Fluid flow overcomes antimicrobial resistance by boosting delivery.

Antimicrobial resistance is an emerging global threat to humanity. As resistance outpaces development, new perspectives are required. For decades, scientists have prioritized chemical optimization, while largely ignoring the physical process of delivery. Here, we used biophysical simulations and microfluidic experiments to explore how fluid flow delivers antimicrobials into communities of the highly resistant pathogen Pseudomonas aeruginosa . We discover that increasing flow overcomes bacterial resistance towards three chemically distinct antimicrobials: hydrogen peroxide, gentamicin, and carbenicillin. Without flow, resistant P. aeruginosa cells generate local zones of depletion by neutralizing all three antimicrobials through degradation or chemical modification. As flow increases, delivery overwhelms neutralization, allowing antimicrobials to regain effectiveness against resistant bacteria. Additionally, we discover that cells on the edge of a community shield internal cells, and cell-cell shielding is abolished in higher flow regimes. Collectively, our quantitative experiments reveal the unexpected result that physical flow and chemical dosage are equally important to antimicrobial effectiveness. Thus, our results should inspire the incorporation of flow into the discovery, development, and implementation of antimicrobials, and could represent a new strategy to combat antimicrobial resistance.

Shear force enhances adhesion of Pseudomonas aeruginosa by counteracting pilus-driven surface departure.

Fluid flow is thought to prevent bacterial adhesion, but some bacteria use adhesins with catch bond properties to enhance adhesion under high shear forces. However, many studies on bacterial adhesion either neglect the influence of shear force or use shear forces that are not typically found in natural systems. In this study, we use microfluidics and single-cell imaging to examine how the human pathogen Pseudomonas aeruginosa interacts with surfaces when exposed to shear forces typically found in the human body (0.1 pN to 10 pN). Through cell tracking, we demonstrate that the angle between the cell and the surface predicts if a cell will depart the surface. We discover that at lower shear forces, type IV pilus retraction tilts cells away from the surface, promoting surface departure. Conversely, we show that higher shear forces counterintuitively enhance adhesion by counteracting type IV pilus retraction-dependent cell tilting. Thus, our results reveal that P. aeruginosa exhibits behavior reminiscent of a catch bond, without having a specific adhesin that is enhanced by force. Instead, P. aeruginosa couples type IV pilus dynamics and cell geometry to tune adhesion to its mechanical environment, which likely provides a benefit in dynamic host environments.

Type-IV pili tune an adhesion-migration trade-off during surface colonization of Pseudomonas aeruginosa.

Bacterial pathogenicity relies on both firm surface adhesion and cell dissemination. How twitching bacteria resolve the fundamental contradiction between adhesion and migration is unknown. To address this question, we employ live-cell imaging of type-IV pili (T4P) and therewith construct a comprehensive mathematical model of Pseudomonas aeruginosa migration. The data show that only 10% to 50% of T4P bind to substrates and contribute to migration through random extension and retraction. Individual T4P do not display a measurable sensory response to surfaces, but their number increases on cellular surface contact. Attachment to surfaces is mediated, besides T4P, by passive adhesive forces acting on the cell body. Passive adhesions slow down cell migration and result in local random motion on short time scales, which is followed by directionally persistent, superdiffusive motion on longer time scales. Moreover, passive adhesions strongly enhance surface attachment under shear flow. Δ pilA mutants, which produce no T4P, robustly stick to surfaces under shear flow. In contrast, rapidly migrating Δ pilH cells, which produce an excessive number of T4P, are easily detached by shear. Wild-type cells sacrifice migration speed for robust surface attachment by maintaining a low number of active pili. The different cell strains pertain to disjunct regimes in a generic adhesion-migration trait space. Depending on the nature of the adhesion structures, adhesion and migration are either compatible or a trade-off is required for efficient bacterial surface colonization under different conditions.

Shear force enhances adhesion of Pseudomonas aeruginosa by counteracting pilus-driven surface departure.

