Reconstitution of a biofilm adhesin system from a sulfate-reducing bacterium in Pseudomonas fluorescens.

Biofilms of sulfate-reducing bacterium (SRB) like Desulfovibrio vulgaris Hildenborough (DvH) can facilitate metal corrosion in various industrial and environmental settings leading to substantial economic losses. Although the mechanisms of biofilm formation by DvH are not yet well understood, recent studies indicate the large adhesin, DvhA, is a key determinant of biofilm formation. The dvhA gene neighborhood resembles the biofilm-regulating Lap system of Pseudomonas fluorescens but is curiously missing the c-di-GMP-binding regulator LapD. Instead, DvH encodes an evolutionarily unrelated c-di-GMP-binding protein (DVU1020) that we hypothesized is functionally analogous to LapD. To study this unusual Lap system and overcome experimental limitations with the slow-growing anaerobe DvH, we reconstituted its predicted SRB Lap system in a P. fluorescens strain lacking its native Lap regulatory components (ΔlapGΔlapD). Our data support the model that DvhA is a cell surface-associated LapA-like adhesin with a N-terminal "retention module" and that DvhA is released from the cell surface upon cleavage by the LapG-like protease DvhG. Further, we demonstrate DVU1020 (named here DvhD) represents a distinct class of c-di-GMP-binding, biofilm-regulating proteins that regulates DvhG activity in response to intracellular levels of this second messenger. This study provides insight into the key players responsible for biofilm formation by DvH, thereby expanding our understanding of Lap-like systems.

Second messenger regulation of biofilm formation: breakthroughs in understanding c-di-GMP effector systems.

The second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) has emerged as a broadly conserved intracellular signaling molecule. This soluble molecule is important for controlling biofilm formation, adhesion, motility, virulence, and cell morphogenesis in diverse bacterial species. But how is the typical bacterial cell able to coordinate the actions of upward of 50 proteins involved in synthesizing, degrading, and binding c-di-GMP? Understanding the specificity of c-di-GMP signaling in the context of so many enzymes involved in making, breaking, and binding the second messenger will be possible only through mechanistic studies of its output systems. Here we discuss three newly characterized c-di-GMP effector systems that are best understood in terms of molecular and structural detail. As they are conserved across many bacterial species, they likely will serve as central paradigms for c-di-GMP output systems and contribute to our understanding of how bacteria control critical aspects of their biology.

From Input to Output: The Lap/c-di-GMP Biofilm Regulatory Circuit.

Biofilms are the dominant bacterial lifestyle. The regulation of the formation and dispersal of bacterial biofilms has been the subject of study in many organisms. Over the last two decades, the mechanisms of Pseudomonas fluorescens biofilm formation and regulation have emerged as among the best understood of any bacterial biofilm system. Biofilm formation by P. fluorescens occurs through the localization of an adhesin, LapA, to the outer membrane via a variant of the classical type I secretion system. The decision between biofilm formation and dispersal is mediated by LapD, a c-di-GMP receptor, and LapG, a periplasmic protease, which together control whether LapA is retained or released from the cell surface. LapA localization is also controlled by a complex network of c-di-GMP-metabolizing enzymes. This review describes the current understanding of LapA-mediated biofilm formation by P. fluorescens and discusses several emerging models for the regulation and function of this adhesin.

Broadcasting of amplitude- and frequency-modulated c-di-GMP signals facilitates cooperative surface commitment in bacterial lineages.

Work on surface sensing in bacterial biofilms has focused on how cells transduce sensory input into cyclic diguanylate (c-di-GMP) signaling, low and high levels of which generally correlate with high-motility planktonic cells and low-motility biofilm cells, respectively. Using Granger causal inference methods, however, we find that single-cell c-di-GMP increases are not sufficient to imply surface commitment. Tracking entire lineages of cells from the progenitor cell onward reveals that c-di-GMP levels can exhibit increases but also undergo oscillations that can propagate across 10 to 20 generations, thereby encoding more complex instructions for community behavior. Principal component and factor analysis of lineage c-di-GMP data shows that surface commitment behavior correlates with three statistically independent composite features, which roughly correspond to mean c-di-GMP levels, c-di-GMP oscillation period, and surface motility. Surface commitment in young biofilms does not correlate to c-di-GMP increases alone but also to the emergence of high-frequency and small-amplitude modulation of elevated c-di-GMP signal along a lineage of cells. Using this framework, we dissect how increasing or decreasing signal transduction from wild-type levels, by varying the interaction strength between PilO, a component of a principal surface sensing appendage system, and SadC, a key hub diguanylate cyclase that synthesizes c-di-GMP, impacts frequency and amplitude modulation of c-di-GMP signals and cooperative surface commitment.

