Many functions of Pseudomonas aeruginosa type IVa pili, including twitching motility and surface sensing, depend on dynamic cycles of filament assembly and disassembly powered by the cytoplasmic ATPases PilB, PilT, and PilU. Deletion of pilT results in loss of twitching and pilus-specific bacteriophage susceptibility, while non-twitching pilU mutants remain susceptible to pilus-specific phages, indicating that they still produce retractable pili. pilU mutants have high basal levels of the secondary messenger cyclic AMP (cAMP) that normally increases following surface contact, suggesting aberrant surface sensing. To better understand PilU’s role in pilus biology, we solved its X-ray crystal structure and used phylogenetic analyses to identify conserved differences between PilT and PilU. Chemical mutagenesis followed by whole-genome sequencing was used to identify suppressors in the ΔpilU mutant background that restored twitching motility. The mutations mapped to the major pilin, PilA, or the pilus tip adhesin, PilY1. Both the position and nature of the substitutions in PilA impacted restoration of motility, and it was dependent on functional PilT. Complementation of most pilU suppressors with PilU in trans further increased motility, while the expression of wild-type PilA in trans decreased motility in a dose-dependent manner. Notably, cAMP levels remained elevated in most twitching pilU suppressor mutants, showing that surface sensing and motility can be uncoupled. Together, our data suggest that the bacterial response to surfaces reflects a complex interaction of PilU function with specific alleles of PilY1 and PilA that together modulate pilus dynamics and function. IMPORTANCE The ability of bacteria to sense and respond to contact with surfaces is important for triggering changes in secondary messenger levels and gene expression, leading to the formation of biofilms and increased production of virulence factors. For Pseudomonas aeruginosa, the expression of functional type IVa pili is important for the accumulation of cyclic AMP (cAMP) following surface contact. Deletion of the PilT retraction ATPase paralog PilU leads to loss of pilus-mediated twitching motility but also high intracellular levels of cAMP, a phenotype mimicking that of surface-adapted cells. Here, we isolated twitching suppressors of a pilU deletion mutant that mapped to the pilin subunit PilA or pilus-tip adhesin PilY1 and showed that for most, elevated cAMP levels did not decrease when motility was restored. Twitching was dependent on functional PilT, and complementation with PilU further increased twitching for most mutants. These data show that in permissive contexts, PilU is not required for twitching motility, providing new insights into mechanisms of bacterial surface sensing and evolution of type IVa pilus motor function.

Surface sensing is a key aspect of the early stage of biofilm formation. For P. aeruginosa, the type IV pili (TFP), the TFP alignment complex and PilY1 were shown to play a key role in c-di-GMP signaling upon surface contact. The role of the flagellar machinery in surface sensing is less well understood in P. aeruginosa. Here we show, consistent with findings from other groups, that a mutation in the gene encoding the flagellar hook protein (ΔflgK) or flagellin (ΔfliC) results in a strain that overproduces the Pel exopolysaccharide (EPS) with a concomitant increase in c-di-GMP levels. We use a candidate gene approach and genetic screens, combined with phenotypic assays, to identify key roles for the MotAB and MotCD stators and the FliG protein, a component of the flagellar switch complex, in stimulating the surface-dependent, increased c-di-GMP level noted for these flagellar mutants. These findings are consistent with previous studies showing a role for the stators in surface sensing. We also show that mutations in the genes coding for the diguanylate cyclases SadC and RoeA as well as SadB, a protein involved in early surface colonization, abrogate the increased c-d-GMP-related phenotypes of the ΔflgK mutant. Together, these data indicate that bacteria monitor the status of flagellar synthesis and/or function during surface sensing as a means to trigger the biofilm program.

Type IV pili (TFP) and Pel polysaccharide contribute to the ability of microbes such as Pseudomonas aeruginosa to engage with and move across surfaces.

Most microbial cells found in nature exist in matrix-covered, surface-attached communities known as biofilms. This mode of growth is initiated by the ability of the microbe to sense a surface on which to grow. The opportunistic pathogen Pseudomonas aeruginosa (Pa) PA14 utilizes a single polar flagellum and type 4 pili (T4P) to sense surfaces. For Pa, T4P-dependent "twitching" motility is characterized by effectively pulling the cell across a surface through a complex process of cooperative binding, pulling, and unbinding. T4P retraction is powered by hexameric ATPases. Pa cells that have engaged a surface increase production of the second messenger cyclic AMP (cAMP) over multiple generations via the Pil-Chp system. This rise in cAMP allows cells and their progeny to become better adapted for surface attachment and activates virulence pathways through the cAMP-binding transcription factor Vfr. While many studies have focused on mechanisms of T4P twitching and regulation of T4P production and function by the Pil-Chp system, the mechanism by which Pa senses and relays a surface-engagement signal to the cell is still an open question. Here we review the current state of the surface sensing literature for Pa, with a focus on T4P, and propose an integrated model of surface sensing whereby the retraction motor PilT senses and relays the signal to the Pil-Chp system via PilJ to drive cAMP production and adaptation to a surface lifestyle.

