Type IV pili and cell motility

Type IV pili (Tfp) mediate the movement of bacteria over surfaces without the use of flagella. These movements are known as social gliding in Myxococcus xanthus and twitching in organisms such as Pseudomonas aeruginosa and Neisseria gonorrhoeae. Tfp are localized polarly. Type IV pilins have a signature N-terminal domain, which forms a coiled-coil with other monomer units to polymerize a pilus fibre. At least 10 more proteins at the base of the fibre are conserved; they are related to the type II secretion system. Movements produced by Tfp range from short, jerky displacements to lengthy, smooth ones. Tfp also participate in cell–cell interactions, pathogenesis, biofilm formation, natural DNA uptake, auto-aggregation of cells and development. What is the means by which Tfp bring about the movement of cells?     

Identification of the pgmG Gene, Encoding a Bifunctional Protein with Phosphoglucomutase and Phosphomannomutase Activities, in the Gellan Gum-Producing Strain Sphingomonas paucimobilis ATCC 31461

The pgmG gene of Sphingomonas paucimobilis ATCC 31461, the industrial gellan gum-producing strain, was cloned and sequenced. It encodes a 50,059-Da polypeptide that has phosphoglucomutase (PGM) and phosphomannomutase (PMM) activities and is 37 to 59% identical to other bifunctional proteins with PGM and PMM activities from gram-negative species, including Pseudomonas aeruginosa AlgC. Purified PgmG protein showed a marked preference for glucose-1-phosphate (G1P); the catalytic efficiency was about 50-fold higher for G1P than it was for mannose-1-phosphate (M1P). The estimated apparent K_m values for G1P and M1P were high, 0.33 and 1.27 mM, respectively. The pgmG gene allowed the recovery of alginate biosynthetic ability in a P. aeruginosa mutant with a defective algC gene. This result indicates that PgmG protein can convert mannose-6-phosphate into M1P in the initial steps of alginate biosynthesis and, together with other results, suggests that PgmG may convert glucose-6-phosphate into G1P in the gellan pathway.     

A simple,switchable pili-labelling method by plasmid-based replacement of pilin

Labelling of Type IV pili (TFP) can greatly improve our understanding of the pivotal roles of TFP in a variety of bacterial activities including motility, surface sensing and DNA-uptake etc. Here we show a simple and switchable pili-labelling method by plasmid-based inducible replacement of PilA without genetic modification in bacterial genome employed by complicated methods. Using this method, we characterized pili morphology and twitching motility of Pseudomonas aeruginosa in details. More importantly, we demonstrate its application in studying the replenishment dynamics of pilin pool of P. aeruginosa.     

Periplasmic Domains of Pseudomonas aeruginosa PilN and PilO Form a Stable Heterodimeric Complex

Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 Å resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.     

Structure–function analysis reveals that the Pseudomonas aeruginosa Tps4 two-partner secretion system is involved in CupB5 translocation

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous with cystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a model organism to study biofilms: a bacterial population embedded in an extracellular matrix that provide protection from environmental pressures and lead to persistence. A number of Chaperone-Usher Pathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili on the bacterial surface. One of these, encoded by the cupB operon, is unique as it contains a nonchaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised of a C-terminal integral membrane β-barrel pore with tandem N-terminal POTRA (POlypeptide TRansport Associated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). Using NMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4 (LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 can translocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observation that suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstrate that tps4/cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa has developed synergy between these systems. Furthermore, we have determined the solution-structure of the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mapping and in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residues for TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for each POTRA domain.     

Binding of cyclic diguanylate in the non-catalytic EAL domain of FimX induces a long-range conformational change

FimX is a multidomain signaling protein required for type IV pilus biogenesis and twitching motility in the opportunistic pathogen Pseudomonas aeruginosa. FimX is localized to the single pole of the bacterial cell, and the unipolar localization is crucial for the correct assembly of type IV pili. FimX contains a non-catalytic EAL domain that lacks cyclic diguanylate (c-di-GMP) phosphodiesterase activity. It was shown that deletion of the EAL domain or mutation of the signature EVL motif affects the unipolar localization of FimX. However, it was not understood how the C-terminal EAL domain could influence protein localization considering that the localization sequence resides in the remote N-terminal region of the protein. Using hydrogen/deuterium exchange-coupled mass spectrometry, we found that the binding of c-di-GMP to the EAL domain triggers a long-range (∼ca. 70 Å) conformational change in the N-terminal REC domain and the adjacent linker. In conjunction with the observation that mutation of the EVL motif of the EAL domain abolishes the binding of c-di-GMP, the hydrogen/deuterium exchange results provide a molecular explanation for the mediation of protein localization and type IV pilus biogenesis by c-di-GMP through a remarkable allosteric regulation mechanism.     