Dpr-mediated H2O2 resistance contributes to streptococcus survival in a cystic fibrosis airway model system.

The cystic fibrosis (CF) lung environment is conducive to the colonization of bacteria as polymicrobial biofilms, which are associated with poor clinical outcomes for persons with CF (pwCF). Streptococcus spp. are highly prevalent in the CF airway, but its role in the CF lung microbiome is poorly understood. Some studies have shown Streptococcus spp. to be associated with better clinical outcomes for pwCF, while others show that high abundance of Streptococcus spp. is correlated with exacerbations. Our lab previously reported a polymicrobial culture system consisting of four CF-relevant pathogens that can be used to study microbial behavior in a more clinically relevant setting. Here, we use this model system to identify genetic pathways that are important for Streptococcus sanguinis survival in the context of the polymicrobial community. We identified genes related to reactive oxygen species as differentially expressed in S. sanguinis monoculture versus growth of this microbe in the mixed community. Genetic studies identified Dpr as important for S. sanguinis survival in the community. We show that Dpr, a DNA-binding ferritin-like protein, and PerR, a peroxide-responsive transcriptional regulator of Dpr, are important for protecting S. sanguinis from phenazine-mediated toxicity in co-culture with Pseudomonas aeruginosa and when exposed to hydrogen peroxide, both of which mimic the CF lung environment. Characterizing such interactions in a clinically relevant model system contributes to our understanding of microbial behavior in the context of polymicrobial biofilm infections. IMPORTANCE: Streptococcus spp. are recognized as a highly prevalent pathogen in cystic fibrosis (CF) airway infections. However, the role of this microbe in clinical outcomes for persons with CF is poorly understood. Here, we leverage a polymicrobial community system previously developed by our group to model CF airway infections as a tool to investigate a Pseudomonas-Streptococcus interaction involving reactive oxygen species (ROS). We show that protection against ROS is required for Streptococcus sanguinis survival in a clinically relevant polymicrobial system. Using this model system to study interspecies interactions contributes to our broader understanding of the complex role of Streptococcus spp. in the CF lung.

An in vitro medium for modeling gut dysbiosis associated with cystic fibrosis.

The gut physiology of pediatric and adult persons with cystic fibrosis (pwCF) is altered relative to healthy persons. The CF gut is characterized, in part, as having excess mucus, increased fat content, acidic pH, increased inflammation, increased antibiotic perturbation, and the potential for increased oxygen availability. These physiological differences shift nutritional availability and the local environment for intestinal microbes, thus likely driving significant changes in microbial metabolism, colonization, and competition with other microbes. The impact of any specific change in this physiological landscape is difficult to parse using human or animal studies. Thus, we have developed a novel culture medium representative of the CF gut environment, inclusive of all the aforementioned features. This medium, called CF-MiPro, maintains CF gut microbiome communities, while significantly shifting nonCF gut microbiome communities toward a CF-like microbial profile, characterized by low Bacteroidetes and high Proteobacteria abundance. This medium is able to maintain this culture composition for up to 5 days of passage. Additionally, microbial communities passaged in CF-MiPro produce significantly less immunomodulatory short-chain fatty acids (SCFA), including propionate and butyrate, than communities passaged in MiPro, a culture medium representative of healthy gut physiology, confirming not only a shift in microbial composition but also altered community function. Our results support the potential for this in vitro culture medium as a new tool for the study of CF gut dysbiosis. IMPORTANCE Cystic fibrosis is an autosomal recessive disease that disrupts ion transport at mucosal surfaces, leading to mucus accumulation and altered physiology of both the lungs and the intestines, among other organs, with the resulting altered environment contributing to an imbalance of microbial communities. Culture media representative of the CF airway have been developed and validated; however, no such medium exists for modeling the CF intestine. Here, we develop and validate a first-generation culture medium inclusive of features that are altered in the CF colon. Our findings suggest this novel medium, called CF-MiPro, as a maintenance medium for CF gut microbiome samples and a flexible tool for studying key drivers of CF-associated gut dysbiosis.

