Bap/Flp/CupE pathway identified in this study provides a new perspective on the process of biofilm formation in carbon-limited environments.IMPORTANCE Typically, the determination of the external signals that can trigger a regulatory system is crucial to understand the regulatory logic and inward function of that system. The PprA-PprB two-component system was reported to be involved in biofilm formation in Pseudomonas aeruginosa, but the signals triggering this system are unknown. In this study, we found that carbon starvation stress (CSS) induces transcription of pprB and genes in the PprB regulon through an RpoS-dependent pathway. Increased PprB expression leads to enhanced cell-cell adhesion (CCA) and cell-surface adhesion (CSA) in P. aeruginosa Both CCA and CSA are largely dependent on the Bap secretion system and are moderately dependent on the CupE fimbriae. Our findings suggest that PprB reinforces the structure of biofilms under carbon-limited conditions, and the Bap secretion system and CupE fimbriae are two potential targets for biofilm treatment.

Visual Multifunctional Aggregation-Induced Emission-Based Bacterial Cellulose for Killing of Multidrug-Resistant Bacteria.

Multidrug-resistant (MDR) bacteria-related wound infections are a thorny issue. It is urgent to develop new antibacterial wound dressings that can not only prevent wounds from MDR bacteria infection but also promote wound healing. Herein, an aggregation-induced emission (AIE) molecule BITT-composited bacterial cellulose (BC) is presented as wound dressings. BC-BITT composites have good transparency, making it easy to monitor the wound healing process through the composite membrane. The BC-BITT composites retain the advantages of biocompatible BC, and display photodynamic and photothermal synergistic antibacterial effects under irradiation of a 660 nm laser. Furthermore, the BC-BITT composites show excellent wound healing performance in a mouse full-thickness skin wound model infected by MDR bacteria, simultaneously with negligible toxicity. This work paves a way for treating clinically troublesome wound infections.

High-throughput, microscopy-based screening and quantification of genetic elements.

Synthetic biology relies on the screening and quantification of genetic components to assemble sophisticated gene circuits with specific functions. Microscopy is a powerful tool for characterizing complex cellular phenotypes with increasing spatial and temporal resolution to library screening of genetic elements. Microscopy-based assays are powerful tools for characterizing cellular phenotypes with spatial and temporal resolution and can be applied to large-scale samples for library screening of genetic elements. However, strategies for high-throughput microscopy experiments remain limited. Here, we present a high-throughput, microscopy-based platform that can simultaneously complete the preparation of an 8 × 12-well agarose pad plate, allowing for the screening of 96 independent strains or experimental conditions in a single experiment. Using this platform, we screened a library of natural intrinsic promoters from Pseudomonas aeruginosa and identified a small subset of robust promoters that drives stable levels of gene expression under varying growth conditions. Additionally, the platform allowed for single-cell measurement of genetic elements over time, enabling the identification of complex and dynamic phenotypes to map genotype in high throughput. We expected that the platform could be employed to accelerate the identification and characterization of genetic elements in various biological systems, as well as to understand the relationship between cellular phenotypes and internal states, including genotypes and gene expression programs.

Optogenetic Modification of Pseudomonas aeruginosa Enables Controllable Twitching Motility and Host Infection.

Cyclic adenosine monophosphate (cAMP) is an important secondary messenger that controls carbon metabolism, type IVa pili biogenesis, and virulence in Pseudomonas aeruginosa. Precise manipulation of bacterial intracellular cAMP levels may enable tunable control of twitching motility or virulence, and optogenetic tools are attractive because they afford excellent spatiotemporal resolution and are easy to operate. Here, we developed an engineered P. aeruginosa strain (termed pactm) with light-dependent intracellular cAMP levels through introducing a photoactivated adenylate cyclase gene (bPAC) into bacteria. On blue light illumination, pactm displayed a 15-fold increase in the expression of the cAMP responsive promoter and an 8-fold increase in its twitching activity. The skin lesion area of nude mouse in a subcutaneous infection model after 2-day pactm inoculation was increased 14-fold by blue light, making pactm suitable for applications in controllable bacterial host infection. In addition, we achieved directional twitching motility of pactm colonies through localized light illumination, which will facilitate the studies of contact-dependent interactions between microbial species.

