17763885
Environmental Salinity Determines the Specificity and Need for Tat-Dependent Secretion of the YwbN Protein in Bacillus subtilis
Twin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described “minimal Tat translocase” consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate.
14462084
Sec- and Tat-mediated protein secretion across the bacterial cytoplasmic membrane--distinct translocases and mechanisms.
In bacteria, two major pathways exist to secrete proteins across the cytoplasmic membrane. The general Secretion route, termed Sec-pathway, catalyzes the transmembrane translocation of proteins in their unfolded conformation, whereupon they fold into their native structure at the trans-side of the membrane. The Twin-arginine translocation pathway, termed Tat-pathway, catalyses the translocation of secretory proteins in their folded state. Although the targeting signals that direct secretory proteins to these pathways show a high degree of similarity, the translocation mechanisms and translocases involved are vastly different.
10266547
The twin-arginine transport system: moving folded proteins across membranes.
The Tat (twin-arginine transport) pathway is a protein-targeting system dedicated to the transmembrane translocation of fully folded proteins. This system is highly prevalent in the cytoplasmic membranes of bacteria and archaea, and is also found in the thylakoid membranes of plant chloroplasts and possibly also in the inner membrane of plant mitochondria. Proteins are targeted to a membrane-embedded Tat translocase by specialized N-terminal twin-arginine signal peptides bearing an SRRXFLK amino acid motif. The genes encoding components of the Tat translocase were discovered approx. 10 years ago, and, since then, research in this area has expanded on a global scale. In this review, the key discoveries in this field are summarized, and recent studies of bacterial twin-arginine signal-peptide-binding proteins are discussed.
45823533
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.
21781262
Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway
To examine the relationship between folding and export competence by the twin-arginine translocation (Tat) pathway we analyzed the subcellular localization of fusions between a set of eight putative Tat leader peptides and alkaline phosphatase in isogenic Escherichia coli strains that either allow or disfavor the formation of protein disulfide bonds in the cytoplasm. We show that export by the Tat translocator is observed only in strains that enable oxidative protein folding in the cytoplasm. Further, we show that other disulfide-containing proteins, namely single-chain Fv and heterodimeric FAB antibody fragments, are export-competent only in strains having an oxidizing cytoplasm. Functional, heterodimeric FAB protein was exported from the cytoplasm by means of a Tat leader peptide fused to the heavy chain alone, indicating that the formation of a disulfide-bonded dimer preceeds export. These results demonstrate that in vivo only proteins that have attained the native conformation are exported by the Tat translocator, indicating that a folding quality-control mechanism is intrinsic to the export process. The ability to export proteins with disulfide bonds and the folding proofing feature of the Tat pathway are of interest for biotechnology applications.
35220802
Following the Path of a Twin-arginine Precursor along the TatABC Translocase of Escherichia coli*
The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Consistent with previous in vivo data, we show that the model Tat substrate TorA-PhoA is translocated by the TatABC translocase of Escherichia coli inner membrane vesicles, only if the PhoA moiety was allowed to fold by disulfide bond formation. Although even unfolded TorA-PhoA was found to physically associate with the Tat translocase of the vesicles, site-specific cross-linking revealed a perturbed interaction of the signal sequence of unfolded TorA-PhoA with the TatBC receptor site. Some of the folded TorA-PhoA precursor accumulated in a partially protease-protected membrane environment, from where it could be translocated into the lumen of the vesicles upon re-installation of an H+-gradient. Translocation arrest occurred in immediate vicinity to TatA. Consistent with a neighborhood to TatA, TorA-PhoA remained protease-resistant in the presence of detergents that are known to preserve the oligomeric structures of TatA. Moreover, entry of TorA-PhoA to the protease-protected environment strictly required the presence of TatA. Collectively, our results are consistent with some degree of quality control by TatBC and a recruitment of TatA to a folded substrate that has functionally engaged the twin-arginine translocase.
41179104
Targeting signals for a bacterial Sec‐independent export system direct plant thylakoid import by the ΔpH pathway
Preproteins targeted to the Sec‐independent protein transport systems of plant thylakoids and of bacteria both have unusual transfer peptides bearing a consensus twin‐arginine motif. Possible mechanistic similarity between the two Sec‐independent transport pathways was investigated by assessing the ability of bacterial twin‐arginine transfer peptides to direct thylakoid import. High efficiency import was observed. This process was demonstrated to occur specifically via the Sec‐independent ΔpH pathway and to depend on an intact twin‐arginine motif on the transfer peptide. These results provide strong evidence for the operation of mechanistically related Sec‐independent protein transport pathways in chloroplasts and bacteria.
45737924
A common export pathway for proteins binding complex redox cofactors?
The precursor polypeptides of periplasmic proteins binding seven types of redox cofactor have unusually long signal sequences bearing a consensus (S/T)‐R‐R‐x‐F‐L‐K motif immediately before the hydrophobic region. Such ‘double‐arginine’ signal sequences are not, in general, found on the precursors of other periplasmic proteins. It is suggested that precursor proteins with double‐arginine signal sequences share a common specialization in their export pathway. The nature of this specialization, the structure of the double‐arginine signal sequences, and the possible relationship with the double‐arginine signal peptide‐dependent thylakoid import pathway are discussed.
25465616
A new type of signal peptide: central role of a twin‐arginine motif in transfer signals for the delta pH‐dependent thylakoidal protein translocase.