Interaction between the type 4 pili machinery and a diguanylate cyclase fine-tune c-di-GMP levels during early biofilm formation.

To initiate biofilm formation, it is critical for bacteria to sense a surface and respond precisely to activate downstream components of the biofilm program. Type 4 pili (T4P) and increasing levels of c-di-GMP have been shown to be important for surface sensing and biofilm formation, respectively; however, mechanisms important in modulating the levels of this dinucleotide molecule to define a precise output response are unknown. Here, using macroscopic bulk assays and single-cell tracking analyses of Pseudomonas aeruginosa, we uncover a role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two-hybrid and bimolecular fluorescence complementation assays, combined with genetic studies, are consistent with a model whereby PilO interacts with SadC and that the PilO-SadC interaction inhibits SadC's activity, resulting in decreased biofilm formation and increased motility. Using single-cell tracking, we monitor both the mean c-di-GMP and the variance of this dinucleotide in individual cells. Mutations that increase PilO-SadC interaction modestly, but significantly, decrease both the average and variance in c-di-GMP levels on a cell-by-cell basis, while mutants that disrupt PilO-SadC interaction increase the mean and variance of c-di-GMP levels. This work is consistent with a model wherein P. aeruginosa uses a component of the T4P scaffold to fine-tune the levels of this dinucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement.

Architecture of cell-cell junctions in situ reveals a mechanism for bacterial biofilm inhibition.

Many bacteria, including the major human pathogen Pseudomonas aeruginosa, are naturally found in multicellular, antibiotic-tolerant biofilm communities, in which cells are embedded in an extracellular matrix of polymeric molecules. Cell-cell interactions within P. aeruginosa biofilms are mediated by CdrA, a large, membrane-associated adhesin present in the extracellular matrix of biofilms, regulated by the cytoplasmic concentration of cyclic diguanylate. Here, using electron cryotomography of focused ion beam-milled specimens, we report the architecture of CdrA molecules in the extracellular matrix of P. aeruginosa biofilms at intact cell-cell junctions. Combining our in situ observations at cell-cell junctions with biochemistry, native mass spectrometry, and cellular imaging, we demonstrate that CdrA forms an extended structure that projects from the outer membrane to tether cells together via polysaccharide binding partners. We go on to show the functional importance of CdrA using custom single-domain antibody (nanobody) binders. Nanobodies targeting the tip of functional cell-surface CdrA molecules could be used to inhibit bacterial biofilm formation or disrupt preexisting biofilms in conjunction with bactericidal antibiotics. These results reveal a functional mechanism for cell-cell interactions within bacterial biofilms and highlight the promise of using inhibitors targeting biofilm cell-cell junctions to prevent or treat problematic, chronic bacterial infections.

The regulator FleQ both transcriptionally and post-transcriptionally regulates the level of RTX adhesins of Pseudomonas fluorescens.

Biofilm formation by the Gram-negative, Gammaproteobacteria Pseudomonas fluorescens relies on the repeats-in-toxin adhesins LapA and MapA in the cytoplasm, secretion of these adhesins through their respective type 1 secretion systems, and retention at the cell surface. Published work has shown that retention of the adhesins occurs via a post-translational mechanism involving the cyclic-di-GMP receptor LapD and the protease LapG. However, little is known about the underlying mechanisms that regulate the level of these adhesins. Here, we demonstrate that the master regulator FleQ modulates biofilm formation by both transcriptionally and post-transcriptionally regulating LapA and MapA. We find that a ΔfleQ mutant has a biofilm formation defect compared to the wild-type (WT) strain, which is attributed in part to a decrease in LapA and MapA abundance in the cell, despite the ΔfleQ mutant having increased levels of lapA and mapA transcripts compared to the WT strain. Through transposon mutagenesis and subsequent genetic analysis, we found that overstimulation of the Gac/Rsm pathway partially rescues biofilm formation in the ΔfleQ mutant background. Collectively, these findings provide evidence that FleQ regulates biofilm formation by both transcriptionally regulating the expression of the lapA and mapA genes and post-transcriptionally regulating the abundance of LapA and MapA, and that activation of the Gac/Rsm pathway can post-transcriptionally enhance biofilm formation by P. fluorescens. IMPORTANCE Biofilm formation is a highly coordinated process that bacteria undergo to colonize a variety of surfaces. For Pseudomonas fluorescens, biofilm formation requires the production and localization of repeats-in-toxin adhesins to the cell surface. To date, little is known about the underlying mechanisms that regulate biofilm formation by P. fluorescens. Here, we identify FleQ as a key regulator of biofilm formation that modulates both gene expression and abundance of LapA and MapA through both a transcriptional and post-transcriptional mechanism. We provide further evidence implicating activation of the Gac/Rsm system in FleQ-dependent regulation of biofilm formation. Together, our findings uncover evidence for a dual mechanism of transcriptional and post-transcriptional regulation of the LapA and MapA adhesins.

