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Tracy Palmer

Publications and source records attributed to Tracy Palmer.

At least 19 recordsLinked to original sources
PubMed2026

A novel reverse lipase toxin substrate of the Staphylococcus aureus type VII secretion system.

The type VII secretion system (T7SS) is found in many Gram-positive bacteria and secretes toxins with antibacterial activity. Most characterized substrates have an N-terminal LXG domain that interacts with other helical partner proteins to form a composite T7SS targeting signal. Here we describe only the second substrate family to have a reverse domain arrangement. We show that TslM has a C-terminal LXG-like domain and an N-terminal lipase domain that has phospholipase activity. Secretion of TslM requires a single helical partner protein that binds to the TslM C-terminus, and its toxic activity is neutralized by a distinct family of membrane proteins. Genome analysis reveals that Staphylococcus aureus strains have the capacity to encode up to seven paralogous copies of this toxin family. Taken together our findings show that lipases are an important component of the staphylococcal T7SS toxin arsenal, and that toxins with a reverse domain arrangement are more widespread than previously appreciated.

Staphylococcus aureus↗
PubMed2007

Export pathway selectivity of Escherichia coli twin arginine translocation signal peptides.

The Escherichia coli genome encodes at least 29 putative signal peptides containing a twin arginine motif characteristic of proteins exported via the twin arginine translocation (Tat) pathway. Fusions of the putative Tat signal peptides plus six to eight amino acids of the mature proteins to three reporter proteins (short-lived green fluorescent protein, maltose-binding protein (MBP), and alkaline phosphatase) and also data from the cell localization of epitope-tagged full-length proteins were employed to determine the ability of the 29 signal peptides to direct export through the Tat pathway, through the general secretory pathway (Sec), or through both. 27/29 putative signal peptides could export one or more reporter proteins through Tat. Of these, 11 signal peptides displayed Tat specificity in that they could not direct the export of Sec-only reporter proteins. The rest (16/27) were promiscuous and were capable of directing export of the appropriate reporter either via Tat (green fluorescent protein, MBP) or via Sec (PhoA, MBP). Mutations that conferred a >or=+1 charge to the N terminus of the mature protein abolished or drastically reduced routing through the Sec pathway without affecting the ability to export via the Tat pathway. These experiments demonstrate that the charge of the mature protein N terminus affects export promiscuity, independent of the effect of the folding state of the mature protein.

Amino Acid Sequence↗
PubMed2006

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.

Arginine↗
PubMed2006

Cysteine-scanning mutagenesis and disulfide mapping studies of the conserved domain of the twin-arginine translocase TatB component.

The cytoplasmic membrane protein TatB is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. Together with the TatC component it forms a complex that functions as a membrane receptor for substrate proteins. Structural predictions suggest that TatB is anchored to the membrane via an N-terminal transmembrane alpha-helix that precedes an amphipathic alpha-helical section of the protein. From truncation analysis it is known that both these regions of the protein are essential for function. Here we construct 31 unique cysteine substitutions in the first 42 residues of TatB. Each of the substitutions results in a TatB protein that is competent to support Tat-dependent protein translocation. Oxidant-induced disulfide cross-linking shows that both the N-terminal and amphipathic helices form contacts with at least one other TatB protomer. For the transmembrane helix these contacts are localized to one face of the helix. Molecular modeling and molecular dynamics simulations provide insight into the possible structural basis of the transmembrane helix interactions. Using variants with double cysteine substitutions in the transmembrane helix, we were able to detect cross-links between up to five TatB molecules. Protein purification showed that species containing at least four cross-linked TatB molecules are found in correctly assembled TatBC complexes. Our results suggest that the transmembrane helices of TatB protomers are in the center rather than the periphery of the TatBC complex.

Amino Acid Sequence↗
PubMed2006

Formation of functional Tat translocases from heterologous components.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Bacterial Proteins↗
PubMed2006

The Tat pathway of the plant pathogen Pseudomonas syringae is required for optimal virulence.

Pseudomonas syringae is a gram-negative bacterium that infects a number of agriculturally important plant species. The ability of the organism to deliver virulence factors across the plant cell wall is a key to its pathogenicity. Deletion mutants in the twin arginine translocation (Tat) pathway of two pathovars of P. syringae, pvs. tomato DC3000 and maculicola ES4326, displayed a range of pleiotropic phenotypic changes, such as defects in fluorescent siderophore production, a decrease in sodium dodecyl sulfate and copper resistance, and a significant loss in fitness using Arabidopsis thaliana or tomato as plant hosts. The genome sequence of P. syringae pv. tomato DC3000 encodes a number of potential virulence factors that are predicted to be translocated via the Tat pathway, including several proteins involved in iron scavenging (two siderophore receptors, PSPTO3474 and PSPTO3294, and an aminotransferase, PSPTO2155, involved in siderophore biosynthesis). Further candidates for Tat-dependent pathogenicity determinants include the homologs of a cell wall amidase (PSPTO5528), an enzyme involved in periplasmic glucans biosynthesis (PSPTO5542), and two putative phospholipases (PSPTO3648 and PSPTOB0005). Translocation of the putative amidase, aminotransferase, glucans biosynthetic enzyme, and the two phospholipases, but not the two siderophore receptors, is shown to be dependent on the Tat pathway. Strains deleted for the genes encoding the probable aminotransferase and amidase enzymes are significantly less infectious than the wild type. We conclude that the incremental effects due to the failure to correctly localize at least two, and possibly more, Tat substrates gives rise to the attenuated fitness phenotype of the P. syringae pv. tomato DC3000 tat strain.