Fluid flow is thought to prevent bacterial adhesion, but some bacteria use adhesins with catch bond properties to enhance adhesion under high shear forces. However, many studies on bacterial adhesion either neglect the influence of shear force or use shear forces that are not typically found in natural systems. In this study, we use microfluidics and single-cell imaging to examine how the human pathogen Pseudomonas aeruginosa interacts with surfaces when exposed to shear forces typically found in the human body (0.1 pN to 10 pN). Through cell tracking, we demonstrate that the angle between the cell and the surface predicts if a cell will depart the surface. We discover that at lower shear forces, type IV pilus retraction tilts cells away from the surface, promoting surface departure. Conversely, we show that higher shear forces counterintuitively enhance adhesion by counteracting type IV pilus retraction-dependent cell tilting. Thus, our results reveal that P. aeruginosa exhibits behavior reminiscent of a catch bond, without having a specific adhesin that is enhanced by force. Instead, P. aeruginosa couples type IV pilus dynamics and cell geometry to tune adhesion to its mechanical environment, which likely provides a benefit in dynamic host environments.

Shear rate sensitizes bacterial pathogens to H2O2 stress.

Cells regularly experience fluid flow in natural systems. However, most experimental systems rely on batch cell culture and fail to consider the effect of flow-driven dynamics on cell physiology. Using microfluidics and single-cell imaging, we discover that the interplay of physical shear rate (a measure of fluid flow) and chemical stress trigger a transcriptional response in the human pathogen Pseudomonas aeruginosa. In batch cell culture, cells protect themselves by quickly scavenging the ubiquitous chemical stressor hydrogen peroxide (H2O2) from the media. In microfluidic conditions, we observe that cell scavenging generates spatial gradients of H2O2. High shear rates replenish H2O2, abolish gradients, and generate a stress response. Combining mathematical simulations and biophysical experiments, we find that flow triggers an effect like "wind-chill" that sensitizes cells to H2O2 concentrations 100 to 1,000 times lower than traditionally studied in batch cell culture. Surprisingly, the shear rate and H2O2 concentration required to generate a transcriptional response closely match their respective values in the human bloodstream. Thus, our results explain a long-standing discrepancy between H2O2 levels in experimental and host environments. Finally, we demonstrate that the shear rate and H2O2 concentration found in the human bloodstream trigger gene expression in the blood-relevant human pathogen Staphylococcus aureus, suggesting that flow sensitizes bacteria to chemical stress in natural environments.

Bacteria in Fluid Flow.

Bacteria thrive in environments rich in fluid flow, such as the gastrointestinal tract, bloodstream, aquatic systems, and the urinary tract. Despite the importance of flow, how flow affects bacterial life is underappreciated. In recent years, the combination of approaches from biology, physics, and engineering has led to a deeper understanding of how bacteria interact with flow. Here, we highlight the wide range of bacterial responses to flow, including changes in surface adhesion, motility, surface colonization, quorum sensing, virulence factor production, and gene expression. To emphasize the diversity of flow responses, we focus our review on how flow affects four ecologically distinct bacterial species: Escherichia coli, Staphylococcus aureus, Caulobacter crescentus, and Pseudomonas aeruginosa. Additionally, we present experimental approaches to precisely study bacteria in flow, discuss how only some flow responses are triggered by shear force, and provide perspective on flow-sensitive bacterial signaling.

Corner Flows Induced by Surfactant-Producing Bacteria Bacillus subtilis and Pseudomonas fluorescens.