Surface-Induced cAMP Signaling Requires Multiple Features of the Pseudomonas aeruginosa Type IV Pili.

Pseudomonas aeruginosa type IV pili (TFP) are important for twitching motility and biofilm formation. TFP have been implicated in surface sensing, a process whereby surface-engaged cells upregulate the synthesis of the second messenger cAMP to propagate a signaling cascade leading to biofilm initiation and repression of motility. Here, we showed that mutations in PilA impairing proteolytic processing of the prepilin into mature pilin as well as the disruption of essential TFP components, including the PilC platform protein and PilB assembly motor protein, fail to induce surface-dependent cAMP signaling. We showed that TFP retraction by surface-engaged cells was required to induce signaling and that the retractile motor PilT was both necessary and sufficient to power surface-specific induction of cAMP. Furthermore, full TFP function required to support twitching motility is not required for robust cAMP signalling. The PilU retraction motor, in contrast, was unable to support full signaling in the absence of PilT. Finally, while we confirmed that PilA and PilJ interacted by bacterial two-hybrid analysis, our data do not support the current model that PilJ-PilA interaction drives cAMP signaling. IMPORTANCE Surface sensing by P. aeruginosa requires TFP. TFP plays a critical role in the induction of the second messenger cAMP upon surface contact; this second messenger is part of a larger cascade involved in the transition from a planktonic to a biofilm lifestyle. Here, we showed that TFP must be deployed and actively retracted by the PilT motor for the full induction of cAMP signaling. Furthermore, the mechanism whereby TFP retraction triggers cAMP induction is not well understood, and our data argue against one of the current models in the field proposed to address this knowledge gap.

The Power of Touch: Type 4 Pili, the von Willebrand A Domain, and Surface Sensing by Pseudomonas aeruginosa.

Most microbes in the biosphere are attached to surfaces, where they experience mechanical forces due to hydrodynamic flow and cell-to-substratum interactions. These forces likely serve as mechanical cues that influence bacterial physiology and eventually drive environmental adaptation and fitness. Mechanosensors are cellular components capable of sensing a mechanical input and serve as part of a larger system for sensing and transducing mechanical signals. Two cellular components in bacteria that have emerged as candidate mechanosensors are the type IV pili (TFP) and the flagellum. Current models posit that bacteria transmit and convert TFP- and/or flagellum-dependent mechanical force inputs into biochemical signals, including cAMP and c-di-GMP, to drive surface adaptation. Here, we discuss the impact of force-induced changes on the structure and function of two eukaryotic proteins, titin and the human von Willebrand factor (vWF), and these proteins' relevance to bacteria. Given the wealth of understanding about these eukaryotic mechanosensors, we can use them as a framework to understand the effect of force on Pseudomonas aeruginosa during the early stages of biofilm formation, with a particular emphasis on TFP and the documented surface-sensing mechanosensors PilY1 and FimH. We also discuss the importance of disulfide bonds in mediating force-induced conformational changes, which may modulate mechanosensing and downstream biochemical signaling. We conclude by sharing our perspective on the state of the field and what we deem exciting frontiers in studying bacterial mechanosensing to better understand the mechanisms whereby bacteria transition from a planktonic to a biofilm lifestyle.

The Diguanylate Cyclase YfiN of Pseudomonas aeruginosa Regulates Biofilm Maintenance in Response to Peroxide.

