Formation of functional Tat translocases from heterologous components
© Hicks et al; licensee BioMed Central Ltd. 2006
Received: 24 April 2006
Accepted: 19 July 2006
Published: 19 July 2006
The Tat pathway transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plants. In Eschericha coli, Tat transport requires the integral membrane proteins TatA, TatB and TatC. In this study we have tested the ability of tat genes from the eubacterial species Pseudomonas syringae, Streptomyces coelicolor and Aquifex aeolicus, to compensate for the absence of the cognate E. coli tat gene, and thus to form functional Tat translocases with E. coli Tat components.
All three subunits of the Tat system from the Gram positive organism Streptomyces coelicolor were able to form heterologous translocases with substantive Tat transport activity. However, only the TatA and TatB proteins of Pseudomonas syringae were able to functionally interact with the E. coli Tat system even though the two organisms are closely related. Of the Tat components from the phylogenetically distant hyperthermophillic bacterium Aquifex aeolicus only the TatA proteins showed any detectable level of heterologous functionality. The heterologously expressed TatA proteins of S. coelicolor and A. aeolicus were found exclusively in the membrane fraction.
Our results show that of the three Tat proteins, TatA is most likely to show cross-species complementation. By contrast, TatB and TatC do not always show cross-complementation, probably because they must recognise heterologous signal peptides. Since heterologously-expressed S. coelicolor TatA protein was functional and found only in the membrane fraction, it suggests that soluble forms of Streptomyces TatA reported by others do not play a role in protein export.
There are two general pathways by which proteins are translocated across the cytoplasmic membranes of bacteria. The Sec pathway, which is ubiquitous, uses a threading mechanism to transport unfolded polypeptides across the membrane, driven by the energy of ATP hydrolysis and the transmembrane proton gradient . By contrast, the Tat pathway, which is encoded in about half of bacterial genomes sequenced so far, exports only pre-folded proteins. Substrate proteins are targeted to the Tat machinery by N-terminal signal peptides that harbour an almost invariant pair of arginine residues, which are critical for transport by the Tat pathway [reviewed in [2, 3]]. Tat transport is driven solely by the protonmotive force .
Much of our understanding of protein transport by the Tat pathway has come from dual studies of the bacterial Tat pathway and the homologous ΔpH/Tat pathway in plant thylakoids. The Tat system of Escherichia coli is comprised of the three major components, TatA, TatB and TatC, along with the minor component TatE which is a poorly expressed TatA orthologue [5–9]. Protein purification and cross-linking studies have identified two major types of Tat protein complexes in the membranes of E. coli, and analogous complexes have also been identified in thylakoid membranes. An equimolar complex of TatB and TatC, which contains multiple copies of each component, acts as the receptor for Tat substrates [10–12]. Site-specific cross-linking studies have implicated TatC as the component that recognizes the twin arginine motif of the substrate signal peptide . The TatA protein forms a separate, highly heterogeneous complex, which varies in size because it contains different numbers of TatA protomers [13–16]. Examination of purified TatA complexes by negative stain electron microscopy reveal that it forms channel complexes with internal diameters large enough to accommodate folded substrate proteins . Cross-linking studies suggest that TatA transiently associates with the substrate-bound TatBC complex during active protein translocation [12, 17, 18].
The Tat systems of some Gram positive bacteria, exemplified by Bacillus subtilis, and some Archaea show a slightly different organization in that they lack TatB and therefore have translocases that are comprised solely of TatA and TatC . The structural arrangement of subunits in these minimal Tat systems is currently unknown. However, a number of reports have indicated that at least some of the TatA protein of Haloferax volcanii, B. subtilis, and of the TatA and TatB proteins of Streptomyces lividans exists in a soluble form in the cytoplasmic fractions of these organisms [20–22]. These findings are significant, because they imply that the Tat systems in these prokaryotes may operate by a somewhat different mechanism to the canonical Tat systems of E. coli and plant thylakoids.
Previous studies looking at heterologous interactions during Tat transport have generally focused on the ability of Tat systems to recognize and transport foreign Tat substrates. Thus tat genes from different bacterial sources have been expressed in an E. coli strain deleted for all Tat components , which inform on the capacity of foreign translocases to recognize and transport E. coli Tat substrate proteins. Conversely, foreign Tat substrates have also been expressed in E. coli, to test the capacity of the system to recognize non-native signal peptides and passenger proteins [e.g. [24–28]]. However, very few studies have looked at the ability of individual Tat subunits to substitute for the absence of the cognate E. coli Tat component. It was reported that Helicobacter pylori tatA could partially complement the Tat defect of an E. coli ΔtatA ΔtatE strain, but that H. pylori tatB could not substitute for E. coli tatB . A very recent study suggested that the P. syringae pv tomato DC3000 tatC gene could also complement the E. coli tatC deletion strain .
Percentage identities between E. coli Tat proteins and those from other eubacteria analysed in this study.
