General Export Pathway Research Articles

P5.105 Identification and Functional Annotation of Secreted Proteins of Chlamydia Trachomatis Using Bioinformatics Tools

BackgroundThe large number of sexually transmitted diseases cases caused globally each year by Chlamydia trachomatis has made this organism a World Health Organization priority for vaccine development. Even after a...

Identification of protein secretion systems and novel secreted proteins in Rhizobium leguminosarum bv. viciae

BackgroundProteins secreted by bacteria play an important role in infection of eukaryotic hosts. Rhizobia infect the roots of leguminous plants and establish a mutually beneficial symbiosis. Proteins secreted during the infection process by some rhizobial strains can influence infection and modify the plant defence signalling pathways. The aim of this study was to systematically analyse protein secretion in the recently sequenced strain Rhizobium leguminosarum bv. viciae 3841.ResultsSimilarity searches using defined protein secretion systems from other Gram-negative bacteria as query sequences revealed that R. l. bv. viciae 3841 has ten putative protein secretion systems. These are the general export pathway (GEP), a twin-arginine translocase (TAT) secretion system, four separate Type I systems, one putative Type IV system and three Type V autotransporters. Mutations in genes encoding each of these (except the GEP) were generated, but only mutations affecting the PrsDE (Type I) and TAT systems were observed to affect the growth phenotype and the profile of proteins in the culture supernatant. Bioinformatic analysis and mass fingerprinting of tryptic fragments of culture supernatant proteins identified 14 putative Type I substrates, 12 of which are secreted via the PrsDE, secretion system. The TAT mutant was defective for the symbiosis, forming nodules incapable of nitrogen fixation.ConclusionNone of the R. l. bv. viciae 3841 protein secretion systems putatively involved in the secretion of proteins to the extracellular space (Type I, Type IV, Type V) is required for establishing the symbiosis with legumes. The PrsDE (Type I) system was shown to be the major route of protein secretion in non-symbiotic cells and to secrete proteins of widely varied size and predicted function. This is in contrast to many Type I systems from other bacteria, which typically secrete specific substrates encoded by genes often localised in close proximity to the genes encoding the secretion system itself.

The twin-arginine translocation pathway is a major route of protein export in Streptomyces coelicolor

The twin-arginine translocation (Tat) pathway is a protein transport system for the export of folded proteins. Substrate proteins are targeted to the Tat translocase by N-terminal signal peptides harboring a distinctive R-R-x-Phi-Phi "twin-arginine" amino acid motif. Using a combination of proteomic techniques, the protein contents from the cell wall of the model Gram-positive bacterium Streptomyces coelicolor were identified and compared with that of mutant strains defective in Tat transport. The proteomic experiments pointed to 43 potentially Tat-dependent extracellular proteins. Of these, 25 were verified as bearing bona fide Tat-targeting signal peptides after independent screening with a facile, rapid, and sensitive reporter assay. The identified Tat substrates, among others, include polymer-degrading enzymes, phosphatases, and binding proteins as well as enzymes involved in secondary metabolism. Moreover, in addition to predicted extracellular substrates, putative lipoproteins were shown to be Tat-dependent. This work provides strong experimental evidence that the Tat system is used as a major general export pathway in Streptomyces.

Positive Selection for Loss-of-Function tat Mutations Identifies Critical Residues Required for TatA Activity

The Tat system, found in the cytoplasmic membrane of many bacteria, is a general export pathway for folded proteins. Here we describe the development of a method, based on the transport of chloramphenicol acetyltransferase, that allows positive selection of mutants defective in Tat function. We have demonstrated the utility of this method by selecting novel loss-of-function alleles of tatA from a pool of random tatA mutations. Most of the mutations that were isolated fall in the amphipathic region of TatA, emphasizing the pivotal role that this part of the protein plays in TatA function.

The general secretory pathway: a general misnomer?

The term general secretory pathway (GSP) has been usurped and misused in the literature over the past few years. The concept of GSP is discussed from an historical perspective, and the definitions of the general export pathway (GEP), the main terminal branch (MTB) of the GSP, the unified GSP nomenclature and the type II, IV and V secretion pathways are also described to show how they have fuelled the confusion. By putting the record straight and using novel findings within the field of bacterial protein secretion, we hope to bring clarity to this area of science and prevent further promulgation of incorrect terminologies.

