The Pseudomonas aeruginosa Pathogenicity Island PAPI-1 Is Transferred via a Novel Type IV Pilus

Pseudomonas aeruginosa is a major cause of nosocomial infections, particularly in immunocompromised patients or in individuals with cystic fibrosis. The notable ability of P. aeruginosa to inhabit a broad range of environments, including humans, is in part due to its large and diverse genomic repertoire. The genomes of most strains contain a significant number of large and small genomic islands, including those carrying virulence determinants (pathogenicity islands). The pathogenicity island PAPI-1 of strain PA14 is a cluster of 115 genes, and some have been shown to be responsible for virulence phenotypes in a number of infection models. We have previously demonstrated that PAPI-1 can be transferred to other P. aeruginosa strains following excision from the chromosome of the donor. Here we show that PAPI-1 is transferred into recipient P. aeruginosa by a conjugative mechanism, via a type IV pilus, encoded in PAPI-1 by a 10-gene cluster which is closely related to the genes in the enterobacterial plasmid R64. We also demonstrate that the precursor of the major pilus subunit, PilS2, is processed by the chromosomally encoded prepillin peptidase PilD but not its paralog FppA. Our results suggest that the pathogenicity island PAPI-1 may have evolved by acquisition of a conjugation system but that because of its dependence on an essential chromosomal determinant, its transfer is restricted to P. aeruginosa or other species capable of providing a functional prepilin peptidase.The genomes of a number of microorganisms, primarily those that have a capability of changing and adapting to a wide range of environments, evolve by acquisition of novel genetic information in blocks of genes via a process referred to as horizontal gene transfer (HGT). Other bacterial species change their genetic repertoire minimally, principally those that have adapted to a particular environment and, in the case of pathogenic bacteria, to a specific host. For HGT-mediated acquisition of genes to occur, a recipient has to be in an environment where donor genetic material is available, such as different strains of the same species cohabitating a shared niche or growing in a large and diverse community of several hundred different microorganisms. Moreover, for bacteria to become successful recipients of foreign genetic material, they have to posses one of three mechanisms of HGT: natural competence for uptake of foreign DNA (transformation), the ability to be infected by transducing bacteriophages (transduction), or serving as recipients during conjugation of plasmids or mobilized chromosomal DNA (conjugation). Acquired genetic material can consist of individual genes, where they recombine into homologous sequences in the recipient genome and thus increase the genetic diversity. However, large blocks of hundreds of contiguous genes in elements called genomic islands can be also transferred between bacteria, allowing the recipient microorganisms to acquire a number of new traits by a single HGT event.Previous studies comparing genomes of the opportunistic pathogen Pseudomonas aeruginosa pointed toward HGT as an important factor in its evolution (23). The genomes of all strains sequenced to date contain a significant fraction of horizontally acquired genes, in genomic islands and prophages, consisting of a few to several hundred. These islands can be recognized by the presence of certain signature features, such as an atypical nucleotide composition relative to the rest of the genome, location within predicted sites of chromosomal integration (att sites), and the presence of genes encoding bacteriophages and conjugation machineries. We have recently demonstrated that PAPI-1, a large P. aeruginosa genomic (pathogenicity) island, can be excised from its tRNA att site and that a copy can be transferred into a recipient, where it integrates into the same tRNA gene (27). Inspection of the genes in PAPI-1 and features of the transfer process, namely, an integrase-dependent excision and formation of a circular intermediate, suggested that PAPI-1 is an integrative and conjugative element and that it is likely transferred by a conjugative mechanism.Here we extended our analysis of PAPI-1 by testing its transfer from a preselected group of P. aeruginosa PA14 mutants with insertions in each of the genes on the island. Among those mutants that were defective in PAPI-1 transfer, one group of genes encode homologs of type IV pilus proteins. While type IV pili have been found to be involved primarily in bacterial adhesion and twitching motility (24), the PAPI-1-encoded pilus is closely related to the conjugative apparatus of plasmid R64 (14). Moreover, we show that an essential posttranslational modification reaction, converting the precursor of the major pilin subunit encoded in PAPI-1 into a mature protein, is carried out by an enzyme encoded in the chromosome of the donor cells. The acquisition and adaptation of groups of genes and subsequent loss of an essential function may represent a novel evolutionary strategy, limiting horizontal transfer to a specific bacterial species.     

