Modulation of flagellar rotation in surface-attached bacteria: A pathway for rapid surface-sensing after flagellar attachment.

Attachment is a necessary first step in bacterial commitment to surface-associated behaviors that include colonization, biofilm formation, and host-directed virulence. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa can initially attach to surfaces via its single polar flagellum. Although many bacteria quickly detach, some become irreversibly attached and express surface-associated structures, such as Type IV pili, and behaviors, including twitching motility and biofilm initiation. P. aeruginosa that lack the GTPase FlhF assemble a randomly placed flagellum that is motile; however, we observed that these mutant bacteria show defects in biofilm formation comparable to those seen for non-motile, aflagellate bacteria. This phenotype was associated with altered behavior of ΔflhF bacteria immediately following surface-attachment. Forward and reverse genetic screens led to the discovery that FlhF interacts with FimV to control flagellar rotation at a surface, and implicated cAMP signaling in this pathway. Although cAMP controls many transcriptional programs in P. aeruginosa, known targets of this second messenger were not required to modulate flagellar rotation in surface-attached bacteria. Instead, alterations in switching behavior of the motor appeared to result from direct or indirect effects of cAMP on switch complex proteins and/or the stators associated with them.

In Situ Structures of Polar and Lateral Flagella Revealed by Cryo-Electron Tomography.

The bacterial flagellum is a sophisticated self-assembling nanomachine responsible for motility in many bacterial pathogens, including Pseudomonas aeruginosa, Vibrio spp., and Salmonella enterica The bacterial flagellum has been studied extensively in the model systems Escherichia coli and Salmonella enterica serovar Typhimurium, yet the range of variation in flagellar structure and assembly remains incompletely understood. Here, we used cryo-electron tomography and subtomogram averaging to determine in situ structures of polar flagella in P. aeruginosa and peritrichous flagella in S Typhimurium, revealing notable differences between these two flagellar systems. Furthermore, we observed flagellar outer membrane complexes as well as many incomplete flagellar subassemblies, which provide additional insight into mechanisms underlying flagellar assembly and loss in both P. aeruginosa and S Typhimurium.IMPORTANCE The bacterial flagellum has evolved as one of the most sophisticated self-assembled molecular machines, which confers locomotion and is often associated with virulence of bacterial pathogens. Variation in species-specific features of the flagellum, as well as in flagellar number and placement, results in structurally distinct flagella that appear to be adapted to the specific environments that bacteria encounter. Here, we used cutting-edge imaging techniques to determine high-resolution in situ structures of polar flagella in Pseudomonas aeruginosa and peritrichous flagella in Salmonella enterica serovar Typhimurium, demonstrating substantial variation between flagella in these organisms. Importantly, we observed novel flagellar subassemblies and provided additional insight into the structural basis of flagellar assembly and loss in both P. aeruginosa and S Typhimurium.

Bacterial cell surface characterization by phage display coupled to high-throughput sequencing.

The remarkable capacity of bacteria to adapt in response to selective pressures drives antimicrobial resistance. Pseudomonas aeruginosa illustrates this point, establishing chronic infections during which it evolves to survive antimicrobials and evade host defenses. Many adaptive changes occur on the P. aeruginosa cell surface but methods to identify these are limited. Here we combine phage display with high-throughput DNA sequencing to create a high throughput, multiplexed technology for surveying bacterial cell surfaces, Phage-seq. By applying phage display panning to hundreds of bacterial genotypes and analyzing the dynamics of the phage display selection process, we capture important biological information about cell surfaces. This approach also yields camelid single-domain antibodies that recognize key P. aeruginosa virulence factors on live cells. These antibodies have numerous potential applications in diagnostics and therapeutics. We propose that Phage-seq establishes a powerful paradigm for studying the bacterial cell surface by identifying and profiling many surface features in parallel.

The GTPase activity of FlhF is dispensable for flagellar localization, but not motility, in Pseudomonas aeruginosa.

The opportunistic human pathogen Pseudomonas aeruginosa uses two surface organelles, flagella and pili, for motility and adhesion in biotic and abiotic environments. Polar flagellar placement and number are influenced by FlhF, which is a signal recognition particle (SRP)-type GTPase. The FlhF proteins of Bacillus subtilis and Campylobacter jejuni were recently shown to have GTPase activity. However, the phenotypes associated with flhF deletion and/or mutation differ between these organisms and P. aeruginosa, making it difficult to generalize a role for FlhF in pseudomonads. In this study, we confirmed that FlhF of P. aeruginosa binds and hydrolyzes GTP. We mutated FlhF residues that we predicted would alter nucleotide binding and hydrolysis and determined the effects of these mutations on FlhF enzymatic activity, protein dimerization, and bacterial motility. Both hydrolytically active and inactive FlhF point mutants restored polar flagellar assembly, as seen for wild-type FlhF. However, differential effects on flagellar function were observed in single-cell assays of swimming motility and flagellar rotation. These findings indicate that FlhF function is influenced by its nucleotide binding and hydrolytic activities and demonstrate that FlhF affects P. aeruginosa flagellar function as well as assembly.

Influence of K+-dependent membrane lipid composition on the expression of the kdpFABC operon in Escherichia coli.