Biofilm formation begins when bacteria contacting a surface induce cellular changes to become better adapted for surface growth. One of the first changes to occur for Pseudomonas aeruginosa after surface contact is an increase in the nucleotide second messenger 3',5'-cyclic adenosine monophosphate (cAMP). It has been demonstrated that this increase in intracellular cAMP is dependent on functional Type IV pili (T4P) relaying a signal to the Pil-Chp system, but the mechanism by which this signal is transduced remains poorly understood. Here, we investigate the role of the Type IV pili retraction motor PilT in sensing a surface and relaying that signal to cAMP production. We show that mutations affecting the structure of PilT and in particular ATPase activity of this motor protein, reduce surface-dependent cAMP production. We identify a novel interaction between PilT and PilJ, a member of the Pil-Chp system, and propose a new model whereby P. aeruginosa uses its retraction motor to sense a surface and to relay that signal via PilJ to increased production of cAMP. We discuss these findings in light of current TFP-dependent surface sensing models for P. aeruginosa . Importance:T4P are cellular appendages that allow P. aeruginosa to sense a surface leading to the production of cAMP. This second messenger not only activates virulence pathways but leads to further surface adaptation and irreversible attachment of cells. Here, we demonstrate the importance of the retraction motor PilT in surface sensing. We also present a new surface sensing model in P. aeruginosa whereby the T4P retraction motor PilT senses and transmits the surface signal, likely via its ATPase domain and interaction with PilJ, to mediate production of the second messenger cAMP.

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.

The mechanisms controlling entry into and exit from the death phase in the bacterial life cycle remain unclear. Although bacterial growth studies in batch cultures traditionally focus on the first three phases during incubation, two additional phases, the death phase and the long-term stationary phase, are less understood. Although there are a number of stressors that arise during long-term batch culture, including nutrient depletion and the accumulation of metabolic toxins such as reactive oxidative species, their roles in cell death are not well-defined. By manipulating the environmental conditions of Escherichia coli incubated in long-term batch culture through chemical and mechanical means, we investigated the role of volatile metabolic toxins in modulating the onset of the death phase. Here, we demonstrate that with the introduction of substrates with high binding affinities for volatile compounds, toxic by-products of normal cell metabolism, into the headspace of batch cultures, cells display a prolonged stationary phase and delayed entry into the death phase. The addition of these substrates allows cultures to maintain a high cell density for hours to days longer than cultures incubated under standard growth conditions. A similar effect is observed when the gaseous headspace in culture flasks is continuously replaced with sterile air, mechanically preventing the accumulation of metabolic by-products in batch cultures. We establish that toxic compound(s) are produced during the exponential phase, demonstrate that buildup of toxic by-products influence entry into the death phase, and present a novel tool for improving high-density growth in batch culture that may be used in future research or industrial or biotechnology applications. IMPORTANCE Bacteria, such as Escherichia coli, are routinely used in the production of biomaterials because of their efficient and sustainable capacity for synthesis of bioproducts. Industrial applications of microbial synthesis typically utilize cells in the stationary phase, when cultures have the greatest density of viable cells. By manipulating culture conditions to delay the transition from the stationary phase to the death phase, we can prolong the stationary phase on a scale of hours to days, thereby maintaining the maximum density of cells that would otherwise quickly decline. Characterization of the mechanisms that control entry into the death phase for the model organism E. coli not only deepens our understanding of the bacterial life cycle but also presents an opportunity to enhance current protocols for batch culture growth and explore similar effects in a variety of widely used bacterial strains.

With a growing body of work directed toward understanding the mechanisms of evolution using experimental systems, it is crucial to decipher what effects the experimental setup has on the outcome. If the goal of experimental laboratory evolution is to elucidate underlying evolutionary mechanisms and trends, these must be demonstrated in a variety of systems and environments. Here, we perform experimental evolution in a complex medium allowing the cells to transition through all five phases of growth, including death phase and long-term stationary phase. We show that the swiftness of selection and the specific targets of adaptive evolution are different in this system compared to others. We also observe parallel evolution where different mutations in the same genes are under positive natural selection. Together, these data show that while some outcomes of microbial evolution experiments may be generalizable, many outcomes will be environment or system specific.