Biomedical subjects

Anthony P Pugsley

Publications and source records attributed to Anthony P Pugsley.

17 recordsLinked to original sources

PubMed2006

Signal recognition particle-dependent inner membrane targeting of the PulG Pseudopilin component of a type II secretion system.

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The beta-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.

Amino Acid Sequence↗

PubMed2006

Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin.

Dodecamerization and insertion of the outer membrane secretin PulD is entirely determined by the C-terminal half of the polypeptide (PulD-CS). In the absence of its cognate chaperone PulS, PulD-CS and PulD mislocalize to the inner membrane, from which they are extractable with detergents but not urea. Electron microscopy of PulD-CS purified from the inner membrane revealed apparently normal dodecameric complexes. Electron microscopy of PulD-CS and PulD in inner membrane vesicles revealed inserted secretin complexes. Mislocalization of PulD or PulD-CS to this membrane induces the phage shock response, probably as a result of a decreased membrane electrochemical potential. Production of PulD in the absence of the phage shock response protein PspA and PulS caused a substantial drop in membrane potential and was lethal. Thus, PulD-CS and PulD assemble in the inner membrane if they do not associate with PulS. We propose that PulS prevents premature multimerization of PulD and accompanies it through the periplasm to the outer membrane. PulD is the first bacterial outer membrane protein with demonstrated ability to insert efficiently into the inner membrane.

Bacterial Outer Membrane Proteins↗

PubMed2006

Secretins take shape.

Secretins are a unique class of bacterial multimeric outer membrane proteins that probably differ considerably from other, less complex outer membrane proteins in their overall structure and organization, and in their requirements for outer membrane targeting and assembly factors. In this MicroCommentary, we discuss these differences with respect to the role of a specific class of lipoproteins, often referred to as pilotins, in secretin complex assembly. We compare them with other lipoproteins that play a role in Omp85/YaeT-mediated assembly of more classical outer membrane proteins. One of the examples we have chosen is the Myxococcus Xanthus lipoprotein Tgl. Coculture of cells with and without Tgl allows secretin assembly (and, hence, type IV pilus assembly) in the cells without Tgl, indicating that it can act by cell-to-cell contact or can transfer between cells.

Bacterial Outer Membrane Proteins↗

PubMed2006

Secretion by numbers: Protein traffic in prokaryotes.

Almost all aspects of protein traffic in bacteria were covered at the ASM-FEMS meeting on the topic in Iraklio, Crete in May 2006. The studies presented ranged from mechanistic analysis of specific events leading proteins to their final destinations to the physiological roles of the targeted proteins. Among the highlights from the meeting that are reviewed here are the molecular dynamics of SecA protein, membrane protein insertion, type III secretion needles and chaperones, type IV secretion, the two partner and autosecretion systems, the 'secretion competent state', and the recently discovered type VI secretion system.

Bacterial Proteins↗

PubMed2006

Direct visualization of red fluorescent lipoproteins indicates conservation of the membrane sorting rules in the family Enterobacteriaceae.

Chimeras created by fusing the monomeric red fluorescent protein (RFP) to a bacterial lipoprotein signal peptide (lipoRFPs) were visualized in the cell envelope by epifluorescence microscopy. Plasmolysis of the bacteria separated the inner and outer membranes, allowing the specific subcellular localization of lipoRFPs to be determined in situ. When equipped with the canonical inner membrane lipoprotein retention signal CDSR, lipoRFP was located in the inner membrane in Escherichia coli, whereas the outer membrane sorting signal CSSR caused lipoRFP to localize to the outer membrane. CFSR-RFP was also routed to the outer membrane, but CFNSR-RFP was located in the inner membrane, consistent with previous data showing that this sequence functions as an inner membrane retention signal. These four lipoproteins exhibited identical localization patterns in a panel of members of the family Enterobacteriaceae, showing that the lipoprotein sorting rules are conserved in these bacteria and validating the use of E. coli as a model system. Although most predicted inner membrane lipoproteins in these bacteria have an aspartate residue after the fatty acylated N-terminal cysteine residue, alternative signals such as CFN can and probably do function in parallel, as indicated by the existence of putative inner membrane lipoproteins with this sequence at their N termini.

Bacterial Proteins↗

PubMed2006

Green fluorescent chimeras indicate nonpolar localization of pullulanase secreton components PulL and PulM.