The membrane-bound sensor kinase KdpD and the cytoplasmic response regulator KdpE regulate the expression of the kdpFABC operon coding for the high affinity potassium uptake system KdpFABC in Escherichia coli. The signal transduction cascade of this two component system is activated under K(+)-limiting conditions in the medium, but is less sensitive to high osmolality. In order to test whether K(+) limitation affects membrane phospholipid composition and whether this change affects kdpFABC expression, we analysed the phospholipid composition of E. coli under these conditions. Our measurements revealed that there is an increase in the cardiolipin (CL) content during the exponential growth phase at the expense of the zwitterionic phospholipid phosphatidylethanolamine. The higher anionic phospholipid content occurs along with an increase of transcriptional activity of the cls gene coding for CL synthase. Furthermore, in vivo studies with E. coli derivatives carrying mutations in genes coding for enzymes involved in phospholipid biosynthesis revealed that the increase in the anionic lipid composition enhances the expression rate of the kdpFABC operon. Finally, we show that kinase activity of KdpD is stimulated in its native membrane environment by fusion with liposomes of anionic, but reduced with liposomes of zwitterionic phospholipids.

So You Want to Start an Institutional Biosafety Committee.

Introduction: The number of institutions engaging in research with potentially biohazardous materials has increased, indicating a need for newly formed Institutional Biosafety Committees (IBCs) in the United States and for similar biorisk management committees located outside the United States. Our institution identified the need for an IBC due to the growth of pertinent activities on campus. Objectives: This article shares our experiences creating a new IBC at our institution from September 2017 to April 2019. Our lessons learned and approaches to the challenges faced may be helpful to others finding themselves with similar needs. Methods: In this case study, we outline IBC membership, documents, relationships with federal agencies and within the institution, creation of registration forms, and the review process. Along with our account, we have included links to helpful resources from federal agencies. Results: At the time of the submission of this article, we have established our IBC and reviewed two registrations. Conclusion: This case report demonstrates the successful creation of an IBC that works for our current institutional needs.

Host suppression of quorum sensing during catheter-associated urinary tract infections.

Chronic bacterial infections on medical devices, including catheter-associated urinary tract infections (CAUTI), are associated with bacterial biofilm communities that are refractory to antibiotic therapy and resistant to host immunity. Previously, we have shown that Pseudomonas aeruginosa can cause CAUTI by forming a device-associated biofilm that is independent of known biofilm exopolysaccharides. Here, we show by RNA-seq that host urine alters the transcriptome of P. aeruginosa by suppressing quorum sensing regulated genes. P. aeruginosa produces acyl homoserine lactones (AHLs) in the presence of urea, but cannot perceive AHLs. Repression of quorum sensing by urine implies that quorum sensing should be dispensable during infection of the urinary tract. Indeed, mutants defective in quorum sensing are able to colonize similarly to wild-type in a murine model of CAUTI. Quorum sensing-regulated processes in clinical isolates are also inhibited by urea. These data show that urea in urine is a natural anti-quorum sensing mechanism in mammals.

Cross-regulation of Pseudomonas motility systems: the intimate relationship between flagella, pili and virulence.

Pseudomonas aeruginosa navigates using two distinct forms of motility, swimming and twitching. A polar flagellum and Type 4 pili power these movements, respectively, allowing P. aeruginosa to attach to and colonize surfaces. Single cell imaging and particle tracking algorithms have revealed a wide range of bacterial surface behaviors which are regulated by second messengers cyclic-di-GMP and cAMP; the production of these signals is, in turn, responsive to the engagement of motility organelles with a surface. Innate immune defense systems, long known to recognize structural components of flagella, appear to respond to motility itself. The association of motility with both upregulation of virulence and induction of host defense mechanisms underlies the complex contributions of flagella and pili to P. aeruginosa pathogenesis.

The extension of the fourth transmembrane helix of the sensor kinase KdpD of Escherichia coli is involved in sensing.

The KdpD sensor kinase and the KdpE response regulator control expression of the kdpFABC operon coding for the KdpFABC high-affinity K+ transport system of Escherichia coli. In search of a distinct part of the input domain of KdpD which is solely responsible for K+ sensing, sequences of kdpD encoding the transmembrane region and adjacent N-terminal and C-terminal extensions were subjected to random mutagenesis. Nine KdpD derivatives were identified that had lost tight regulation of kdpFABC expression. They all carried single amino acid replacements located in a region encompassing the fourth transmembrane helix and the adjacent arginine cluster of KdpD. All mutants exhibited high levels of kdpFABC expression regardless of the external K+ concentration. However, 3- to 14-fold induction was observed under extreme K+-limiting conditions and in response to an osmotic upshift when sucrose was used as an osmolyte. These KdpD derivatives were characterized by a reduced phosphatase activity in comparison to the autokinase activity in vitro, which explains constitutive expression. Whereas for wild-type KdpD the autokinase activity and also, in turn, the phosphotransfer activity to KdpE were inhibited by increasing concentrations of K+, both activities were unaffected in the KdpD derivatives. These data clearly show that the extension of the fourth transmembrane helix encompassing the arginine cluster is mainly involved in sensing both K+ limitation and osmotic upshift, which may not be separated mechanistically.