T4P Assembly Research Articles

PlzR regulates type IV pili assembly in Pseudomonas aeruginosa via PilZ binding

Type IV pili (T4P) are thin, flexible filaments exposed on the cell surface of gram-negative bacteria and are involved in pathogenesis-related processes, including cell adsorption, biofilm formation, and twitching motility. Bacteriophages often use these filaments as receptors to infect host cells. Here, we describe the identification of a protein that inhibits T4P assembly in Pseudomonas aeruginosa, discovered during a screen for host factors influencing phage infection. We show that expression of PA2560 (renamed PlzR) in P. aeruginosa inhibits adsorption of T4P-dependent phages. PlzR does this by directly binding the T4P chaperone PilZ, which in turn regulates the ATPase PilB and results in disturbed T4P assembly. As the plzR promoter is induced by cyclic di-GMP, PlzR might play a role in coupling T4P function to levels of this second messenger.

Myxococcus xanthus PilB interacts with c-di-GMP and modulates motility and biofilm formation.

The regulation of biofilm and motile states as alternate bacterial lifestyles has been studied extensively in flagellated bacteria, where the second messenger cyclic-di-GMP (cdG) plays a crucial role. However, much less is known about the mechanisms of such regulation in motile bacteria without flagella. The bacterial type IV pilus (T4P) serves as a motility apparatus that enables Myxococcus xanthus to move on solid surfaces. PilB, the T4P assembly ATPase, is, therefore, required for T4P-dependent motility in M. xanthus. Interestingly, T4P is also involved in the regulation of exopolysaccharide as the biofilm matrix material in this bacterium. A newly discovered cdG-binding domain, MshEN, is conserved in the N-terminus of PilB (PilBN) in M. xanthus and other bacteria. This suggests that cdG may bind to PilB to control the respective outputs that regulate biofilm development and T4P-powered motility. In this study, we aimed to validate M. xanthus PilB as a cdG effector protein. We performed a systematic mutational analysis of its cdG-binding domain to investigate its relationship with motility, piliation, and biofilm formation. Excluding those resulting in low levels of PilB protein, all other substitution mutations in PilBN resulted in pilB mutants with distinct and differential phenotypes in piliation and biofilm levels in M. xanthus. This suggests that the PilBN domain plays dual roles in modulating motility and biofilm levels, and these two functions of PilB can be dependent on and independent of each other in M. xanthus. IMPORTANCE The regulation of motility and biofilm by cyclic-di-GMP in flagellated bacteria has been extensively investigated. However, our knowledge regarding this regulation in motile bacteria without flagella remains limited. Here, we aimed to address this gap by investigating a non-flagellated bacterium with motility powered by bacterial type-IV pilus (T4P). Previous studies hinted at the possibility of Myxococcus xanthus PilB, the T4P assembly ATPase, serving as a cyclic-di-GMP effector involved in regulating both motility and biofilm. Our findings strongly support the hypothesis that PilB directly interacts with cyclic-di-GMP to act as a potential switch to promote biofilm formation or T4P-dependent motility. These results shed light on the bifurcation of PilB functions and its pivotal role in coordinating biofilm formation and T4P-mediated motility.

Pseudomonas aeruginosa Resists Phage Infection via Eavesdropping on Indole Signaling.

Phage therapy is challenged by the frequent emergence of bacterial resistance to phages. As an interspecies signaling molecule, indole plays important roles in regulating bacterial behaviors. However, it is unclear whether indole is involved in the phage-bacterium interactions. Here, we report that indole modulated phage resistance of Pseudomonas aeruginosa PAO1. Specifically, we found that the type IV pilus (T4P) acts as an important receptor for P. aeruginosa phages vB_Pae_S1 and vB_Pae_TR, and indole could protect P. aeruginosa against phage infection via decreasing the T4P-mediated phage adsorption. Further investigation demonstrated that indole downregulated the expression of genes pilA, pilB, and pilQ, which are essential for T4P assembly and activity. Indole inhibits phage attacks, but our data suggest that indole functions not through interfering with the AHL-based QS pathway, although las quorum sensing (QS) of P. aeruginosa PAO1 were reported to promote phage infection. Our finding confirms the important roles of indole in virus-host interactions, which will provide important enlightenment in promoting phage therapy for P. aeruginosa infections. IMPORTANCE Our finding is significant with respect to the study of the interactions between phage and host. Although the important roles of indole in bacterial physiology have been revealed, no direct examples of indole participating in phage-host interactions were reported. This study reports that indole could modulate the phage resistance of indole-nonproducing Pseudomonas aeruginosa PAO1 through inhibition of phage adsorption mechanism. Our finding will be significant for guiding phage therapy and fill some gaps in the field of phage-host interactions.