Expression and localization of the Mycobacterium tuberculosis protein tyrosine phosphatase PtpA

The Mycobacterium tuberculosis open reading frame Rv2234 encodes a low molecular weight tyrosine phosphatase named PtpA. Kinetic analyses of PtpA activity reveal that it is capable of dephosphorylation of p-nitrophenyl phosphate, as well as a variety of phosphotyrosine-containing substrates. In contrast, PtpA showed no detectable activity towards substrates containing phosphoserine or -threonine residues. Transcriptional analysis reveals that the M. tuberculosis ptpA promoter is expressed in the slow-growing Mycobacterium species M. bovis BCG, but not in the fast-growing species M. smegmatis. Furthermore, ptpA expression is upregulated upon entry of BCG cultures into stationary phase and increases upon infection of human monocytes. We also show that, despite the lack of a general export pathway signal sequence, the M. tuberculosis PtpA protein can be released from both M. tuberculosis and M. smegmatis during growth.

Involvement of the twin-arginine translocation system in protein secretion via the type II pathway

The general secretory pathway (GSP) is a two-step process for the secretion of proteins by Gram-negative bacteria. The translocation across the outer membrane is carried out by the type II system, which involves machinery called the secreton. This step is considered to be an extension of the general export pathway, i.e. the export of proteins across the inner membrane by the Sec machinery. Here, we demonstrate that two substrates for the Pseudomonas aeruginosa secreton, both phospholipases, use the twin-arginine translocation (Tat) system, instead of the Sec system, for the first step of translocation across the inner membrane. These results challenge the previous vision of the GSP and suggest for the first time a mosaic model in which both the Sec and the Tat systems feed substrates into the secreton. Moreover, since P.aeruginosa phospholipases are secreted virulence factors, the Tat system appears to be a novel determinant of bacterial virulence.

GSP-dependent protein secretion in gram-negative bacteria: the Xcp system of Pseudomonas aeruginosa.

Bacteria have evolved several secretory pathways to release proteins into the extracellular medium. In Gram-negative bacteria, the exoproteins cross a cell envelope composed of two successive hydrophobic barriers, the cytoplasmic and outer membranes. In some cases, the protein is translocated in a single step across the cell envelope, directly from the cytoplasm to the extracellular medium. In other cases, outer membrane translocation involves an extension of the signal peptide-dependent pathway for translocation across the cytoplasmic membrane via the Sec machinery. By analogy with the so-called general export pathway (GEP), this latter route, including two separate steps across the inner and the outer membrane, was designated as the general secretory pathway (GSP) and is widely conserved among Gram-negative bacteria. In their great majority, exoproteins use the main terminal branch (MTB) of the GSP, namely the Xcp machinery in Pseudomonas aeruginosa, to reach the extracellular medium. In this review, we will use the P. aeruginosa Xcp system as a basis to discuss multiple aspects of the GSP mechanism, including machinery assembly, exoprotein recognition, energy requirement and pore formation for driving through the outer membrane.

Need for TolC, anEscherichia coliouter membrane protein, in the secretion of heat-stable enterotoxin I across the outer membrane

Escherichia coliheat-stable enterotoxin Ip (STIp) is a typical extracellular toxin consisting of 18 amino acid residues synthesized as a precursor of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions. STIp synthesized in the cytoplasm must cross the inner and outer membranes to migrate into the extracellular environment. Previous studies showed that the precursor translocates across the inner membrane utilizing the general export pathway consisting of Sec proteins. However, it remains unclear how it crosses the outer membrane. In this study, we examined the effects of mutation of thetolCgene which encodes anE. coliouter membrane protein, TolC, on the release of STIp into the extracellular environment. The mutation reduced the amount of STIp released into culture supernatant and increased the amount of STIp accumulated in the periplasm. This indicates that TolC mediates the translocation of STIp across the outer membrane. The inability to transfer STIp in the periplasm into the culture supernatant was restored by introduction of thetolCgene into the mutant cells. In the mouse intestinal loop assay, living cells of the mutants did not show a positive response, but wild-type cells did. These results showed that TolC is involved in the translocation of STIp across the outer membrane.

