In vivo dissection of the Tat translocation pathway in Escherichia coli

doi:10.1006/jmbi.2002.5431

Journal of Molecular Biology

Volume 317, Issue 3, 29 March 2002, Pages 327-335

https://doi.org/10.1006/jmbi.2002.5431 Get rights and content

Abstract

The bacterial Tat pathway is capable of exporting folded proteins carrying a special twin arginine (RR) signal peptide. By using two in vivo reporter proteins, we assessed factors that affect Tat pathway transport. We observed that, like the intact RR signal peptide, those with a KR or RK substitution were still capable of mediating the translocation of the folded green fluorescent protein (GFP). However, the translocation efficiency decreased in the order of RR>KR>RK. The KK motif was unable to mediate GFP translocation. The translocation of the RR-GFP fusion required TatA, TatB and TatC proteins. By exploiting the periplasmic bactericidal property of colicin V (ColV), we constructed a translocation-suicide probe, RR-ColV. The translocation of RR-ColV fully inhibited the growth of wild-type Escherichia coli and those of the ΔtatD and ΔtatE mutants. In contrast, the deletion of the tatC gene blocked RR-ColV in the cytoplasm and this strain exhibited a normal growth phenotype. Interestingly, the growth of ΔtatA and tatB mutants was inhibited partially by RR-ColV. Moreover, KR, RK and KK motifs were capable of mediating the ColV translocation with a decreasing RR=KR>RK>KK efficiency. In addition to TatE and TatC proteins, either TatA or TatB was sufficient for the translocation of RR-ColV or KR-ColV. In contrast, TatA plus the conserved N-terminal domain of TatB were required to mediate the killing effect of ColV fused to the less-efficient RK signal peptide. Taken together, these results suggest that a fully efficient Tat pathway transport is determined by the sequence of the signal peptide, the composition of the Tat apparatus, and the intrinsic characteristics of exported proteins.

Introduction

The bacterial twin arginine translocation or Tat (also called MTT) system has the unusual ability to export folded enzymes across the bacterial plasma membrane.1, 2 The protein composition of the Tat system varies from one organism to another, and a minimal Tat system requires one copy of tatC and one copy of a tatA-like gene.2 The Tat system is not ubiquitous in bacterial genomes and is unlikely to be among the minimal genes required for life.2 It is essential for bacteria only under certain growth conditions. Escherichia coli Tat components are encoded by the tatABCD operon and the tatE gene.3 TatD is not required for Tat function.4 TatC is an integral membrane protein with six predicted transmembrane segments. The deletion of tatC leads to mislocation of all substrates analyzed.5 Therefore, TatC is essential for Tat function. TatA, TatB and TatE share sequence homology at their N termini, including one transmembrane segment and an adjacent amphipathic domain, whereas their C termini vary in sequence and in length.6 The sequence homology suggests the three proteins perform a similar function. Indeed, TatA and TatE exhibit functional overlap and deletion of the tatA and tatE genes affects only partially, or has no detectable effect on, the translocation of the Tat substrates studied, respectively.3 In contrast to TatA and TatE, the importance of TatB for Tat function has not been established clearly. The deletion of the entire tatB gene in the ΔtatB mutant seems to abolish the export of all Tat substrates analyzed, and therefore shows the same phenotype as the ΔtatC mutant.7 The tatB∷Kn strain carries an interruption of the tatB gene at amino acid position 88, after the conserved N-terminal domain by the insertion of a kanamycin-resistant cassette. This strain still shows export of hydrogenase-2.6

The Tat system is structurally and mechanistically similar to the ΔpH-driven thylakoid protein import pathway.8, 9, 10 The signal peptides of the proteins transported by these pathways contain a conserved twin arginine motif and are functionally interchangeable.9, 11, 12, 13 They resemble Sec-dependent signal peptides in their overall structures, but possess a twin arginine motif in the positively charged n-region, a weakly hydrophobic h-region and a positively charged Sec-avoidance signal in the c-region.14, 15 The twin arginine motif is critical for protein targeting by both bacterial Tat and thylakoid ΔpH pathways.12, 15, 16, 17, 18 Interestingly, recent studies showed that mutated19 or naturally occurring twin arginine signal peptides20 with a conservative substitution (Lys for Arg) for the first arginine residue are still able to mediate Tat pathway transport. Therefore, the importance of the RR motif and the capacity of the Tat signal peptide to export various folded proteins remain to be established.