Amino Acid Sequence↗
PubMed2006

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.

Bacterial Proteins↗
PubMed2006

Pathfinders and trailblazers: a prokaryotic targeting system for transport of folded proteins.

The twin-arginine (Tat) protein translocase is a highly unusual protein transport machine that is dedicated to the movement of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat pathway by means of N-terminal signal peptides harbouring a distinctive twin-arginine motif. In this minireview, we describe our current knowledge of the Tat system, paying particular attention to the function of the TatA protein and to the often overlooked step of signal peptide cleavage.

Amino Acid Sequence↗
PubMed2006

Subunit composition and in vivo substrate-binding characteristics of Escherichia coli Tat protein complexes expressed at native levels.

The Tat system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Substrates are targeted to the Tat pathway by signal peptides containing a pair of consecutive arginine residues. The membrane proteins TatA, TatB and TatC are the essential components of this pathway in Escherichia coli. The complexes that these proteins form at native levels of expression have been investigated by the use of affinity tag-coding sequences fused to chromosomal tat genes. Distinct TatA and TatBC complexes were identified using size-exclusion chromatography and shown to have apparent molecular masses of approximately 700 and 500 kDa, respectively. Following in vivo expression, the Tat substrate protein SufI was found to copurify with the TatBC, but not the TatA, complex. This binding required the SufI signal peptide. Substitution of the twin-arginine residues in the SufI signal peptide by either twin lysine or twin alanine residues abolished export. However, both variant SufI proteins still copurified with the TatBC complex. These data show that the twin-arginine residues of the Tat consensus motif are not essential for binding of precursor to the TatBC complex but are required for the successful entry of the precursor into the transport cycle. The effect on substrate binding of single amino acid substitutions in TatC that affect Tat transport were studied using TatC variants Phe94Ala, Glu103Ala, Glu103Arg and Asp211Ala. Only variant Glu103Arg showed reduced copurification of SufI with TatBC. The transport defects associated with the other TatC variants do not, therefore, arise from an inability to bind substrate proteins.

Amino Acid Sequence↗
PubMed2005

The TatA component of the twin-arginine protein transport system forms channel complexes of variable diameter.

The Tat system mediates Sec-independent transport of folded precursor proteins across the bacterial plasma membrane or the chloroplast thylakoid membrane. Tat transport involves distinct high-molecular-weight TatA and TatBC complexes. Here we report the 3D architecture of the TatA complex from Escherichia coli obtained by single-particle electron microscopy and random conical tilt reconstruction. TatA forms ring-shaped structures of variable diameter in which the internal channels are large enough to accommodate known Tat substrate proteins. This morphology strongly supports the proposal that TatA forms the protein-conducting channel of the Tat system. One end of the channel is closed by a lid that might gate access to the channel. On the basis of previous protease accessibility measurements, the lid is likely to be located at the cytoplasmic side of the membrane. The observed variation in TatA diameter suggests a model for Tat transport in which the number of TatA protomers changes to match the size of the channel to the size of the substrate being transported. Such dynamic close packing would provide a mechanism to maintain the membrane permeability barrier during transport.

Escherichia coli↗
PubMed2005

Prediction of twin-arginine signal peptides.

BACKGROUND: Proteins carrying twin-arginine (Tat) signal peptides are exported into the periplasmic compartment or extracellular environment independently of the classical Sec-dependent translocation pathway. To complement other methods for classical signal peptide prediction we here present a publicly available method, TatP, for prediction of bacterial Tat signal peptides. RESULTS: We have retrieved sequence data for Tat substrates in order to train a computational method for discrimination of Sec and Tat signal peptides. The TatP method is able to positively classify 91% of 35 known Tat signal peptides and 84% of the annotated cleavage sites of these Tat signal peptides were correctly predicted. This method generates far less false positive predictions on various datasets than using simple pattern matching. Moreover, on the same datasets TatP generates less false positive predictions than a complementary rule based prediction method. CONCLUSION: The method developed here is able to discriminate Tat signal peptides from cytoplasmic proteins carrying a similar motif, as well as from Sec signal peptides, with high accuracy. The method allows filtering of input sequences based on Perl syntax regular expressions, whereas hydrophobicity discrimination of Tat- and Sec-signal peptides is carried out by an artificial neural network. A potential cleavage site of the predicted Tat signal peptide is also reported. The TatP prediction server is available as a public web server at http://www.cbs.dtu.dk/services/TatP/.