Electrogenic degradation of Reactive Red 152 dye by Niallia circulans DC10 and its genome sequence analysis reveals genes mediating dye degradation and anodic electron transfer

This study demonstrates the application of a newly isolated bacterium N. circulans DC10 for the degradation of azo dye Reactive Red 152 (RR152) and bio-electricity generation in a microbial fuel cell (MFC) system. When used as an anodic biocatalyst, N. circulans DC10 achieved 94.78 ± 1.17% decolourization and 89.55 ± 2.28% COD reduction within 72 h in the MFC system along with 166.48 mW/m2 maximum power density generation. The FT-IR and GC–MS analysis suggested that N. circulans DC10 utilizes the phenylacetic acid catabolic pathway for degradation of RR152 in the dual chamber MFC system. The genome sequence analysis of N. circulans DC10 revealed the presence of flavin mononucleotide (FMN) dependant-NADH azoreductase gene azoR and a cluster of genes, most of which participate in the biosynthesis of type IV pili (T4P), an extracellular appendage. The RT-qPCR analysis showed that the expression levels of azoR gene increased around seven folds and T4P assembly genes pilT/pilZ expression levels increased more than two folds in N. circulans DC10 during active biodegradation of RR152 in the anode of MFC systems as compared to the static control flask cultures. The obtained results suggest that T4P has a role in promoting cell adherence to the surface of anode by N. circulans DC10 for exo-electron transfer generating bioelectricity. The cumulative effect of azoR and type IV pilus assembly genes resulted in efficient bio-electrogenic degradation of azo dye RR152 in the MFC system.

Natural Transformation in Acinetobacter baumannii W068: A Genetic Analysis Reveals the Involvements of the CRP, XcpV, XcpW, TsaP, and TonB2.

Acinetobacter baumannii is a serious threat to public health, and there is increasing attention to the development of antibiotic resistance in this bacterium. Natural transformation is a major horizontal gene transfer mechanism that can lead to antibiotic resistance. To better understand the mechanism of natural transformation in A. baumannii, we selected a clinical isolate that was transformable but had no visible extracellular type IV pili (T4P) filaments and then examined the effects of multiple single-gene knockouts on natural plasmid transformation. Among 33 candidate genes, 28 knockout mutants had severely or completely impaired transformability. Some of these genes had established roles in T4P biogenesis; DNA transfer across the outer membrane, periplasm, or inner membrane; and protection of intracellular single-stranded DNA (ssDNA). Other genes had no previously reported roles in natural transformation of A. baumannii, including competence activator cAMP receptor protein (CRP), a periplasmic protein that may function in T4P assembly (TonB2), a T4P secretin-associated protein (TsaP), and two type II secretion system (T2SS) minor pseudopilus assembly prime complex competent proteins (XcpV and XcpW). The deletion of the T2SS assembly platform protein X had no effect on transformation, and the minor pseudopilins were capable of initiating major pilin assembly. Thus, we speculate that XcpV and XcpW may function in DNA uptake with major pilin assembly, a non-T2SS-dependent mechanism and that a competence pseudopilus similar to T4P constituted the central part of the DNA uptake complex. These results may help guide future research on the alarming increase of antibiotic resistance in this pathogen.

High-Throughput Screen for Inhibitors of the Type IV Pilus Assembly ATPase PilB.