Extracellular secretion of Escherichia coli heat-stable enterotoxin I across the outer membrane.

Escherichia coli heat-stable enterotoxin Ip (STIp) is an extracellular toxin consisting of 18 amino acid residues that is synthesized as a precursor of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions. The precursor synthesized in the cytoplasm is translocated across the inner membrane by the general export pathway consisting of Sec proteins. The pre region functions as a leader peptide and is cleaved during translocation. However, it remains unknown how the resulting peptide (pro-mature peptide) translocates across the outer membrane. In this study, we investigated the structure of the STIp that passes through the outer membrane to determine how it translocates through the outer membrane. The results showed that the pro region is cleaved in the periplasmic space. The generated peptide becomes the mature form of STIp, which happens to have disulfide bonds, which then passes through the outer membrane. We also showed that STIp with a carboxy-terminal peptide consisting of 3 amino acid residues passes through the outer membrane, whereas STIp with a peptide composed of 37 residues does not. Amino acid analysis of mutant STIp purified from culture supernatant revealed that the peptide composed of 37 amino acid residues was cleaved into fragments of 5 amino acid residues. In addition, analyses of STIps with a mutation at the cysteine residue and the dsbA mutant strain revealed that the formation of an intramolecular disulfide bond within STIp is not absolutely required for the mature region of STIp to pass through the outer membrane.

Escherichia coli SecB stimulates export without maintaining export competence of ribose-binding protein signal sequence mutants.

Ribose-binding protein (RBP) is exported to the periplasm of Escherichia coli via the general export pathway. An rbsB-lacZ gene fusion was constructed and used to select mutants defective in RBP export. The spontaneous Lac+ mutants isolated in this selection contained either single-amino-acid substitutions or a deletion of the RBP signal sequence. Intact rbsB genes containing eight different point mutations in the signal sequence were reconstructed, and the effects of the mutations on RBP export were examined. Most of the mutations caused severe defects in RBP export. In addition, different suppressor mutations in SecY/PrlA protein were analyzed for their effects on the export of RBP signal sequence mutants in the presence or absence of SecB. Several RBP signal sequence mutants were efficiently suppressed, but others were not suppressed. Export of an RBP signal sequence mutant in prlA mutant strains was partially dependent on SecB, which is in contrast to the SecB independence of wild-type RBP export. However, the kinetics of export of an RBP signal sequence mutant point to a rapid loss of pre-RBP export competence, which occurs in strains containing or lacking SecB. These results suggest that SecB does not stabilize the export-competent conformation of RBP and may affect translocation by stabilizing the binding of pre-RBP at the translocation site.

PrtD, the integral membrane ATP-binding cassette component of the Erwinia chrysanthemi metalloprotease secretion system, exhibits a secretion signal-regulated ATPase activity.

We have overproduced, partially purified, and characterized PrtD, the ATP-binding cassette (ABC) integral membrane component from the metalloproteases secretion system of the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi. These metalloproteases are secreted independently of the general export pathway encoded by the sec genes. They are secreted via a C-terminal secretion signal and by a secretion apparatus composed of two inner membrane proteins, PrtD and PrtE, and one outer membrane protein PrtF. PrtD is specifically labeled by 8-azido-ATP both in whole membrane vesicles and upon purification. The purified protein displays a low level of P-type ATPase activity. This activity is almost completely and specifically inhibited by the cognate C-terminal secretion signal of the PrtG and PrtB metalloproteases (half inhibition at 0.1 microM) but not by a C-terminal secretion signal of a protein not secreted by the Prt translocator. A mutant PrtD protein bearing a point mutation in the ATP binding site (conserved lysine 370 of the Walker A box changed to arginine) has also been purified. It displays a lower level of ATPase activity which correlates with the lower level of secretion of the metalloproteases by a strain expressing this mutated protein.

MxiJ, a lipoprotein involved in secretion of Shigella Ipa invasins, is homologous to YscJ, a secretion factor of the Yersinia Yop proteins.