A mechanistic understanding of bacterial spreading in soil, which has both air and water in angular pore spaces, is critical to control pathogenic contamination of soil and to design bioremediation projects. A recent study (J. Q. Yang, J. E. Sanfilippo, N. Abbasi, Z. Gitai, et al., Proc Natl Acad Sci U S A 118:e2111060118, 2021, https://doi.org/10.1073/pnas.2111060118) shows that Pseudomonas aeruginosa can self-generate flows along sharp corners by producing rhamnolipids, a type of biosurfactants that change the hydrophobicity of solid surfaces. We hypothesize that other types of biosurfactants and biosurfactant-producing bacteria can also generate corner flows. Here, we first demonstrate that rhamnolipids and surfactin, biosurfactants with different chemical structures, can generate corner flows. We identify the critical concentrations of these two biosurfactants to generate corner flow. Second, we demonstrate that two common soil bacteria, Pseudomonas fluorescens and Bacillus subtilis (which produce rhamnolipids and surfactin, respectively), can generate corner flows along sharp corners at the speed of several millimeters per hour. We further show that a surfactin-deficient mutant of B. subtilis cannot generate corner flow. Third, we show that, similar to the finding for P. aeruginosa, the critical corner angle for P. fluorescens and B. subtilis to generate corner flows can be predicted from classic corner flow theories. Finally, we show that the height of corner flows is limited by the roundness of corners. Our results suggest that biosurfactant-induced corner flows are prevalent in soil and should be considered in the modeling and prediction of bacterial spreading in soil. The critical biosurfactant concentrations we identify and the mathematical models we propose will provide a theoretical foundation for future predictions of bacterial spreading in soil. IMPORTANCE The spread of bacteria in soil is critical in soil biogeochemical cycles, soil and groundwater contamination, and the efficiency of soil-based bioremediation projects. However, the mechanistic understanding of bacterial spreading in soil remains incomplete due to a lack of direct observations. Here, we simulate confined spaces of hydrocarbon-covered soil using a transparent material with similar hydrophobicity and visualize the spread of two common soil bacteria, Pseudomonas fluorescens and Bacillus subtilis. We show that both bacteria can generate corner flows at the velocity of several millimeters per hour by producing biosurfactants, soap-like chemicals. We provide quantitative equations to predict the critical corner angle for bacterial corner flow and the maximum distance of the corner spreading. We anticipate that bacterial corner flow is prevalent because biosurfactant-producing bacteria and angular pores are common in soil. Our results will help improve predictions of bacterial spreading in soil and facilitate the design of soil-related bioremediation projects.

Multiple Photolyases Protect the Marine Cyanobacterium Synechococcus from Ultraviolet Radiation.

Marine cyanobacteria depend on light for photosynthesis, restricting their growth to the photic zone. The upper part of this layer is exposed to strong UV radiation (UVR), a DNA mutagen that can harm these microorganisms. To thrive in UVR-rich waters, marine cyanobacteria employ photoprotection strategies that are still not well defined. Among these are photolyases, light-activated enzymes that repair DNA dimers generated by UVR. Our analysis of genomes of 81 strains of Synechococcus, Cyanobium, and Prochlorococcus isolated from the world's oceans shows that they possess up to five genes encoding different members of the photolyase/cryptochrome family, including a photolyase with a novel domain arrangement encoded by either one or two separate genes. We disrupted the putative photolyase-encoding genes in Synechococcus sp. strain RS9916 and discovered that each gene contributes to the overall capacity of this organism to survive UVR. Additionally, each conferred increased survival after UVR exposure when transformed into Escherichia coli lacking its photolyase and SOS response. Our results provide the first evidence that this large set of photolyases endows Synechococcus with UVR resistance that is far superior to that of E. coli, but that, unlike for E. coli, these photolyases provide Synechococcus with the vast majority of its UVR tolerance. IMPORTANCE Cells use DNA photolyases to protect their DNA from the damaging effects of UV radiation. Marine cyanobacteria possess many genes that appear to encode photolyases, but the function of the proteins encoded by these genes is unclear. The study uses comparative genomics and molecular genetic approaches to describe and characterize the roles of these proteins in DNA damage repair in the marine cyanobacterium Synechococcus. This study identifies the important role of DNA photolyases in DNA repair for these cells and describes a previously undescribed structural class of DNA of these enzymes.

Evidence for biosurfactant-induced flow in corners and bacterial spreading in unsaturated porous media.

The spread of pathogenic bacteria in unsaturated porous media, where air and liquid coexist in pore spaces, is the major cause of soil contamination by pathogens, soft rot in plants, food spoilage, and many pulmonary diseases. However, visualization and fundamental understanding of bacterial transport in unsaturated porous media are currently lacking, limiting the ability to address the above contamination- and disease-related issues. Here, we demonstrate a previously unreported mechanism by which bacterial cells are transported in unsaturated porous media. We discover that surfactant-producing bacteria can generate flows along corners through surfactant production that changes the wettability of the solid surface. The corner flow velocity is on the order of several millimeters per hour, which is the same order of magnitude as bacterial swarming, one of the fastest known modes of bacterial surface translocation. We successfully predict the critical corner angle for bacterial corner flow to occur based on the biosurfactant-induced change in the contact angle of the bacterial solution on the solid surface. Furthermore, we demonstrate that bacteria can indeed spread by producing biosurfactants in a model soil, which consists of packed angular grains. In addition, we demonstrate that bacterial corner flow is controlled by quorum sensing, the cell-cell communication process that regulates biosurfactant production. Understanding this previously unappreciated bacterial transport mechanism will enable more accurate predictions of bacterial spreading in soil and other unsaturated porous media.