Single-Residue Changes in the C-Terminal Disulfide-Bonded Loop of the Pseudomonas aeruginosa Type IV Pilin Influence Pilus Assembly and Twitching Motility

PilA, the major pilin subunit of Pseudomonas aeruginosa type IV pili (T4P), is a principal structural component. PilA has a conserved C-terminal disulfide-bonded loop (DSL) that has been implicated as the pilus adhesinotope. Structural studies have suggested that DSL is involved in intersubunit interactions within the pilus fiber. PilA mutants with single-residue substitutions, insertions, or deletions in the DSL were tested for pilin stability, pilus assembly, and T4P function. Mutation of either Cys residue of the DSL resulted in pilins that were unable to assemble into fibers. Ala replacements of the intervening residues had a range of effects on assembly or function, as measured by changes in surface pilus expression and twitching motility. Modification of the C-terminal P-X-X-C type II beta-turn motif, which is one of the few highly conserved features in pilins across various species, caused profound defects in assembly and twitching motility. Expression of pilins with suspected assembly defects in a pilA pilT double mutant unable to retract T4P allowed us to verify which subunits were physically unable to assemble. Use of two different PilA antibodies showed that the DSL may be an immunodominant epitope in intact pili compared with pilin monomers. Sequence diversity of the type IVa pilins likely reflects an evolutionary compromise between retention of function and antigenic variation. The consequences of DSL sequence changes should be evaluated in the intact protein since it is technically feasible to generate DSL-mimetic peptides with mutations that will not appear in the natural repertoire due to their deleterious effects on assembly.The gram-negative opportunistic pathogen Pseudomonas aeruginosa uses polar type IV pili (T4P) to attach to various materials, to move across surfaces via twitching motility, and to initiate host colonization and biofilm formation. T4P are widely distributed among bacteria and have been most extensively studied in Neisseria spp., Escherichia coli, Vibrio cholerae, and P. aeruginosa (8, 16, 42). T4P are divided into two major groups, type IVa and type IVb pili (T4aP and T4bP, respectively); there are several differences that distinguish these subfamilies (reviewed in reference 16). Most P. aeruginosa strains express T4aP composed of one of five different variants of the 15- to 17-kDa PilA protein (37).The crystal structures of N-terminally truncated or full-length forms of PilA from P. aeruginosa strains PAK and K122-4 have been solved (17, 18, 28, 34), as has the structure of the type IVa pilin from Neisseria gonorrhoeae MS11, called PilE (45). The pilins have a ladle-like structure, with a long, hydrophobic, kinked N-terminal alpha helix joined to a C-terminal domain of antiparallel beta-sheet architecture, terminating in a characteristic disulfide-bonded loop (DSL; also called the D-region). In a recent report describing the cryo-electron microscopy-derived ultrastructure of an assembled type IV pilus from N. gonorrhoeae, Craig and colleagues confirmed the predictions of earlier models that the N-terminal alpha helices of the subunits form the hydrophobic core of the fiber, with the hydrophilic C-terminal beta sheet and loop domains forming its outer surface (17).P. aeruginosa T4P mediate attachment to, and twitching motility on, an astonishing array of living and nonliving surfaces, from stainless steel and plastic to living cells (15, 20, 22, 25, 27, 44), contributing to the ability of this organism to cause opportunistic infections in a wide range of hosts. Twitching motility involves cycles of pilus extension, adherence, and subsequent pilus retraction that pulls the cell body forward (51). For twitching to occur, the pilus must adhere with sufficient strength that retraction of the pilus will result in translocation of the cell, overcoming the combination of surface tension and other cell surface adhesins that hold the cell body in place.Most bacterial pili, such as the types 1 and P pili of uropathogenic E. coli, are composed of separate structural (FimA and PapA) and adhesive (FimH and PapG) subunits, with the adhesive subunit present only at the tip of the pilus fiber (7, 32). P. aeruginosa T4P are unusual in this respect, in that the PilA subunit has been reported to act as both the main structural component and the tip adhesin (39, 50). The C-terminal DSL of the PilA subunit has been shown to mediate attachment of piliated P. aeruginosa to host cells and to abiotic surfaces such as stainless steel (25, 39, 50). This subdomain of PilA was shown by immunogold labeling studies to be exposed only at the pilus tip, suggesting that it is otherwise masked by adjacent subunits in the assembled pilus (39). These data are consistent with recent ultrastructural studies of N. gonorrhoeae T4P, which suggest that the C termini of the pilins are involved in intersubunit contacts throughout the length of the pilus fiber (17).To address the roles of specific residues within the DSL in host cell attachment, Wong and colleagues synthesized peptides corresponding to C-terminal residues 128 to 144 of the pilins from strains PAK and KB7, as well as analogues thereof containing Ala substitutions at each position (57). The peptides were oxidized to allow disulfide bond formation and used in a competition assay, measuring their ability to block binding of biotinylated PAK pili to buccal epithelial cells. Their study confirmed earlier observations that the Cys residues involved in disulfide bond formation contributed significantly to adhesin function and implicated a number of other residues in binding. However, a single adhesinotope common to both peptides could not be defined since they have only partial sequence identity. Conserved residues contributing to conformational elements, particularly type I and type II beta turns, were found to be important while a conserved hydrophobic residue (F137 in the PAK pilin) was not crucial for binding (57).As a prelude to studies examining the effects of sequence variation within the key DSL region on the adhesive capacity of the pilin subunit, we investigated the effects of PilA mutations on its multiple functions, including participation in protein secretion via the structurally related type II secretion (T2S) system in P. aeruginosa. A previous study (41) reported that PilA could form heterodimers with XcpT, the major pseudopilin of the Xcp T2S, and that PilA mutants were defective in T2S of proteases. In this work, the pilA gene from the laboratory strain PAO1 was mutagenized to generate single-residue variants of PilA that were expressed from an l-arabinose-inducible promoter in a pilA mutant background. This approach permitted the simultaneous interrogation of the effects of the mutations on pilin stability, assembly, and function in terms of twitching motility and pilus-specific bacteriophage susceptibility, as well as potential dominant-negative effects in the wild type upon induction. Here, we show that it is possible to identify single-residue variants of PilA that are affected in each step of pilus assembly and function.     