ABSTRACTThe bacterial type IV pilus (T4P) is a prominent virulence factor in many significant human pathogens, some of which have become increasingly antibiotic resistant. Antivirulence chemotherapeutics are considered a promising alternative to antibiotics because they target the disease process instead of bacterial viability. However, a roadblock to the discovery of anti-T4P compounds is the lack of a high-throughput screen (HTS) that can be implemented relatively easily and economically. Here, we describe the first HTS for the identification of inhibitors specifically against the T4P assembly ATPase PilB in vitro. Chloracidobacterium thermophilum PilB (CtPilB) had been demonstrated to have robust ATPase activity and the ability to bind its expected ligands in vitro. We utilized CtPilB and MANT-ATP, a fluorescent ATP analog, to develop a binding assay and adapted it for an HTS. As a proof of principle, we performed a pilot screen with a small compound library of kinase inhibitors and identified quercetin as a PilB inhibitor in vitro. Using Myxococcus xanthus as a model bacterium, we found quercetin to reduce its T4P-dependent motility and T4P assembly in vivo. These results validated our HTS as effective in identifying PilB inhibitors. This assay may prove valuable in seeking leads for the development of antivirulence chemotherapeutics against PilB, an essential and universal component of all bacterial T4P systems.IMPORTANCE Many bacterial pathogens use their type IV pili (T4P) to facilitate and maintain infection of a human host. Small chemical compounds that inhibit the production or assembly of T4P hold promise in the treatment and prevention of infections, especially in the era of increasing threats from antibiotic-resistant bacteria. However, few chemicals are known to have inhibitory or anti-T4P activity. Their identification has not been easy due to the lack of a method for the screening of compound collections or libraries on a large scale. Here, we report the development of an assay that can be scaled up to screen compound libraries for inhibitors of a critical T4P assembly protein. We further demonstrate that it is feasible to use whole cells to examine potential inhibitors for their activity against T4P assembly in a bacterium.

Cyclic-di-GMP and ADP bind to separate domains of PilB as mutual allosteric effectors.

PilB is the assembly ATPase for the bacterial type IV pilus (T4P), and as a consequence, it is essential for T4P-mediated bacterial motility. In some cases, PilB has been demonstrated to regulate the production of exopolysaccharide (EPS) during bacterial biofilm development independently of or in addition to its function in pilus assembly. While the ATPase activity of PilB resides at its C-terminal region, the N terminus of a subset of PilBs forms a novel cyclic-di-GMP (cdG)-binding domain. This multi-domain structure suggests that PilB binds cdG and adenine nucleotides through separate domains which may influence the functionality of PilB in both motility and biofilm development. Here, Chloracidobacterium thermophilum PilB is used to investigate ligand binding by its separate domains and by the full-length protein. Our results confirm the specificity of these individual domains for their respective ligands and demonstrate communications between these domains in the full-length protein. It is clear that when the N- and the C-terminal domains of PilB bind to cdG and ADP, respectively, they mutually influence each other in conformation and in their binding to ligands. We propose that the interactions between these domains in response to their ligands play critical roles in modulating or controlling the functions of PilB as a regulator of EPS production and as the T4P assembly ATPase.

Two direct gene targets contribute to Clp-dependent regulation of type IV pilus-mediated twitching motility in Lysobacter enzymogenes OH11

Lysobacter enzymogenes is an agriculturally important Gram-negative bacterium that employs a multitude of antifungal mechanisms to inhibit and infect filamentous fungal pathogens, through secretion of antifungal antibiotic HSAF (heat-stable antifungal factor), formation of T4P (type IV pilus)-mediated twitching motility, and production of extracellular chitinase. Interestingly, all such key antifungal factors seem to be controlled by Clp, a master regulator in L. enzymogenes; however, the underlying mechanisms are poorly understood. Here, employing strain OH11 as a working model, we show that Clp plays a dual role in controlling OH11 twitching motility. It controls transcription of pilA, a major T4P structure pilin gene, via directly binding to its promoter region, as well as regulates the gene transcription of pilMONOPQ operon, whose products were essential for T4P assembly, by directly binding to a similar promoter sequence. We also truncated the Clp-binding region of the pilA promoter fragment down to 41bp to identify the potential Clp-binding sequence. In addition, the Clp-recognized pilM promoter motif of the L. enzymogenes strains is similarly conserved as the pilA promoter, both with a conserved 5'-GTG and a conserved CAC-3', spaced by ten highly variable nucleotides. Thus, this study identified two direct and previously uncharacterized gene targets of Clp contributing to its regulation in the L. enzymogenes twitching motility. Overall, our findings further elucidate the molecular genetics of Clp-dependent twitching motility in Lysobacter.

The type IV pilus assembly motor PilB is a robust hexameric ATPase with complex kinetics.

