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Significance In their natural environments, bacteria frequently transition from a free-swimming state to a surface-associated state, attached to a substratum. As they encounter a surface, they may initiate developmental programs to optimally colonize this new environment and induce pathways such as virulence. Here we demonstrate that the pathogen Pseudomonas aeruginosa uses fiber-like motorized appendages called type IV pili to sense initial contact with surfaces. This leads to a signaling cascade that results in the expression of hundreds of genes associated with pathogenicity and surface-specific twitching motility. Thus, bacteria use pili not only to attach and move, but also to sense mechanical features of their environment and regulate cellular processes of surface-associated lifestyles. Bacteria have evolved a wide range of sensing systems to appropriately respond to environmental signals. Here we demonstrate that the opportunistic pathogen Pseudomonas aeruginosa detects contact with surfaces on short timescales using the mechanical activity of its type IV pili, a major surface adhesin. This signal transduction mechanism requires attachment of type IV pili to a solid surface, followed by pilus retraction and signal transduction through the Chp chemosensory system, a chemotaxis-like sensory system that regulates cAMP production and transcription of hundreds of genes, including key virulence factors. Like other chemotaxis pathways, pili-mediated surface sensing results in a transient response amplified by a positive feedback that increases type IV pili activity, thereby promoting long-term surface attachment that can stimulate additional virulence and biofilm-inducing pathways. The methyl-accepting chemotaxis protein-like chemosensor PilJ directly interacts with the major pilin subunit PilA. Our results thus support a mechanochemical model where a chemosensory system measures the mechanically induced conformational changes in stretched type IV pili. These findings demonstrate that P. aeruginosa not only uses type IV pili for surface-specific twitching motility, but also as a sensor regulating surface-induced gene expression and pathogenicity.

Key Points Gram-negative bacteria have evolved a wide array of secretion systems to transport small molecules, proteins and DNA into the extracellular space or target cells. In this Review, we describe insights into the structural and mechanistic features of the six secretion systems (types I–VI) of Gram-negative bacteria, the unique mycobacterial type VII secretion system, the chaperone–usher pathway and the curli biogenesis machinery. These systems are remarkably varied in size, composition and architecture. Double-membrane-spanning secretion systems are composed of many tens of protein subunits and can reach multi-megadalton sizes, whereas outer-membrane-spanning systems are relatively simple and are usually composed of only one type of subunit. These systems can transport folded or unfolded substrates and use various energy sources to power transport, from ATP to proton or entropy gradients. Recent structural and molecular advances have uncovered remarkable structural and functional similarities between secretion systems that have the potential to be exploited for the development of novel antibacterial compounds. In this Review, Waksman and colleagues describe the structural and mechanistic details of the six secretion systems (types I–VI) of Gram-negative bacteria, the unique mycobacterial type VII secretion system, the chaperone–usher pathway and the curli biogenesis machinery. They discuss both conserved and divergent properties of these systems and their potential as targets of novel antibacterial compounds. Bacteria have evolved a remarkable array of sophisticated nanomachines to export various virulence factors across the bacterial cell envelope. In recent years, considerable progress has been made towards elucidating the structural and molecular mechanisms of the six secretion systems (types I–VI) of Gram-negative bacteria, the unique mycobacterial type VII secretion system, the chaperone–usher pathway and the curli secretion machinery. These advances have greatly enhanced our understanding of the complex mechanisms that these macromolecular structures use to deliver proteins and DNA into the extracellular environment or into target cells. In this Review, we explore the structural and mechanistic relationships between these single- and double-membrane-embedded systems, and we briefly discuss how this knowledge can be exploited for the development of new antimicrobial strategies.

Bacteria have developed a large array of motility mechanisms to exploit available resources and environments. These mechanisms can be broadly classified into swimming in aqueous media and movement over solid surfaces. Swimming motility involves either the rotation of rigid helical filaments through the external medium or gyration of the cell body in response to the rotation of internal filaments. On surfaces, bacteria swarm collectively in a thin layer of fluid powered by the rotation of rigid helical filaments, they twitch by assembling and disassembling type IV pili, they glide by driving adhesins along tracks fixed to the cell surface and, finally, non-motile cells slide over surfaces in response to outward forces due to colony growth. Recent technological advances, especially in cryo-electron microscopy, have greatly improved our knowledge of the molecular machinery that powers the various forms of bacterial motility. In this Review, we describe the current understanding of the physical and molecular mechanisms that allow bacteria to move around.In this Review, Wadhwa and Berg explore the most common bacterial motility mechanisms and summarize the current understanding of the molecular machines that enable bacteria to swim in aqueous media and move on solid surfaces.

The opportunistic pathogen Pseudomonas aeruginosa explores surfaces using twitching motility powered by retractile extracellular filaments called type IV pili (T4P). Single cells twitch by sequential T4P extension, attachment, and retraction. How single cells coordinate T4P to efficiently navigate surfaces remains unclear. We demonstrate that P. aeruginosa actively directs twitching in the direction of mechanical input from T4P in a process called mechanotaxis. The Chp chemotaxis-like system controls the balance of forward and reverse twitching migration of single cells in response to the mechanical signal. Collisions between twitching cells stimulate reversals, but Chp mutants either always or never reverse. As a result, while wild-type cells colonize surfaces uniformly, collision-blind Chp mutants jam, demonstrating a function for mechanosensing in regulating group behavior. On surfaces, Chp senses T4P attachment at one pole, thereby sensing a spatially resolved signal. As a result, the Chp response regulators PilG and PilH control the polarization of the extension motor PilB. PilG stimulates polarization favoring forward migration, while PilH inhibits polarization, inducing reversal. Subcellular segregation of PilG and PilH efficiently orchestrates their antagonistic functions, ultimately enabling rapid reversals upon perturbations. The distinct localization of response regulators establishes a signaling landscape known as local excitation–global inhibition in higher-order organisms, identifying a conserved strategy to transduce spatially resolved signals.