Length (amino acids)
Percentage identity to E. coli homologue
E. coli TatA
P. syringae TatA
60% over 48 residue overlap
S. coelicolor TatA
36% over 40 residue overlap
A. aeolicus TatA1
41% over 46 residue overlap
A. aeolicus TatA2
38% over 55 residue overlap
E. coli TatB
P. syringae TatB
40% over 71 residue overlap
S. coelicolor TatB
33% over 55 residue overlap
A. aeolicus TatB
23% over 102 residue overlap
E. coli TatC
P. syringae TatC
62% over 245 residue overlap
S. coelicolor TatC
27% over 247 residue overlap
A. aeolicus TatC
41% over 234 residue overlap
Results and discussion
Throughout these experiments we have used three different tests to assess functionality of the Tat system, each of which depends upon the transport of one or more native E. coli Tat substrates. E. coli tat mutants show a pleiotropic cell envelope defect due to an inability to export two Tat-dependent periplasmic amidases, AmiA and AmiC, which are involved in cell wall remodelling [31, 32]. Strains with an inactive Tat system are unable to grow on solid media in the presence of 2% SDS and therefore the ability to grow in the presence of this detergent is a qualitative indication of Tat function . Likewise, E. coli is able to grow anaerobically using trimethylamine-N-oxide (TMAO) as an electron acceptor due to the Tat-dependent export of two enzymes, TMAO reductase (TorA, which is a soluble periplasmic protein) and dimethylsulphoxide (DMSO) reductase (DmsABC, which is membrane-bound, with its active site facing the periplasm ). Therefore the ability of strains to grow anaerobically on minimal media with glycerol as a carbon source and TMAO as sole electron acceptor is also a qualitative indicator of Tat functionality. Finally we have also assayed the activity of TMAO reductase in periplasmic extracts as a more quantitative assessment of the level of Tat functionality.
We have observed previously that the stoichiometry of Tat subunit expression is critical for activity of the Tat translocase. In particular, high level expression of TatB relative to TatA and TatC has a severe inactivating effect on the E. coli Tat system . Thus in the following experiments we have routinely used pcnB derivatives of each of the tat mutant strains, which drastically lowers the copy number of plasmids with the ColE1 replicon, typically to less than 5 copies per cell [35, 36].
Heterologous expression of TatA proteins
We have previously demonstrated that residue F39 of E. coli TatA is critical for TatA function and that mutation to anything other than tyrosine or tryptophan not only inactivates TatA function but also shows a dominant phenotype such that in co-expression studies it grossly affects the function of wild type TatA [37, 38]. Surprisingly, S. coelicolor is one of a very few organisms that does not have phenylalanine at that position. Instead the amino acid at residue 40, which is the equivalent position in S. coelicolor TatA, is leucine. Although a leucine substitution of E. coli F39 is barely tolerated , the S. coelicolor TatA protein shows a significant level of functionality in E. coli. We tested whether the activity of S. coelicolor TatA could be enhanced by mutagenesis of L40 to phenylalanine. However, as shown in Fig 1C, this substitution did not significantly alter the functionality of S. coelicolor TatA in the E. coli system. We conclude that other residues in S. coelicolor TatA compensate for the presence of a leucine at this position.
Heterologous expression of TatB
The TatB protein, where present, is an essential component of the Tat system. It forms an equimolar complex with TatC and site-specific cross-linking analysis has indicated that it contacts Tat signal peptides close to the twin arginine motif, but also within the hydrophobic core [10, 12]. In addition TatB has also been shown to co-purify with TatA, with which it shares some sequence homology . It has been suggested to function as the adaptor between the TatBC complex and the TatA channel complex and therefore high level expression of TatB may disrupt Tat function by interfering with the co-assembly of the two individual complexes . Since it interacts with each of the other Tat components and with Tat signal peptides, cross-complementation with TatB might be expected to be less efficient than with other Tat proteins. As shown in Fig 2A and 2B, the TatB proteins from either S. coelicolor or P. syringae permitted significant growth of the E. coli tatB strain, BØD-P, on selective media containing either SDS or TMAO. By contrast A. aeolicus TatB (co-expressed on a construct that also contains tatC) did not allow any significant growth of the E. coli tatB mutant on either medium. It should, however, be noted that we were not able to demonstrate expression of the A. aeolicus TatB protein in E. coli (data not shown) so it is possible that A. aeolicus TatB does not complement the E. coli tatB mutant because the protein is not produced. Fractionation of the tatB strain harbouring the different tatB genes and assay for periplasmic TMAO reductase activity, shown in Fig 2C, demonstrated that, as seen for TatA, the S. coelicolor TatB homologue gave the highest level of cross-species complementation.