Minor Pilins of the Type IV Pilus System Participate in the Negative Regulation of Swarming Motility

Pseudomonas aeruginosa exhibits distinct surface-associated behaviors, including biofilm formation, flagellum-mediated swarming motility, and type IV pilus-driven twitching. Here, we report a role for the minor pilins, PilW and PilX, components of the type IV pilus assembly machinery, in the repression of swarming motility. Mutating either the pilW or pilX gene alleviates the inhibition of swarming motility observed for strains with elevated levels of the intracellular signaling molecule cyclic di-GMP (c-di-GMP) due to loss of BifA, a c-di-GMP-degrading phosphodiesterase. Blocking PilD peptidase-mediated processing of PilW and PilX renders the unprocessed proteins defective for pilus assembly but still functional in c-di-GMP-mediated swarming repression, indicating our ability to separate these functions. Strains with mutations in pilW or pilX also fail to exhibit the increase in c-di-GMP levels observed when wild-type (WT) or bifA mutant cells are grown on a surface. We also provide data showing that c-di-GMP levels are increased upon PilY1 overexpression in surface-grown cells and that this c-di-GMP increase does not occur in the absence of the SadC diguanylate cyclase. Increased levels of endogenous PilY1, PilX, and PilA are observed when cells are grown on a surface compared to liquid growth, linking surface growth and enhanced signaling via SadC. Our data support a model wherein PilW, PilX, and PilY1, in addition to their role(s) in type IV pilus biogenesis, function to repress swarming via modulation of intracellular c-di-GMP levels. By doing so, these pilus assembly proteins contribute to P. aeruginosa's ability to coordinately regulate biofilm formation with its two surface motility systems.     