The twin arginine signal peptides are capable of targeting most passenger proteins to the Tat pathway.2 However, the Sec-targeting information of some proteins, which is present outside of the signal peptide, can override the Tat-targeting information in the twin arginine signal peptide.15, 21 Moreover, when fused to a twin arginine signal peptide, the folded holocytochrome c is translocated via the Tat pathway, but the unfolded apocytochrome is transferred across the cytoplasmic membrane through the Sec pathway of E. coli.22 In addition, most known Tat substrates are enzymes containing various cofactors and the acquisition of the cofactors, leading to protein folding, seems to be a prerequisite for their translocation exclusively through the Tat pathway.17, 23, 24, 25 Therefore, the intrinsic characteristics of the reporter proteins could have an important impact on our understanding of the Tat system.

In this study, we assessed factors that could affect the Tat pathway function by using two in vivo reporter proteins. Recently, we showed that the twin arginine signal peptide of the trimethylamine N-oxide (TMAO) reductase (TorA) is capable of translocating the folded green fluorescent protein (GFP) via the Tat system into the periplasm, where GFP is recruited to the two poles of the cell under osmotic up-shock conditions.26 Therefore, the RR-GFP fusion provides a powerful tool for dissection of the Tat translocation pathway. Colicin V (ColV) is a peptide antibiotic encoded by the cvaC gene and is synthesized as a 103 amino acid residue precursor (pre-ColV). It is secreted by a dedicated ABC-exporter composed of three proteins, CvaA, CvaB, and TolC, and is processed to an 88 amino acid residue polypeptide.27 ColV kills sensitive cells by disrupting the membrane potential.28 Importantly, it is bactericidal only when it gains access to the inner membrane from the periplasmic face.29 Moreover, the immunity protein, Cvi, is sufficient to fully protect a target cell from the bactericidal activity of ColV. To create the second reporter protein used in this study, we re-routed ColV to the Tat pathway by replacing its double glycine signal peptide with the twin arginine signal peptide of TorA. The translocation of ColV into the periplasm via the Tat pathway inhibits cell growth severely. By using the fluorescent reporter RR-GFP and the translocation-suicide probe RR-ColV, we found that RR, KR and RK motifs were all capable of exporting GFP and ColV. Significantly, we found that tatB mutants have a phenotype distinct from that of the ΔtatC mutant, and that TatB and TatA may substitute for each other partially or operate cooperatively in the export of ColV.

Section snippets

Export of folded GFP mediated by mutated RR signal peptides in various genetic backgrounds

Recently we showed that the twin arginine signal peptide of TorA conducts the export of the folded GFP via the Tat pathway very efficiently.26 Moreover, the periplasmic GFP could be visualized by the formation of polar fluorescent spots in the wild-type strain (Figure 1(a)). In contrast, RR-GFP blocked in the cytoplasm of the ΔtatC mutant is distributed uniformly (Figure 1(b)).26 Therefore, GFP could be used as a powerful tool to monitor the Tat-dependent protein translocation directly. Like

Discussion

Genomic analysis reveals that the composition and copy number of the tat genes vary in different microbes and that a minimal Tat system requires one copy of tatA-like gene and one copy of the tatC gene.2 Genetic studies show a functional overlap between TatA and TatE,3 and suggest a substrate specificity regarding TatB.6 TatAB complexes with various TatA:TatB ratios32 or containing a large excess of the TatA subunit,33 and a TatABC complex with an approximately equimolar ratio of each subunit34