Heterologous expression of TatC
Heterologous expression of tatABC genes in a strain of E. coli devoid of all Tat components
The experiments described above where individual Tat subunits are expressed in E. coli examine the ability of that particular subunit to interact with E. coli Tat components, and, in addition, to recognize E. coli Tat signal peptides and substrate proteins. Under these circumstances, it is apparent that whilst all of the TatA proteins displayed some level of heterologous function, the A. aeolicus tatB and tatC genes, and P. syringae tatC, failed to complement the cognate E. coli tat mutant strains. In order to determine whether this was because of an inability to form functional complexes with E. coli Tat components rather than an inability to interact with the test E. coli substrates, we examined whether transport activity was observed when the full set of heterologous Tat proteins were co-expressed. As shown in Fig 4, the P. syringae tatABC operon showed a low but detectable level of Tat function, indicating that the P. syringae TatC protein alone was not functional in the E. coli tatC strain most likely because it could not either interact with E. coli TatB and/or TatA, or because it was expressed at an inappropriate ratio with these proteins. An alternative explanation is that P. syringae tatC in vector pUniprom-PC fails to express, although we note that it clearly does express from the same vector in the presence of P. syringae tatA and tatB. Conversely, the A. aeolicus tatA1, tatA2, tatB and tatC genes when expressed together from an artificial operon could not complement the E. coli total tat deletion strain. With the proviso that all of the A. aeolicus tatA, tatB and tatC genes express in E. coli, this result suggests that the A. aeolicus Tat proteins are unable to recognize any of the AmiA, AmiC, TorA or DmsA signal peptides.
Summary of Tat activity observed after heterologous expression of tat genes from different bacteria in E. coli tat mutant strains.
E. coli strain
Functionality of the Tat system1
Heterologously expressed TatA proteins from S. coelicolor and A. aeolicus are found exclusively in the membrane fraction
In this report we have tested the ability of Tat proteins from three different eubacterial species to form complexes with E. coli Tat components. Surprisingly, we found that it was the individual Tat proteins of S. coelicolor that consistently gave the highest level of Tat activity with the cognate E. coli tat deletion strains. Indeed each of S. coelicolor TatA, TatB and TatC formed heterologous Tat systems that were able to support 40–50% of the wild type E. coli Tat activity. The P. syringae TatA protein was able to restore reasonable Tat function to the E. coli tatA/E mutant strain, but the tatB gene complemented very poorly and P. syringae TatC was apparently completely inactive.
For each of the S. coelicolor and P. syringae cross-complementation experiments, individual Tat subunits were expressed from identical constructs, with transcription being driven by the constitutive E. coli tat promoter. It is probable that the heterologously expressed S. coelicolor tat genes are translated less efficiently than the P. syringae genes because of codon bias resulting from the high G+C content of S. coelicolor DNA. Whilst we cannot completely rule out the possibility that there is a deleteriously high level of production of the individual P. syringae Tat proteins (but see below), it is none-the-less, striking that the S. coelicolor Tat proteins show a high level of cross-species activity. One possible explanation for this might be linked to the fact that the S. coelicolor has by far the largest predicted Tat secretome. Thus the S. coelicolor Tat system probably transports in excess of 150 Tat substrates, including some of the largest Tat substrates ever identified [43–45]. It therefore must recognize many different twin arginine signal peptides and as a result one might expect that the S. coelicolor Tat components are less stringent than the P. syringae system in terms of signal peptide recognition and substrate size.
It should be noted that TatC from a different pathovar of P. syringae to that tested here [but with which it shares 97% amino acid sequence identity; ] was reported to show at least some minimal Tat activity in the E. coli tatC strain for transport of the Tat substrates AmiA and/or AmiC . However in those experiments, the expression of P. syringae tatC was from the strong ptac promoter on plasmid pTrc99A and was carried out in a pcnB+ background where plasmid copy number would be considerably higher. Thus it is likely that the levels of TatC between the two experiments would be significantly different which may account for the discrepancy in the observed activities.
It is striking to note that wherever cross-species complementation has been tested, TatA proteins always seem to retain some level of function in the heterologous host [8, 47]. This suggests that the constraints on TatA function are less severe than those for TatB or TatC and is consistent with the fact that most of the interactions of heterologously expressed TatA would be self-oligomerisation to assemble into channel-forming multimers. By contrast, the constraints on cross-complementation with heterologously expressed TatB or TatC proteins are likely to be much more stringent since this would require the formation of equimolar complexes with the appropriate E. coli partner subunit, recognition of non-native signal peptides and associated conformational changes to promote assembly of the active translocase. It is notable that the A. aeolicus TatA proteins, which would normally be operating at temperatures of 80°C, retain function when expressed in E. coli cells growing at 37°C. Thus either the TatA protein does not change conformation during the transport cycle or the protein retains sufficient flexibility for function even at temperatures considerably lower than physiological.
A number of groups have reported in the literature the presence of soluble forms of the TatA and TatB proteins from Gram positive bacteria and archaea [20–22]. Fractionation and Western blotting showed quite clearly here that all of the heterologously expressed TatA proteins from S. coelicolor and A. aeolicus are found only in the membrane fraction. It is not clear why extra-membraneous forms of TatA/TatB are present in some organisms, but clearly since the S. coelicolor TatA protein [which is 100% identical to the S. lividans TatA protein; ] supports such a high level of Tat activity in E. coli then it is difficult to imagine how the mechanism of Tat transport at least between E. coli and Streptomycetes cannot be anything other than highly similar.