Outside-In Assembly Pathway of the Type IV Pilus System in Myxococcus xanthus

Type IV pili (T4P) are ubiquitous bacterial cell surface structures that undergo cycles of extension, adhesion, and retraction. T4P function depends on a highly conserved envelope-spanning macromolecular machinery consisting of 10 proteins that localizes polarly in Myxococcus xanthus. Using this localization, we investigated the entire T4P machinery assembly pathway by systematically profiling the stability of all and the localization of eight of these proteins in the absence of other T4P machinery proteins as well as by mapping direct protein-protein interactions. Our experiments uncovered a sequential, outside-in pathway starting with the outer membrane (OM) PilQ secretin ring. PilQ recruits a subcomplex consisting of the inner membrane (IM) lipoprotein PilP and the integral IM proteins PilN and PilO by direct interaction with the periplasmic domain of PilP. The PilP/PilN/PilO subcomplex recruits the cytoplasmic PilM protein, by direct interaction between PilN and PilM, and the integral IM protein PilC. The PilB/PilT ATPases that power extension/retraction localize independently of other T4P machinery proteins. Thus, assembly of the T4P machinery initiates with formation of the OM secretin ring and continues inwards over the periplasm and IM to the cytoplasm.     

Genetic Analysis of Phenylalanine-Responding Mutants of Pseudomonas aeruginosa

A transduction analysis of phenylalanine-responding mutants of Pseudomonas aeruginosa revealed the existence of six unlinked genes. Enzyme assays showed that one gene was involved in the terminal production of phenylalanine (chorismate mutase), and the remaining five genes were involved in the common pathway of aromatic amino acid biosynthesis.     

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.     

Regulation of Pseudomonas aeruginosa chemotaxis by the nitrogen source.

The regulation of amino acid chemotaxis by nitrogen was investigated in the gram-negative bacterium Pseudomonas aeruginosa. The quantitative capillary tube technique was used to measure chemotactic responses of bacteria to spatial gradients of amino acids and other attractants. Chemotaxis toward serine, arginine, and alpha-aminoisobutyrate was sharply dependent on the form in which nitrogen was presented to the bacteria. Bacteria grown on mineral salts-succinate with potassium nitrate gave responses to amino acids that were 2 to 3 times those of cells grown on ammonium sulfate and 10 to 20 times those of cells grown in mineral salts-succinate with Casamino Acids as the nitrogen source. A combination of ammonium sulfate and glutamate was as effective as Casamino Acids in depressing serine taxis. The threshold concentration for alpha-aminoisobutyrate taxis was consistently lower in nitrate-grown bacteria than in ammonia-grown bacteria. Responsiveness to sodium succinate, however, was not subject to regulation by nitrogen, and glucose chemotaxis was inhibited, rather than enhanced, in nitrate-grown bacteria. These results indicate that chemotaxis of P. aeruginosa toward amino acids is subject to regulation by nitrogen and that this regulation probably is expressed at the level of the chemoreceptors or transducers.     

Regulation of the mandelate pathway in Pseudomonas aeruginosa

The pathway of mandelate metabolism in Pseudomonas aeruginosa is composed of the following steps: l(+)-mandelate --> benzoylformate --> benzaldehyde --> benzoate. These three steps are unique to mandelate oxidation; the benzoate formed is further metabolized via the beta-ketoadipate pathway. The first enzyme, l(+)-mandelate dehydrogenase, is induced by its substrate. The second and third enzymes, benzoylformate decarboxylase and benzaldehyde dehydrogenase, are both induced by benzoylformate. The same benzaldehyde dehydrogenase, or one very similar to it, is also induced by beta-ketoadipate, an intermediate in the subsequent metabolism of benzoate. This dehydrogenase may also be induced by adipate or a metabolite of adipate. These conclusions have been drawn from the physiological and genetic properties of wild-type P. aeruginosa strains and from the study of mutants lacking the second and third enzyme activities.     