Bacterial strains, plasmids, and media used in this work

E. coli strains used in this study were: MC4100 (F′ lacΔU169 araD139 rpsL150 thi flbB5301 deoC7 ptsF25 relA1, laboratory stock) and its derivatives ELV16 (ΔtatA),3 BØD (ΔtatB),7 J1M1 (ΔtatE),3 JARV16 (ΔtatAE),3 B1LK0 (ΔtatC),5 TDD7 (ΔtatD, ΔyjjV, ΔycfH),4 DADE (ΔtatABCDE),4 MCMTA (tatB∷Kan),6 SC44 (tolC∷Tn5) and WB591 (tonB). Strain BED (MC4100 ΔtatBE) was constructed by transfer of the mutant ΔtatB allele present on plasmid pFAT1647 onto the chromosome of the ΔtatE mutant strain J1M13 using

Acknowledgements

We thank R. Lloubes, L.-M. Guzman and R. Kolter for the tolC and tonB mutants, and for plasmids pBAD24 and pHK22, respectively. We thank A. Bernadac for assistance in the fluorescence microscope observation. We are grateful to D. Duché, V. Géli and D. Cavard for discussions and suggestions. B.I., G.F. and A.C. were supported by the Minister of Research and Technology, R.V. by the “Fondation pour la recherche Médicale” (FRM) and M.Z. by China Scholarship Council and INCO fellowship. L.F.W. and

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Proteins are targeted to the Tat apparatus by an N-terminal signal sequence containing a highly-conserved RR motif that is critical for efficient recognition of Tat substrates (Alami et al., 2003; Stanley et al., 2000). However, it has been shown that signal sequences with a single mutation of an arginine residue are still transported by the Tat system (Ize et al., 2002; Summer et al., 2000). In E. coli, at least 29 secreted proteins are predicted to be Tat-dependent.
In Escherichia coli, the Twin-arginine translocation (Tat) pathway secretes a set of folded proteins with important physiological functions to the periplasm and outer membrane. The loss of Tat secretion impairs outer membrane integrity and leads to decreased cell growth. Only recently, the Tat pathway has gained more attention due to its essential role in bacterial virulence and applications in the production of fully folded heterologous proteins. In this study, we investigated the influence of the deletion of all active Tat pathway components on the E. coli cells. The comprehensive proteomic analysis revealed activation of several stress responses and experimentally confirmed the dependence of certain proteins on the Tat system for export. We observed that a tat deletion triggers protein aggregation, membrane vesiculation, synthesis of colanic acid and biofilm formation. Furthermore, the mislocalization of Tat-dependent proteins disturbs iron and molybdenum homeostasis and impairs the cell envelope integrity. The results show that the functional Tat pathway is important for the physiological stability and that its dysfunction leads to a series of severe changes in E. coli cells.
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No single mutation has thus far been identified that would completely inactivate E. coli TatB (Fröbel et al., 2012b). A lack of TatB abolishes the transport of endogenous Tat substrates in E. coli, while allowing low levels of export of some fusions between an RR-signal sequence and reporter proteins (Blaudeck et al., 2005; Chanal et al., 1998; Ize et al., 2002b). These findings could reflect some residual TatB-like activity of E. coli TatA, consistent with the idea that ancestral orthologues of TatA were as bifunctional as those of nowadays Gram-positive bacteria.
Gram negative bacteria possess a large variety of protein transport systems, by which proteins that are synthesised in the cytosol are exported to destinations in the cell envelope or entirely secreted into the extracellular environment. The inner membrane (IM) contains three major transport systems for the translocation and insertion of signal sequence containing proteins: the Sec translocon, the YidC insertase, and the Tat system. The heterotrimeric SecYEG translocon forms a narrow channel in the membrane that serves a dual function; it allows the translocation of unfolded proteins across the pore and the integration of α-helical proteins into the IM. The YidC insertase is a multi-spanning membrane protein that cooperates with the SecYEG translocon during the integration of membrane proteins but also functions as an independent insertase. Depending upon the type of protein cargo that needs to be transported, the Signal Recognition Particle (SRP), the SRP receptor, SecA and chaperones are required to coordinate translation with transport and to target and energise the different transport systems. The Tat system consists of three membrane proteins (TatA, TatB and TatC) which in a still unknown manner accomplish the transmembrane passage of completely folded proteins and protein complexes.
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The twin arginine protein transport (Tat) system transports folded proteins across cytoplasmic membranes of bacteria and thylakoid membranes of plants, and in Escherichia coli it comprises TatA, TatB and TatC components. In this study we show that the membrane extrinsic domain of TatB forms parallel contacts with at least one other TatB protein. Truncation of the C-terminal two thirds of TatB still allows complex formation with TatC, although protein transport is severely compromised. We were unable to isolate transport-inactive single codon substitution mutations in tatB suggesting that the precise amino acid sequence of TatB is not critical to its function.
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TatB physically interacts with TatB by two hybrid (View Interaction 1, 2)
Heterologous production of Escherichia coli penicillin G acylase in Pseudomonas aeruginosa
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Penicillin G acylase (PGA) is a widely studied bacterial enzyme of great industrial importance. Since its overproduction in the original organisms is mostly limited to the intracellular bacterial spaces which may lead to aggregation and cell toxicity, we have set out to explore the host organism Pseudomonas aeruginosa that possesses the Xcp machinery for secretion of folded proteins to the extracellular medium. We have made fusion proteins, consisting of Pseudomonas Sec- or Tat-specific signal peptides, the elastase propeptide and the mature penicillin G acylase. With all constructs we obtained production of PGA in P. aeruginosa, but we observed that processing of the PGA was temperature dependent and that the active enzyme could only be found after growth at 25 °C or lower temperatures. Remarkably, the mature protein, expressed from a TatProPGA hybrid, was not only found in the extracellular medium and the periplasm, but also in the cytoplasm as assessed by comparison to the reporter beta-lactamase protein. The unusual cytoplasmic localization of the mature protein strongly suggests that processing of PGA can also occur in the cytoplasm of P. aeruginosa. The extracellular localization of the TatProPGA hybrid was found not to be dependent on the tatABC-genes. The elastase signal sequence/propeptide combination appeared to be an inadequate carrier for transporting penicillin G acylase across the outer membrane of P. aeruginosa.
Twin-arginine-dependent translocation of folded proteins
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¹: Edited by G. von Heijne