Bacterial strains and growth conditions
Strains and plasmids used in this study
F-ΔlacU169 araD139 rpsL150 relA1 ptsF rbs flbB5301
φ80dlac ZΔM15, recA1, endA1, gyrA96, thi-1, hsdR 17(rK-,mK+) supE44, relA1, deoR, Δ(lacZYA-argF) U169
F-, lac, ara, gal, mtl
As MC4100 ΔtatA ΔtatE pcn B1 zad-981::Tn10 d (Kmr)
As MC4100 ΔtatB pcn B1 zad-981::Tn10 d (Kmr)
As MC4100 ΔtatC pcn B1 zad-981::Tn10 d (Kmr)
As MC4100 ΔtatABCD ΔtatE pcn B1 zad-981::Tn10 d (Kmr)
pBluescript carrying E. coli tatA
pBluescript carrying E. coli tatB
pBluescript carrying E. coli tatC
AmpR contains E. coli tat promoter and multiple cloning site
pUniprom carrying P. syringae tatA gene
pUniprom carrying P. syringae tatB gene
pUniprom carrying P. syringae tatC gene
pUniprom carrying P. syringae tatABC genes
pUniprom carrying S. coelicolor tatA gene
pUniprom carrying S. coelicolor tatB gene
pUniprom carrying S. coelicolor tatC gene
pBluescript carrying A. aeolicus tatA1 and tatA2
pBluescript carrying A. aeolicus tatBC
pBluescript carrying A. aeolicus tatA, tatA2 and tatBC
C-terminal His-tag expression vector
pQE70 carrying A. aeolicus tatA1
pQE70 carrying A. aeolicus tatA2
pQE70 carrying A. aeolicus tatBC
pQE70 carrying A. aeolicus tatA1 and tatA2
Plasmids pUniprom-PA, pUniprom-PB and pUniprom-PC carry the P. syringae pv maculicola ES4326 tatA, tatB and tatC genes, respectively, under the control of the E. coli tat promoter. They were cloned following amplification with primers GCGGCCGGATCC ATGGGTATTTTTGACTGG and GCGCTCTAGA TTAAACC TGGTCTTTGCGG to amplify tatA, GCGCGGGGATCC ATGTTCGGTATCAGC and GCGCGCTCTAGA TCATGGGGCTCGCGGTGGC to amplify tatB, and GCGC GCGGATCC ATGAGCGCTGATATCCCG and GCGCGCTCTAGA TCACGGTGT GGTGGCGGGCGG to amplify tatC (restriction sites shown in bold) and plasmid pKS-PSMtatABC  as template. Each product was digested with Bam HI and Xba I and cloned into pUniprom  that had been similarly digested. Plasmids pUniprom-SA, pUniprom-SB and pUniprom-SC carry the S. coelicolor tatA, tatB and tatC genes, respectively, under the control of the E. coli tat promoter. They were cloned following amplification with primers GCGCGCGGATCC ATGTTCGGAAGGCTCG GC and GCGCGCTCTAGA TCAGCGCTTGGTCGTGTC to amplify tatA, GCGCG CGGATTC GTGTTCAATGACATAGGC and GCGCGCTCTAGA TCAGGTGGCG TCCATGTC to amplify tatB, and GCGCGCGGATCC ATGCCGCTCGCGGAACA C and GCGCGCTCTAGA TCAGGTCACGTCGTCG to amplify tatC with S. coelicolor chromosomal DNA as template. Each product was digested with Bam HI and Xba I and cloned into pUniprom that had been similarly digested.