Pseudomonas aeruginosa forms surface-attached communities that persist in the face of antimicrobial agents and environmental perturbation. Published work has found that extracellular polysaccharide (EPS) production, regulation of motility, and induction of stress response pathways contribute to biofilm tolerance during such insults. However, little is known regarding the mechanism(s) whereby biofilm maintenance is regulated when exposed to such environmental challenges. Here, we provide evidence that the diguanylate cyclase YfiN is important for the regulation of biofilm maintenance when exposed to peroxide. We find that compared to the wild type (WT), static biofilms of the ΔyfiN mutant exhibit a maintenance defect, which can be further exacerbated by exposure to peroxide (H2O2); this defect can be rescued through genetic complementation. Additionally, we found that the ΔyfiN mutant biofilms produce less c-di-GMP than WT and that H2O2 treatment enhanced motility of surface-associated bacteria and increased cell death for the ΔyfiN mutant grown as a biofilm compared to WT biofilms. These data provide evidence that YfiN is required for biofilm maintenance by P. aeruginosa, via c-di-GMP signaling, to limit motility and protect viability in response to peroxide stress. These findings add to the growing recognition that biofilm maintenance by P. aeruginosa is an actively regulated process that is controlled, at least in part, by the wide array of c-di-GMP metabolizing enzymes found in this microbe. IMPORTANCE We build on previous findings that suggest that Pseudomonas aeruginosa utilizes c-di-GMP metabolizing enzymes to actively maintain a mature biofilm. Here, we explore how the diguanylate cyclase YfiN contributes to the regulation of biofilm maintenance during peroxide exposure. We find that mature P. aeruginosa biofilms require YfiN to synthesize c-di-GMP, regulate motility, and ensure viability during peroxide stress. These findings provide further evidence that the modulation of c-di-GMP in response to environmental signals is an important mechanism by which biofilms are maintained.

Nonmotile Subpopulations of Pseudomonas aeruginosa Repress Flagellar Motility in Motile Cells through a Type IV Pilus- and Pel-Dependent Mechanism.

The downregulation of Pseudomonas aeruginosa flagellar motility is a key event in biofilm formation, host colonization, and the formation of microbial communities, but the external factors that repress motility are not well understood. Here, we report that on soft agar, swarming motility can be repressed by cells that are nonmotile due to the absence of a flagellum or flagellar rotation. Mutants that lack either flagellum biosynthesis or rotation, when present at as little as 5% of the total population, suppressed swarming of wild-type cells. Non-swarming cells required functional type IV pili and the ability to produce Pel exopolysaccharide to suppress swarming by the flagellated wild type. Flagellated cells required only type IV pili, but not Pel production, for their swarming to be repressed by non-flagellated cells. We hypothesize that interactions between motile and nonmotile cells may enhance the formation of sessile communities, including those involving multiple genotypes, phenotypically diverse cells, and perhaps other species. IMPORTANCE Our study shows that, under the conditions tested, a small population of non-swarming cells can impact the motility behavior of a larger population. The interactions that lead to the suppression of swarming motility require type IV pili and a secreted polysaccharide, two factors with known roles in biofilm formation. These data suggest that interactions between motile and nonmotile cells may enhance the transition to sessile growth in populations and promote interactions between cells with different genotypes.

A Symphony of Cyclases: Specificity in Diguanylate Cyclase Signaling.

Cyclic diguanylate (c-di-GMP) is a near universal signaling molecule produced by diguanylate cyclases that can direct a variety of bacterial behaviors. A major area of research over the last several years has been aimed at understanding how a cell with dozens of diguanylate cyclases can deploy a given subset of them to produce a desired phenotypic outcome without undesired cross talk between c-di-GMP-dependent systems. Several models have been put forward to address this question, including specificity of cyclase activation, tuned binding constants of effector proteins, and physical interaction between cyclases and effectors. Additionally, recent evidence has suggested that there may be a link between the catalytic state of a cyclase and its physical contact with an effector. This review highlights several key studies, examines the proposed global and local models of c-di-GMP signaling specificity in bacteria, and attempts to identify the most fruitful steps that can be taken to better understand how dynamic networks of sibling cyclases and effector proteins result in sensible outputs that govern cellular behavior.

The Gut-Lung Axis in Cystic Fibrosis.

Cystic fibrosis (CF) is a heritable, multiorgan disease that impacts all tissues that normally express cystic fibrosis transmembrane conductance regulator (CFTR) protein. While the importance of the airway microbiota has long been recognized, the intestinal microbiota has only recently been recognized as an important player in both intestinal and lung health outcomes for persons with CF (pwCF). Here, we summarize current literature related to the gut-lung axis in CF, with a particular focus on three key ideas: (i) mechanisms through which microbes influence the gut-lung axis, (ii) drivers of microbiota alterations, and (iii) the potential for intestinal microbiota remediation.

Differential Surface Competition and Biofilm Invasion Strategies of Pseudomonas aeruginosa PA14 and PAO1.

