Pseudomonas aeruginosa surface motility and invasion into competing communities enhance interspecies antagonism.

Chronic polymicrobial infections involving Pseudomonas aeruginosa and Staphylococcus aureus are prevalent, difficult to eradicate, and associated with poor health outcomes. Therefore, understanding interactions between these pathogens is important to inform improved treatment development. We previously demonstrated that P. aeruginosa is attracted to S. aureus using type IV pili (TFP)-mediated chemotaxis, but the impact of attraction on S. aureus growth and physiology remained unknown. Using live single-cell confocal imaging to visualize microcolony structure, spatial organization, and survival of S. aureus during coculture, we found that interspecies chemotaxis provides P. aeruginosa a competitive advantage by promoting invasion into and disruption of S. aureus microcolonies. This behavior renders S. aureus susceptible to P. aeruginosa antimicrobials. Conversely, in the absence of TFP motility, P. aeruginosa cells exhibit reduced invasion of S. aureus colonies. Instead, P. aeruginosa builds a cellular barrier adjacent to S. aureus and secretes diffusible, bacteriostatic antimicrobials like 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) into the S. aureus colonies. Reduced invasion leads to the formation of denser and thicker S. aureus colonies with increased HQNO-mediated lactic acid fermentation, a physiological change that could complicate treatment strategies. Finally, we show that P. aeruginosa motility modifications of spatial structure enhance competition against S. aureus. Overall, these studies expand our understanding of how P. aeruginosa TFP-mediated interspecies chemotaxis facilitates polymicrobial interactions, highlighting the importance of spatial positioning in mixed-species communities. IMPORTANCE: The polymicrobial nature of many chronic infections makes their eradication challenging. Particularly, coisolation of Pseudomonas aeruginosa and Staphylococcus aureus from airways of people with cystic fibrosis and chronic wound infections is common and associated with severe clinical outcomes. The complex interplay between these pathogens is not fully understood, highlighting the need for continued research to improve management of chronic infections. Our study unveils that P. aeruginosa is attracted to S. aureus, invades into neighboring colonies, and secretes anti-staphylococcal factors into the interior of the colony. Upon inhibition of P. aeruginosa motility and thus invasion, S. aureus colony architecture changes dramatically, whereby S. aureus is protected from P. aeruginosa antagonism and responds through physiological alterations that may further hamper treatment. These studies reinforce accumulating evidence that spatial structuring can dictate community resilience and reveal that motility and chemotaxis are critical drivers of interspecies competition.

Pseudomonas aeruginosa surface motility and invasion into competing communities enhances interspecies antagonism.

Chronic polymicrobial infections involving Pseudomonas aeruginosa and Staphylococcus aureus are prevalent, difficult to eradicate, and associated with poor health outcomes. Therefore, understanding interactions between these pathogens is important to inform improved treatment development. We previously demonstrated that P. aeruginosa is attracted to S. aureus using type IV pili-mediated chemotaxis, but the impact of attraction on S. aureus growth and physiology remained unknown. Using live single-cell confocal imaging to visualize microcolony structure, spatial organization, and survival of S. aureus during coculture, we found that interspecies chemotaxis provides P. aeruginosa a competitive advantage by promoting invasion into and disruption of S. aureus microcolonies. This behavior renders S. aureus susceptible to P. aeruginosa antimicrobials. Conversely, in the absence of type IV pilus motility, P. aeruginosa cells exhibit reduced invasion of S. aureus colonies. Instead, P. aeruginosa builds a cellular barrier adjacent to S. aureus and secretes diffusible, bacteriostatic antimicrobials like 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) into the S. aureus colonies. P. aeruginosa reduced invasion leads to the formation of denser and thicker S. aureus colonies with significantly increased HQNO-mediated lactic acid fermentation, a physiological change that could complicate the effective treatment of infections. Finally, we show that P. aeruginosa motility modifications of spatial structure enhance competition against S. aureus. Overall, these studies build on our understanding of how P. aeruginosa type IV pili-mediated interspecies chemotaxis mediates polymicrobial interactions, highlighting the importance of spatial positioning in mixed-species communities.

Spatial propagation of temperate phages within and among biofilms.