Plasmids pFATAQ1 contains the tatA1 and tatA2 genes of A. aeolicus, under control of the lac promoter. The tatA1 and tatA2 genes, whose reading frames overlap, were amplified with the following primers: GCGCGCGAATTC CCCTTAAATTATTCTC TAAGGAGGC and GCGCGCGGATCC GCTGAGTTAAGCCTCTACCTTTTCC, with A. aeolicus chromosomal DNA (a kind gift of R. Huber) as template. The product was digested with Eco RI and Bam HI and cloned into pBluescript that had been digested with the same enzymes. The A. aeolicus tatB gene is not annotated on the genome , however, a gene encoding a protein with 23% identity (over 102 amino acid overlap) to E. coli TatB is found immediately upstream of the tatC gene (see Table 1 for identities of Tat proteins studied in this work with the paralogous E. coli protein), and we herein refer to it as tatB. Plasmid pFATAQ2 contains the tatB and tatC genes of A. aeolicus, under control of the lac promoter. The genes, whose reading frames also overlap, were amplified with primers GCGCGGATCC GGAAA GCAATCCCTATTAACGGAAG and GCGCTCTAGA GCTTATGCCTTTTGAATT TCCTTC, digested with Bam HI and Xba I and cloned into pBluescript that had been pre-digested with the same enzymes. Plasmid pFATAQ3 contains an artificial operon encoding the tatA1, -A2, -B and -C genes of A. aeolicus and was generated by subcloning the tatBC coding region from pFATAQ2 by digestion with Bam HI and Xba I and ligation into pFATAQ1 that had been similarly digested. For overproduction of the A. aeolicus Tat proteins with C-terminal hexahistidine affinity tags, the encoding tat genes were re-amplified and cloned into the overexpression vector pQE70. Plasmid pQEAQ1 contains the gene encoding the TatA1 protein with a C-terminal histag and was amplified using primers GCGCGCATGC ACTTTCCTCTGC CGTGGC and GCGCAGATCT TTTCACCCTCCTTTTTAACTTCC, and pFATAQ1 as template. The product was digested with Sph I and Bgl II, and cloned into similarly digested pQE70. Plasmid pQEAQ2 is analogous to pQEAQ1 but encodes a tagged version of the TatA2 protein. It was cloned in a similar manner, using primers GCGC ACATGT TTTCCCGGCGGAATATCTATG and GCGCAGATCT AGCCTCTACC TTTTCCTTCTC to amplify the encoding gene. Plasmid pQEAQ12 carries both of the A. aeolicus tatA genes, with just TatA2 supplied with the C-terminal histag. The genes were amplified using primers GCGCGCATGC ACTTTCCTCTGCCGTGGC and GC GCAGATCT AGCCTCTACCTTTTCCTTCTC, the product digested with Sph I and Bgl II, and cloned into similarly digested pQE70.
All clones obtained from PCR amplified DNA were sequenced to ensure that no mutations (other than site-specific mutations) had been introduced.
SDS-PAGE and immunoblotting were carried out as described [53, 54] and immunoreactive bands were visualized with the ECL detection system (Amersham Biosciences). Peptide antibodies to the S. coelicolor TatA protein were raised in rabbits, using a peptide of amino acid sequence CTSSRPVTEPTDTTKR, by Davids biotechnology (Regensburg, Germany). Antibodies to E. coli TatA have been described previously , the anti-pentahis antibody was obtained from Qiagen. Subcellular fractions for TMAO reductase activity measurements were prepared from small (30 ml) cultures using the cold osmotic shock protocol . TMAO:benzyl viologen oxidoreductase activity was measured as described previously . Protein concentrations were estimated according to the method of Lowry et al. . Preparation of membrane fractions was as described previously .
- Sec -general secretory pathway:
Tat – Twin arginine translocation pathway, TMAO -trimethylamine-N-oxide, TorA – TMAO reductase DMSO – dimethylsulphoxide, DmsABC – membrane-bound DMSO reductase.
We thank Drs Frank Sargent and Govind Chandra for helpful discussion, and Prof Robert Huber of the University of Regensburg for the gift of Aquifex aeolicus chromosomal DNA. This work was supported by the CEU project LHSG-CT-2004-005257, the BBSRC through a grant-in-aid to the John Innes Centre and a BBSRC-funded PhD studentship to MGH, the Swiss National Science Foundation through the award of a fellowship to IC and the Medical Research Council via a Senior Non-Clinical Fellowship award to TP.
- Manting EH, Driessen AJ: Escherichia coli translocase: the unravelling of a molecular machine. Mol Microbiol. 2000, 37: 226-238. 10.1046/j.1365-2958.2000.01980.x.View ArticlePubMedGoogle Scholar
- Berks BC, Palmer T, Sargent F: The Tat protein translocation pathway and its role in microbial physiology. Adv Microb Physiol. 2003, 47: 187-254.View ArticlePubMedGoogle Scholar