CpeY is a phycoerythrobilin lyase for cysteine 82 of the phycoerythrin I α-subunit in marine Synechococcus.

Marine Synechococcus are widespread in part because they are efficient at harvesting available light using their complex antenna, or phycobilisome, composed of multiple phycobiliproteins and bilin chromophores. Over 40% of Synechococcus strains are predicted to perform a type of chromatic acclimation that alters the ratio of two chromophores, green-light-absorbing phycoerythrobilin and blue-light-absorbing phycourobilin, to optimize light capture by phycoerythrin in the phycobilisome. Lyases are enzymes which catalyze the addition of bilin chromophores to specific cysteine residues on phycobiliproteins and are involved in chromatic acclimation. CpeY, a candidate lyase in the model strain Synechococcus sp. RS9916, added phycoerythrobilin to cysteine 82 of only the α subunit of phycoerythrin I (CpeA) in the presence or absence of the chaperone-like protein CpeZ in a recombinant protein expression system. These studies demonstrated that recombinant CpeY attaches phycoerythrobilin to as much as 72% of CpeA, making it one of the most efficient phycoerythrin lyases characterized to date. Phycobilisomes from a cpeY- mutant showed a near native bilin composition in all light conditions except for a slight replacement of phycoerythrobilin by phycourobilin at CpeA cysteine 82. This demonstrates that CpeY is not involved in any chromatic acclimation-driven chromophore changes and suggests that the chromophore attached at cysteine 82 of CpeA in the cpeY- mutant is ligated by an alternative phycoerythrobilin lyase. Although loss of CpeY does not greatly inhibit native phycobilisome assembly in vivo, the highly active recombinant CpeY can be used to generate large amounts of fluorescent CpeA for biotechnological uses.

Chromatic Acclimation in Cyanobacteria: A Diverse and Widespread Process for Optimizing Photosynthesis.

Chromatic acclimation (CA) encompasses a diverse set of molecular processes that involve the ability of cyanobacterial cells to sense ambient light colors and use this information to optimize photosynthetic light harvesting. The six known types of CA, which we propose naming CA1 through CA6, use a range of molecular mechanisms that likely evolved independently in distantly related lineages of the Cyanobacteria phylum. Together, these processes sense and respond to the majority of the photosynthetically relevant solar spectrum, suggesting that CA provides fitness advantages across a broad range of light color niches. The recent discoveries of several new CA types suggest that additional CA systems involving additional light colors and molecular mechanisms will be revealed in coming years. Here we provide a comprehensive overview of the currently known types of CA and summarize the molecular details that underpin CA regulation.

Microfluidic-based transcriptomics reveal force-independent bacterial rheosensing.

Multiple cell types sense fluid flow as an environmental cue. Flow can exert shear force (or stress) on cells, and the prevailing model is that biological flow sensing involves the measurement of shear force1,2. Here, we provide evidence for force-independent flow sensing in the bacterium Pseudomonas aeruginosa. A microfluidic-based transcriptomic approach enabled us to discover an operon of P. aeruginosa that is rapidly and robustly upregulated in response to flow. Using a single-cell reporter of this operon, which we name the flow-regulated operon (fro), we establish that P. aeruginosa dynamically tunes gene expression to flow intensity through a process we call rheosensing (as rheo- is Greek for flow). We further show that rheosensing occurs in multicellular biofilms, involves signalling through the alternative sigma factor FroR, and does not require known surface sensors. To directly test whether rheosensing measures force, we independently altered the two parameters that contribute to shear stress: shear rate and solution viscosity. Surprisingly, we discovered that rheosensing is sensitive to shear rate but not viscosity, indicating that rheosensing is a kinematic (force-independent) form of mechanosensing. Thus, our findings challenge the dominant belief that biological mechanosensing requires the measurement of forces.