Bacteria form groups comprised of cells and a secreted polymeric matrix that controls their spatial organization. These groups - termed biofilms - can act as refuges from environmental disturbances and from biotic threats, including phages. Despite the ubiquity of temperate phages and bacterial biofilms, live propagation of temperate phages within biofilms has never been characterized on cellular spatial scales. Here, we leverage several approaches to track temperate phages and distinguish between lytic and lysogenic host infections. We determine that lysogeny within E. coli biofilms initially occurs within a predictable region of cell group packing architecture on the biofilm periphery. Because lysogens are generally found on the periphery of large cell groups, where lytic viral infections also reduce local biofilm cell packing density, lysogens are predisposed to disperse into the passing liquid and are over-represented in biofilms formed from the dispersal pool of the original biofilm-phage system. Comparing our results with those for virulent phages reveals that temperate phages have previously unknown advantages in propagating over long spatial and time scales within and among bacterial biofilms.

Breakdown of clonal cooperative architecture in multispecies biofilms and the spatial ecology of predation.

Biofilm formation, including adherence to surfaces and secretion of extracellular matrix, is common in the microbial world, but we often do not know how interaction at the cellular spatial scale translates to higher-order biofilm community ecology. Here we explore an especially understudied element of biofilm ecology, namely predation by the bacterium Bdellovibrio bacteriovorus. This predator can kill and consume many different Gram-negative bacteria, including Vibrio cholerae and Escherichia coli. V. cholerae can protect itself from predation within densely packed biofilm structures that it creates, whereas E. coli biofilms are highly susceptible to B. bacteriovorus. We explore how predator-prey dynamics change when V. cholerae and E. coli are growing in biofilms together. We find that in dual-species prey biofilms, E. coli survival under B. bacteriovorus predation increases, whereas V. cholerae survival decreases. E. coli benefits from predator protection when it becomes embedded within expanding groups of highly packed V. cholerae. But we also find that the ordered, highly packed, and clonal biofilm structure of V. cholerae can be disrupted if V. cholerae cells are directly adjacent to E. coli cells at the start of biofilm growth. When this occurs, the two species become intermixed, and the resulting disordered cell groups do not block predator entry. Because biofilm cell group structure depends on initial cell distributions at the start of prey biofilm growth, the surface colonization dynamics have a dramatic impact on the eventual multispecies biofilm architecture, which in turn determines to what extent both species survive exposure to B. bacteriovorus.

Multispecies biofilm architecture determines bacterial exposure to phages.

Numerous ecological interactions among microbes-for example, competition for space and resources, or interaction among phages and their bacterial hosts-are likely to occur simultaneously in multispecies biofilm communities. While biofilms formed by just a single species occur, multispecies biofilms are thought to be more typical of microbial communities in the natural environment. Previous work has shown that multispecies biofilms can increase, decrease, or have no measurable impact on phage exposure of a host bacterium living alongside another species that the phages cannot target. The reasons underlying this variability are not well understood, and how phage-host encounters change within multispecies biofilms remains mostly unexplored at the cellular spatial scale. Here, we study how the cellular scale architecture of model 2-species biofilms impacts cell-cell and cell-phage interactions controlling larger scale population and community dynamics. Our system consists of dual culture biofilms of Escherichia coli and Vibrio cholerae under exposure to T7 phages, which we study using microfluidic culture, high-resolution confocal microscopy imaging, and detailed image analysis. As shown previously, sufficiently mature biofilms of E. coli can protect themselves from phage exposure via their curli matrix. Before this stage of biofilm structural maturity, E. coli is highly susceptible to phages; however, we show that these bacteria can gain lasting protection against phage exposure if they have become embedded in the bottom layers of highly packed groups of V. cholerae in co-culture. This protection, in turn, is dependent on the cell packing architecture controlled by V. cholerae biofilm matrix secretion. In this manner, E. coli cells that are otherwise susceptible to phage-mediated killing can survive phage exposure in the absence of de novo resistance evolution. While co-culture biofilm formation with V. cholerae can confer phage protection to E. coli, it comes at the cost of competing with V. cholerae and a disruption of normal curli-mediated protection for E. coli even in dual species biofilms grown over long time scales. This work highlights the critical importance of studying multispecies biofilm architecture and its influence on the community dynamics of bacteria and phages.