- Mori H, Cline K: Post-translational protein translocation into thylakoids by the Sec and ΔpH-dependent pathways. Biochim Biophys Acta. 2001, 1541: 80-90. 10.1016/S0167-4889(01)00150-1.View ArticlePubMedGoogle Scholar
- Yahr TL, Wickner WT: Functional reconstitution of bacterial Tat translocation in vitro. EMBO J. 2001, 20: 2472-2479. 10.1093/emboj/20.10.2472.PubMed CentralView ArticlePubMedGoogle Scholar
- Weiner JH, Bilous PT, Shaw GM, Lubitz SP, Frost L, Thomas GH, Cole JA, Turner RJ: A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins. Cell. 1998, 93: 93-101. 10.1016/S0092-8674(00)81149-6.View ArticlePubMedGoogle Scholar
- Sargent F, Bogsch E, Stanley NR, Wexler M, Robinson C, Berks BC, Palmer T: Overlapping functions of components of a bacterial Sec-independent protein export pathway. EMBO J. 1998, 17: 3640-3650. 10.1093/emboj/17.13.3640.PubMed CentralView ArticlePubMedGoogle Scholar
- Bogsch E, Sargent F, Stanley NR, Berks BC, Robinson C, Palmer T: An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria. J Biol Chem. 1998, 273: 18003-18006. 10.1074/jbc.273.29.18003.View ArticlePubMedGoogle Scholar
- Sargent F, Stanley NR, Berks BC, Palmer T: Sec-independent protein translocation in Escherichia coli: a distinct and pivotal role for the TatB protein. J Biol Chem. 1999, 274: 36073-36083. 10.1074/jbc.274.51.36073.View ArticlePubMedGoogle Scholar
- Jack RL, Sargent F, Berks BC, Sawers G, Palmer T: Constitutive expression of Escherichia coli tat genes indicates an important role for the twin-arginine translocase during aerobic and anaerobic growth. J Bacteriol. 2001, 183: 1801-1804. 10.1128/JB.183.5.1801-1804.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Bolhuis A, Mathers JE, Thomas JD, Barrett CM, Robinson C: TatB and TatC form a functional and structural unit of the twin-arginine translocase from Escherichia coli. J Biol Chem. 2001, 276: 20213-20219. 10.1074/jbc.M100682200.View ArticlePubMedGoogle Scholar
- Cline K, Mori H: Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport. J Cell Biol. 2001, 154: 719-729. 10.1083/jcb.200105149.PubMed CentralView ArticlePubMedGoogle Scholar
- Alami M, Luke I, Deitermann S, Eisner G, Koch HG, Brunner J, Muller M: Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli. Mol Cell. 2003, 12: 937-946. 10.1016/S1097-2765(03)00398-8.View ArticlePubMedGoogle Scholar
- Porcelli I, de Leeuw E, Wallis R, van den Brink-van der Laan E, de Kruijff B, Wallace BA, Palmer T, Berks BC: Characterisation and membrane assembly of the TatA component of the Escherichia coli twin-arginine protein transport system. Biochemistry. 2002, 41: 13690-13697. 10.1021/bi026142i.View ArticlePubMedGoogle Scholar
- de Leeuw E, Granjon T, Porcelli I, Alami M, Carr SB, Müller M, Sargent F, Palmer T, Berks BC: Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes. J Mol Biol. 2002, 322: 1135-1146. 10.1016/S0022-2836(02)00820-3.View ArticlePubMedGoogle Scholar
- Oates J, Barrett CM, Barnett JP, Byrne KG, Bolhuis A, Robinson C: The Escherichia coli twin-arginine translocation apparatus incorporates a distinct form of TatABC complex, spectrum of modular TatA complexes and minor TatAB complex. J Mol Biol. 2005, 346: 295-305. 10.1016/j.jmb.2004.11.047.View ArticlePubMedGoogle Scholar
- Gohlke U, Pullan L, McDevitt CA, Porcelli I, de Leeuw E, Palmer T, Saibil H, Berks BC: The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter. Proc Natl Acad Sci USA. 2005, 102: 10482-10486. 10.1073/pnas.0503558102.PubMed CentralView ArticlePubMedGoogle Scholar
- Mori J, Cline K: A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase. J Cell Biol. 2002, 157: 205-210. 10.1083/jcb.200202048.PubMed CentralView ArticlePubMedGoogle Scholar
- Dabney-Smith C, Mori H, Cline K: Oligomers of Tha4 organize at the thylakoid Tat translocase during protein transport. J Biol Chem. 2006, 281: 5476-5483. 10.1074/jbc.M512453200.View ArticlePubMedGoogle Scholar
- Jongbloed JDH, Grieger U, Antelmann H, Hecker M, Nijland R, Bron S, van Dijl JM: Two minimal Tat translocases in Bacillus. Mol Microbiol. 2004, 54: 1319-1325. 10.1111/j.1365-2958.2004.04341.x.View ArticlePubMedGoogle Scholar
- Pop O, Westermann M, Volkmer-Engert R, Schulz D, Lemke C, Schreiber S, Gerlach R, Wetzker R, Müller JP: Sequence-specific binding of prePhoD to soluble TatAd indicates protein-mediated targeting of the Tat export in Bacillus subtilis. J Biol Chem. 2003, 278: 38428-38436. 10.1074/jbc.M306516200.View ArticlePubMedGoogle Scholar
