Identification of a system required for the functional surface localization of sugar binding proteins with class III signal peptides in Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus contains an unusual large number of sugar binding proteins that are synthesized as precursors with a class III signal peptide. Such signal peptides are commonly used to direct archaeal flagellin subunits or bacterial (pseudo)pilins into extracellular macromolecular surface appendages. Likewise, S. solfataricus binding proteins have been suggested to assemble in higher ordered surface structures as well, tentatively termed the bindosome. Here we show that S. solfataricus contains a specific system that is needed for the functional surface localization of sugar binding proteins. This system, encoded by the bas (bindosome assembly system) operon, is composed of five proteins: basABC, three homologues of so-called bacterial (pseudo)pilins; BasE, a cytoplasmic ATPase; and BasF, an integral membrane protein. Deletion of either the three (pseudo)pilin genes or the basEF genes resulted in a severe defect of the cells to grow on substrates which are transported by sugar binding proteins containing class III signal peptides, while growth on glucose and maltose was restored when the corresponding genes were reintroduced in these cells. Concomitantly, ΔbasABC and ΔbasEF cells were severely impaired in glucose uptake even though the sugar binding proteins were normally secreted across the cytoplasmic membrane. These data underline the hypothesis that the bas operon is involved in the functional localization of sugar binding proteins at the cell surface of S. solfataricus. In contrast to surface structure assembly systems of Gram-negative bacteria, the bas operon seems to resemble an ancestral simplified form of these machineries.

Structures of type IV pilins from Thermus thermophilus demonstrate similarities with type II secretion system pseudopilins

AbstractType IV pilins are proteins which form polymers that extend from the surface of the bacterial cell; they are involved in mediating a wide variety of functions, including adhesion, motility and natural competence. Here we describe the determination of the crystal structures of three type IVa pilins proteins from the thermophile Thermus thermophilus. They form part of a cluster of pilus-like proteins within the genome; our results show that one, Tt1222, is very closely related to the main structural type IV pilin, PilA4. The other two, Tt1218 and Tt1219, also adopt canonical pilin-like folds but, interestingly, are most closely related to the structures of the type II secretion system pseudopilins, EpsI/GspI and XcpW/GspJ. GspI and GspJ have been shown to form a complex with another pseudopilin, GspK, and this heterotrimeric complex is known to play a key role in initiating assembly of a pseudopilus which is thought to drive the secretion process. The structural similarity of Tt1218 and Tt1219 to GspI and GspJ suggests that they might work in a similar way, to deliver functions associated with type IV pili in T. thermophilus, such as natural competence

Protein Secretion Systems in Pseudomonas aeruginosa: An Essay on Diversity, Evolution, and Function

Protein secretion systems are molecular nanomachines used by Gram-negative bacteria to thrive within their environment. They are used to release enzymes that hydrolyze complex carbon sources into usable compounds, or to release proteins that capture essential ions such as iron. They are also used to colonize and survive within eukaryotic hosts, causing acute or chronic infections, subverting the host cell response and escaping the immune system. In this article, the opportunistic human pathogen Pseudomonas aeruginosa is used as a model to review the diversity of secretion systems that bacteria have evolved to achieve these goals. This diversity may result from a progressive transformation of cell envelope complexes that initially may not have been dedicated to secretion. The striking similarities between secretion systems and type IV pili, flagella, bacteriophage tail, or efflux pumps is a nice illustration of this evolution. Differences are also needed since various secretion configurations call for diversity. For example, some proteins are released in the extracellular medium while others are directly injected into the cytosol of eukaryotic cells. Some proteins are folded before being released and transit into the periplasm. Other proteins cross the whole cell envelope at once in an unfolded state. However, the secretion system requires conserved basic elements or features. For example, there is a need for an energy source or for an outer membrane channel. The structure of this review is thus quite unconventional. Instead of listing secretion types one after each other, it presents a melting pot of concepts indicating that secretion types are in constant evolution and use basic principles. In other words, emergence of new secretion systems could be predicted the way Mendeleïev had anticipated characteristics of yet unknown elements

The Vibrio cholerae Minor Pilin TcpB Initiates Assembly and Retraction of the Toxin- Coregulated Pilus