The Incidence of Fusarium graminearum in Wild Grasses is Associated With Rainfall and Cumulative Host Density in New York.

The movement of plant pathogens between cultivated and natural host communities can result in lost agricultural production and altered microbial or plant biodiversity. Fusarium graminearum incidence was studied in wild grass hosts for 3 years to better understand the ecology of this plant pathogen at the interface of crop fields and nonagricultural environments. Research sites (n = 23) were spread between regions of high and low agricultural production and included both agricultural and nonagricultural fields. Pathogen incidence in living grass spikes and senesced, overwintered stems varied between regions of New York and was lowest in a region with sparser agricultural production (P = 0.001). However, pathogen incidence within regions was similar at both agricultural and nonagricultural sites. The groundcover of crop and wild hosts within 1 km of sample sites were equally effective predictors of pathogen incidence, indicating either host group may drive pathogen spread. Rainfall in the 8 weeks preceding sample collection was strongly correlated with F. graminearum incidence in grasses, as well as an increased prevalence of F. graminearum in Fusarium spp. communities (P = 0.001). Grass species diversity was not associated with a reduction in pathogen incidence, and F. graminearum incidence did not vary among the most well-sampled grasses. These results indicate the pathogen colonizes and spreads in noncultivated grasses in a manner consistent with existing concepts of pathogen epidemiology in cereal crops. Increasing host acreage, whether cultivated or not, could drive the colonization of grasses in remote or protected environments, potentially altering their microbial communities.

Population Genetics of Fusarium graminearum at the Interface of Wheat and Wild Grass Communities in New York.

Fusarium graminearum is primarily understood as an agricultural pathogen affecting cereal crops, but its host range also includes diverse, noncultivated grasses ubiquitous across agricultural and natural environments. Wild grasses may select for the production of diverse toxin variants (chemotypes) and serve as reservoirs of genetic diversity or sources of disease-inciting inoculum. Populations at the intersection of wheat and wild grass communities were described using 909 isolates collected from wheat spikes, wild grass spikes, and overwintered wild grass stems found at natural and agricultural sites in regions of high and low crop production. Trichothecene (TRI) genotypes correlated to pathogen chemotype were predicted from two loci, and multilocus genotypes (MLGs) were determined using eight microsatellite loci. The genetic diversity of wild grass and wheat-derived populations was comparable, and their differentiation was low. Duplicate MLGs were rare even in samples collected from a single square meter, although they could be found in multiple hosts, environments, regions, and years. TRI genotype frequencies differed between region and land use. Admixture between TRI genotype-defined populations, which correspond to three previously described sympatric North American populations, was detected and was highest in a region with remote host communities and little agricultural production. Nonagricultural environments may maintain different pathogen TRI genotypes than wheat fields and provide an opportunity for recombination between isolates from different F. graminearum populations. A lack of structural barriers suggests that pathogen gene flow is uninhibited between wheat and wild grass communities, and the recovery of putative clones from multiple hosts and environments provides initial evidence that noncultivated grasses are a source of local and regional inoculum.

Crystal structure of the ligand-binding domain of a LysR-type transcriptional regulator: transcriptional activation via a rotary switch.

LysR-type transcriptional regulators (LTTRs) generally bind to target promoters in two conformations, depending on the availability of inducing ligands. OccR is an LTTR that regulates the octopine catabolism operon of Agrobacterium tumefaciens. OccR binds to a site located between the divergent occQ and occR promoters. Octopine triggers a conformational change that activates the occQ promoter, and does not affect autorepression. This change shortens the length of bound DNA and relaxes a high-angle DNA bend. Here, we describe the crystal structure of the ligand-binding domain (LBD) of OccR apoprotein and holoprotein. Pairs of LBDs form dimers with extensive hydrogen bonding, while pairs of dimers interact via a single helix, creating a tetramer interface. Octopine causes a 70° rotation of each dimer with respect to the opposite dimer, precisely at the tetramer interface. We modeled the DNA binding domain (DBD), linker helix and bound DNA onto the apoprotein and holoprotein. The two DBDs of the modeled apoprotein lie far apart and the bound DNA between them has a high-angle DNA bend. In contrast, the two DBDs of the holoprotein lie closer to each other, with a low DNA bend angle. This inter-dimer pivot fully explains earlier studies of this LTTR.