- Dilks K, Giménez MI, Pohlschröder M: Genetic and biochemical analysis of the twin-arginine translocation pathway in halophilic archaea. J Bacteriol. 2005, 187: 8104-8113. 10.1128/JB.187.23.8104-8113.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- De Keersmaeker S, Van Mellaert L, Schaerlaekens K, Van Dessel W, Vrancken K, Lammertyn E, Anné J, Geukens N: Structural organization of the twin-arginine translocation system in Streptomyces lividans. FEBS Letts. 2005, 579: 797-802. 10.1016/j.febslet.2004.12.059.View ArticleGoogle Scholar
- Oates J, Mathers J, Mangels D, Kuhlbrandt W, Robinson C, Model K: Consensus structural features of purified bacterial TatABC complexes. J Mol Biol. 2003, 330: 277-286. 10.1016/S0022-2836(03)00621-1.View ArticlePubMedGoogle Scholar
- Bruser T, Deutzmann R, Dahl C: Evidence against the double-arginine motif as the only determinant for protein translocation by a novel Sec-independent pathway in Escherichia coli. FEMS Microbiol Lett. 1998, 164: 329-336. 10.1111/j.1574-6968.1998.tb13106.x.View ArticlePubMedGoogle Scholar
- Angelini S, Moreno R, Gouffi K, Santini C, Yamagishi A, Berenguer J, Wu L-F: Export of Thermus thermophilus alkaline phosphatase via the twin-arginine translocation pathway in Escherichia coli. FEBS Letts. 2001, 506: 103-107. 10.1016/S0014-5793(01)02890-3.View ArticleGoogle Scholar
- Blaudeck N, Sprenger GA, Freudl R, Wiegert T: Specificity of signal peptide recognition in Tat-dependent bacterial protein translocation. J Bacteriol. 2001, 183: 604-610. 10.1128/JB.183.2.604-610.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Pop O, Martin U, Abel C, Müller JP: The twin-arginine signal peptide of PhoS and the TatAd/Cd proteins of Bacillus subtilis form an autonomous Tat translocation system. J Biol Chem. 2002, 277: 3268-3273. 10.1074/jbc.M110829200.View ArticlePubMedGoogle Scholar
- Snyder A, Vasil AI, Zajdowicz SL, Wilson ZR, Vasil ML: Role of the Pseudomonas aeruginosa PlcH Tat signal peptide in protein secretion, transcription, and cross-species Tat secretion system compatibility. J Bacteriol. 2006, 188: 1762-1774. 10.1128/JB.188.5.1762-1774.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Bronstein PA, Marrichi M, Cartinhour S, Schneider DJ, DeLisa MP: Identification of a twin-arginine translocation system in Pseudomonas syringae pv. tomato DC3000 and its contribution to pathogenicity and fitness. J Bacteriol. 2005, 187: 8450-8461. 10.1128/JB.187.24.8450-8461.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Battistuzzi FU, Feijao A, Hedges SB: A genomic timescale of prokaryotic evolution: insights into the origin of methanogenesis, phototrophy, and the colonization of land. BMC Evol Biol. 2004, 4: 44-58. 10.1186/1471-2148-4-44.PubMed CentralView ArticlePubMedGoogle Scholar
- Ize B, Stanley NR, Buchanan G, Palmer T: Role of the Escherichia coli Tat pathway in outer membrane integrity. Mol Microbiol. 2003, 48: 1183-1193. 10.1046/j.1365-2958.2003.03504.x.View ArticlePubMedGoogle Scholar
- Bernhardt TG, de Boer PA: The Escherichia coli amidase AmiC is a periplasmic septal ring component exported via the twin-arginine transport pathway. Mol Microbiol. 2003, 48: 1171-1182. 10.1046/j.1365-2958.2003.03511.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Buchanan G, de Leeuw E, Stanley NR, Wexler M, Berks BC, Sargent F, Palmer T: Functional complexity of the twin-arginine translocase TatC component revealed by site-directed mutagenesis. Mol Microbiol. 2002, 43: 1457-1470. 10.1046/j.1365-2958.2002.02853.x.View ArticlePubMedGoogle Scholar
- Stanley NR, Sargent F, Buchanan G, Shi J, Stewart V, Palmer T, Berks BC: Behaviour of topological marker proteins targeted to the Tat protein transport pathway. Mol Microbiol. 2002, 43: 1005-1021. 10.1046/j.1365-2958.2002.02797.x.View ArticlePubMedGoogle Scholar
- Liu JD, Parkinson JS: Genetics and sequence analysis of the pcnB locus, an Escherichia coli gene involved in plasmid copy number control. J Bacteriol. 1989, 171: 1254-1261.PubMed CentralPubMedGoogle Scholar
- University of East Anglia, Lee P: Characterisation of the TatA and TatB proteins of the Escherichia coli Tat translocase. PhD thesis. 2003, University of East AngliaGoogle Scholar
- Hicks MG, de Leeuw E, Porcelli I, Buchanan G, Berks BC, Palmer T: The Escherichia coli twin-arginine translocase: conserved residues of TatA and TatB family components involved in protein transport. FEBS Letts. 2003, 539: 61-67. 10.1016/S0014-5793(03)00198-4.View ArticleGoogle Scholar