The delta pH‐driven and Sec‐related thylakoidal protein translocases recognise distinct types of thylakoid transfer signal, yet all transfer signals resemble bacterial signal peptides in structural terms. Comparison of known transfer signals reveals a single concrete difference: signals for the delta pH‐dependent system contain a common twin‐arginine motif immediately before the hydrophobic region. We show that this motif is critical for the delta pH‐driven translocation process; substitution of the arg‐arg by gln‐gln or even arg‐lys totally blocks translocation across the thylakoid membrane, and replacement by lys‐arg reduces the rate of translocation by > 100‐fold. The targeting information in this type of signal thus differs fundamentally from that of bacterial signal peptides, where the required positive charge can be supplied by any basic amino acid. Insertion of a twin‐arg motif into a Sec‐dependent substrate does not alter the pathway followed but reduces translocation efficiency, suggesting that the motif may also repel the Sec‐type system. Other information must help to specify the choice of translocation mechanism, but this information is unlikely to reside in the hydrophobic region because substitution by a hydrophobic section from an integral membrane protein does not affect the translocation pathway.
45115952
Targeting of thylakoid proteins by the ΔpH‐driven twin‐arginine translocation pathway requires a specific signal in the hydrophobic domain in conjunction with the twin‐arginine motif
Superficially similar cleavable targeting signals specify whether lumenal proteins are transported across the thylakoid membrane by a Sec‐ or ΔpH‐dependent pathway. A twin‐arginine motif is essential but not sufficient to direct ΔpH‐dependent targeting; here we show that a second determinant is located in the hydrophobic region. A highly hydrophobic amino acid is found either two or three residues C‐terminal to the twin‐arginine in all known transfer peptides for the ΔpH‐dependent system, and substitution of this residue in the 23‐kDa (23K) peptide markedly inhibits translocation. Further, whereas the insertion of twin‐arginine in a Sec‐dependent precursor does not permit efficient ΔpH‐dependent targeting, the simultaneous presence of a leucine at the +3 position (relative to the RR) enables the peptide to function as efficiently as an authentic transfer peptide. RRNVL, RRAAL and RRALA within a Sec targeting signal all support efficient ΔpH‐dependent targeting, RRNVA is less effective and RRNAA/RRNAG are totally ineffective. We conclude that the core signal for this pathway is a twin‐arginine together with an adjacent hydrophobic determinant.
41829374
The Twin Arginine Consensus Motif of Tat Signal Peptides Is Involved in Sec-independent Protein Targeting in Escherichia coli *
In Escherichia coli a subset of periplasmic proteins is exported through the Tat pathway to which substrates are directed by an NH2-terminal signal peptide containing a consensus SRRXFLK “twin arginine” motif. The importance of the individual amino acids of the consensus motif forin vivo Tat transport has been assessed by site-directed mutagenesis of the signal peptide of the Tat substrate pre-SufI. Although the invariant arginine residues are crucial for efficient export, we find that slow transport of SufI is still possible if a single arginine is conservatively substituted by a lysine residue. Thus, in at least one signal peptide context there is no absolute dependence of Tat transport on the arginine pair. The consensus phenylalanine residue was found to be a critical determinant for efficient export but could be functionally substituted by leucine, another amino acid with a highly hydrophobic side chain. Unexpectedly, the consensus lysine residue was found to retard Tat transport. These observations and others suggest that the sequence conservation of the Tat consensus motif is a reflection of the functional importance of the consensus residues. Tat signal peptides characteristically have positively charged carboxyl-terminal regions. However, changing the sign of this charge does not affect export of SufI.
3223586
Competition between Sec‐ and TAT‐dependent protein translocation in Escherichia coli
Recently, a new protein translocation pathway, the twin‐arginine translocation (TAT) pathway, has been identified in both bacteria and chloroplasts. To study the possible competition between the TAT‐ and the well‐characterized Sec translocon‐dependent pathways in Escherichia coli, we have fused the TorA TAT‐targeting signal peptide to the Sec‐dependent inner membrane protein leader peptidase (Lep). We find that the soluble, periplasmic P2 domain from Lep is re‐routed by the TorA signal peptide into the TAT pathway. In contrast, the full‐length TorA–Lep fusion protein is not re‐routed into the TAT pathway, suggesting that Sec‐targeting signals in Lep can override TAT‐targeting information in the TorA signal peptide. We also show that the TorA signal peptide can be converted into a Sec‐targeting signal peptide by increasing the hydrophobicity of its h‐region. Thus, beyond the twin‐arginine motif, the overall hydrophobicity of the signal peptide plays an important role in TAT versus Sec targeting. This is consistent with statistical data showing that TAT‐targeting signal peptides in general have less hydrophobic h‐regions than Sec‐targeting signal peptides.
25887367
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.
2056736
Overlapping functions of components of a bacterial Sec‐independent protein export pathway
We describe the identification of two Escherichia coli genes required for the export of cofactor‐containing periplasmic proteins, synthesized with signal peptides containing a twin arginine motif. Both gene products are homologous to the maize HCF106 protein required for the translocation of a subset of lumenal proteins across the thylakoid membrane. Disruption of either gene affects the export of a range of such proteins, and a complete block is observed when both genes are inactivated. The Sec protein export pathway was unaffected, indicating the involvement of the gene products in a novel export system. The accumulation of active cofactor‐containing proteins in the cytoplasm of the mutant strains suggests a role for the gene products in the translocation of folded proteins. One of the two HCF106 homologues is encoded by the first gene of a four cistron operon, tatABCD, and the second by an unlinked gene, tatE. A mutation previously assigned to the hcf106 homologue encoded at the tatABCD locus, mttA, lies instead in the tatB gene.
