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.     

A genetic screen for suppressors of Escherichia coli Tat signal peptide mutations establishes a critical role for the second arginine within the twin-arginine motif

The Escherichia coli Tat protein export pathway transports folded proteins synthesized with N-terminal twin-arginine signal peptides. Twin-arginine signal sequences contain a conserved SRRxFLK "twin-arginine" amino acid sequence motif which is required for protein export by the Tat pathway. The E. coli trimethylamine N-oxide reductase (TorA) is a Tat-dependent periplasmic molybdoenzyme that facilitates anaerobic respiration with trimethylamine N-oxide as terminal electron acceptor. Here, we describe mutant strains constructed with modified TorA twin-arginine signal peptides. Substitution of the second arginine residue of the TorA signal peptide twin-arginine motif with either lysine or aspartate, or the simultaneous substitution of both arginines with lysine residues, completely abolished export. In each case, the now cytoplasmically localised TorA retained full enzymatic activity with the artificial electron donor benzyl viologen. However, the mutant strains were incapable of anaerobic growth with trimethylamine N-oxide and the non-fermentable carbon-source glycerol. The growth phenotype of the mutant strains was exploited in a genetic screen with the aim of identifying second-site suppressor mutations that allowed export of the modified TorA precursors.     

Escherichia coli twin arginine (Tat) mutant translocases possessing relaxed signal peptide recognition specificities

The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across the cytoplasmic membrane of bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive arginine residues that are thought to be involved in the recognition of the signal peptides by the Tat translocase. Here, we have analyzed the specificity of Tat signal peptide recognition by using a genetic approach. Replacement of the two arginine residues in a Tat-specific precursor protein by lysine-glutamine resulted in an export-defective mutant precursor that was no longer accepted by the wild-type translocase. Selection for restored export allowed for the isolation of Tat translocases possessing single mutations in either the amino-terminal domain of TatB or the first cytosolic domain of TatC. The mutant Tat translocases still efficiently accepted the unaltered precursor protein, indicating that the substrate specificity of the translocases was not strictly changed; rather, the translocases showed an increased tolerance toward variations of the amino acids occupying the positions of the twin arginine residues in the consensus motif of a Tat signal peptide.     

Secretion of active xylanase C from Streptomyces lividans is exclusively mediated by the Tat protein export system

The bacterial twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane. The precursors targeted to the Tat pathway have signal peptides bearing the consensus motif (S/T-R-R-X-F-L-K). The xylanase C (XlnC) of Streptomyces lividans is a 20-kDa secreted enzyme. The XlnC signal peptide is 49 amino acids long and contains the S-R-R-G-F-L-G sequence, which is similar to the twin-arginine consensus motif. In S. lividans, XlnC secretion was impaired in a tatC insertion mutant, which is unable to secrete proteins that are dependent on the Tat system. When the signal peptide of XlnC was replaced by the Sec-dependent signal peptide of xylanase A, XlnC was secreted as an inactive form and demonstrated rapid proteolytic degradation in the culture supernatant, thus indicating that XlnC was specifically secreted through the Tat system. Deletions of the n-region of the XlnC signal sequence showed that a minimum of six amino acids residues preceding the twin-arginine motif was required to secrete XlnC. Replacement of one or both arginines by lysine residues in the twin arginine motif decreased four- and sevenfold, respectively, the enzyme production but did not abolish it. However, pulse chase experiments showed that the half-life of the precursor was from 2 to 3 h instead of 11 min for the wild- type precursor. Since XlnC is not associated with cofactors to exhibit activity, it is therefore a newly identified prokaryotic non-redox Tat substrate.     

Unusual signal peptide directs penicillin amidase from Escherichia coli to the Tat translocation machinery

The recently described Tat protein translocation system in Escherichia coli recognizes its protein substrates by the consensus twin arginine (SRRXFLK) motif in the signal peptide. The signal sequence of E. coli pre-pro-penicillin amidase bears two arginine residues separated by one aspargine and does not resemble the Tat-targeting motif but can nevertheless target the precursor to the Tat pathway. Mutational studies have shown that the hydrophobic core region acts in synergism with the positive charged N-terminal part of the signal peptide as a Tat recognition signal and contributes to the efficient Tat targeting of the pre-pro-penicillin amidase.     