- Hicks MG, Lee PA, Georgiou G, Berks BC, Palmer T: Positive selection for loss-of-function tat mutations identifies critical residues required for TatA activity. J Bacteriol. 2005, 187: 2920-2925. 10.1128/JB.187.8.2920-2925.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Sargent F, Gohlke U, de Leeuw E, Stanley NR, Palmer T, Saibil HR, Berks BC: Purified components of the Escherichia coli Tat protein transport system form a double-layered ring structure. Eur J Biochem. 2001, 268: 3361-3367. 10.1046/j.1432-1327.2001.02263.x.View ArticlePubMedGoogle Scholar
- De Keersmaeker S, Van Mellaert L, Lammertyn E, Vrancken K, Anné J, Geukens N: Functional analysis of TatA and TatB in Streptomyces lividans. Biochem Biophys Res Comm. 2005, 335: 973-982. 10.1016/j.bbrc.2005.07.165.View ArticlePubMedGoogle Scholar
- Jongbloed JD, Martin U, Antelmann H, Hecker M, Tjalsma H, Venema G, Bron S, van Dijl JM, Muller J: TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway. J Biol Chem. 2000, 275: 41350-41357. 10.1074/jbc.M004887200.View ArticlePubMedGoogle Scholar
- Lee PA, Buchanan G, Stanley NR, Berks BC, Palmer T: Truncation analysis of TatA and TatB defines the minimal functional units required for protein translocation. J Bacteriol. 2002, 184: 5871-5879. 10.1128/JB.184.21.5871-5879.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Dilks K, Rose RW, Hartmann E, Pohlschroder M: Prokaryotic utilization of the twin-arginine translocation pathway: a genomic survey. J Bacteriol. 2003, 185: 1478-1483. 10.1128/JB.185.4.1478-1483.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Bendtsen JD, Nielsen H, Widdick D, Palmer T, Brunak S: Prediction of twin-arginine signal peptides. BMC Bioinformatics. 2005, 6: 167-175. 10.1186/1471-2105-6-167.PubMed CentralView ArticlePubMedGoogle Scholar
- Widdick DA, Dilks K, Chandra G, Bottrill A, Naldrett M, Pohlschroder M, Palmer T: The twin-arginine translocation pathway is a major route of protein export in Streptomyces coelicolor. 2006, submittedGoogle Scholar
- Caldelari I, Mann S, Crooks C, Palmer T: The Tat pathway of the plant pathogen Pseudomonas syringae is required for optimal virulence. Mol Plant-Microbe Interact. 2006, 19: 200-212.View ArticlePubMedGoogle Scholar
- Bernhard M, Friedrich B, Siddiqui RA: Ralstonia eutropha TF93 is blocked in Tat-mediated protein export. J Bacteriol. 2000, 182: 581-588. 10.1128/JB.182.3.581-588.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Schaerlaekens K, Schierová M, Lammertyn E, Geukens N, Anné J, van Mellaert L: Twin-arginine translocation pathway in Streptomyces lividans. J Bacteriol. 2001, 183: 6727-6732. 10.1128/JB.183.23.6727-6732.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. 1989, Cold Spring Harbor Press, Cold Spring Harbor, NY, 2Google Scholar
- Cohen GN, Rickenberg HW: Concentration specifique reversible des amino acids chez Escherichia coli. Ann Inst Pasteur (Paris). 1956, 91: 693-720.Google Scholar
- Jack RL, Buchanan G, Dubini A, Hatzixanthis K, Palmer T, Sargent F: Coordinating assembly and export of complex bacterial proteins. EMBO J. 2004, 23: 3962-3972. 10.1038/sj.emboj.7600409.PubMed CentralView ArticlePubMedGoogle Scholar
- Berks BC, Sargent F, Palmer T: The Tat protein export pathway. Mol Microbiol. 2000, 35: 260-274. 10.1046/j.1365-2958.2000.01719.x.View ArticlePubMedGoogle Scholar
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 277: 680-685. 10.1038/227680a0.View ArticleGoogle Scholar
- Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA. 1979, 76: 4350-4354. 10.1073/pnas.76.9.4350.PubMed CentralView ArticlePubMedGoogle Scholar
- Stanley NR, Palmer T, Berks BC: The twin arginine consensus motif of Tat signal peptides is involved in Sec-independent protein targeting in Escherichia coli. J Biol Chem. 2000, 257: 11591-11596. 10.1074/jbc.275.16.11591.View ArticleGoogle Scholar
- Silvestro A, Pommier J, Giordano G: The inducible trimethylamine-N-oxide reductase of Escherichia coli K12: biochemical and immunological studies. Biochim Biophys Acta. 1988, 954: 1-13.View ArticlePubMedGoogle Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurements with the Folin phenol reagent. J Biol Chem. 1951, 193: 265-275.PubMedGoogle Scholar
- Casadaban MJ, Cohen SN: Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci USA. 1979, 76: 4530-4533. 10.1073/pnas.76.9.4530.PubMed CentralView ArticlePubMedGoogle Scholar
- Villarejo MR, Zabin I: Beta-galactosidase from termination and deletion mutant strains. J Bacteriol. 1974, 120: 466-474.PubMed CentralPubMedGoogle Scholar
- Lee PA, Greene NP, Buchanan G, Bond PJ, Punginelli C, Jack RL, Sansom MSP, Berks BC, Palmer T: Cysteine-scanning mutagenesis of the conserved domain of the twin-arginine translocase TatB component. 2006,Google Scholar
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