22644025
An Essential Component of a Novel Bacterial Protein Export System with Homologues in Plastids and Mitochondria*
Proteins are transported across the bacterial plasma membrane and the chloroplast thylakoid membrane by means of protein translocases that recognize N-terminal targeting signals in their cognate substrates. Transport of many of these proteins involves the well defined Sec apparatus that operates in both membranes. We describe here the identification of a novel component of a bacterial Sec-independent translocase. The system probably functions in a similar manner to a Sec-independent translocase in the thylakoid membrane, and substrates for both systems bear a characteristic twin-arginine motif in the targeting peptide. The translocase component is encoded inEscherichia coli by an unassigned reading frame,yigU, disruption of which blocks the export of at least five twin-Arg-containing precursor proteins that are predicted to bind redox cofactors, and hence fold, prior to translocation. The Sec pathway remains unaffected in the deletion strain. The gene has been designated tatC (for twin-argininetranslocation), and we show that homologous genes are present in a range of bacteria, plastids, and mitochondria. These findings suggest a central role for TatC-type proteins in the translocation of tightly folded proteins across a spectrum of biological membranes.
21943141
TatC Is a Specificity Determinant for Protein Secretion via the Twin-arginine Translocation Pathway*
The recent discovery of a ubiquitous translocation pathway, specifically required for proteins with a twin-arginine motif in their signal peptide, has focused interest on its membrane-bound components, one of which is known as TatC. Unlike most organisms of which the genome has been sequenced completely, the Gram-positive eubacterium Bacillus subtilis contains twotatC-like genes denoted tatCd andtatCy. The corresponding TatCd and TatCy proteins have the potential to be involved in the translocation of 27 proteins with putative twin-arginine signal peptides of which ∼6–14 are likely to be secreted into the growth medium. Using a proteomic approach, we show that PhoD of B. subtilis, a phosphodiesterase belonging to a novel protein family of which all known members are synthesized with typical twin-arginine signal peptides, is secreted via the twin-arginine translocation pathway. Strikingly, TatCd is of major importance for the secretion of PhoD, whereas TatCy is not required for this process. Thus, TatC appears to be a specificity determinant for protein secretion via the Tat pathway. Based on our observations, we hypothesize that the TatC-determined pathway specificity is based on specific interactions between TatC-like proteins and other pathway components, such as TatA, of which three paralogues are present inB. subtilis.
22603430
Selective Contribution of the Twin-Arginine Translocation Pathway to Protein Secretion in Bacillus subtilis *
The availability of the complete genome sequence of Bacillus subtilis has allowed the prediction of all exported proteins of this Gram-positive eubacterium. Recently, ∼180 secretory and 114 lipoprotein signal peptides were predicted to direct protein export from the cytoplasm. Whereas most exported proteins appear to use the Sec pathway, 69 of these proteins could potentially use the Tat pathway, as their signal peptides contain RR- or KR-motifs. In the present studies, proteomic techniques were applied to verify how many extracellular B. subtilis proteins follow the Tat pathway. Strikingly, the extracellular accumulation of 13 proteins with potential RR/KR-signal peptides was Tat-independent, showing that their RR/KR-motifs are not recognized by the Tat machinery. In fact, only the phosphodiesterase PhoD was shown to be secreted in a strictly Tat-dependent manner. Sodium azide-inhibition of SecA strongly affected the extracellular appearance of de novosynthesized proteins, including the lipase LipA and two other proteins with predicted RR/KR-signal peptides. The SecA-dependent export of pre-LipA is particularly remarkable, because its RR-signal peptide conforms well to stringent criteria for the prediction of Tat-dependent export in Escherichia coli. Taken together, our observations show that the Tat pathway makes a highly selective contribution to the extracellular proteome of B. subtilis.
21854601
Two minimal Tat translocases in Bacillus
Activity of the Tat machinery for protein transport across the inner membrane of Escherichia coli and the chloroplast thylakoidal membrane requires the presence of three membrane proteins: TatA, TatB and TatC. Here, we show that the Tat machinery of the Gram‐positive bacterium Bacillus subtilis is very different because it contains at least two minimal Tat translocases, each composed of one specific TatA and one specific TatC component. A third, TatB‐like component is apparently not required. This implies that TatA proteins of B. subtilis perform the functions of both TatA and TatB of E. coli and thylakoids. Notably, the two B. subtilis translocases named TatAdCd and TatAyCy both function as individual, substrate‐specific translocases for the twin‐arginine preproteins PhoD and YwbN, respectively. Importantly, these minimal TatAC translocases of B. subtilis are representative for the Tat machinery of the vast majority of Gram‐positive bacteria, Streptomycetes being the only known exception with TatABC translocases.
45201145
Bifunctional TatA subunits in minimal Tat protein translocases.
Funding for the project, of which this work is a part, was provided by the CEU project LSHG-CT-2004-005257.
34094397
TatB and TatC Form a Functional and Structural Unit of the Twin-arginine Translocase from Escherichia coli *
In Escherichia coli, a subset of periplasmic proteins is exported via the twin-arginine translocation (Tat) pathway. In the present study, we have purified the Tat complex from E. coli, and we show that it contains only TatA, TatB, and TatC. Within the purified complex, TatB and TatC are present in a strict 1:1 ratio, suggesting a functional association. This has been confirmed by expression of a translational fusion between TatB and TatC. This Tat(BC) chimera supports efficient Tat-dependent export, indicating that TatB and TatC act as a unit in both structural and functional terms. The purified Tat complex contains varying levels of TatA, suggesting a gradual loss during isolation and a looser association. The molecular mass of the complex is ∼600 kDa, demonstrating the presence of multiple copies of TatA, B, and C. Co-immunoprecipitation experiments show that TatC is required for the interaction of TatA with TatB, suggesting that TatA may interact with the complex via binding to TatC.