²: Present addresses:Bérengère Ize, Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK; R. Voulhoux, Department of Molecular Microbiology, Utrecht University, Padualaan, 8, 3584 CH Utrecht, The Netherlands.

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Journal of Molecular Biology

Regular articleIn vivo dissection of the Tat translocation pathway in Escherichia coli1

Abstract

Introduction

Section snippets

Export of folded GFP mediated by mutated RR signal peptides in various genetic backgrounds

Discussion

Bacterial strains, plasmids, and media used in this work

Acknowledgements

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Trends Biochem. Sci

Trends Cell Biol

J. Biol. Chem

FEBS Letters

FEBS Letters

J. Biol. Chem

FEBS Letters

FEMS Microbiol. Letters

FEBS Letters

J. Biol. Chem

J. Biol. Chem

FEBS Letters

J. Biol. Chem

FEBS Letters

Gene

The Tat protein export pathway

Mol. Microbiol

Bacterial twin arginine signal peptide-dependent protein translocation pathwayevolution and mechanism

J. Mol. Microbiol. Biotechnol

Overlapping functions of components of a bacterial Sec-independent protein export pathway

EMBO J

Potential receptor function of three homologous components, TatA, TatB and TatE, of the twin arginine signal sequence-dependent metalloenzyme translocation pathway in Escherichia coli

Mol. Microbiol

Regular article
In vivo dissection of the Tat translocation pathway in Escherichia coli¹