A Synthetic Genetic Circuit Enables Precise Quantification of Direct Repeat Deletion in Bacteria.

Quantification of the rate of direct repeat deletion (DRD) is an important aspect in the research of DNA rearrangement. The widely used tetracycline selection method usually introduces antibiotic pressure to the tested organism, which may interfere with the DRD process. Also the length of repeat arm (LRA) is limited by the length of the TetR coding sequence. On the basis of the fluorescent microscopy and high-throughput imaging processing, here we developed a two-module genetic circuit, termed TFDEC (which stands for three-color fluorescence-based deletion event counter), to quantify the DRD rate under neutral conditions. DRD events were determined from the state of a three-state fluorescent logic gate constructed through coupling of an OR gate and an AND gate. TFDEC was applied in Pseudomonas aeruginosa, and we found that the DRD rate was RecA-dependent for long repeat arms (>500 bp) and RecA-independent for short repeat arms (<500 bp), which was consistent with the case in Escherichia coli. In addition, the increase of DRD rate followed an S-shaped curve with the increase of LRA, while treating cells with ciprofloxacin did not change the LRA-dependence of DRD. We also detected a significant increased DRD rate for long repeat arms in the uvrD (8-fold) and radA (4-fold) mutants. Our results show that the TFDEC method could be used as a complement tool for quantification of the DRD rate in the future.

Simultaneous Visualization of Multiple Gene Expression in Single Cells Using an Engineered Multicolor Reporter Toolbox and Approach of Spectral Crosstalk Correction.

Synthetic biology aims to make biology easier to engineer and focuses on the design and construction of core components that can be modeled, understood, and tuned to meet specific performance criteria, and the assembly of these smaller parts and devices into larger integrated systems to solve specific problems. Here, we designed and engineered a multicolor fluorescent reporter toolbox to simultaneously monitor the activities of multiple genes in single cells. The toolbox contained standardized and well-characterized genetic building blocks for the convenient and reproducible assembly of multiple promoter-reporter fusions (ranging from 1 to 4) into a single plasmid. Given the common problem of spectral crosstalk among multiple fluorescent proteins, we deciphered multiple spectral signatures within cells through a deduced linear unmixing algorithm. Our approach enabled the quantification of gene expression with direct FP concentrations, instead of mix-contributed fluorescence intensities, thus enabling true signal separation with high confidence. This approach performed well in the imaging of mixing cells with single FP labels. Additionally, combining with the multicolor toolbox, we succeeded in simultaneously monitoring the genetic dynamics of four selected quorum-sensing genes in response to the induction of two exogenously added autoinducers and were able to examine gene regulatory connections within the QS signaling network in Pseudomonas aeruginosa. Overall, this synthetic framework (i.e., the genetic toolbox and the well-evaluated approach of spectral correction) will be useful for applied synthetic biology projects, multicolor imaging, and analyzing interactions of multiple genes of natural genetic networks or assembling synthetic ones.

Bioprinting Living Biofilms through Optogenetic Manipulation.

In this paper, we present a new strategy for microprinting dense bacterial communities with a prescribed organization on a substrate. Unlike conventional bioprinting techniques that require bioinks, through optogenetic manipulation, we directly manipulated the behaviors of Pseudomonas aeruginosa to allow these living bacteria to autonomically form patterned biofilms following prescribed illumination. The results showed that through optogenetic manipulation, patterned bacterial communities with high spatial resolution (approximately 10 µm) could be constructed in 6 h. Thus, optogenetic manipulation greatly increases the range of available bioprinting techniques.