PilJ Localizes to Cell Poles and Is Required for Type IV Pilus Extension in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Twitching motility allows Pseudomonas aeruginosa to respond to stimuli by extending and retracting its type IV pili (TFP). PilJ is a protein necessary for this surface-associated twitching motility and bears high sequence identity with Escherichia coli methyl-accepting chemotaxis proteins (MCP). Here, we report that whereas wild-type P. aeruginosa PAO1 cells have extended pili at a single pole, pilJ mutant cells have shortened pili often at both poles despite normal levels of pilin accumulation, suggesting that PilJ is required for full TFP assembly/extension. Using yellow fluorescent protein fusions (pilJ-yfp), both plasmid born and in-frame chromosomal constructs, we determined that PilJ localizes to both poles of the cell. Overexpression of pilJ-yfp resulted in the protein accumulating between the poles. Paul DeLange and Tracy Collins contributed equally to this work.     

Identification and characterization of pilG, a highly conserved pilus-assembly gene in pathogenic Neisseria

Expression of type IV pili appears to be a requisite determinant of infectivity for the strict human pathogens Neisseria gonorrhoeae and Neisseria meningitidis. The assembly of these colonization factors is a complex process. This report describes a new pilus-assembly gene, pilG, that immediately precedes the gonococcal (Gc) pilD gene encoding the pre-pilin leader peptidase. The nucleotide sequence of this region revealed a single complete open reading frame whose derived polypeptide displayed significant identities to the pilus-assembty protein PilC of Pseudomonas aeruginosa and other polytopic integral cytoplasmic membrane constituents involved in protein export and competence. A unique polypeptide of M_r 38kDa corresponding to the gene product was identified. A highly related gene and flanking sequences were cloned from a group E polysaccharide-producing strain of N. meningitidis (Mc). The results indicate that the pilG genes and genetic organization at these loci in Gc and Me are extremely conserved. Hybridization studies strongly suggest that pilG-related genes exist in commensal Neisseria species and other species known to express type IV pili. Defined genetic lesions were created by using insertional and transposon mutagenesis and moved into the Gc and Me chromosomes by allelic replacement. Chromosomal pilG insertion mutants were devoid of pili and displayed dramatically reduced competence for transformation. These findings could not be ascribed to pilin-gene alterations or to polarity exerted on pilD expression. The results indicated that PilG exerts its own independent role in neisserial pilus biogenesis.     

Crystal structure and oligomeric state of the RetS signaling kinase sensory domain

The opportunistic pathogen Pseudomonas aeruginosa may cause both acute and chronic‐persistent infections in predisposed individuals. Acute infections require the presence of a functional type III secretion system (T3SS), whereas chronic P. aeruginosa infections are characterized by the formation of drug‐resistant biofilms. The T3SS and biofilm formation are reciprocally regulated by the signaling kinases LadS, RetS, and GacS. RetS downregulates biofilm formation and upregulates expression of the T3SS through a unique mechanism. RetS forms a heterodimeric complex with GacS and thus prevents GacS autophosphorylation and downstream signaling. The signals that regulate RetS are not known but RetS possesses a distinctive periplasmic sensor domain that is believed to serve as receptor for the regulatory ligand. We have determined the crystal structure of the RetS sensory domain at 2.0 Å resolution. The structure closely resembles those of carbohydrate binding modules of other proteins, suggesting that the elusive ligands are likely carbohydrate moieties. In addition to the conserved beta‐sandwich structure, the sensory domain features two alpha helices which create a unique surface topology. Protein–protein crosslinking and fluorescence energy transfer experiments also revealed that the sensory domain dimerizes with a dissociation constant of K_d = 580 ± 50 nM, a result with interesting implications for our understanding of the underlying signaling mechanism. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Flow directs surface-attached bacteria to twitch upstream

Bacteria inhabit a wide variety of environments in which fluid flow is present, including healthcare and food processing settings and the vasculature of animals and plants. The motility of bacteria on surfaces in the presence of flow has not been well characterized. Here we focus on Pseudomonas aeruginosa, an opportunistic human pathogen that thrives in flow conditions such as in catheters and respiratory tracts. We investigate the effects of flow on P. aeruginosa cells and describe a mechanism in which surface shear stress orients surface-attached P. aeruginosa cells along the flow direction, causing cells to migrate against the flow direction while pivoting in a zig-zag motion. This upstream movement is due to the retraction of type IV pili by the ATPase motors PilT and PilU and results from the effects of flow on the polar localization of type IV pili. This directed upstream motility could be beneficial in environments where flow is present, allowing bacteria to colonize environments that cannot be reached by other surface-attached bacteria.     