The Klebsiella oxytoca pullulanase secreton (type II secretion system) components PulM and PulL were tagged at their N termini with green fluorescent protein (GFP), and their subcellular location was examined by fluorescence microscopy and fractionation. When produced at moderate levels without other secreton components in Escherichia coli, both chimeras were envelope associated, as are the native proteins. Fluorescent GFP-PulM was evenly distributed over the cell envelope, with occasional brighter foci. Under the same conditions, GFP-PulL was barely detectable in the envelope by fluorescence microscopy. When produced together with all other secreton components, GFP-PulL exhibited circumferential fluorescence, with numerous brighter patches. The envelope-associated fluorescence of GFP-PulL was almost completely abolished when native PulL was also produced, suggesting that the chimera cannot compete with PulL for association with other secreton components. The patches of GFP-PulL might represent functional secretons, since GFP-PulM also appeared in similar patches. GFP-PulM and GFP-PulL both appeared in spherical polar foci when made at high levels. In K. oxytoca, GFP-PulM was evenly distributed over the cell envelope, with few patches, whereas GFP-PulL showed only weak envelope-associated fluorescence. These data suggest that, in contrast to their Vibrio cholerae Eps secreton counterparts (M. Scott, Z. Dossani, and M. Sandkvist, Proc. Natl. Acad. Sci. USA 98:13978-13983, 2001), PulM and PulL do not localize specifically to the cell poles and that the Pul secreton is distributed over the cell surface.

Artificial Gene Fusion↗

PubMed2005

Structural insights into the secretin PulD and its trypsin-resistant core.

Limited proteolysis, secondary structure and biochemical analyses, mass spectrometry, and mass measurements by scanning transmission electron microscopy were combined with cryo-electron microscopy to generate a three-dimensional model of the homomultimeric complex formed by the outer membrane secretin PulD, an essential channel-forming component of the type II secretion system from Klebsiella oxytoca. The complex is a dodecameric structure composed of two rings that sandwich a closed disc. The two rings form chambers on either side of a central plug that is part of the middle disc. The PulD polypeptide comprises two major, structurally quite distinct domains; an N domain, which forms the walls of one of the chambers, and a trypsin-resistant C domain, which contributes to the outer chamber, the central disc, and the plug. The C domain contains a lower proportion of potentially transmembrane beta-structure than classical outer membrane proteins, suggesting that only a small part of it is embedded within the outer membrane. Indeed, the C domain probably extends well beyond the confines of the outer membrane bilayer, forming a centrally plugged channel that penetrates both the peptidoglycan on the periplasmic side and the lipopolysaccharide and capsule layers on the cell surface. The inner chamber is proposed to constitute a docking site for the secreted exoprotein pullulanase, whereas the outer chamber could allow displacement of the plug to open the channel and permit the exoprotein to escape.

Bacterial Outer Membrane Proteins↗

PubMed2005

Measure for measure in the control of type III secretion hook and needle length.

Bacterial flagella and injectisomes are supramolecular structures that are responsible for motility and for delivering toxic proteins into the cytosol of eukaryotic cells, respectively. They look very similar to each other. Both systems are called type III secretion pathways, and their components share substantial sequence similarities. One remarkable feature of the type III systems is that the length of their substructure is fairly well controlled by a secretion switching machinery, which consists of at least two proteins, a length control protein and an integral membrane secretion component. Here, we review how and why the length of these structures must be accurately controlled.

Adhesins, Bacterial↗

PubMed2005

Towards the identification of type II secretion signals in a nonacylated variant of pullulanase from Klebsiella oxytoca.

Pullulanase (PulA) from the gram-negative bacterium Klebsiella oxytoca is a 116-kDa surface-anchored lipoprotein of the isoamylase family that allows growth on branched maltodextrin polymers. PulA is specifically secreted via a type II secretion system. PelBsp-PulA, a nonacylated variant of PulA made by replacing the lipoprotein signal peptide (sp) with the signal peptide of pectate lyase PelB from Erwinia chrysanthemi, was efficiently secreted into the medium. Two 80-amino-acid regions of PulA, designated A and B, were previously shown to promote secretion of beta-lactamase (BlaM) and endoglucanase CelZ fused to the C terminus. We show that A and B fused to the PelB signal peptide can also promote secretion of BlaM and CelZ but not that of nuclease NucB or several other reporter proteins. However, the deletion of most of region A or all of region B, either individually or together, had only a minor effect on PelBsp-PulA secretion. Four independent linker insertions between amino acids 234 and 324 in PelBsp-PulA abolished secretion. This part of PulA, region C, could contain part of the PulA secretion signal or be important for its correct presentation. Deletion of region C abolished PelBsp-PulA secretion without dramatically affecting its stability. PelBsp-PulA-NucB chimeras were secreted only if the PulA-NucB fusion point was located downstream from region C. The data show that at least three regions of PulA contain information that influences its secretion, depending on their context, and that some reporter proteins might contribute to the secretion of chimeras of which they are a part.