17227955
Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC–Hcf106 complex before Tha4-dependent transport
The thylakoid ΔpH-dependent pathway transports folded proteins with twin arginine–containing signal peptides. Identified components of the machinery include cpTatC, Hcf106, and Tha4. The reaction occurs in two steps: precursor binding to the machinery, and transport across the membrane. Here, we show that a cpTatC–Hcf106 complex serves as receptor for specific binding of twin arginine–containing precursors. Antibodies to either Hcf106 or cpTatC, but not Tha4, inhibited precursor binding. Blue native gel electrophoresis and coimmunoprecipitation of digitonin-solubilized thylakoids showed that Hcf106 and cpTatC are members of an ∼700-kD complex that lacks Tha4. Thylakoid-bound precursor proteins were also associated with an ∼700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106. Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane. Precursor binding to the cpTatC–Hcf106 complex required both the twin arginine and the hydrophobic core of the signal peptide. Precursors remained bound to the complex when Tha4 was sequestered by antibody, even in the presence of ΔpH. These results indicate that precursor binding to the cpTatC–Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.
34223273
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.
6087396
A twin arginine signal peptide and the pH gradient trigger reversible assembly of the thylakoid ΔpH/Tat translocase
The thylakoid ΔpH-dependent/Tat pathway is a novel system with the remarkable ability to transport tightly folded precursor proteins using a transmembrane ΔpH as the sole energy source. Three known components of the transport machinery exist in two distinct subcomplexes. A cpTatC–Hcf106 complex serves as precursor receptor and a Tha4 complex is required after precursor recognition. Here we report that Tha4 assembles with cpTatC–Hcf106 during the translocation step. Interactions among components were examined by chemical cross-linking of intact thylakoids followed by immunoprecipitation and immunoblotting. cpTatC and Hcf106 were consistently associated under all conditions tested. In contrast, Tha4 was only associated with cpTatC and Hcf106 in the presence of a functional precursor and the ΔpH. Interestingly, a synthetic signal peptide could replace intact precursor in triggering assembly. The association of all three components was transient and dissipated upon the completion of protein translocation. Such an assembly–disassembly cycle could explain how the ΔpH/Tat system can assemble translocases to accommodate folded proteins of varied size. It also explains in part how the system can exist in the membrane without compromising its ion and proton permeability barrier.
25129008
Sequence and phylogenetic analyses of the twin-arginine targeting (Tat) protein export system
Abstract. Twin-arginine targeting (Tat) protein secretion systems consist of two protein types, members of the TatA and TatC families. Homologues of these proteins are found in many archaea, bacteria, chloroplasts and mitochondria. Every prokaryotic organism with a fully sequenced genome exhibits either neither family member, or between one and three paralogues of these two family members. The Arabidopsis thaliana genome encodes three of each. Although many mitochondrially encoded TatC homologues have been identified, corresponding TatA homologues have not been found in this organelle. Phylogenetic analyses reveal that most prokaryotic Tat systems consist of one TatC homologue and two sequence-divergent TatA homologues (TatA and TatB). When only one TatA homologue is present, TatB is missing, and when three TatA homologues are present, the third one arose by duplication of TatA, not TatB. Further, homologues most resembling TatB are more sequence-divergent than those more closely resembling TatA. In contrast to the TatA family, the TatC family shows phylogenetic clustering in strict accordance with organismal type. These results are discussed in terms of their probable structural, functional and evolutionary significance.
25333723
YcdB from Escherichia coli Reveals a Novel Class of Tat-dependently Translocated Hemoproteins*
The Tat (twin-arginine translocation) system of Escherichia coli serves to translocate folded proteins across the cytoplasmic membrane. The reasons established so far for the Tat dependence are cytoplasmic cofactor assembly and/or heterodimerization of the respective proteins. We were interested in the reasons for the Tat dependence of novel Tat substrates and focused on two uncharacterized proteins, YcdO and YcdB. Both proteins contain predicted Tat signal sequences. However, we found that only YcdB was indeed Tat-dependently translocated, whereas YcdO was equally well translocated in a Tat-deficient strain. YcdB is a dimeric protein and contains a heme cofactor that was identified to be a high-spin FeIII-protoporphyrin IX complex. In contrast to all other periplasmic hemoproteins analyzed so far, heme was assembled into YcdB in the cytoplasm, suggesting that heme assembly could take place prior to translocation. The function of YcdB in the periplasm may be related to a detoxification reaction under specific conditions because YcdB had peroxidase activity at acidic pH, which coincides well with the known acid-induced expression of the gene. The data demonstrate the existence of a class of heme-containing Tat substrates, the first member of which is YcdB.
16498987
A facile reporter system for the experimental identification of twin-arginine translocation (Tat) signal peptides from all kingdoms of life.