Structural and functional characterization of TgpA,a critical protein for the viability of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen associated with severe diseases, such as cystic fibrosis. During an extensive search for novel essential genes, we identified tgpA (locus PA2873) in P. aeruginosa PAO1, as a gene playing a critical role in bacterial viability. TgpA, the translated protein, is an internal membrane protein with a periplasmic soluble domain, predicted to be endowed with a transglutaminase-like fold, hosting the Cys404, His448, and Asp464 triad. We report here that Cys404 mutation hampers the essential role of TgpA in granting P. aeruginosa viability. Moreover, we present the crystal structure of the TgpA periplasmic domain at 1.6?Å resolution as a first step towards structure–activity analysis of a new potential target for the discovery of antibacterial compounds.     

Evolutionary History and Functional Diversification of Phosphomannomutase Genes

Phosphomannomutases (PMMs) catalyze the interconversion of mannose-6-phosphate to mannose-1-phosphate. In humans, two PMM enzymes exist—PMM1 and PMM2; yet, they have different functional specificities. PMM2 presents PMM activity, and its deficiency causes a Congenital Disorder of Glycosylation (PMM2-CDG). On the other hand, PMM1 can also act as glucose-1,6-bisphosphatase in the brain after stimulation with inosine monophosphate and thus far has not been implicated in any human disease. This study aims to refine the evolutionary time frame at which gene duplication gave rise to PMM1 and PMM2, and to identify the most likely amino acid positions underlying the proteins’ different functions. The phylogenetic analysis using available protein sequences, allowed us to establish that duplication occurred early in vertebrate evolution. In order to understand the molecular basis underlying the functional divergence, conserved and most likely functional divergence-related sites were identified, through the analysis of site-specific evolutionary rates. This analysis indicates that most of the sites known to be important in the homodimer formation and in the catalytic activity are conserved in both proteins. Among those potentially related to functional divergence, two positions (183 and 186 in human PMM1) emerge as the most interesting ones. The residues at these positions have different side-chain conformations in the protein structure in the unbound and bound states, and are highly but differently conserved in PMM1 and in PMM2 proteins. Altogether, these results provide new data into the evolutionary history of PMM1 and PMM2 duplicates and highlight the most probable sites that evolved to distinct functional specificities.     

A Novel Alginate Lyase with High Activity on Acetylated Alginate of Pseudomonas aeruginosa FRD1 from Pseudomonas sp. QD03

Summary To exploit alginate lyase which could degrade bacterial alginates, degenerate PCR and long range-inverse PCR (LR-IPCR) were used to isolate alginate lyase genes from soil bacteria. Gene algL, an alginate lyase-encoding gene from Pseudomonas sp. QD03 was cloned, and it was composed of a 1122 bp open reading frame (ORF) encoding 373 amino acid residues with the calculated molecular mass of 42.2 kDa. The deduced protein had a potential N-terminal signal peptide of 20 amino acid residues that was consistent with its proposed periplasmic location. Gene algL was expressed in pET24a (+)/E. coli BL21 (DE3) system. The recombinant AlgL was purified to electrophoretic homogeneity using affinity chromatography. The molecular weight of AlgL was estimated to be 42.8 kDa by SDS-PAGE. AlgL exhibited maximal activity at pH 7.5 and 37 °C. Na⁺, K⁺, Ca²⁺ and Ba²⁺ significantly enhanced the activity of AlgL. AlgL could degrade alginate and mannuronate blocks, but hardly degrade guluronate blocks. In particular, AlgL could degrade acetylated alginate of Pseudomonas aeruginosa FRD1 (approximately 0.54 mol of O-acetyl group per mol of alginate). It might be possible to use alginate lyase AlgL as an adjuvant therapeutic medicine for the treatment of disease associated with P. aeruginosa infection.     