The bacterial type IV pilus (T4P) is a versatile nanomachine that functions in pathogenesis, biofilm formation, motility, and horizontal gene transfer. T4P assembly is powered by the motor ATPase PilB which is proposed to hydrolyze ATP by a symmetrical rotary mechanism. This mechanism, which is deduced from the structure of PilB, is untested. Here, we report the first kinetic studies of the PilB ATPase, supporting co-ordination among the protomers of this hexameric enzyme. Analysis of the genome sequence of Chloracidobacterium thermophilum identified a pilB gene whose protein we then heterologously expressed. This PilB formed a hexamer in solution and exhibited highly robust ATPase activity. It displays complex steady-state kinetics with an incline followed by a decline over an ATP concentration range of physiological relevance. The incline is multiphasic and the decline signifies substrate inhibition. These observations suggest that variations in intracellular ATP concentrations may regulate T4P assembly and T4P-mediated functions in vivo in accordance with the physiological state of bacteria with unanticipated complexity. We also identified a mutant pilB gene in the genomic DNA of C. thermophilum from an enrichment culture. The mutant PilB variant, which is significantly less active, exhibited similar inhibition of its ATPase activity by high concentrations of ATP. Our findings here with the PilB ATPase from C. thermophilum provide the first line of biochemical evidence for the co-ordination among PilB protomers consistent with the symmetrical rotary model of catalysis based on structural studies.

Cryoelectron Microscopy Reconstructions of the Pseudomonas aeruginosa and Neisseria gonorrhoeae Type IV Pili at Sub-nanometer Resolution

We report here cryoelectron microscopy reconstructions of type IV pili (T4P) from two important human pathogens, Pseudomonas aeruginosa and Neisseria gonorrhoeae, at ∼ 8 and 5Å resolution, respectively. The two structures reveal distinct arrangements of the pilin globular domains on the pilus surfaces, which impart different helical parameters, but similar packing of the conserved N-terminal α helices, α1, in the filament core. In contrast to the continuous α helix seen in the X-ray crystal structures of the P.aeruginosa and N.gonorrhoeae pilin subunits, α1 in the pilus filaments has a melted segment located between conserved helix-breaking residues Gly14 and Pro22, as seen for the Neisseria meningitidis T4P. Using mutagenesis we show that Pro22 is critical for pilus assembly, as are Thr2 and Glu5, which are positioned to interact in the hydrophobic filament core. These structures provide a framework for understanding T4P assembly, function, and biophysical properties.

Interaction of the cyclic-di-GMP binding protein FimX and the Type 4 pilus assembly ATPase promotes pilus assembly.

Type IVa pili (T4P) are bacterial surface structures that enable motility, adhesion, biofilm formation and virulence. T4P are assembled by nanomachines that span the bacterial cell envelope. Cycles of T4P assembly and retraction, powered by the ATPases PilB and PilT, allow bacteria to attach to and pull themselves along surfaces, so-called “twitching motility”. These opposing ATPase activities must be coordinated and T4P assembly limited to one pole for bacteria to show directional movement. How this occurs is still incompletely understood. Herein, we show that the c-di-GMP binding protein FimX, which is required for T4P assembly in Pseudomonas aeruginosa, localizes to the leading pole of twitching bacteria. Polar FimX localization requires both the presence of T4P assembly machine proteins and the assembly ATPase PilB. PilB itself loses its polar localization pattern when FimX is absent. We use two different approaches to confirm that FimX and PilB interact in vivo and in vitro, and further show that point mutant alleles of FimX that do not bind c-di-GMP also do not interact with PilB. Lastly, we demonstrate that FimX positively regulates T4P assembly and twitching motility by promoting the activity of the PilB ATPase, and not by stabilizing assembled pili or by preventing PilT-mediated retraction. Mutated alleles of FimX that no longer bind c-di-GMP do not allow rapid T4P assembly in these assays. We propose that by virtue of its high-affinity for c-di-GMP, FimX can promote T4P assembly when intracellular levels of this cyclic nucleotide are low. As P. aeruginosa PilB is not itself a high-affinity c-di-GMP receptor, unlike many other assembly ATPases, FimX may play a key role in coupling T4P mediated motility and adhesion to levels of this second messenger.