The predatory bacterium, Myxococcus xanthus , kills its prey by contact, using a putative Tight Adherence pilus, known as the Kil system, along with a protein complex resembling the basal body a type-III secretion system, named the “needleless” T3SS*. In this work, we provide direct evidence that Myxococcus polymerizes a Kil pilus at the prey contact site, which is constituted by the major pilin KilP. We also genetically demonstrate that the predation function of this pilus is linked to four different minor pilin complexes, which work in specific combinations to detect and kill phylogenetically diverse bacterial species. Structural models of the Kil pilus suggest that these minor pilin complexes form interchangeable “Tips”, exposing variable domains at the extremity of the pilus to interact with prey cells. Remarkably, the activity of these Tips also depends on the T3SS*, revealing a tight functional connection between the Kil system and the T3SS*. While these Tips are mostly restricted to predatory bacteria, genomic and structural analyses suggest that in other bacteria, including pathogens, Tad pili are also customized and functionalized by similar minor pilin complexes exposing variable domains. The predatory bacterium Myxococcus xanthus kills other bacteria by contact. Here, Herrou et al. show that the predator uses an extensible appendage, or pilus, that is functionalized by four distinct minor pilin complexes which work in association with a needleless type-III secretion system to kill various prey species.

Bifidobacteria represent one of the dominant groups of microorganisms colonizing the human infant intestine. Commensal bacteria that interact with a eukaryotic host are believed to express adhesive molecules on their cell surface that bind to specific host cell receptors or soluble macromolecules. Whole-genome transcription profiling of Bifidobacterium bifidum PRL2010, a strain isolated from infant stool, revealed a small number of commonly expressed extracellular proteins, among which were genes that specify sortase-dependent pili. Expression of the coding sequences of these B. bifidum PRL2010 appendages in nonpiliated Lactococcus lactis enhanced adherence to human enterocytes through extracellular matrix protein and bacterial aggregation. Furthermore, such piliated L. lactis cells evoked a higher TNF-α response during murine colonization compared with their nonpiliated parent, suggesting that bifidobacterial sortase-dependent pili not only contribute to adherence but also display immunomodulatory activity.

Amongst the major types of archaeal filaments, several have been shown to closely resemble bacterial homologues of the Type IV pili (T4P). Within Sulfolobales , member species encode for three types of T4P, namely the archaellum, the UV-inducible pilus system (Ups) and the archaeal adhesive pilus (Aap). Whereas the archaellum functions primarily in swimming motility, and the Ups in UV-induced cell aggregation and DNA-exchange, the Aap plays an important role in adhesion and twitching motility. Here, we present a cryoEM structure of the Aap of the archaeal model organism Sulfolobus acidocaldarius . We identify the component subunit as AapB and find that while its structure follows the canonical T4P blueprint, it adopts three distinct conformations within the pilus. The tri-conformer Aap structure that we describe challenges our current understanding of pilus structure and sheds new light on the principles of twitching motility. The cells of many archaeal species display surface appendages that closely resemble bacterial Type IV pili (T4P). Here, Gaines et al. present a cryoEM structure of the archaeal adhesive pilus from Sulfolobus acidocaldarius , showing that the structure of the component subunit follows the canonical T4P blueprint but adopts three distinct conformations within the pilus.

The ability of eukaryotic cells to differentiate surface stiffness is fundamental for many processes like stem cell development. Bacteria were previously known to sense the presence of surfaces, but the extent to which they could differentiate stiffnesses remained unclear. Here we establish that the human pathogen Pseudomonas aeruginosa actively measures surface stiffness using type IV pili (TFP). Stiffness sensing is nonlinear, as induction of the virulence factor regulator is peaked with stiffness in a physiologically important range between 0.1 kPa (similar to mucus) and 1,000 kPa (similar to cartilage). Experiments on surfaces with distinct material properties establish that stiffness is the specific biophysical parameter important for this sensing. Traction force measurements reveal that the retraction of TFP is capable of deforming even stiff substrates. We show how slow diffusion of the pilin PilA in the inner membrane yields local concentration changes at the base of TFP during extension and retraction that change with substrate stiffness. We develop a quantitative biomechanical model that explains the transcriptional response to stiffness. A competition between PilA diffusion in the inner membrane and a loss/gain of monomers during TFP extension/retraction produces substrate stiffness-dependent dynamics of the local PilA concentration. We validated this model by manipulating the ATPase activity of the TFP motors to change TFP extension and retraction velocities and PilA concentration dynamics, altering the stiffness response in a predictable manner. Our results highlight stiffness sensing as a shared behavior across biological kingdoms, revealing generalizable principles of environmental sensing across small and large cells.

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.

Development of the human gut microbiota commences at birth, with bifidobacteria being among the first colonizers of the sterile newborn gastrointestinal tract. To date, the genetic basis of Bifidobacterium colonization and persistence remains poorly understood. Transcriptome analysis of the Bifidobacterium breve UCC2003 2.42-Mb genome in a murine colonization model revealed differential expression of a type IVb tight adherence (Tad) pilus-encoding gene cluster designated \"tad₂₀₀₃.\" Mutational analysis demonstrated that the tad₂₀₀₃ gene cluster is essential for efficient in vivo murine gut colonization, and immunogold transmission electron microscopy confirmed the presence of Tad pili at the poles of B. breve UCC2003 cells. Conservation of the Tad pilus-encoding locus among other B. breve strains and among sequenced Bifidobacterium genomes supports the notion of a ubiquitous pili-mediated host colonization and persistence mechanism for bifidobacteria.