We have developed a reporter protein system for the experimental verification of twin-arginine signal peptides. This reporter system is based on the Streptomyces coelicolor agarase protein, which is secreted into the growth medium by the twin-arginine translocation (Tat) pathway and whose extracellular activity can be assayed colorimetrically in a semiquantitative manner. Replacement of the native agarase signal peptide with previously characterized twin-arginine signal peptides from other Gram-positive and Gram-negative bacteria resulted in efficient Tat-dependent export of agarase. Candidate twin-arginine signal peptides from archaeal proteins as well as plant thylakoid-targeting sequences were also demonstrated to mediate agarase translocation. A naturally occurring variant signal peptide with an arginine-glutamine motif instead of the consensus di-arginine was additionally recognized as a Tat-targeting sequence by Streptomyces. Application of the agarase assay to previously uncharacterized candidate Tat signal peptides from Bacillus subtilis identified two further probable Tat substrates in this organism. This is the first versatile reporter system for Tat signal peptide identification.
39518767
Overflow of a hyper‐produced secretory protein from the Bacillus Sec pathway into the Tat pathway for protein secretion as revealed by proteogenomics
Bacteria secrete numerous proteins into their environment for growth and survival under complex and ever‐changing conditions. The highly different characteristics of secreted proteins pose major challenges to the cellular protein export machinery and, accordingly, different pathways have evolved. While the main secretion (Sec) pathway transports proteins in an unfolded state, the twin‐arginine translocation (Tat) pathway transports folded proteins. To date, these pathways were believed to act in strictly independent ways. Here, we have employed proteogenomics to investigate the secretion mechanism of the esterase LipA of Bacillus subtilis, using a serendipitously obtained hyper‐producing strain. While LipA is secreted Sec‐dependently under standard conditions, hyper‐produced LipA is secreted predominantly Tat‐dependently via an unprecedented overflow mechanism. Two previously identified B. subtilis Tat substrates, PhoD and YwbN, require each a distinct Tat translocase for secretion. In contrast, hyper‐produced LipA is transported by both Tat translocases of B. subtilis, showing that they have distinct but overlapping specificities. The identified overflow secretion mechanism for LipA focuses interest on the possibility that secretion pathway choice can be determined by environmental and intracellular conditions. This may provide an explanation for the previous observation that many Sec‐dependently transported proteins have potential twin‐arginine signal peptides for export via the Tat pathway.
42257053
Adaptation of protein secretion to extremely high‐salt conditions by extensive use of the twin‐arginine translocation pathway
Halophilic archaea thrive in environments with salt concentrations approaching saturation. However, little is known about the way in which these organisms stabilize their secreted proteins in such ‘hostile’ conditions. Here, we present data suggesting that the utilization of protein translocation pathways for protein secretion by the Halobacteriaceae differs significantly from that of non‐haloarchaea, and most probably represents an adaptation to the high‐salt environment. Although most proteins are secreted via the general secretion (Sec) machinery, the twin‐arginine translocation (Tat) pathway is mainly used for the secretion of redox proteins and is distinct from the Sec pathway, in that it allows cytoplasmic folding of secreted proteins. tatfind (developed in this study) was used for systematic whole‐genome analysis of Halobacterium sp. NRC‐1 and several other prokaryotes to identify putative Tat substrates. Our analyses revealed that the vast majority of haloarchaeal secreted proteins were predicted substrates of the Tat pathway. Strikingly, most of these putative Tat substrates were non‐redox proteins, the homologues of which in non‐haloarchaea were identified as putative Sec substrates. We confirmed experimentally that the secretion of one such putative Tat substrate depended on the twin‐arginine motif in its signal sequence. This extensive utilization of the Tat pathway in haloarchaea suggests an evolutionary adaptation to high‐salt conditions by allowing cytoplasmic folding of secreted proteins before their secretion.
19488478
Protein transport in the halophilic archaeon Halobacterium sp. NRC-1: a major role for the twin-arginine translocation pathway?
Trafficking of proteins, which is an essential process for all living organisms, has been extensively studied in bacteria and eukarya. Very little, however, is known about protein transport in the third domain of life, the Archaea. These organisms are prokaryotes, thus have the same cellular organization as bacteria, but phylogenetic analyses showed that archaea are evolutionarily distinct from both bacteria and eukarya (Woese et al., 1990). A unique aspect of archaea is the composition of their cytoplasmic membrane: whereas bacterial and eukaryal membranes are composed of fatty acids linked to glycerol by an ester bond, archaeal phospholipids are composed of branched isoprene units linked to glycerol by an ether group. These lipids play an important role in surviving the extreme environments in which archaea are frequently found, as they are less permeable to ions and protons and more resistant to extreme temperatures or high salt concentrations (van de Vossenberg et al., 1998). Because of the unique cytoplasmic membrane and the extreme environment in which many archaea thrive, it is likely that protein translocation machineries of archaea contain specific adaptations or even completely novel components that are important for protein transport.
26224478
The tatC gene cluster is essential for viability in halophilic archaea.
In prokaryotes the twin-arginine translocase (Tat) is a unique transport system for the export of folded proteins. The Tat pathway is usually involved in the export of a small proportion of extracytoplasmic proteins. An exception is found in halophilic archaea, in which the majority of secretory proteins have been predicted to be Tat-dependent. All haloarchaea analysed to date contain two genes encoding homologues of the Tat-component TatC. In all of these cases both genes are located adjacently on the chromosome, indicating that they form a functional unit. We show that this gene cluster is essential for viability in haloarchaea, which is in complete contrast to all other prokaryotes that have been tested thus far.
384164
The archaeal twin-arginine translocation pathway.