The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin-arginine translocase

The Rieske [2Fe-2S] protein (ISP) is an essential subunit of cytochrome bc(1) complexes in mitochondrial and bacterial respiratory chains. Based on the presence of two consecutive arginines, it was argued that the ISP of Paracoccus denitrificans, a Gram-negative soil bacterium, is inserted into the cytoplasmic membrane via the twin-arginine translocation (Tat) pathway. Here, we provide experimental evidence that membrane integration of the bacterial ISP indeed relies on the Tat translocon. We show that targeting of the ISP depends on the twin-arginine motif. A strict requirement is established particularly for the second arginine residue (R16); conservative replacement of the first arginine (R15K) still permits substantial ISP transport. Comparative sequence analysis reveals characteristics common to Tat signal peptides in several bacterial ISPs; however, there are distinctive features relating to the fact that the presumed ISP Tat signal simultaneously serves as a membrane anchor. These differences include an elevated hydrophobicity of the h-region compared with generic Tat signals and the absence of an otherwise well-conserved '+5'-consensus motif lysine residue. Substitution of the +5 lysine (Y20K) compromises ISP export and/or cytochrome bc(1) stability to some extent and points to a specific role for this deviation from the canonical Tat motif. EPR spectroscopy confirms cytosolic insertion of the [2Fe-2S] cofactor. Mutation of an essential cofactor binding residue (C152S) decreases the ISP membrane levels, possibly indicating that cofactor insertion is a prerequisite for efficient translocation along the Tat pathway.     

Genetic analysis of the twin arginine translocator secretion pathway in bacteria

The twin arginine translocation (Tat) pathway of bacteria and plant chloroplasts mediates translocation of essentially folded proteins across the cytoplasmic membrane. The detailed understanding of the mechanism of protein targeting to the Tat pathway has been hampered by the lack of screening or selection systems suitable for genetic analysis. We report here the development of a highly quantitative protein reporter for genetic analysis of Tat-specific export. Specifically, export via the Tat pathway rescues green fluorescent protein (GFP) fused to an SsrA peptide from degradation by the cytoplasmic proteolytic ClpXP machinery. As a result, cellular fluorescence is determined by the amount of GFP in the periplasmic space. We used the GFP-SsrA reporter to isolate gain-of-function mutants of a Tat-specific leader peptide and for the genetic analysis of the "invariant" signature RR dipeptide motif. Flow cytometric screening of trimethylamine N-oxide reductase (TorA) leader peptide libraries resulted in isolation of six gain-of function mutants that conferred significantly higher steady-state levels of export relative to the wild-type TorA leader. All the gain-of-function mutations occurred within or near the (S/T)RRXFLK consensus motif, highlighting the significance of this region in interactions with the Tat export machinery. Randomization of the consensus RR dipeptide in the TorA leader revealed that a basic side chain (R/K) is required at the first position whereas the second position can also accept Gln and Asn in addition to basic amino acids. This result indicates that twin arginine translocation does not require the presence of an arginine dipeptide within the conserved sequence motif.     

Genetic evidence for a tight cooperation of TatB and TatC during productive recognition of twin-arginine (Tat) signal peptides in Escherichia coli

The twin arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane of bacteria. Tat signal peptides contain a consensus motif (S/T-R-R-X-F-L-K) that is thought to play a crucial role in substrate recognition by the Tat translocase. Replacement of the phenylalanine at the +2 consensus position in the signal peptide of a Tat-specific reporter protein (TorA-MalE) by aspartate blocked export of the corresponding TorA(D(+2))-MalE precursor, indicating that this mutation prevents a productive binding of the TorA(D(+2)) signal peptide to the Tat translocase. Mutations were identified in the extreme amino-terminal regions of TatB and TatC that synergistically suppressed the export defect of TorA(D(+2))-MalE when present in pairwise or triple combinations. The observed synergistic suppression activities were even more pronounced in the restoration of membrane translocation of another export-defective precursor, TorA(KQ)-MalE, in which the conserved twin arginine residues had been replaced by lysine-glutamine. Collectively, these findings indicate that the extreme amino-terminal regions of TatB and TatC cooperate tightly during recognition and productive binding of Tat-dependent precursor proteins and, furthermore, that TatB and TatC are both involved in the formation of a specific signal peptide binding site that reaches out as far as the end of the TatB transmembrane segment.     