The type IV pilus assembly ATPase PilB functions as a signaling protein to regulate exopolysaccharide production in Myxococcus xanthus

Myxococcus xanthus possesses a form of surface motility powered by the retraction of the type IV pilus (T4P). Additionally, exopolysaccharide (EPS), the major constituent of bacterial biofilms, is required for this T4P-mediated motility in M. xanthus as the putative trigger of T4P retraction. The results here demonstrate that the T4P assembly ATPase PilB functions as an intermediary in the EPS regulatory pathway composed of the T4P upstream of the Dif signaling proteins in M. xanthus. A suppressor screen isolated a pilB mutation that restored EPS production to a T4P− mutant. An additional PilB mutant variant, which is deficient in ATP hydrolysis and T4P assembly, supports EPS production without the T4P, indicating PilB can regulate EPS production independently of its function in T4P assembly. Further analysis confirms that PilB functions downstream of the T4P filament but upstream of the Dif proteins. In vitro studies suggest that the nucleotide-free form of PilB assumes the active signaling conformation in EPS regulation. Since M. xanthus PilB possesses conserved motifs with high affinity for c-di-GMP binding, the findings here suggest that c-di-GMP can regulate both motility and biofilm formation through a single effector in this surface-motile bacterium.

Crystal Structure of a Type IV Pilus Assembly ATPase: Insights into the Molecular Mechanism of PilB from Thermus thermophilus

Type IV pili (T4P) mediate bacterial motility and virulence. The PilB/GspE family ATPases power the assembly of T4P and type 2 secretion systems. We determined the structure of the ATPase region of PilB (PilBATP) in complex with ATPγS to provide a model of a T4P assembly ATPase and a view of a PilB/GspE family hexamer at better than 3-Å resolution. Spatial positioning and conformations of the protomers suggest a mechanism of force generation. All six PilBATP protomers contain bound ATPγS. Two protomers form a closed conformation poised for ATP hydrolysis. The other four molecules assume an open conformation but separate into two pairs with distinct active-site accessibilities. We propose that one pair represents the post-hydrolysis phase while the other pair appears poised for ADP/ATP exchange. Collectively, the data suggest that T4P assembly is powered by coordinating concurrent substrate binding with ATP hydrolysis across the PilB hexamer.

Type IV Pilus Alignment Subcomplex Proteins PilN and PilO Form Homo- and Heterodimers in Vivo

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Type IV pili (T4P) are among the key virulence factors used by P. aeruginosa for host cell attachment, biofilm formation, and twitching motility, making this system a promising target for novel therapeutics. Point mutations in the conserved PilMNOP alignment subcomplex were previously shown to have distinct effects on assembly and disassembly of T4P, suggesting that it may function in a dynamic manner. We introduced mutations encoding Cys substitutions into pilN and/or pilO on the chromosome to maintain normal stoichiometry and expression levels and captured covalent PilNO heterodimers, as well as PilN and PilO homodimers, in vivo Most covalent PilN or PilO homodimers had minimal functional impact in P. aeruginosa, suggesting that homodimers are a physiologically relevant state. However, certain covalent homo- or heterodimers eliminated twitching motility, suggesting that specific PilNO configurations are essential for T4P function. These data were verified using soluble N-terminal truncated fragments of PilN and PilO Cys mutants, which purified as a mixture of homo- and heterodimers at volumes consistent with a tetramer. Deletion of genes encoding alignment subcomplex components, PilM or PilP, but not other T4P components, including the motor ATPases PilB or PilT, blocked in vivo formation of disulfide-bonded PilNO heterodimers, suggesting that both PilM and PilP influence the heterodimer interface. Combined, our data suggest that T4P function depends on rearrangements at PilN and PilO interfaces.

Type IV-pili dependent motility is co-regulated by PilSR and PilS2R2 two-component systems via distinct pathways in Myxococcus xanthus.

Myxococcus xanthus is an environmental bacterium with two forms of motility. One type, known as social motility, is dependent on extension and retraction of Type-IV pili (T4P) and production of extracellular polysaccharides (EPS). Several signaling systems have been linked to regulation of T4P-dependent motility. In particular, expression of the pilin subunit pilA requires the PilSR two-component signaling system (TCS). A second TCS, PilS2R2, encoded within the same locus that encodes PilSR, has also been linked to M. xanthus T4P-dependent motility. We demonstrate that PilSR and PilS2R2 regulate M. xanthus T4P-dependent motility through distinct pathways. Consistent with known roles of PilSR, our results indicate that the primary function of PilSR is to regulate expression of pilA. In contrast, PilS2 and PilR2 have little to no affect on PilA protein levels. However, deletion of pilR2 resulted in a reduction of assembled pili, significant decreases in EPS production and loss of T4P-dependent motility. Furthermore, the pilR2 mutation led to increased production of outer membrane vesicles (OMV). Collectively, we propose that PilS2R2 is required for proper assembly of T4P and regulation of OMV production, and hypothesize that production of these vesicles is related to M. xanthus motility.