Acylation↗

PubMed2004

Depletion of apolipoprotein N-acyltransferase causes mislocalization of outer membrane lipoproteins in Escherichia coli.

Lipoproteins in Gram-negative Enterobacteriaceae carry three fatty acids on the N-terminal cysteine residue, two as a diacylglyceride and one through an N-linkage following signal peptide cleavage. Most lipoproteins are anchored in the outer membrane, facing the periplasm, but some lipoproteins remain in the plasma membrane, depending on the amino acid at position +2, immediately after the fatty-acylated cysteine. In vitro, the last step in lipoprotein maturation, N-acylation of apolipoproteins by the plasma membrane apolipoprotein N-acyltransferase (Lnt), is necessary for efficient recognition of outer membrane lipoproteins by the Lol system, which transports them from the plasma to the outer membrane (Fukuda, A., Matsuyama, S.-I., Hara, T., Nakayama, J., Nagasawa, H., and Tokuda, H. (2002) J. Biol. Chem. 277, 43512-43518). To study the role of Lnt in vivo, we constructed a conditional lnt mutant of Escherichia coli. The apo-form of peptidoglycan-anchored major lipoprotein (Lpp) and two other outer membrane lipoproteins accumulated in the plasma membrane when lnt expression was reduced. We also found that Lnt is an essential protein in E. coli and that the lethality is partially because of the retention of apoLpp in the plasma membrane. Topology mapping of Lnt with beta-galactosidase and alkaline phosphatase fusions indicated the presence of six membrane-spanning segments. The lnt gene in a mutant of Salmonella enterica displaying thermosensitive Lnt activity (Gupta, S. D., Gan, K., Schmid, M. B., and Wu, H. C. (1993) J. Biol. Chem. 268, 16551-16556) was found to carry a mutation causing a single glutamate to lysine substitution at a highly conserved position in the last predicted periplasmic loop of the protein.

Acyltransferases↗

PubMed2004

Getting out: protein traffic in prokaryotes.

Protein secretion systems in prokaryotes are increasingly shifting from being considered as experimental models for 'more complex' processes (i.e. eukaryotes) to being a major source of key biological questions in their own right. The pathways by which proteins move between compartments or insert into membranes in prokaryotic cells are certainly less numerous than in eukaryotes (though not dramatically so). However, the quality and complexity of bacterial protein targeting systems indicate that virtually all mechanistic problems associated with protein traffic were solved very efficiently well before eukaryotes appeared on the Earth crust. Indeed, recent studies have both increased the number of known prokaryotic protein traffic systems and indicated new layers of complexity for those that were already well characterized. This report describes some recent developments in bacterial protein traffic that were presented at two meetings in the autumn of 2003.

Archaea↗

PubMed2004

Traffic spotting: poles apart.

Finding out where specific functions are carried out within a bacterial cell has now become technically feasible. Here we consider recent experiments aimed at determining where bacteria translocate proteins across the cytoplasmic membrane using the Sec machinery.

Bacterial Physiological Phenomena↗

PubMed2004

Structure and assembly of the pseudopilin PulG.

The pseudopilin PulG is one of several essential components of the type II pullulanase secretion machinery (the Pul secreton) of the Gram-negative bacterium Klebsiella oxytoca. The sequence of the N-terminal 25 amino acids of the PulG precursor is hydrophobic and very similar to the corresponding region of type IV pilins. The structure of a truncated PulG (lacking the homologous region), as determined by X-ray crystallography, was found to include part of the long N-terminal alpha-helix and the four internal anti-parallel beta-strands that characterize type IV pilins, but PulG lacks the highly variable loop region with a disulphide bond that is found in the latter. When overproduced, PulG forms flexible pili whose structural features, as visualized by electron microscopy, are similar to those of bacterial type IV pili. The average helical repeat comprises 17 PulG subunits and four helical turns. Electron microscopy and molecular modelling show that PulG probably assembles into left-handed helical pili with the long N-terminal alpha-helix tightly packed in the centre of the pilus. As in the type IV pilins, the hydrophobic N-terminal part of the PulG alpha-helix is necessary for its assembly. Subtle sequence variations within this highly conserved segment seem to determine whether or not a type IV pilin can be assembled into pili by the Pul secreton.