The twin-arginine translocation (Tat) pathway is a system with the unique ability to export proteins in a fully folded conformation. Its main components are TatA, TatB and TatC, all of which are required for Tat-dependent export. The Tat pathway is found in several Archaea, and in most of them a moderate number of predicted Tat-dependent substrates are present. Putative substrates include those binding cofactors such as iron-sulphur clusters and molybdopterin. In these Archaea, the role of the Tat pathway seems to be similar to that of bacteria: the export of a small subset of proteins that fold before translocation across the cytoplasmic membrane. The exception to this is the Tat system of the halophilic archaeon Halobacterium sp. NRC-1. In this organism, the majority of extra-cytoplasmic proteins are predicted to use the Tat pathway, which is, most likely, a specific adaptation to its particular lifestyle in highly saline conditions.
204477
Genetic and Biochemical Analysis of the Twin-Arginine Translocation Pathway in Halophilic Archaea
ABSTRACT The twin-arginine translocation (Tat) pathway is present in a wide variety of prokaryotes and is capable of exporting partially or fully folded proteins from the cytoplasm. Although diverse classes of proteins are transported via the Tat pathway, in most organisms it facilitates the secretion of a relatively small number of substrates compared to the Sec pathway. However, computational evidence suggests that haloarchaea route nearly all secreted proteins to the Tat pathway. We have expanded previous computational analyses of the haloarchaeal Tat pathway and initiated in vivo characterization of the Tat machinery in a model haloarchaeon, Haloferax volcanii. Consistent with the predicted usage of the this pathway in the haloarchaea, we determined that three of the four identified tat genes in Haloferax volcanii are essential for viability when grown aerobically in complex medium. This represents the first report of an organism that requires the Tat pathway for viability when grown under such conditions. Deletion of the nonessential gene had no effect on the secretion of a verified substrate of the Tat pathway. The two TatA paralogs TatAo and TatAt were detected in both the membrane and cytoplasm and could be copurified from the latter fraction. Using size exclusion chromatography to further characterize cytoplasmic and membrane TatA proteins, we find these proteins present in high-molecular-weight complexes in both cellular fractions.
10143400
Haloferax volcanii twin‐arginine translocation substates include secreted soluble, C‐terminally anchored and lipoproteins
Recent in silico and in vivo studies have suggested that the majority of proteins destined for secretion in the haloarchaea are trafficked through the twin‐arginine translocation (Tat) pathway. The presence of lipobox motifs in most haloarchaeal Tat signal sequences is intriguing as: (i) bioinformatic searches of archaeal genomes have not identified lipoprotein biogenesis enzymes and (ii) there are no known Tat substrates containing both a twin‐arginine and a bona fide lipobox. We have examined six computationally designated Tat substrates in the haloarchaeon Haloferax volcanii to verify previous computational predictions and to initiate studies of lipoprotein biogenesis via the Tat pathway. Our results confirmed that the six candidate proteins were not only Tat substrates, but also belonged to diverse classes of secretory proteins. Analysis of predicted lipoprotein Tat substrates revealed that they are anchored to the archaeal membrane in a cysteine‐dependent manner. Interestingly, despite the absence of an archaeal lipoprotein signal peptidase II (SPase II) homologue, the SPase II inhibitor globomycin impeded cell growth and specifically prevented maturation of lipoproteins. Together, this work not only represents the first experimental demonstration of a lipoprotein Tat substrate, but also indicates the presence of an unidentified lipoprotein biogenesis pathway in archaea.
15133260
The transcriptionally active regions in the genome of Bacillus subtilis
The majority of all genes have so far been identified and annotated systematically through in silico gene finding. Here we report the finding of 3662 strand‐specific transcriptionally active regions (TARs) in the genome of Bacillus subtilis by the use of tiling arrays. We have measured the genome‐wide expression during mid‐exponential growth on rich (LB) and minimal (M9) medium. The identified TARs account for 77.3% of the genes as they are currently annotated and additionally we find 84 putative non‐coding RNAs (ncRNAs) and 127 antisense transcripts. One ncRNA, ncr22, is predicted to act as a translational control on cstA and an antisense transcript was observed opposite the housekeeping sigma factor sigA. Through this work we have discovered a long conserved 3′ untranslated region (UTR) in a group of membrane‐associated genes that is predicted to fold into a large and highly stable secondary structure. One of the genes having this tail is efeN, which encodes a target of the twin‐arginine translocase (Tat) protein translocation system.
34469216
Role of the Twin-Arginine Translocation Pathway in Staphylococcus
ABSTRACT In Staphylococcus, the twin-arginine translocation (Tat) pathway is present only in some species and is composed of TatA and TatC. The tatAC operon is associated with the fepABC operon, which encodes homologs to an iron-binding lipoprotein, an iron-dependent peroxidase (FepB), and a high-affinity iron permease. The FepB protein has a typical twin-arginine (RR) signal peptide. The tat and fep operons constitute an entity that is not present in all staphylococcal species. Our analysis was focused on Staphylococcus aureus and S. carnosus strains. Tat deletion mutants (ΔtatAC) were unable to export active FepB, indicating that this enzyme is a Tat substrate. When the RR signal sequence from FepB was fused to prolipase and protein A, their export became Tat dependent. Since no other protein with a Tat signal could be detected, the fepABC-tatAC genes comprise not only a genetic but also a functional unit. We demonstrated that FepABC drives iron import, and in a mouse kidney abscess model, the bacterial loads of ΔtatAC and Δtat-fep mutants were decreased. For the first time, we show that the Tat pathway in S. aureus is functional and serves to translocate the iron-dependent peroxidase FepB.