An Escherichia coli twin-arginine signal peptide switches between helical and unstructured conformations depending on the hydrophobicity of the environment.

The Tat system catalyzes the transport of folded globular proteins across the bacterial plasma membrane and the chloroplast thylakoid. It recognizes cleavable signal peptides containing a critical twin-arginine motif but little is known of the overall structure of these peptides. In this report, we have analyzed the secondary structure of the SufI signal peptide, together with those of two nonfunctional variants in which the region around the twin-arginine, RRQFI, is replaced by KKQFI or RRQAA. Circular dichroism studies show that the SufI peptide exists as an unstructured peptide in aqueous solvent with essentially no stable secondary structure. In membrane-mimetic environments such as SDS micelles or water/trifluoroethanol, however, the peptide adopts a structure containing up to about 40% alpha-helical content. Secondary structure predictions and molecular modelling programs strongly suggest that the helical region begins at, or close to, the twin-arginine motif. Studies on the thermal stability of the helix demonstrate a sharp transition between the unstructured and helical states, suggesting that the peptide exists in one of two distinct states. The two nonfunctional peptides exhibit almost identical spectra and properties to the wild-type SufI peptide, indicating that it is the arginine sidechains, and not their contribution to the helical structure, that are critical in this class of peptide.     

Differential interactions between a twin-arginine signal peptide and its translocase in Escherichia coli

The twin-arginine translocation (Tat) machinery of the Escherichia coli inner membrane is dedicated to the export of proteins harboring a conserved SRRxFLK motif in their signal sequence. TatA, TatB, and TatC are the functionally essential constituents of the Tat machinery, but their precise function is unknown. Using site-specific crosslinking, we have analyzed interactions of the twin-arginine precursor preSufI with the Tat proteins upon targeting to inner membrane vesicles. TatA association is observed only in the presence of a transmembrane H(+) gradient. TatB is found in contact with the entire signal sequence and adjacent parts of mature SufI. Interaction of TatC with preSufI is, however, restricted to a discrete area around the consensus motif. The results reveal a hierarchy in targeting of a Tat substrate such that for the primary interaction, TatC is both necessary and sufficient while a subsequent association with TatB likely mediates transfer from TatC to the actual Tat pore.     

Specificity of signal peptide recognition in tat-dependent bacterial protein translocation

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of Zymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC and tatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.     

The Tat protein export pathway

The Tat (twin-arginine translocation) system is a bacterial protein export pathway with the remarkable ability to transport folded proteins across the cytoplasmic membrane. Preproteins are directed to the Tat pathway by signal peptides that bear a characteristic sequence motif, which includes consecutive arginine residues. Here, we review recent progress on the characterization of the Tat system and critically discuss the structure and operation of this major new bacterial protein export pathway.     

Sequence-specific binding of prePhoD to soluble TatAd indicates protein-mediated targeting of the Tat export in Bacillus subtilis

The Tat (twin-arginine protein translocation) system initially discovered in the thylakoid membrane of chloroplasts has been described recently for a variety of eubacterial organisms. Although in Escherichia coli four Tat proteins with calculated membrane spanning domains have been demonstrated to mediate Tat-dependent transport, a specific transport system for twin-arginine signal peptide containing phosphodiesterase PhoD of Bacillus subtilis consists of one TatA/TatC (TatAd/TatCd) pair of proteins. Here, we show that TatAd was found beside its membrane-integrated localization in the cytosol were it interacted with prePhoD. prePhoD was efficiently co-immunoprecipitated by TatAd. Inefficient co-immunoprecipitation of mature PhoD and missing interaction to Sec-dependent and cytosolic peptides by TatAd demonstrated a particular role of the twin-arginine signal peptide for this interaction. Affinity of prePhoD to TatAd was interfered by peptides containing the twin-arginine motif but remained active when the arginine residues were substituted. The selective binding of TatAd to peptides derived from the signal peptide of PhoD elucidated the function of the twin-arginine motif as a target site for pre-protein TatAd interaction. Substitution of the binding motif demonstrated the pivotal role of basic amino acid residues for TatA binding. These features suggest that TatA interacts prior to membrane integration with its pre-protein substrate and could therefore assist targeting of twin-arginine pre-proteins.     