High-Force Generation Is a Conserved Property of Type IV Pilus Systems

The type IV pilus (T4P) system of Neisseria gonorrhoeae is the strongest linear molecular motor reported to date, but it is unclear whether high-force generation is conserved between bacterial species. Using laser tweezers, we found that the average stalling force of single-pilus retraction in Myxococcus xanthus of 149 ± 14 pN exceeds the force generated by N. gonorrhoeae. Retraction velocities including a bimodal distribution were similar between M. xanthus and N. gonorrhoeae, but force-dependent directional switching was not. Force generation by pilus retraction is energized by the ATPase PilT. Surprisingly, an M. xanthus mutant lacking PilT apparently still retracted T4P, although at a reduced frequency. The retraction velocity was comparable to the high-velocity mode in the wild type at low forces but decreased drastically when the force increased, with an average stalling force of 70 ± 10 pN. Thus, M. xanthus harbors at least two different retraction motors. Our results demonstrate that the major physical properties are conserved between bacteria that are phylogenetically distant and pursue very different lifestyles.Type IV pili (T4P) are among the most widespread cell surface appendages in bacteria and have been found in beta-, gamma-, delta-, and epsilonproteobacteria and cyanobacteria, as well as in firmicutes (27). As opposed to other filamentous surface structures, T4P are highly dynamic structures and undergo cycles of extension and retraction (22, 30, 34). During the retraction step, sufficient force is generated to pull a bacterial cell forward in a type of surface movement referred to as twitching motility (30). The dynamic behavior is central to most of the functions of T4P, which in addition to cell motility, include surface adhesion, horizontal gene transfer, biofilm formation, and protein secretion (3).T4P are thin (5- to 8-nm) flexible filaments with a length of several micrometers (7). A core set of 10 proteins is conserved between different T4P systems and is required for T4P dynamics in Myxococcus xanthus, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Neisseria meningitidis, and Synechocystis sp. strain PCC6803 (24, 27). Genetic and biochemical data suggest that the proteins required for T4P function interact to form a complex that spans the cell envelope (2, 9, 10, 14, 28). The molecular mechanism underlying the assembly of T4P involves the incorporation of pilin subunits in the base of the pilus (8) from a reservoir in the cytoplasmic membrane (15, 30), and retraction involves the removal and transfer of pilin subunits from the pilus base into the cytoplasmic membrane (23). Genetic and biochemical evidence suggest that assembly of T4P is energized by ATP hydrolysis by the assembly ATPase PilB (PilF in Neisseria spp.) (15, 29) and that T4P retraction is energized by ATP hydrolysis by the retraction ATPase PilT (5, 11, 15).The soil-dwelling bacterium M. xanthus (a rod-shaped bacterium belonging to the deltaproteobacteria) requires T4P-dependent motility for the formation of spreading colonies in vegetative cells and fruiting bodies in starving cells. T4P extension and retraction have not been quantified in M. xanthus; however, indirect evidence for T4P retraction was obtained by characterizing the “jiggling” movement of isolated, individual M. xanthus cells adhering to polystyrene-coated surfaces (34).The dynamics and force generation of individual T4P have been characterized in detail in the human pathogen N. gonorrhoeae (6, 19-21), a diplococcus belonging to the betaproteobacteria. Generation of high forces in the range of 110 pN is a remarkable quality of T4P retractions in N. gonorrhoeae (21). It has been suggested that high-force generation may have evolved with the “lifestyle” of N. gonorrhoeae to induce signaling processes in the host cells during infections and to induce cytoprotection and cytoskeletal rearrangements (13). Here, we show that T4P retractions in M. xanthus, which lives in an entirely different habitat, has a different morphology, and is phylogenetically distant from N. gonorrhoeae, generate high forces in the range of 150 pN. On the basis of these observations, we suggest that high-force generation and bimodal velocity distributions are inherent properties of all T4P systems independent of phylogeny and bacterial lifestyle. Intriguingly, retractions still occurred at a low frequency in an M. xanthus strain lacking PilT, providing evidence for a PilT-independent retraction mechanism in M. xanthus. The physical characteristics of the PilT-independent T4P retractions were distinct from those in a PilT⁺ strain.     

群体感应对铜绿假单胞菌生物被膜形成的调控

生物被膜是一种与浮游细胞相对应的生长方式,由细菌和自身分泌的包外基质组成。铜绿假单胞菌是研究这一生长方式的模式生物。在过去十年,对铜绿假单胞菌生物被膜的研究已取得显著进展。群体感应(QS)的细胞沟通机制在铜绿假单胞菌生物被膜形成中发挥着重要作用。介绍生物被膜的特点,并重点讨论了QS和生物被膜之间的关系。