Pseudomonas aeruginosa strains PA14 and PAO1 are among the two best-characterized model organisms used to study the mechanisms of biofilm formation while also representing two distinct lineages of P. aeruginosa. Previous work has shown that PA14 and PAO1 use different strategies for surface colonization; they also have different extracellular matrix composition and different propensities to disperse from biofilms back into the planktonic phase surrounding them. We expand on this work here by exploring the consequences of these different biofilm production strategies during direct competition. Using differentially labeled strains and microfluidic culture methods, we show that PAO1 can outcompete PA14 in direct competition during early colonization and subsequent biofilm growth, that they can do so in constant and perturbed environments, and that this advantage is specific to biofilm growth and requires production of the Psl polysaccharide. In contrast, P. aeruginosa PA14 is better able to invade preformed biofilms and is more inclined to remain surface-associated under starvation conditions. These data together suggest that while P. aeruginosa PAO1 and PA14 are both able to effectively colonize surfaces, they do so in different ways that are advantageous under different environmental settings. IMPORTANCE Recent studies indicate that P. aeruginosa PAO1 and PA14 use distinct strategies to initiate biofilm formation. We investigated whether their respective colonization and matrix secretion strategies impact their ability to compete under different biofilm-forming regimes. Our work shows that these different strategies do indeed impact how these strains fair in direct competition: PAO1 dominates during colonization of a naive surface, while PA14 is more effective in colonizing a preformed biofilm. These data suggest that even for very similar microbes there can be distinct strategies to successfully colonize and persist on surfaces during the biofilm life cycle.

Roadmap on emerging concepts in the physical biology of bacterial biofilms: from surface sensing to community formation.

Bacterial biofilms are communities of bacteria that exist as aggregates that can adhere to surfaces or be free-standing. This complex, social mode of cellular organization is fundamental to the physiology of microbes and often exhibits surprising behavior. Bacterial biofilms are more than the sum of their parts: single-cell behavior has a complex relation to collective community behavior, in a manner perhaps cognate to the complex relation between atomic physics and condensed matter physics. Biofilm microbiology is a relatively young field by biology standards, but it has already attracted intense attention from physicists. Sometimes, this attention takes the form of seeing biofilms as inspiration for new physics. In this roadmap, we highlight the work of those who have taken the opposite strategy: we highlight the work of physicists and physical scientists who use physics to engage fundamental concepts in bacterial biofilm microbiology, including adhesion, sensing, motility, signaling, memory, energy flow, community formation and cooperativity. These contributions are juxtaposed with microbiologists who have made recent important discoveries on bacterial biofilms using state-of-the-art physical methods. The contributions to this roadmap exemplify how well physics and biology can be combined to achieve a new synthesis, rather than just a division of labor.

Multigenerational memory and adaptive adhesion in early bacterial biofilm communities.

Using multigenerational, single-cell tracking we explore the earliest events of biofilm formation by Pseudomonas aeruginosa During initial stages of surface engagement (≤20 h), the surface cell population of this microbe comprises overwhelmingly cells that attach poorly (∼95% stay <30 s, well below the ∼1-h division time) with little increase in surface population. If we harvest cells previously exposed to a surface and direct them to a virgin surface, we find that these surface-exposed cells and their descendants attach strongly and then rapidly increase the surface cell population. This "adaptive," time-delayed adhesion requires determinants we showed previously are critical for surface sensing: type IV pili (TFP) and cAMP signaling via the Pil-Chp-TFP system. We show that these surface-adapted cells exhibit damped, coupled out-of-phase oscillations of intracellular cAMP levels and associated TFP activity that persist for multiple generations, whereas surface-naïve cells show uncorrelated cAMP and TFP activity. These correlated cAMP-TFP oscillations, which effectively impart intergenerational memory to cells in a lineage, can be understood in terms of a Turing stochastic model based on the Pil-Chp-TFP framework. Importantly, these cAMP-TFP oscillations create a state characterized by a suppression of TFP motility coordinated across entire lineages and lead to a drastic increase in the number of surface-associated cells with near-zero translational motion. The appearance of this surface-adapted state, which can serve to define the historical classification of "irreversibly attached" cells, correlates with family tree architectures that facilitate exponential increases in surface cell populations necessary for biofilm formation.

Pseudomonas aeruginosa PA14 Enhances the Efficacy of Norfloxacin against Staphylococcus aureus Newman Biofilms.