21483353
The dhb Operon of Bacillus subtilisEncodes the Biosynthetic Template for the Catecholic Siderophore 2,3-Dihydroxybenzoate-Glycine-Threonine Trimeric Ester Bacillibactin*
Bacillus subtilis was reported to produce the catecholic siderophore itoic acid (2,3-dihydroxybenzoate (DHB)-glycine) in response to iron deprivation. However, by inspecting the DNA sequences of the genes dhbE, dhbB, anddhbF as annotated by the B. subtilis genome project to encode the synthetase complex for the siderophore assembly, various sequence errors within the dhbF gene were predicted and confirmed by re-sequencing. According to the corrected sequence, dhbF encodes a dimodular instead of a monomodular nonribosomal peptide synthetase. We have heterologously expressed, purified, and assayed the substrate selectivity of the recombinant proteins DhbB, DhbE, and DhbF. DhbE, a stand-alone adenylation domain of 59.9 kDa, activates, in an ATP-dependent reaction, DHB, which is subsequently transferred to the free thiol group of the cofactor phosphopantetheine of the bifunctional isochorismate lyase/aryl carrier protein DhbB. The third synthetase, DhbF, is a dimodular nonribosomal peptide synthetase of 264 kDa that specifically adenylates threonine and, to a lesser extent, glycine and that covalently loads both amino acids onto their corresponding peptidyl carrier domains. To functionally link the dhb gene cluster to siderophore synthesis, we have disrupted the dhbF gene. Comparative mass spectrometric analysis of culture extracts from both the wild type and the dhbF mutant led to the identification of a mass peak at m/z 881 ([M-H]1−) that corresponds to a cyclic trimeric ester of DHB-glycine-threonine.
42946871
Ferrous iron uptake by a magnesium transport system is toxic for Escherichia coli and Salmonella typhimurium
At low magnesium concentrations, Escherichia coli and Salmonella typhimurium LT2 accumulate ferrous iron independent of the ferrous iron transport system feo. Mutant strains with mutations in the magnesium transport gene corA accumulated less ferrous iron than the parent strains. corA+ and corA strains also differed in their sensitivity to ferrous iron under oxic conditions. corA mutants were more resistant to ferrous iron than their parent corA+ strains. Part of the ferrous iron accumulated can be chased by the addition of magnesium. Much less iron was chased when ferric iron was taken up by the siderophore ferrichrome. These results may indicate that the intracellular metabolism of the iron taken up by these systems differs and that it depends on the uptake route of the iron.
11707504
Metal ion homeostasis in Bacillus subtilis.
Bacillus subtilis, a Gram-positive soil bacterium, provides a model system for the study of metal ion homeostasis. Metalloregulatory proteins serve as the arbiters of metal ion sufficiency and regulate the expression of metal homeostasis pathways. In B. subtilis, uptake systems are regulated by the highly selective metal-sensing repressors Fur (iron), Zur (zinc), and MntR (manganese). Metal efflux systems are regulated by MerR and ArsR family homologs which, by contrast, can be rather non-specific with regard to metal selectivity. A Fur homolog, PerR, functions as an Fe(II)-dependent peroxide stress sensor and regulates putative metal transport and storage functions.
26173147
Role of the Fur Regulon in Iron Transport in Bacillus subtilis
ABSTRACT The Bacillus subtilis ferric uptake regulator (Fur) protein mediates the iron-dependent repression of at least 20 operons encoding ∼40 genes. We investigated the physiological roles of Fur-regulated genes by the construction of null mutations in 14 transcription units known or predicted to function in siderophore biosynthesis or iron uptake. We demonstrate that ywbLMN, encoding an elemental iron uptake system orthologous to the copper oxidase-dependent Fe(III) uptake system of Saccharomyces cerevisiae, is essential for growth in low iron minimal medium lacking citric acid. 2,3-Dihydroxybenzoyl-glycine (Itoic acid), the siderophore precursor produced by laboratory strains of B. subtilis, is of secondary importance. In the presence of citrate, the YfmCDEF ABC transporter is required for optimal growth. B. subtilis is unable to grow in minimal medium containing the iron chelator EDDHA unless the ability to synthesize the intact bacillibactin siderophore is restored (by the introduction of a functional sfp gene) or exogenous siderophores are provided. Utilization of the catecholate siderophores bacillibactin and enterobactin requires the FeuABC importer and the YusV ATPase. Utilization of hydroxamate siderophores requires the FhuBGC ABC transporter together with the FhuD (ferrichrome) or YxeB (ferrioxamine) substrate-binding proteins. Growth with schizokinen or arthrobactin is at least partially dependent on the YfhA YfiYZ importer and the YusV ATPase. We have also investigated the effects of a fur mutation on the proteome and documented the derepression of 11 Fur-regulated proteins, including a newly identified thioredoxin reductase homolog, YcgT.
