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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Studies the encoding of functional proteins by the cryptic general secretory pathway operon of Escherichia coli K-12. GspO's processing of prePulG and prePilE; GspO prepilin peptidase's processing of GspG; Expression of chromosomal copies of gspG and gspO.

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. [ABSTRACT FROM AUTHOR]

It has been proposed that the four type IV pilin-like proteins that are required for extracellular protein secretion by the general secretory pathway (GSP) might assemble into a trans-periplasm complex resembling a type IV pilus. To test this idea, we examined the subcellular distribution and oligomeric state of PulG, one of the type IV pilin-like proteins required for pullulanase secretion in Klebsieila oxytoca. Fractionation of Escherichia coli cells carrying a single copy of each pul gene showed that PulG protein was located in two distinct envelope fractions corresponding to the outer and cytoplasmic membranes. The protein was partially released by treating the membranes with Triton X-100 + EDTA or at high pH, but not by Triton X-100 alone or by 8M urea, 6M guanidine hydrochloride or 1M NaCl. Like type IV pilins, non-sedimentable PulG that had been released from the membranes at high pH could be sedimented by centrifugation when the pH was lowered. Treatment of whole cells, sphaeroplasts or isolated membranes with a cleavable cross-linking agent produced mainly PulG homodimers. Previous studies showed that both PulO, which cleaves and N-methylates the PulG precursor, and PulE, a putative ATP-binding protein, share extensive sequence identity with proteins known to be required for type IV pilus processing and assembly. However, mutations which disrupted either pulE or pulO, or indeed the complete absence of all other components of the pullulanase secretion apparatus, had little or no effect on any of the properties of PulG protein described above. We conclude that there is no evidence that PulG protein assembles into a stable multiprotein complex or that processing of the PulG precursor causes a detectable change in its subcellular distribution. [ABSTRACT FROM AUTHOR]

The analyses of hybrid proteins and of deletion and insertion mutations reveal that the only amino acid at the amino-proximal end of the cell surface lipoprotein pullulanase that is specifically required for its extra. cellular secretion is an aspartate at position +2, immediately after the fatty acylated amino-terminal cysteine. To see whether the requirement for this amino acid is related to its proposed role as a cytoplasmic membrane lipoprotein sorting signal, we used sucrose gradient floatation analysis to determine the subcellular location of pullulanase variants (with or without the aspartate residue) that accumulated in cells lacking the pullulanase-specific secretion genes. A non-secretable pullulanase variant with a serine at position +2 cofractionated mainly with the major peak of outer membrane porin. In contrast, most (55%) of a pullulanase variant with an aspartate at position +2 cofractionated with slightly lighter fractions that contained small proportions of both outer membrane porin and the cytoplasmic membrane marker NADH oxidase. Only 5% of this pullulanase variant cofractionated with the major NADH oxidase peak, while the rest (c. 40%) remained at the bottom of the gradient in fractions totally devoid of porin and NADH oxidase. When analysed by sedimentation through sucrose gradients, however, a large proportion of this variant was recovered from fractions near the top of the gradient that also contained the major NADH oxidase peak. When this peak fraction was applied to a floatation gradient, the pullulanase activity remained at the bottom while the NADH oxidase floated to the top. Thus, there is no evidence that lipoproteins that cofractionate with the cytoplasmic membrane under certain conditions are actually associated with the membrane. Instead, the results support our previous proposal that lipoproteins with an aspartate +2 residue are specifically enriched in a distinct domain of the cell envelope that contains material from... [ABSTRACT FROM AUTHOR]

The PulO protein required for extracellular secretion of puliulanase by Klebsislla oxytoca is known to be highly homologous to two type IV prepilin peptidases, namely XcpA(PilD) (Pseudomonas aeruginosa) and TcpJ (Vibrio cholerae). The predicted prepilln peptidase activity of PulO was confirmed by showing that it could correctly process the product of the cloned pilE.1 type IV pilin structural gene from Nsisseria gonorrhoese in Escherichla coli. The P. aeruginose prepilin peptidase and another putative prepilin peptidase, ComC from Bacillus subtilis, also processed prePilE. Subcellular fractionation showed that the pilE gene product that had been processed by PulO remained associated with the cytoplasmic membrane, as did the unprocessed precursor. PulO was also shown to process three of the four prePilE--PhoA hybrids tested. Southern hybridization experiments suggest that a pulO homologue is present in the N. gonorrhoeae chromosome. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (prefiagellins), (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. [ABSTRACT FROM AUTHOR]

Discusses the catalysis of type III protein secretion (TTS) by translocases which span both membranes of gram-negative bacteria. Importance of hydrophilic TTS for secretion; Assembly of the peripheral protein in clusters at the membrane; Findings of hydrodynamic analysis, crosslinking and electron microscopy.

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 pill, 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). [ABSTRACT FROM AUTHOR]

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 β-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. [ABSTRACT FROM AUTHOR]