Identification, Cloning, and Expression of Pseudomonas aeruginosa Phosphorylcholine Phosphatase Gene

Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) is a periplasmic enzyme produced simultaneously with the hemolytic phospholipase C (PLc-H) when the bacteria are grown in the presence of choline, betaine, dimethylglycine or carnitine. Molecular analysis of the P. aeruginosa mutant JUF8-00, after Tn5-751 mutagenesis, revealed that the PA5292 gene in the P. aeruginosa PAO1 genome was responsible for the synthesis of PChP. The enzyme expressed in E. coli, rPChP-Ec, purified by a chitin-binding column (IMPACT-CN system, New England BioLabs) was homogeneous after SDS-PAGE analysis. PChP was also expressed in P. aeruginosa PAO1-LAC, rPChP-Pa. Both recombinant enzymes exhibited a molecular mass of approximately 40 kDa, as expected for the size of the PA5292 gene, and catalyzed the hydrolysis of phosphorylcholine, phosphorylethanolamine, and p-nitrophenylphosphate. The saturation curve of rPChP-Ec and rPChP-Pa by phosphorylcholine revealed that these recombinant enzymes, like the purified native PChP, also contained the high- and low-affinity sites for phosphorylcholine and that the enzyme activity was inhibited by high substrate concentration.     

The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip-associated event involving the C-terminal region of the structural pilin subunit

Pili are one of the adhesins of Pseudomonas aeruginosa that mediate adherence to epithelial cell-surface receptors. The pili of P. aeruginosa strains PAK and PAO were examined and found to bind gangliotetraosyl ceramide (asialo-GM₁) and, to a lesser extend, ll³N-acetylneuraminosylgangliotetraosyl ceramide (GM₁) in solid-phase binding assays. Asialo-GM₁, but not GM₁, inhibited both PAK and PAK pili binding to immobilized asialo-GM₁ on the microtitre plate. PAO pili competitively inhibited PAK pili binding to asialo-GM₁, suggesting the presence of a structurally similar receptor-binding domain in both pilus types. The interaction between asialo-GM₁ and pili occurs at the pilus tip as asialo-GM₁ coated colloidal gold only decorates the tip of purified pili. Three sets of evidence suggest that the C-terminal disulphide-bonded region of the Pseudomonas pilin is exposed at the tip of the pilus: (i) immunocytochemical studies indicate that P. aeruginosa pili have a basal-tip structural differentiation where the monoclonal antibody (mAb) PK3B recognizes an antigenic epitope displayed only on the basal ends of pili (produced by shearing) while the mAb PK99H, whose antigenic epitope resides in residues 134–140 (Wong et al., 1992), binds only to the tip of PAK pili; (ii) synthetic peptides, PAK(128–144)^ox-OH and PAO(128–144)^ox-OH, which correspond to the C-terminal disulphide-bonded region of Pseudomonas pilin are able to bind to asialo-GM₁ and inhibit the binding of pili to the glycolipid; (iii) PK99H was shown to block PAK pilus binding to asialo-GM₁ Monoclonal antibody PK3B had no effect on PAK pili binding to asialo-GM₁ Thus, the adherence of the Pseudomonas pilus to glycosphingolipid receptors is a tip-associated phenomenon Involving a tip-exposed C-terminal region of the pilin structural subunit.     

Pseudomonas aeruginosa Bacteriophage PA1O Requires Type IV Pili for Infection and Shows Broad Bactericidal and Biofilm Removal Activities

We isolated a new lytic Pseudomonas aeruginosa phage that requires type IV pili for infection. PA1Ø has a broad bactericidal spectrum, covering Gram-positive and Gram-negative bacteria, and can eradicate biofilm cells. PA1Ø may be developed as a therapeutic agent for biofilm-related mixed infections with P. aeruginosa and Staphylococcus aureus.     

Specificity of the lipase-specific foldases of gram-negative bacteria and the role of the membrane anchor

Folding of lipases that are secreted by Pseudomonads and other gram-negative bacteria via the type II secretion pathway is facilitated by dedicated chaperones, called lipase-specific foldases (Lifs). Lifs are membrane-anchored proteins with a large periplasmic domain. The functional interaction between the Lif and its cognate lipase is specific, since the Pseudomonas aeruginosa Lif was found not to substitute for Lifs from Burkholderia glumae or Acinetobacter calcoaceticus. However, the P. aeruginosa Lif was able to activate the lipase from the closely related species P. alcaligenes. Hybrid proteins constructed from parts of the P. aeruginosa and B. glumae Lifs revealed that the C-terminal 138 amino acids of the B. glumae Lif determine the specificity of the interaction with the cognate lipase. Furthermore, the periplasmic domain of the B. glumae Lif was functional when cloned in frame with a cleavable signal sequence, which demonstrates that the membrane anchor is not essential for Lif function in vivo. However, the recombinant Lif was released into the medium, indicating that the function of the membrane anchor is to prevent secretion of the Lif together with the lipase. Received: 12 November 1998 / Accepted: 19 February 1999