Spatial control of sensory adaptation modulates mechanosensing in Pseudomonas aeruginosa.

Sensory signaling pathways use adaptation to dynamically respond to changes in their environment. Here, we report the mechanism of sensory adaptation in the Pil-Chp mechanosensory system, which the important human pathogen Pseudomonas aeruginosa uses to sense mechanical stimuli during surface exploration. Using biochemistry, genetics, and cell biology, we discovered that the enzymes responsible for adaptation, a methyltransferase and a methylesterase, are segregated to opposing cell poles as P. aeruginosa explore surfaces. By coordinating the localization of both enzymes, we found that the Pil-Chp response regulators influence local receptor methylation, the molecular basis of bacterial sensory adaptation. We propose a model in which adaptation during mechanosensing spatially resets local receptor methylation, and thus Pil-Chp signaling, to modulate the pathway outputs, which are involved in P. aeruginosa virulence. Despite decades of bacterial sensory adaptation studies, our work has uncovered an unrecognized mechanism that bacteria use to achieve adaptation to sensory stimuli.

Two antagonistic response regulators control Pseudomonas aeruginosa polarization during mechanotaxis.

The opportunistic pathogen Pseudomonas aeruginosa adapts to solid surfaces to enhance virulence and infect its host. Type IV pili (T4P), long and thin filaments that power surface-specific twitching motility, allow single cells to sense surfaces and control their direction of movement. T4P distribution is polarized to the sensing pole by the chemotaxis-like Chp system via a local positive feedback loop. However, how the initial spatially resolved mechanical signal is translated into T4P polarity is incompletely understood. Here, we demonstrate that the two Chp response regulators PilG and PilH enable dynamic cell polarization by antagonistically regulating T4P extension. By precisely quantifying the localization of fluorescent protein fusions, we show that phosphorylation of PilG by the histidine kinase ChpA controls PilG polarization. Although PilH is not strictly required for twitching reversals, it becomes activated upon phosphorylation and breaks the local positive feedback mechanism established by PilG, allowing forward-twitching cells to reverse. Chp thus uses a main output response regulator, PilG, to resolve mechanical signals in space and employs a second regulator, PilH, to break and respond when the signal changes. By identifying the molecular functions of two response regulators that dynamically control cell polarization, our work provides a rationale for the diversity of architectures often found in non-canonical chemotaxis systems.

Mechanotaxis directs Pseudomonas aeruginosa twitching motility.

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.

Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi.

The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans1-6. However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.

A scaffold protein connects type IV pili with the Chp chemosensory system to mediate activation of virulence signaling in Pseudomonas aeruginosa.

Type IV pili (TFP) function as mechanosensors to trigger acute virulence programs in Pseudomonas aeruginosa. On surface contact, TFP retraction activates the Chp chemosensory system phosphorelay to upregulate 3', 5'-cyclic monophosphate (cAMP) production and transcription of virulence-associated genes. To dissect the specific interactions mediating the mechanochemical relay, we used affinity purification/mass spectrometry, directed co-immunoprecipitations in P. aeruginosa, single cell analysis of contact-dependent transcriptional reporters, subcellular localization and bacterial two hybrid assays. We demonstrate that FimL, a Chp chemosensory system accessory protein of unknown function, directly links the integral component of the TFP structural complex FimV, a peptidoglycan binding protein, with one of the Chp system output response regulators PilG. FimL and PilG colocalize at cell poles in a FimV-dependent manner. While PilG phosphorylation is required for TFP function and mechanochemical signaling, it is not required for polar localization or binding to FimL. Phylogenetic analysis reveals other bacterial species simultaneously encode TFP, the Chp system, FimL, FimV and adenylate cyclase homologs, suggesting that surface sensing may be widespread among TFP-expressing bacteria. We propose that FimL acts as a scaffold enabling spatial colocalization of TFP and Chp system components to coordinate signaling leading to cAMP-dependent upregulation of virulence genes on surface contact.

Type IV pili mechanochemically regulate virulence factors in Pseudomonas aeruginosa.

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.