Bacterial Proteins↗

PubMed2003

Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization.

Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F1/V1-ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, cross-linking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa (monomer); II, approximately 300 kDa (putative hexamer); III, 575 kDa (dodecamer); and IV, approximately 3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (>700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane.

Adenosine Triphosphatases↗

PubMed2003

An intramolecular disulphide bond reduces the efficacy of a lipoprotein plasma membrane sorting signal.

To study lipoprotein sorting in Escherichia coli, we devised a novel screen in which sensitivity or resistance to bacteriophage T5 and colicin M reflects the membrane localization of the bacteriophage T5-encoded lipoprotein Llp, which inactivates the outer membrane (OM) T5 receptor (FhuA). When processed by lipoprotein signal peptidase, Llp has a serine at position +2, immediately after the fatty acylated N-terminal cysteine. As predicted by the '+2 lipoprotein sorting rule' that determines the localization of lipoproteins in the cell envelope, Llp is located in the OM. However, contrary to expectations, when serine +2 was replaced by aspartate, the canonical plasma membrane lipoprotein retention signal, Llp was still > or =40% targeted to the OM and protected cells against colicin M and phage T5. OM association of this Llp derivative was abolished when a peptide spacer was inserted between the aspartate and the rest of Llp or when the formation of an intramolecular disulphide bond in Llp was prevented by substituting one or other of the cysteines involved. Furthermore, analysis of a MalE-Llp hybrid protein with or without a lipid moiety demonstrated that fatty acylation of Llp is essential for its OM association and for protection against colicin M and bacteriophage T5. These data suggest (i) that phage-encoded Llp uses the endogenous E. coli Lol pathway for lipoprotein sorting to the OM and (ii) that the conformation of a lipoprotein can affect its sorting within the cell envelope.

Amino Acid Sequence↗

PubMed2003

Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella.

Homologues of the protein constituents of the Klebsiella pneumoniae (Klebsiella oxytoca) type II secreton (T2S), the Pseudomonas aeruginosa type IV pilus/fimbrium biogenesis machinery (T4P) and the Methanococcus voltae flagellum biogenesis machinery (Fla) have been identified. Known constituents of these systems include (1). a major prepilin (preflagellin), (2). several minor prepilins (preflagellins), (3). a prepilin (preflagellin) peptidase/methylase, (4). an ATPase, (5). a multispanning transmembrane (TM) protein, (6). an outer-membrane secretin (lacking in Fla) and (7). several functionally uncharacterized envelope proteins. Sequence and phylogenetic analyses led to the conclusion that, although many of the protein constituents are probably homologous, extensive sequence divergence during evolution clouds this homology so that a common ancestry can be established for all three types of systems for only two constituents, the ATPase and the TM protein. Sequence divergence of the individual T2S constituents has occurred at characteristic rates, apparently without shuffling of constituents between systems. The same is probably also true for the T4P and Fla systems. The family of ATPases is much larger than the family of TM proteins, and many ATPase homologues function in capacities unrelated to those considered here. Many phylogenetic clusters of the ATPases probably exhibit uniform function. Some of these have a corresponding TM protein homologue although others probably function without one. It is further shown that proteins that compose the different phylogenetic clusters in both the ATPase and the TM protein families exhibit unique structural characteristics that are of probable functional significance. The TM proteins are shown to have arisen by at least two dissimilar intragenic duplication events, one in the bacterial kingdom and one in the archaeal kingdom. The archaeal TM proteins are twice as large as the bacterial TM proteins, suggesting an oligomeric structure for the latter.

Adenosine Triphosphatases↗

PubMed2003

Type IV-like pili formed by the type II secreton: specificity, composition, bundling, polar localization, and surface presentation of peptides.

The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown Escherichia coli K-12 cells expressing the Klebsiella oxytoca pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in E. coli or to PulG. For example, high-level expression of the endogenous E. coli gsp secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic "pseudopilus" and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).

Bacterial Proteins↗