Type IV pilus (T4P) systems are complex molecular machines that polymerize major pilin proteins into thin filaments displayed on bacterial surfaces. Pilus functions require rapid extension and depolymerization of the pilus, powered by the assembly and retraction ATPases, respectively. A set of low abundance minor pilins influences pilus dynamics by unknown mechanisms. The Vibrio cholerae toxin-coregulated pilus (TCP) is among the simplest of the T4P systems, having a single minor pilin TcpB and lacking a retraction ATPase. Here we show that TcpB, like its homolog CofB, initiates pilus assembly. TcpB co-localizes with the pili but at extremely low levels, equivalent to one subunit per pilus. We used a micropillars assay to demonstrate that TCP are retractile despite the absence of a retraction ATPase, and that retraction relies on TcpB, as a V. cholerae tcpB Glu5Val mutant is fully piliated but does not induce micropillars movements. This mutant is impaired in TCP-mediated autoagglutination and TcpF secretion, consistent with retraction being required for these functions. We propose that TcpB initiates pilus retraction by incorporating into the growing pilus in a Glu5-dependent manner, which stalls assembly and triggers processive disassembly. These results provide a framework for understanding filament dynamics in more complex T4P systems and the closely related Type II secretion system

The role of intrinsic disorder and dynamics in the assembly and function of the type II secretion system

International audienceMany Gram-negative commensal and pathogenic bacteria use a type II secretion system (T2SS) to transport proteins out of the cell. These exported proteins or substrates play a major role in toxin delivery, maintaining biofilms, replication in the host and subversion of host immune responses to infection. We review the current structural and functional work on this system and argue that intrinsically disordered regions and protein dynamics are central for assembly, exo-protein recognition, and secretion competence of the T2SS. The central role of intrinsic disorder-order transitions in these processes may be a particular feature of type secretion

Type II secretion: from structure to function

Gram-negative bacteria use the type II secretion system to transport a large number of secreted proteins from the periplasmic space into the extracellular environment. Many of the secreted proteins are major virulence factors in plants and animals. The components of the type II secretion system are located in both the inner and outer membranes where they assemble into a multi-protein, cell-envelope spanning, complex. This review discusses recent progress, particularly newly published structures obtained by X-ray crystallography and electron microscopy that have increased our understanding of how the type II secretion apparatus functions and the role that individual proteins play in this complex system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74575/1/j.1574-6968.2006.00102.x.pd

Pseudomonas aeruginosa is capable of natural transformation in biofilms

Natural transformation is a mechanism that enables competent bacteria to acquire naked, exogenous DNA from the environment. It is a key process that facilitates the dissemination of antibiotic resistance and virulence determinants throughout bacterial populations. Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that produces large quantities of extracellular DNA (eDNA) that is required for biofilm formation. P. aeruginosa has a remarkable level of genome plasticity and diversity that suggests a high degree of horizontal gene transfer and recombination but is thought to be incapable of natural transformation. Here we show that P. aeruginosa possesses homologues of all proteins known to be involved in natural transformation in other bacterial species. We found that P. aeruginosa in biofilms is competent for natural transformation of both genomic and plasmid DNA. Furthermore, we demonstrate that type-IV pili (T4P) facilitate but are not absolutely essential for natural transformation in P. aeruginosa

Etude de la transformation plasmidique naturelle d'Escherichia coli et de ses relations éventuelles avec la compétence programmée pour la transformation génétique et la compétence dite nutritionnelle