The thick mucus within the airways of individuals with cystic fibrosis (CF) promotes frequent respiratory infections that are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent pathogens that cause CF pulmonary infections, and both are among the most common etiologic agents of chronic wound infections. Furthermore, the ability of P. aeruginosa and S. aureus to form biofilms promotes the establishment of chronic infections that are often difficult to eradicate using antimicrobial agents. In this study, we found that multiple LasR-regulated exoproducts of P. aeruginosa, including 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), siderophores, phenazines, and rhamnolipids, likely contribute to the ability of P. aeruginosa PA14 to shift S. aureus Newman norfloxacin susceptibility profiles. Here, we observe that exposure to P. aeruginosa exoproducts leads to an increase in intracellular norfloxacin accumulation by S. aureus We previously showed that P. aeruginosa supernatant dissipates the S. aureus membrane potential, and furthermore, depletion of the S. aureus proton motive force recapitulates the effect of the P. aeruginosa PA14 supernatant on shifting norfloxacin sensitivity profiles of biofilm-grown S. aureus Newman. From these results, we hypothesize that exposure to P. aeruginosa PA14 exoproducts leads to increased uptake of the drug and/or an impaired ability of S. aureus Newman to efflux norfloxacin. Surprisingly, the effect observed here of P. aeruginosa PA14 exoproducts on S. aureus Newman susceptibility to norfloxacin seemed to be specific to these strains and this antibiotic. Our results illustrate that microbially derived products can alter the ability of antimicrobial agents to kill bacterial biofilms.IMPORTANCEPseudomonas aeruginosa and Staphylococcus aureus are frequently coisolated from multiple infection sites, including the lungs of individuals with cystic fibrosis (CF) and nonhealing diabetic foot ulcers. Coinfection with P. aeruginosa and S. aureus has been shown to produce worse outcomes compared to infection with either organism alone. Furthermore, the ability of these pathogens to form biofilms enables them to cause persistent infection and withstand antimicrobial therapy. In this study, we found that P. aeruginosa-secreted products dramatically increase the ability of the antibiotic norfloxacin to kill S. aureus biofilms. Understanding how interspecies interactions alter the antibiotic susceptibility of bacterial biofilms may inform treatment decisions and inspire the development of new therapeutic strategies.

Availability of Zinc Impacts Interactions between Streptococcus sanguinis and Pseudomonas aeruginosa in Coculture.

Airway infections associated with cystic fibrosis (CF) are polymicrobial. We reported previously that clinical isolates of Pseudomonas aeruginosa promote the growth of a variety of streptococcal species. To explore the mechanistic basis of this interaction, we performed a genetic screen to identify mutants of Streptococcus sanginuis SK36 whose growth was no longer enhanced by P. aeruginosa PAO1. Mutations in the zinc uptake systems of S. sanguinis SK36 reduced growth of these strains by 1 to 3 logs compared to that of wild-type S. sanguinis SK36 when grown in coculture with P. aeruginosa PAO1, and exogenous zinc (0.1 to 10 µM) rescued the coculture defect of zinc uptake mutants of S. sanguinis SK36. Zinc uptake mutants of S. sanguinis SK36 had no obvious growth defect in monoculture. Consistent with competition for zinc driving coculture dynamics, S. sanguinis SK36 grown in coculture with P. aeruginosa showed increased expression of zinc uptake genes compared to that of S. sanguinis grown alone. Strains of P. aeruginosa PAO1 defective in zinc transport also supported ∼2-fold more growth by S. sanguinis compared to that in coculture with wild-type P. aeruginosa PAO1. An analysis of 118 CF sputum samples revealed that total zinc levels varied from ∼5 to 145 µM. At relatively low zinc levels, Pseudomonas and Streptococcus spp. were found in approximately equal abundance; at higher zinc levels, we observed a decline in relative abundance of Streptococcus spp., perhaps as a result of increasing zinc toxicity. Together, our data indicate that the relative abundances of these microbes in the CF airway may be impacted by zinc levels.IMPORTANCE Polymicrobial infections in CF cases likely impact patient health, but the mechanism(s) underlying such interactions is poorly understood. Here, we show using an in vitro model system that interactions between Pseudomonas and Streptococcus are modulated by zinc availability, and clinical data are consistent with this model. Together with previous studies, our work supports a role for metal homeostasis as a key factor driving microbial interactions.