Interplay between differentially expressed enzymes contributes to light color acclimation in marine Synechococcus.

Marine Synechococcus, a globally important group of cyanobacteria, thrives in various light niches in part due to its varied photosynthetic light-harvesting pigments. Many Synechococcus strains use a process known as chromatic acclimation to optimize the ratio of two chromophores, green-light-absorbing phycoerythrobilin (PEB) and blue-light-absorbing phycourobilin (PUB), within their light-harvesting complexes. A full mechanistic understanding of how Synechococcus cells tune their PEB to PUB ratio during chromatic acclimation has not yet been obtained. Here, we show that interplay between two enzymes named MpeY and MpeZ controls differential PEB and PUB covalent attachment to the same cysteine residue. MpeY attaches PEB to the light-harvesting protein MpeA in green light, while MpeZ attaches PUB to MpeA in blue light. We demonstrate that the ratio of mpeY to mpeZ mRNA determines if PEB or PUB is attached. Additionally, strains encoding only MpeY or MpeZ do not acclimate. Examination of strains of Synechococcus isolated from across the globe indicates that the interplay between MpeY and MpeZ uncovered here is a critical feature of chromatic acclimation for marine Synechococcus worldwide.

Adaptation to Blue Light in Marine Synechococcus Requires MpeU, an Enzyme with Similarity to Phycoerythrobilin Lyase Isomerases.

Marine Synechococcus has successfully adapted to environments with different light colors, which likely contributes to this genus being the second most abundant group of microorganisms worldwide. Populations of Synechococcus that grow in deep, blue ocean waters contain large amounts of the blue-light absorbing chromophore phycourobilin (PUB) in their light harvesting complexes (phycobilisomes). Here, we show that all Synechococcus strains adapted to blue light possess a gene called mpeU. MpeU is structurally similar to phycobilin lyases, enzymes that ligate chromophores to phycobiliproteins. Interruption of mpeU caused a reduction in PUB content, impaired phycobilisome assembly and reduced growth rate more strongly in blue than green light. When mpeU was reintroduced in the mpeU mutant background, the mpeU-less phenotype was complemented in terms of PUB content and phycobilisome content. Fluorescence spectra of mpeU mutant cells and purified phycobilisomes revealed red-shifted phycoerythrin emission peaks, likely indicating a defect in chromophore ligation to phycoerythrin-I (PE-I) or phycoerythrin-II (PE-II). Our results suggest that MpeU is a lyase-isomerase that attaches a phycoerythrobilin to a PEI or PEII subunit and isomerizes it to PUB. MpeU is therefore an important determinant in adaptation of Synechococcus spp. to capture photons in blue light environments throughout the world's oceans.

Self-regulating genomic island encoding tandem regulators confers chromatic acclimation to marine Synechococcus.

The evolutionary success of marine Synechococcus, the second-most abundant phototrophic group in the marine environment, is partly attributable to this group's ability to use the entire visible spectrum of light for photosynthesis. This group possesses a remarkable diversity of light-harvesting pigments, and most of the group's members are orange and pink because of their use of phycourobilin and phycoerythrobilin chromophores, which are attached to antennae proteins called phycoerythrins. Many strains can alter phycoerythrin chromophore ratios to optimize photon capture in changing blue-green environments using type IV chromatic acclimation (CA4). Although CA4 is common in most marine Synechococcus lineages, the regulation of this process remains unexplored. Here, we show that a widely distributed genomic island encoding tandem master regulators named FciA (for type four chromatic acclimation island) and FciB plays a central role in controlling CA4. FciA and FciB have diametric effects on CA4. Interruption of fciA causes a constitutive green light phenotype, and interruption of fciB causes a constitutive blue light phenotype. These proteins regulate all of the molecular responses occurring during CA4, and the proteins' activity is apparently regulated posttranscriptionally, although their cellular ratio appears to be critical for establishing the set point for the blue-green switch in ecologically relevant light environments. Surprisingly, FciA and FciB coregulate only three genes within the Synechococcus genome, all located within the same genomic island as fciA and fciB These findings, along with the widespread distribution of strains possessing this island, suggest that horizontal transfer of a small, self-regulating DNA region has conferred CA4 capability to marine Synechococcus throughout many oceanic areas.