The Escherichia coli TatABC system and a Bacillus subtilis TatAC-type system recognise three distinct targeting determinants in twin-arginine signal peptides

The Tat system transports folded proteins across bacterial and thylakoid membranes. In Gram-negative organisms, it is encoded by tatABC genes and the system recognizes substrates bearing signal peptides with a conserved twin-arginine motif. Most Gram-positive organisms lack a tatB gene, indicating major differences in organisation and/or mechanism. Here, we have characterized the essential targeting determinants that are recognized by a Bacillus subtilis TatAC-type system, TatAdCd. Substitution by lysine of either of the twin-arginine residues in the TorA signal peptide can be tolerated, but the presence of twin-lysine residues blocks export completely. We show that additional determinants can be as important as the twin-arginine motif. Replacement of the −1 serine by alanine, in either the TorA or DmsA signal peptide, almost blocks export by either the B. subtilis TatAdCd or Escherichia coli TatABC systems, firmly establishing the importance of this −1 residue in these signal peptides. Surprisingly, the +2 leucine in the DmsA signal peptide (sequence SRRGLV) appears to play an equally important role and substitution by alanine or phenylalanine blocks export by both the B. subtilis and E. coli systems. These data identify three distinct determinants, whose importance varies depending on the signal peptide in question. The data also show that the B. subtilis TatAdCd and E. coli TatABC systems recognize very similar determinants within their target peptides, and exhibit surprisingly similar responses to mutations within these determinants.     

Conserved signal peptide recognition systems across the prokaryotic domains

The twin-arginine translocation (Tat) pathway is a protein targeting system found in bacteria, archaea, and chloroplasts. Proteins are directed to the Tat translocase by N-terminal signal peptides containing SRRxFLK "twin-arginine" amino acid motifs. The key feature of the Tat system is its ability to transport fully folded proteins across ionically sealed membranes. For this reason the Tat pathway has evolved for the assembly of extracytoplasmic redox enzymes that must bind cofactors, and so fold, prior to export. It is important that only cofactor-loaded, folded precursors are presented for export, and cellular processes have been unearthed that regulate signal peptide activity. One mechanism, termed "Tat proofreading", involves specific signal peptide binding proteins or chaperones. The archetypal Tat proofreading chaperones belong to the TorD family, which are dedicated to the assembly of molybdenum-dependent redox enzymes in bacteria. Here, a gene cluster was identified in the archaeon Archaeoglobus fulgidus that is predicted to encode a putative molybdenum-dependent tetrathionate reductase. The gene cluster also encodes a TorD family chaperone (AF0160 or TtrD) and in this work TtrD is shown to bind specifically to the Tat signal peptide of the TtrA subunit of the tetrathionate reductase. In addition, the 3D crystal structure of TtrD is presented at 1.35 ? resolution and a nine-residue binding epitope for TtrD is identified within the TtrA signal peptide close to the twin-arginine targeting motif. This work suggests that archaea may employ a chaperone-dependent Tat proofreading system that is similar to that utilized by bacteria.     

Analysis of p300 acetyltransferase substrate specificity by MALDI TOF mass spectrometry

Acetyltransferase enzymes target specific lysine residues in substrate proteins. While the list of histone and nonhistone substrates is growing, the mechanisms of substrate selection remain unclear. Here, we describe a mass spectrometric approach to examine the site selection of the acetyltransferase p300 in the HIV-1 protein Tat. Tat is acetylated by p300 at a single lysine (K50) within its basic RNA-binding domain. To determine the sequence requirements for K50 recognition within this domain, we synthesized mixtures of "degenerated" Tat peptides, in which one of the surrounding residues was substituted by all proteinogenic amino acids. Peptide mixtures were assembled based on nonoverlapping peptide masses and acetylated by p300 in a standard in vitro acetylation reaction. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified amino acid substitutions that prevented acetylation by p300. This approach represents a fast and comprehensive screening method that was applied to the six surrounding residues of K50 in Tat. It can be applied to any known acetyltransferase substrate and might help to define consensus recognition sequences for individual acetyltransferase enzymes.     