Homo-trimeric Structure of the Type IVb Minor Pilin CofB Suggests Mechanism of CFA/III Pilus Assembly in Human Enterotoxigenic Escherichia coli

In gram-negative bacteria, the assembly of type IV pilus (T4P) and the evolutionally related pseudopilus of type II secretion system involves specialized structural proteins called pilins and pseudopilins, respectively, and is dynamically regulated to promote bacterial pathogenesis. Previous studies have suggested that a structural “tip”-like hetero-complex formed through the interaction of at least three minor (pseudo) pilins plays an important role in this process, while some members of the pathogenic type IVb subfamily are known to have only one such minor pilin subunit whose function is still unknown. Here, we determined the crystal structure of the type IVb minor pilin CofB of colonization factor antigen/III from human enterotoxigenic Escherichia coli at 1.88-Å resolution. The crystal structure, in conjunction with physicochemical analysis in solution, reveals a symmetrical homo-trimeric arrangement distinct from the hetero-complexes of minor (pseudo) pilins observed in other T4P and type II secretion systems. Each CofB monomer adopts a unique three-domain architecture, in which the C-terminal β-sheet-rich lectin domain can effectively initiate trimer association of its pilin-like N-terminal domain through extensive hydrophobic interactions followed by domain swapping at the central hinge-like domain. Deletion of cofB produces a phenotype with no detectable pili formation on the cell surface, while molecular modeling indicates that the characteristic homo-trimeric structure of CofB is well situated at the pilus tip of colonization factor antigen/III formed by the major pilin CofA, suggesting a role for the minor pilin in the efficient initiation of T4P assembly.

Cyclic Di-GMP Regulates Type IV Pilus-Dependent Motility in Myxococcus xanthus.

ABSTRACTThe nucleotide-based second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is involved in regulating a plethora of processes in bacteria that are typically associated with lifestyle changes. Myxococcus xanthus undergoes major lifestyle changes in response to nutrient availability, with the formation of spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. Here, we investigated the function of c-di-GMP in M. xanthus and show that this bacterium synthesizes c-di-GMP during growth. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase correlated with defects in type IV pilus (T4P)-dependent motility, whereas gliding motility was unaffected. An increased level of c-di-GMP correlated with reduced transcription of the pilA gene (which encodes the major pilin of T4P), reduced the assembly of T4P, and altered cell agglutination, whereas a decreased c-di-GMP level correlated with altered cell agglutination. The systematic inactivation of the 24 genes in M. xanthus encoding proteins containing GGDEF, EAL, or HD-GYP domains, which are associated with c-di-GMP synthesis, degradation, or binding, identified three genes encoding proteins important for T4P-dependent motility, whereas all mutants had normal gliding motility. Purified DmxA had diguanylate cyclase activity, whereas the hybrid histidine protein kinases TmoK and SgmT, each of which contains a GGDEF domain, did not have diguanylate cyclase activity. These results demonstrate that c-di-GMP is important for T4P-dependent motility in M. xanthus.IMPORTANCE We provide the first direct evidence that M. xanthus synthesizes c-di-GMP and demonstrate that c-di-GMP is important for T4P-dependent motility, whereas we did not obtain evidence that c-di-GMP regulates gliding motility. The data presented uncovered a novel mechanism for regulation of T4P-dependent motility, in which increased levels of c-di-GMP inhibit transcription of the pilA gene (which encodes the major pilin of T4P), ultimately resulting in the reduced assembly of T4P. Moreover, we identified an enzymatically active diguanylate cyclase that is important for T4P-dependent motility.

Novel Role for PilNO in Type IV Pilus Retraction Revealed by Alignment Subcomplex Mutations.

