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The Klebsiella lipoprotein pullulanase (PulA) is exported to the periplasm, triacylated, and anchored via lipids in the inner membrane (IM) prior to its transport to the bacterial surface through a type II secretion system (T2SS). X-Ray crystallography and atomistic molecular dynamics (MD) simulations of PulA in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) model membrane provided an unprecedented molecular view of an N-terminal unstructured tether and the IM lipoprotein retention signal, and revealed novel interactions with the IM via N-terminal immunoglobulin-like domains in PulA. An efficiently secreted nonacylated variant (PulANA) showed similar peripheral membrane association during MD simulations, consistent with the binding of purified PulANA to liposomes. Remarkably, combined X-ray, MD, and functional studies identified a novel subdomain, Ins, inserted in the α-amylase domain, which is required for PulA secretion. Available data support a model in which PulA binding to the IM promotes interactions with the T2SS, possibly via the Ins subdomain., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The outer membrane portal of the Klebsiella oxytoca type II secretion system, PulD, is a prototype of a family of proteins, the secretins, which are essential components of many bacterial secretion and pilus assembly machines. PulD is a homododecamer with a periplasmic vestibule and an outer chamber on either side of a membrane-spanning region that is poorly resolved by electron microscopy. Membrane insertion involves the formation of a dodecameric membrane-embedded intermediate. Here, we describe an amino acid substitution in PulD that blocks its assembly at this intermediate "prepore" stage. Electron microscopy indicated that the prepore has an apparently normal periplasmic vestibule but a poorly organized outer chamber. A peptide loop around this amino acid appears to be important for the formation/stability of the fully folded complex. A similar assembly intermediate results from creation of the same amino acid substitution in the Pseudomonas aeruginosa secretin XcpQ., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The gene (cpo) encoding the extracellular protease CPI produced by the moderately halophilic bacterium Pseudoalteromonas ruthenica CP76 was cloned, and its nucleotide sequence was analyzed. The cpo gene encodes a 733-residue protein showing sequence similarity to metalloproteases of the M4 family. The type II secretion apparatus was shown to be responsible for secretion of the haloprotease CPI.

The DNA-binding protein Sac7d was previously modified to bind with high affinity to the N domain of the outer membrane secretin PulD from the bacterium Klebsiella oxytoca. Here, we show that binding of the Sac7d derivatives (affitins) to PulD is sensitive to conformational changes caused by denaturant and by the zwitterionic detergent Zwittergent 3-14 routinely used to extract secretins from outer membranes. This sensitivity to the conformational state of PulD allowed us to use the affitins as probes for the native structure of PulD and to devise protocols for examining in vitro synthesized protein in nonionic detergent and for the affinity purification of native PulD using affitins as ligands. When fused to periplasmic PhoA, three affitins inhibited PulD multimerization in vivo and caused loss of function. In two cases, this was likely to be due to dimerization of the affitin by the bound PhoA, as the effect was absent when the affitins were fused to monomeric MalE. In the third case, the MalE and PhoA moieties probably interfered sterically with PulD protomer interactions and, thereby, inhibited multimerization. None of the affitins tested interacted with PulD at sites of protomer interaction or blocked the secretin channel through which exoproteins cross the outer membrane in the Type II secretion pathway of which PulD is a key component.

The ultimate membrane localization and function of most of the 185 predicted Pseudomonas aeruginosa PAO1 lipoproteins remain unknown. We constructed a fluorescent lipoprotein, CSFP(OmlA)-ChFP, by fusing the signal peptide and the first four amino acids of the P. aeruginosa outer membrane lipoprotein OmlA to the monomeric red fluorescent protein mCherry (ChFP). When cells were plasmolyzed with 0.5 M NaCl, the inner membrane separated from the outer membrane and formed plasmolysis bays. This permits the direct observation of fluorescence in either the outer or inner membrane. CSFP(OmlA)-ChFP was shown to localize in the outer membrane by fluorescence microscopy and immunoblotting analysis of inner and outer membrane fractions. The site-directed substitution of the amino acids at positions +2, +3, and +4 in CSFP(OmlA)-ChFP was performed to test the effects on lipoprotein localization of a series of amino acid sequences selected from a panel of predicted lipoproteins. We confirmed Asp(+2) and Lys(+3) Ser(+4) function as inner membrane retention signals and identified four novel inner membrane retention signals: CK(+2) V(+3) E(+4), CG(+2) G(+3) G(+4), CG(+2) D(+3) D(+4), and CQ(+2) G(+3) S(+4). These inner membrane retention signals are found in 5% of the 185 predicted P. aeruginosa lipoproteins. Full-length chimeras of predicted lipoproteins PA4370 and PA3262 fused to mCherry were shown to reside in the inner membrane and showed a nonuniform or patchy distribution in the membrane. The optical sectioning of cells producing PA4370(CGDD)-ChFP and PA3262(CDSQ)-ChFP by confocal microscopy improved the resolution and indicated a helix-like localization pattern in the inner membrane. The method described here permits the in situ visualization of lipoprotein localization and should work equally well for other membrane-associated proteins.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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. [ABSTRACT FROM AUTHOR]

Secretins form large multimeric complexes in the outer membranes of many Gram-negative bacteria, where they function as dedicated gateways that allow proteins to access the extracellular environment. Despite their overall relatedness, different secretins use different specific and general mechanisms for their targeting, assembly, and membrane insertion. We report that all tested secretins from several type II secretion systems and from the filamentous bacteriophage fl can spontaneously multimer and insert into liposomes in an in vitro transcription-translation system. Phylogenetic analyses indicate that these secretins form a group distinct from the secretins of the type IV piliation and type III secretion systems, which do not autoassemble in vitro. A mutation causing a proline-to-leucine substitution allowed PilQ secretins from two different type IV piliation systems assemble in vitro, albeit with very low efficiency, suggesting that autoassembly is an inherent property of all secretins. [ABSTRACT FROM AUTHOR]