Arginine↗
PubMed2005

Characterisation of Tat protein transport complexes carrying inactivating mutations.

The Tat system functions to transport folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of plant chloroplasts. Tat transport involves a high molecular weight TatBC-containing complex that transiently associates with TatA during protein translocation. Sedimentation equilibrium experiments were used to determine a protein-only molecular mass for the TatBC complex of 630+/-30kDa, suggesting that it contains approximately 13 copies of the TatB and TatC protomers. Point mutations that inactivate Tat transport have previously been identified in each of TatA, TatB, and TatC. Analysis of the TatBC complexes formed by these inactive variants demonstrates that the amino acid substitutions neither affect the composition of the TatBC complex nor cause accumulation of the assembled TatABC translocation site. In addition, the TatA protein is shown not to be required for the assembly or stability of the TatBC complex.

Amino Acid Sequence↗
PubMed2005

Protein targeting by the bacterial twin-arginine translocation (Tat) pathway.

The Tat (twin-arginine translocation) protein export system is found in the cytoplasmic membrane of most prokaryotes and is dedicated to the transport of folded proteins. The Tat system is now known to be essential for many bacterial processes including energy metabolism, cell wall biosynthesis, the nitrogen-fixing symbiosis and bacterial pathogenesis. Recent studies demonstrate that substrate-specific accessory proteins prevent improperly assembled substrates from interacting with the Tat transporter. During the transport cycle itself substrate proteins bind to a receptor complex in the membrane which then recruits a protein-translocating channel to carry out the transport reaction.

Bacteria↗
PubMed2005

Export of complex cofactor-containing proteins by the bacterial Tat pathway.

The twin-arginine (Tat) protein translocase is a highly unusual protein transport machine that is dedicated to the movement of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat pathway by means of N-terminal signal peptides harbouring a distinctive twin-arginine motif. In the model organism Escherichia coli, many of the Tat substrates bind redox cofactors that are inserted into apo-proteins before they engage with the Tat machinery. Here we review recent advances in understanding the events involved in the coordination of cofactor insertion with the export process. Current models for Tat protein transport are also discussed.

Bacteria↗
PubMed2005

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.

Escherichia coli↗
PubMed2004

Coordinating assembly and export of complex bacterial proteins.

The Escherichia coli twin-arginine protein transport (Tat) system is a molecular machine dedicated to the translocation of fully folded substrate proteins across the energy-transducing inner membrane. Complex cofactor-containing Tat substrates, such as the model (NiFe) hydrogenase-2 and trimethylamine N-oxide reductase (TorA) systems, acquire their redox cofactors prior to export from the cell and require to be correctly assembled before transport can proceed. It is likely, therefore, that cellular mechanisms exist to prevent premature export of immature substrates. Using a combination of genetic and biochemical approaches including gene knockouts, signal peptide swapping, complementation, and site-directed mutagenesis, we highlight here this crucial 'proofreading' or 'quality control' activity in operation during assembly of complex endogenous Tat substrates. Our experiments successfully uncouple the Tat transport and cofactor-insertion activities of the TorA-specific chaperone TorD and demonstrate unequivocally that TorD recognises the TorA twin-arginine signal peptide. It is proposed that some Tat signal peptides operate in tandem with cognate binding chaperones to orchestrate the assembly and transport of complex enzymes.

Bacteria↗
PubMed2004

Novel phenotypes of Escherichia coli tat mutants revealed by global gene expression and phenotypic analysis.

The Tat protein export system serves to export folded proteins harboring an N-terminal twin arginine signal peptide across the cytoplasmic membrane. In this study, we have used gene expression profiling of Escherichia coli supported by phenotypic analysis to investigate how cells respond to a defect in the Tat pathway. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are defective in the integrity of their cell envelope because of the mislocalization of two amidases involved in cell wall metabolism (Ize, B., Stanley, N. R., Buchanan, G., and Palmer, T. (2003) Mol. Microbiol. 48, 1183-1193). To distinguish between genes that are differentially expressed specifically because of the cell envelope defect and those that result from other effects of the tatC deletion, we also analyzed two different transposon mutants of the DeltatatC strain that have their outer membrane integrity restored. Approximately 50% of the genes that were differentially expressed in the tatC mutant are linked to the envelope defect, with the products of many of these genes involved in self-defense or protection mechanisms, including the production of exopolysaccharide. Among the changes that were not explicitly linked to envelope integrity, we characterized a role for the Tat system in iron acquisition and copper homeostasis. Finally, we have demonstrated that overproduction of the Tat substrate SufI saturates the Tat translocon and produces effects on global gene expression that are similar to those resulting from the DeltatatC mutation.

Amino Acid Sequence↗