Type IV pili (T4P) are dynamic protein filaments that mediate bacterial adhesion, biofilm formation, and twitching motility. The highly conserved PilMNOP proteins form an inner membrane alignment subcomplex required for function of the T4P system, though their exact roles are unclear. Three potential interaction interfaces for PilNO were identified: core-core, coiled coils (CC), and the transmembrane segments (TMSs). A high-confidence PilNO heterodimer model was used to select key residues for mutation, and the resulting effects on protein-protein interactions were examined both in a bacterial two-hybrid (BTH) system and in their native Pseudomonas aeruginosa context. Mutations in the oppositely charged CC regions or the TMS disrupted PilNO heterodimer formation in the BTH assay, while up to six combined mutations in the core failed to disrupt the interaction. When the mutations were introduced into the P. aeruginosa chromosome at the pilN or pilO locus, specific changes at each of the three interfaces--including core mutations that failed to disrupt interactions in the BTH system--abrogated surface piliation and/or impaired twitching motility. Unexpectedly, specific CC mutants were hyperpiliated but nonmotile, a hallmark of pilus retraction defects. These data suggest that PilNO participate in both the extension and retraction of T4P. Our findings support a model of multiple, precise interaction interfaces between PilNO; emphasize the importance of studying protein function in a minimally perturbed context and stoichiometry; and highlight potential target sites for development of small-molecule inhibitors of the T4P system. Pseudomonas aeruginosa is an opportunistic pathogen that uses type IV pili (T4P) for host attachment. The T4P machinery is composed of four cell envelope-spanning subcomplexes. PilN and PilO heterodimers are part of the alignment subcomplex and essential for T4P function. Three potential PilNO interaction interfaces (the core-core, coiled-coil, and transmembrane segment interfaces) were probed using site-directed mutagenesis followed by functional assays in an Escherichia coli two-hybrid system and in P. aeruginosa. Several mutations blocked T4P assembly and/or motility, including two that revealed a novel role for PilNO in pilus retraction, while other mutations affected extension dynamics. These critical PilNO interaction interfaces represent novel targets for small-molecule inhibitors with the potential to disrupt T4P function.

Pseudomonas aeruginosa Minor Pilins Prime Type IVa Pilus Assembly and Promote Surface Display of the PilY1 Adhesin

Type IV pili (T4P) contain hundreds of major subunits, but minor subunits are also required for assembly and function. Here we show that Pseudomonas aeruginosa minor pilins prime pilus assembly and traffic the pilus-associated adhesin and anti-retraction protein, PilY1, to the cell surface. PilV, PilW, and PilX require PilY1 for inclusion in surface pili and vice versa, suggestive of complex formation. PilE requires PilVWXY1 for inclusion, suggesting that it binds a novel interface created by two or more components. FimU is incorporated independently of the others and is proposed to couple the putative minor pilin-PilY1 complex to the major subunit. The production of small amounts of T4P by a mutant lacking the minor pilin operon was traced to expression of minor pseudopilins from the P. aeruginosa type II secretion (T2S) system, showing that under retraction-deficient conditions, T2S minor subunits can prime T4P assembly. Deletion of all minor subunits abrogated pilus assembly. In a strain lacking the minor pseudopilins, PilVWXY1 and either FimU or PilE comprised the minimal set of components required for pilus assembly. Supporting functional conservation of T2S and T4P minor components, our 1.4 Å crystal structure of FimU revealed striking architectural similarity to its T2S ortholog GspH, despite minimal sequence identity. We propose that PilVWXY1 form a priming complex for assembly and that PilE and FimU together stably couple the complex to the major subunit. Trafficking of the anti-retraction factor PilY1 to the cell surface allows for production of pili of sufficient length to support adherence and motility.

Peptidoglycan-binding protein TsaP functions in surface assembly of type IV pili

Type IV pili (T4P) are ubiquitous and versatile bacterial cell surface structures involved in adhesion to host cells, biofilm formation, motility, and DNA uptake. In Gram-negative bacteria, T4P pass the outer membrane (OM) through the large, oligomeric, ring-shaped secretin complex. In the β-proteobacterium Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which we name T4P secretin-associated protein (TsaP), was identified as a phylogenetically widely conserved component of the secretin complex that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the OM in a PilQ-dependent manner. In the δ-proteobacterium Myxococcus xanthus, TsaP is also important for surface assembly of T4P, and it accumulates and localizes in a PilQ-dependent manner to the cell poles. Our results show that TsaP is a novel protein associated with T4P function and suggest that TsaP functions to anchor the secretin complex to the peptidoglycan.