Genetic analysis of pathway specificity during posttranslational protein translocation across the Escherichia coli plasma membrane

In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the "Sec avoidance signal," the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.     

Signal peptide for peroxisomal targeting: replacement of an essential histidine residue by certain amino acids converts the amino-terminal presequence of peroxisomal 3-ketoacyl-CoA thiolase to a mitochondrial signal peptide.

The role of the histidine residue at position -17 of the amino-terminal signal peptide of rat peroxisomal 3-ketoacyl-CoA thiolase was studied in vivo, employing site-directed mutagenesis. Among the nine amino acids tested, only glutamine could partially substitute for the histidine. Mutants carrying basic amino acids, arginine and lysine, and hydrophobic residues, leucine and valine, in place of histidine were all translocated to mitochondria, but not to peroxisomes. These results indicate that the signal peptide of the thiolase is recognized by a mechanism totally different from that for the SKL motif, a known peroxisomal targeting signal. Relationship of the thiolase signal peptide to those of mitochondrial proteins is discussed.     

A method of mapping protein sumoylation sites by mass spectrometry using a modified small ubiquitin-like modifier 1 (SUMO-1) and a computational program

Post-translational modification by small ubiquitin-like modifier 1 (SUMO-1) is a highly conserved process from yeast to humans and plays important regulatory roles in many cellular processes. Sumoylation occurs at certain internal lysine residues of target proteins via an isopeptide bond linkage. Unlike ubiquitin whose carboxyl-terminal sequence is RGG, the tripeptide at the carboxyl terminus of SUMO is TGG. The presence of the arginine residue at the carboxyl terminus of ubiquitin allows tryptic digestion of ubiquitin conjugates to yield a signature peptide containing a diglycine remnant attached to the target lysine residue and rapid identification of the ubiquitination site by mass spectrometry. The absence of lysine or arginine residues in the carboxyl terminus of mammalian SUMO makes it difficult to apply this approach to mapping sumoylation sites. We performed Arg scanning mutagenesis by systematically substituting amino acid residues surrounding the diglycine motif and found that a SUMO variant terminated with RGG can be conjugated efficiently to its target protein under normal sumoylation conditions. We developed a Programmed Data Acquisition (PDA) mass spectrometric approach to map target sumoylation sites using this SUMO variant. A web-based computational program designed for efficient identification of the modified peptides is described.     

Role of the Pseudomonas aeruginosa PlcH Tat signal peptide in protein secretion, transcription, and cross-species Tat secretion system compatibility

The secretion of PlcH and its homolog PlcN of Pseudomonas aeruginosa through the inner membrane depends upon a functional twin arginine translocase (Tat) system and a Tat signal sequence. Conserved twin arginine (Arg) residues within the Tat signal sequence consensus motif (S/TRRxFLK) are considered essential for the secretion of Tat substrates, but some exceptions (e.g., Lys and Arg) to the twin Arg residues in this motif have been noted. The roles of all three Arg residues within the PlcH RRRTFLK consensus motif were examined. Data are presented which indicate that Arg-9 and Arg-10 are essential for PlcH secretion across the inner membrane, but the mutation of Arg-8 (e.g., to Ala or Ser) had no observable effect on the localization of PlcH. In the signal sequence of PlcH and in all of its homologs in other bacteria, there are basic amino acid residues (Arg, Lys, and Gln) immediately adjacent to the signal peptidase cleavage site (Ala-X-Ala) that are not seen in Sec-dependent signal sequences. The mutation of these basic residues to Ala caused slightly decreased levels of extracellular PlcH, but normal localization was still observed. Deletion of the entire Tat signal sequence of PlcH not only resulted in the absence of detectable extracellular PlcH activity and protein but also caused a substantial decrease in the detectable level of plcH mRNA. Finally, data are presented which indicate that P. aeruginosa PlcH exhibits cross-species compatibility with the Escherichia coli Tat secretion machinery, but only when the E. coli Tat machinery is expressed in a P. aeruginosa host.