Anthony P. Pugsley

Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein.

Only one of the characterized components of the main terminal branch of the general secretory pathway (GSP) in Gram‐negative bacteria, GspD, is an integral outer membrane protein that could conceivably form a channel to permit protein transport across this membrane. PulD, a member of the GspD protein family required for pullulanase secretion by Klebsiella oxytoca, is shown here to form outer membrane‐associated complexes which are not readily dissociated by SDS treatment. The outer membrane association of PulD is absolutely dependent on another component of the GSP, the outer membrane‐anchored lipoprotein PulS. Furthermore, the absence of PulS resulted in limited proteolysis of PulD and caused induction of the so‐called phage shock response, as measured by increased expression of the pspA gene. We propose that PulS may be the first member of a new family of periplasmic chaperones that are specifically required for the insertion of a group of outer membrane proteins into this membrane. PulS is only the second component of the main terminal branch of the GSP for which a precise function can be proposed.

Pilus formation and protein secretion by the same machinery in Escherichia coli

The secreton (type II secretion) and type IV pilus biogenesis branches of the general secretory pathway in Gram‐negative bacteria share many features that suggest a common evolutionary origin. Five components of the secreton, the pseudopilins, are similar to subunits of type IV pili. Here, we report that when the 15 genes encoding the pullulanase secreton of Klebsiella oxytoca were expressed on a high copy number plasmid in Escherichia coli, one pseudopilin, PulG, was assembled into pilus‐like bundles. Assembly of the ‘secreton pilus’ required most but not all of the secreton components that are essential for pullulanase secretion, including some with no known homologues in type IV piliation machineries. Two other pseudopilins, pullulanase and two outer membrane‐associated secreton components were not associated with pili. Thus, PulG is probably the major component of the pilus. Expression of a type IV pilin gene, the E.coli K‐12 gene ppdD, led to secreton‐dependent incorporation of PpdD pilin into pili without diminishing pullulanase secretion. This is the first demonstration that pseudopilins can be assembled into pilus‐like structures.

Testing the ‘+2 rule’ for lipoprotein sorting in the Escherichia coli cell envelope with a new genetic selection

We report a novel strategy for selecting mutations that mislocalize lipoproteins within the Escherichia coli cell envelope and describe the mutants obtained. A strain carrying a deletion of the chromosomal malE gene, coding for the periplasmic maltose‐binding protein (MalE), cannot use maltose unless a wild‐type copy of malE is present in trans. Replacement of the natural signal peptide of preMalE by the signal peptide and the first four amino acids of a cytoplasmic membrane‐anchored lipoprotein resulted in N‐terminal fatty acylation of MalE (lipoMalE) and anchoring to the periplasmic face of the cytoplasmic membrane, where it could still function. When the aspartate at position +2 of this protein was replaced by a serine, lipoMalE was sorted to the outer membrane, where it could not function. Chemical mutagenesis followed by selection for maltose‐using mutants resulted in the identification of two classes of mutations. The single class I mutant carried a plasmid‐borne mutation that replaced the serine at position +2 by phenylalanine. Systematic substitutions of the amino acid at position +2 revealed that, besides phenylalanine, tryptophan, tyrosine, glycine and proline could all replace classical cytoplasmic membrane lipoprotein sorting signal (aspartate +2). Analysis of known and putative lipoproteins encoded by the E. coli K‐12 genome indicated that these amino acids are rarely found at position +2. In the class II mutants, a chromosomal mutation caused small and variable amounts of lipoMalE to remain associated with the cytoplasmic membrane. Similar amounts of another, endogenous outer membrane lipoprotein, NlpD, were also present in the cytoplasmic membrane in these mutants, indicating a minor, general defect in the sorting of outer membrane lipoproteins. Four representative class II mutants analysed were shown not to carry mutations in the lolA or lolB genes, known to be involved in the sorting of lipoproteins to the outer membrane.

Cloning and expression in Escherichia coli of the Klebsiella pneumoniae genes for production, surface localization and secretion of the lipoprotein pullulanase.