Bien que la bactérie Escherichia coli ne soit pas connue pour être naturellement transformable, mon travail de thèse montre que l'on peut obtenir des transformants plasmidiques spontanément sur boîte. Cette transformation n'est pas induite par les cations divalents au contraire de la transformation ‘artificielle chimique’ (Sun et al., FEMS Microbiol. Lett. 2006. 265: 249–255). Les bactéries transformables utilisent une machinerie protéique transmembranaire, évolutivement conservée, pour internaliser l'ADN exogène sous forme simple brin. E.coli possède l'ensemble des gènes codant pour cette machinerie. J'ai inactivé les gènes clés de cette machinerie, dont hofQ (canal transmembranaire externe) et ycaI (canal transmembranaire interne), et observé qu'aucun de ces mutants n'est affecté pour la transformation sur boîte. L'ADN plasmidique ne pénètre donc pas via la machinerie de transformation, mais plutôt sous forme double brin ce que suggèrent les courbes de réponse à la concentration d'ADN (Sun et al., J. Bacteriol. 2009. 191: 713-719). Le troisième volet de ma thèse a consisté à tenter de mieux caractériser un phénomène appelé 'compétence nutritionnelle', appellation qui désigne la capacité d'utiliser l'ADN comme source de carbone. Pour établir si différents gènes de la machinerie de transformation étaient impliqués, j'ai cherché à reproduire les expériences publiées de croissance de la souche ZK126 sur milieu minimum M63 contenant de l'ADN. Malgré de nombreuses tentatives et contrôles, je n'ai pas pu reproduire ces expériences, ce qui m'a amené à clore mon mémoire de thèse par une discussion critique des données publiées relatives à la compétence nutritionnelle de E. coli.While Escherichia coli is not considered to belong to naturally transformable species, I established a transformation system allowing spontaneous plasmid transformation on plate (Sun et al., FEMS Microbiol. Lett. 2006. 265: 249–255). Transformation is not induced by divalent cations in contrast to chemically-induced 'artificial transformation' (Sun et al., J. Bacteriol. 2009. 191: 713-719). As DNA uptake in naturally transformable bacteria relies on a conserved multiprotein machinery and the E. coli genome contains all genes encoding this machinery, I investigated whether key genes are required for plasmid transformation. None of the mutants I constructed, including hofQ and ycaI which encode putative outer and inner membrane channel proteins were affected, indicating that plasmid DNA is not taken up via the transformation machinery. We proposed that plasmid DNA instead enters the cytoplasm as double stranded material as suggested by response curves to DNA concentration (Sun et al., J. Bacteriol. 2009. Ibid.). In the last part of my thesis, I reinvestigated so-called ‘nutritional competence’ of E. coli. Previously work reported that E. coli cells are able to use DNA as the sole carbon source. I wished to establish whether this phenomenon relies on the above-mentioned DNA uptake machinery. I therefore tried to reproduce the published growth experiments of E. coli ZK126 on M63 minimal medium with DNA. Despite numerous attempts and controls, I could not observe any growth. This failure to reproduce published observations led me to conclude my thesis by an in-depth discussion of the three articles dedicated to so-called nutritional competence of E. coli

Interactions between exeA and peptidoglycan in the type II secretion system of aeromonas hydrophila

Aeromonas hydrophila uses the type II secretion system to transport protein toxins across the outer membrane. The trans-envelope system is comprised of more than ten proteins, including ExeA and ExeB, which form a complex in the inner membrane and are required for assembly of the ExeD secretion channel multimer, called the secretin, into the outer membrane. A putative peptidoglycan binding domain (Pfam protein families database number PF01471) is present in the periplasmic region of ExeA (pExeA), leading to the hypothesis that ExeA generates gaps in peptidoglycan, a barrier for trans-envelope transport and apparatus assembly, to allow ExeD to assemble into the outer membrane. In this study, interactions between ExeA and peptidoglycan were examined both in vivo and in vitro. Wild type ExeA, but not the mutants containing substitution mutations of three highly conserved amino acid residues in the putative peptidoglycan binding domain, was cross-linked to peptidoglycan in vivo with DTSSP. Furthermore, the presence of wild type ExeA was also required for co-crosslinking of ExeB and ExeC to peptidoglycan. In vitro cosedimentation revealed that purified pExeA was able to bind to highly purified peptidoglycan. The protein assembled into large multimers in the presence of peptidoglycan fragments, as shown in cross-linking and co-gel filtration experiments. The requirement of peptidoglycan for multimerization was abrogated when the protein was incubated at temperatures above 25 °C. Two pExeA constructs, which disrupted the putative peptidoglycan binding domain, greatly reduced the cosedimentation, accompanied by decreased multimerization in the presence of peptidoglycan fragments. These results provide evidence that the putative peptidoglycan binding domain of ExeA is involved in physical contact with peptidoglycan. The interactions cause ExeA to multimerize, possibly forming a ring-like structure on the peptidoglycan, to generate a gap large enough to accommodate the secretion apparatus and/or to form an assembly scaffold. The putative peptidoglycan binding domain of ExeA was also analyzed by comparing its amino acid sequence with that of other homologues. The highly conserved amino acid residues were found to cluster at one pocket on the surface in the crystal structure of hydrolase metallo (Zn) DD-peptidase that also contains this domain. We propose that this pocket is the binding site for the peptidoglycan ligand