Shigella flexneri causes bacillary dysentery by invading epithelial cells of the colonic mucosa. The invasion process requires the synthesis and secretion of the virulence plasmid-encoded Ipa proteins. Using TnphoA mutagenesis, we have identified two virulence plasmid genes, mxiJ and mxiM, that encode proteins exported by the general export pathway. Analysis of the MxiJ and MxiM deduced amino acid sequences suggested that mxiJ and mxiM might encode lipoproteins, which was confirmed by [3H]palmitate labeling of MxiJ:PhoA and MxiM:PhoA fusion proteins. A mxiJ mutant was unable to invade HeLa cells, to induce the formation of plaques on confluent monolayers of HeLa cells, and to provoke keratoconjunctivitis in guinea pigs. In addition, secretion of seven polypeptides, including IpaA, IpaB, and IpaC, was abolished in the mxiJ mutant. Sequence comparisons indicated that MxiJ and MxiH, which is encoded by a gene located upstream from mxiJ, are homologous to the Yersinia enterocolitica YscJ and YscF proteins, respectively.

Two distinct steps in pullulanase secretion by Escherichia coli K12

Two distinct steps in the secretion of the extracellular, cell-surface-anchored lipoprotein pullulanase by Escherichia coli were uncoupled by allowing export of the enzyme to the cytoplasmic membrane via the signal peptide/sec-gene-dependent general export pathway, and then inducing the pulC-O operon of genes required for translocation to the cell surface. The secretion intermediate cofractionated mainly with intermediate-density vesicles when cells were gently lysed and the resulting vesicles were separated by isopycnic sucrose density centrifugation. Cytoplasmic forms of pullulanase (which are not exported because they lack a functional signal peptide) are more sensitive to heat inactivation, denaturation by sodium dodecyl sulphate and carboxymethylation than the intermediate and cell-surface forms. The latter are distinguished only by the fact that the secretion intermediate is less susceptible to proteinase K and trypsin, and is partially inaccessible to substrate or in an inactive conformation in sphaeroplasts. These and other results indicate that the secretion intermediate can acquire considerable higher-ordered structure, including disulphide bridges, before it is transported to the cell surface; this seems to rule out the possibility that it is threaded through this membrane as a locally unfolded polypeptide.

The general protein‐export pathway is directly required for extracellular pullulanase secretion in Escherichia coli k12

Pullulanase is an extracellular, cell surface-anchored lipoprotein produced by Gram-negative bacteria belonging to the genus Klebsiella. Its correct localization in recombinant Escherichia coli requires the products of 14 genes that are linked to the enzyme structural gene in the Klebsiella chromosome. In addition, we show here that six sec genes (secA, secB, secD, secE, secF and secY) are all required for processing of the prepullulanase signal peptide to occur. This implies that pullulanase crosses the cytoplasmic membrane via the general export pathway of which the sec gene products are essential components. Removal or drastic alteration of the prepullulanase signal peptide cause the enzyme to remain cytoplasmic. We propose that pullulanase secretion occurs in two steps, the first of which is common to all signal peptide-bearing precursors of exported and secreted proteins, whereas the second is specifically involved in translocating pullulanase to the cell surface.

Five genes at the 3′ end of the Klebsiella pneumoniae pulC operon are required for pullulanase secretion

The nucleotide sequence of a 5082bp fragment of chromosomal DNA from Klebsiella pneumoniae strain UNF5023 is reported. The sequence includes the last four genes of an operon of genes specifically required for the secretion of the enzyme pullulanase. All four genes (pulL, pulM, pulN and pulO) are shown to be required for pullulanase secretion, as is a fifth gene (pulK) which extends beyond the 5' end of the sequenced DNA. The products of the pulL, pulM, pulN and pulO genes (44 kD, 18 kD, 27 kD and 24 kD, respectively) are all predicted to have one or more hydrophobic domains typical of signal sequences and/or membrane anchors, and were all found mainly associated with the inner membranes of subfractionated cells in which the corresponding genes had been expressed from the bacteriophage T7 gene 10 promoter. The results of this study increase the number of genes which have been identified as required for pullulanase secretion to eight, in addition to genes coding for components of the general export pathway.