This article describes the reconstitution in Escherichia coli of a heterologous protein secretion system comprising a gene for an extracellular protein together with its cognate secretion genes. The protein concerned, pullulanase, is a secreted lipoprotein of the Gram‐negative bacterium Klebsiella pneumoniae. It is initially localized to the cell surface before being specifically released into the medium. E. coli carrying the cloned pullulanase structural gene (pulA) produces pullulanase but does not expose or secrete it. Secretion genes were cloned together with pulA in an 18.8 kbp fragment of K. pneumoniae chromosomal DNA. E. coli carrying this fragment exhibited maltose‐inducible production, exposition and specific secretion of pullulanase. Transposon mutagenesis showed that the secretion genes are located on both sides of pulA. Secretion genes located 5′ to pulA were transcribed in the opposite orientation to pulA under the control of the previously identified, malT‐regulated malX promoter. Thus these secretion genes are part of the maltose regulon and are therefore co‐expressed with pulA. Transposon mutagenesis suggested that secretion genes located 3′ of pulA are not co‐transcribed with pulA, raising the possibility that some secretion functions are not maltose regulated.

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 β-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.

Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy

Secretins, a superfamily of multimeric outer membrane proteins, mediate the transport of large macromolecules across the outer membrane of Gram‐negative bacteria. Limited proteolysis of secretin PulD from the Klebsiella oxytoca pullulanase secretion pathway showed that it consists of an N‐terminal domain and a protease‐resistant C‐terminal domain that remains multimeric after proteolysis. The stable C‐terminal domain starts just before the region in PulD that is highly conserved in the secretin superfamily and apparently lacks the region at the C‐terminal end to which the secretin‐specific pilot protein PulS binds. Electron microscopy showed that the stable fragment produced by proteolysis is composed of two stacked rings that encircle a central channel and that it lacks the peripheral radial spokes that are seen in the native complex. Moreover, the electron microscopic images suggest that the N‐terminal domain folds back into the large cavity of the channel that is formed by the C‐terminal domain of the native complex, thereby occluding the channel, consistent with previous electrophysiological studies showing that the channel is normally closed.

Multiple Interactions between Pullulanase Secreton Components Involved in Stabilization and Cytoplasmic Membrane Association of PulE

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.

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.

Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion

Escherichia coli K‐12, the most widely used laboratory bacterium, does not secrete proteins into the extracellular medium under standard growth conditions, despite possessing chromosomal genes encoding a putative type II secretion machinery (secreton). We show that in wild‐type E.coli K‐12, divergent transcription of the two operons in the main chromosomal gsp locus, encoding the majority of the secreton components, is silenced by the nucleoid‐structuring protein H‐NS. In mutants lacking H‐NS, the secreton genes cloned on a moderate‐copy‐number plasmid are expressed and promote efficient secretion of the endogenous, co‐regulated endochitinase ChiA. This is the first time that secretion of an endogenous extracellular protein has been demonstrated in E.coli K‐12.

The secretin-specific, chaperone-like protein of the general secretory pathway: separation of proteolytic protection and piloting functions

The chaperone‐like protein of the main terminal branch of the general secretory pathway from Klebsiella oxytoca, the outer membrane lipoprotein PulS, protects the multimeric secretin PulD from degradation and promotes its correct localization to the outer membrane. To determine whether these are separable functions, or whether resistance to proteolysis results simply from correct localization of PulD, we replaced the lipoprotein‐type signal peptide of PulS by the signal peptide of periplasmic maltose‐binding protein. The resulting periplasmic PulS retained its ability to protect PulD, but not its ability to localize PulD to the outer membrane and to function in pullulanase secretion. Periplasmic PulS competed with wild‐type PulS to prevent pullulanase secretion, presumably again by causing mislocalization of PulD. A hybrid protein comprising the mature part of PulS fused to the C‐terminus of full‐length maltose‐binding protein (MalE–PulS) had similar properties to the periplasmic PulS protein. Moreover, MalE–PulS was shown to associate with PulD by amylose‐affinity chromatography. The MalE–PulS hybrid was rendered completely functional (i.e. it restored pullulanase secretion in a pulS mutant) by replacing its signal peptide with a lipoprotein‐type signal peptide. However, this fatty‐acylated hybrid protein was only functional if it also carried a lipoprotein sorting signal that targeted it to the outer membrane. Thus, the two functions of PulS are separate and fully dissociable. Incorrect localization, rather than proteolysis, of PulD in the absence of PulS was shown to be the factor that causes high‐level induction of the phage shock response. The Erwinia chrysanthemi PulS homologue, OutS, can substitute for PulS, and PulS can protect the secretin OutD from proteolysis in Escherichia coli, indicating the possible existence of a family of PulS‐like chaperone proteins.