FimL regulates cAMP synthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a ubiquitous bacteria found in diverse ecological niches, is an important cause of acute infections in immunocompromised individuals and chronic infections in patients with Cystic Fibrosis. One signaling molecule required for the coordinate regulation of virulence factors associated with acute infections is 3', 5'-cyclic adenosine monophosphate, (cAMP), which binds to and activates a catabolite repressor homolog, Vfr. Vfr controls the transcription of many virulence factors, including those associated with Type IV pili (TFP), the Type III secretion system (T3SS), the Type II secretion system, flagellar-mediated motility, and quorum sensing systems. We previously identified FimL, a protein with histidine phosphotransfer-like domains, as a regulator of Vfr-dependent processes, including TFP-dependent motility and T3SS function. In this study, we carried out genetic and physiologic studies to further define the mechanism of action of FimL. Through a genetic screen designed to identify suppressors of FimL, we found a putative cAMP-specific phosphodiesterase (CpdA), suggesting that FimL regulates cAMP levels. Inactivation of CpdA increases cAMP levels and restores TFP-dependent motility and T3SS function to fimL mutants, consistent with in vivo phosphodiesterase activity. By constructing combinations of double and triple mutants in the two adenylate cyclase genes (cyaA and cyaB), fimL, and cpdA, we show that ΔfimL mutants resemble ΔcyaB mutants in TM defects, decreased T3SS transcription, and decreased cAMP levels. Similar to some of the virulence factors that they regulate, we demonstrate that CyaB and FimL are polarly localized. These results reveal new complexities in the regulation of diverse virulence pathways associated with acute P. aeruginosa infections.

The receiver domain of FrzE, a CheA-CheY fusion protein, regulates the CheA histidine kinase activity and downstream signalling to the A- and S-motility systems of Myxococcus xanthus.

The Frz chemosensory system is a two-component signal transduction pathway that controls cell reversals and directional movements for the two motility systems in Myxococcus xanthus. To trigger cell reversals, FrzE, a hybrid CheA-CheY fusion protein, autophosphorylates the kinase domain at His-49, and phosphoryl groups are transferred to aspartate residues (Asp-52 and Asp-220) in the two receiver domains of FrzZ, a dual CheY-like protein that serves as the pathway output. The role of the receiver domain of FrzE was unknown. In this paper, we characterize the FrzE protein in vitro and show that the receiver domain of FrzE negatively regulates the autophosphorylation activity of the kinase domain of FrzE. Unexpectedly, it does not appear to play a direct role in phospho-relay as in most other histidine kinase receiver domain hybrid systems. The regulatory role of the FrzE receiver domain suggests that it may interact with or be phosphorylated by an unknown protein. We also show the dynamics of motility system-specific marker proteins in FrzE mutants as cells move forward and reverse. Our studies indicate that the two motility systems are functionally co-ordinated and that any system-specific branching of the pathway most likely occurs downstream of FrzE.

FrzZ, a dual CheY-like response regulator, functions as an output for the Frz chemosensory pathway of Myxococcus xanthus.

Myxococcus xanthus utilizes two distinct motility systems for movement (gliding) on solid surfaces: adventurous motility (A-motility) and social motility (S-motility). Both systems are regulated by the Frz signal transduction pathway, which controls cell reversals required for directed motility and fruiting body formation. The Frz chemosensory system, unlike the Escherichia coli chemotaxis system, contains proteins with multiple response regulator domains: FrzE, a CheA-CheY hybrid protein, and FrzZ, a CheY-CheY hybrid protein. Previously, the CheY domain of FrzE was hypothesized to act as the response regulator output of the Frz system. In this study, using a genetic suppressor screen, we identified FrzZ and showed FrzZ is epistatic to FrzE, demonstrating that FrzZ is the principal output component of the pathway. We constructed M. xanthus point mutations in the phosphoaccepting aspartate residues of FrzZ and demonstrated the respective roles of these residues in group and single cell motility. We also performed in vitro assays and showed rapid phosphotransfer between the CheA domain of FrzE and each of the CheY domains of FrzZ. These experiments showed that FrzZ plays a direct role as an output of the Frz chemosensory pathway and that both CheY domains of FrzZ are functional.

How to make a spiral bacterium.

The motility of some kinds of bacteria depends on their spiral form, as does the virulence of certain pathogenic species. We propose a novel mechanism for the development of spiral shape in bacteria and the supercoiling of chains ('filaments') of many cells. Recently discovered actin-like proteins lying just under the cell wall form fibers that play a role in maintaining cell shape. Some species have a single actin-like fiber helically wrapped around the cell, while others have two fibers wrapped in the same direction. Here, we show that if these fibers elongate more slowly than growth lengthens the cell, the cell both twists and bends, taking on a spiral shape. We tested this mechanism using a mathematical model of expanding fiber-wound structures and via experiments that measure the shape changes of elongating physical models. Comparison of the model with in vivo experiments on stationary phase Caulobacter crescentus filaments provide the first evidence that mechanical stretching of cytoskeletal fibers influences cell morphology. Any hydraulic cylinder can spiral by this mechanism if it is reinforced by stretch-resistant fibers wrapped helically in the same direction, or shortened by contractile elements. This might be useful in the design of man-made actuators.