10271743
The Twin-Arginine Signal Peptide of Bacillus subtilis YwbN Can Direct either Tat- or Sec-Dependent Secretion of Different Cargo Proteins: Secretion of Active Subtilisin via the B. subtilis Tat Pathway
ABSTRACT Proteins that are produced for commercial purposes in Bacillus subtilis are commonly secreted via the Sec pathway. Despite its high secretion capacity, the secretion of heterologous proteins via the Sec pathway is often unsuccessful. Alternative secretion routes, like the Tat pathway, are therefore of interest. Two parallel Tat pathways with distinct specificities have previously been discovered in B. subtilis. To explore the application potential of these Tat pathways, several commercially relevant or heterologous model proteins were fused to the signal peptides of the known B. subtilis Tat substrates YwbN and PhoD. Remarkably, the YwbN signal peptide directed secretion of active subtilisin, a typical Sec substrate, via the B. subtilis TatAyCy route. In contrast, the same signal peptide directed Tat-independent secretion of the Bacillus licheniformis α-amylase (AmyL). Moreover, the YwbN signal peptide directed secretion of SufI, an Escherichia coli Tat substrate, in a Tat-independent manner, most likely via Sec. Our results suggest that cytoplasmic protein folding prior to translocation is probably a major determinant of Tat-dependent protein secretion in B. subtilis, as is the case with E. coli. We conclude that future applications for the Tat system of B. subtilis will most likely involve commercially interesting proteins that are Sec incompatible.
15281033
Salt stress adaptation of Bacillus subtilis: A physiological proteomics approach
The adaptation to osmotic stress is crucial for growth and survival of Bacillus subtilis in its natural ecosystem. Dual channel imaging and warping of 2‐D protein gels were used to visualize global changes in the protein synthesis pattern of cells in response to osmotic stress (6% NaCl). Many vegetative enzymes were repressed in response to salt stress and derepressed after resumption of growth. The enzymes catalyzing the metabolic steps from glucose to 2‐oxoglutarate, however, were almost constantly synthesized during salt stress despite the growth arrest. This indicates an enhanced need for the proline precursor glutamate. The synthesis of enzymes involved in sulfate assimilation and in the formation of Fe‐S clusters was also induced, suggesting an enhanced need for the formation or repair of Fe‐S clusters in response to salt stress. One of the most obvious changes in the protein synthesis profile can be followed by the very strong induction of the SigB regulon. Furthermore, members of the SigW regulon and of the PerR regulon, indicating oxidative stress after salt challenge, were also induced. This proteomic approach provides an overview of cell adaptation to an osmotic upshift in B. subtilis visualizing the most dramatic changes in the protein synthesis pattern.
23567220
Osmotically Induced Synthesis of the Compatible Solute Hydroxyectoine Is Mediated by an Evolutionarily Conserved Ectoine Hydroxylase*
By using natural abundance 13C NMR spectroscopy, we investigated the types of compatible solutes synthesized in a variety of Bacilli under high salinity growth conditions. Glutamate, proline, and ectoine were the dominant compatible solutes synthesized by the various Bacillus species. The majority of the inspected Bacilli produced the tetrahydropyrimidine ectoine in response to high salinity stress, and a subset of these also synthesized a hydroxylation derivative of ectoine, 5-hydroxyectoine. In Salibacillus salexigens, a representative of the ectoine- and 5-hydroxyectoine-producing species, ectoine production was linearly correlated with the salinity of the growth medium and dependent on an ectABC biosynthetic operon. The formation of 5-hydroxyectoine was primarily a stationary growth phase phenomenon. The enzyme responsible for ectoine hydroxylation (EctD) was purified from S. salexigens to apparent homogeneity. The EctD protein was shown in vitro to directly hydroxylate ectoine in a reaction dependent on iron(II), molecular oxygen, and 2-oxoglutarate. We identified the structural gene (ectD) for the ectoine hydroxylase in S. salexigens. Northern blot analysis showed that the transcript levels of the ectABC and ectD genes increased as a function of salinity. Many EctD-related proteins can be found in data base searches in various Bacteria. Each of these bacterial species also contains an ectABC ectoine biosynthetic gene cluster, suggesting that 5-hydroxyectoine biosynthesis strictly depends on the prior synthesis of ectoine. Our data base searches and the biochemical characterization of the EctD protein from S. salexigens suggest that the EctD-related ectoine hydroxylases are members of a new subfamily within the non-heme-containing, iron(II)- and 2-oxoglutarate-dependent dioxygenase superfamily (EC 1.14.11).
25197589
A new ferrous iron‐uptake transporter, EfeU (YcdN), from Escherichia coli
Escherichia coli possesses multiple routes for iron uptake. Here we present EfeU (YcdN), a novel iron acquisition system of E. coli strain Nissle 1917. Laboratory strains of E. coli such as K12 lack a functional (efeU) ycdN gene caused by a frameshift mutation. EfeU, a member of the oxidase‐dependent iron transporters (OFeT), is a homologue of the iron permease Ftr1p from yeast. The ycdN gene is part of the ycdNOB tricistronic operon which is expressed in response to iron deprivation in a Fur‐dependent manner. Expression of efeU resulted in improved growth of an E. coli mutant lacking all known iron‐uptake systems and mediated increased iron uptake into cells. Furthermore, the presence of other divalent metal cations did not impair growth of strains expressing efeU. The EfeU protein functioned as ferrous iron permease in proteoliposomes in vitro. Topology analysis indicated that EfeU is an integral cytoplasmic membrane protein exhibiting seven transmembrane helices. Two REXXE motifs within transmembrane helices of OFeT family members are implicated in iron translocation. Site‐directed mutagenesis of each REGLE motif of EfeU diminished iron uptake in vivo and growth yield. In vitro the EfeU variant protein with an altered first REGLE motif was impaired in iron permeation, whereas activity of the EfeU variant with a mutation in the second motif was similar to the wild‐type protein.
21866464
Molecular mechanisms of iron uptake in fungi
Fungi, like all free‐living organisms, are in competition for limiting nutrients. In accumulating iron, fungi are faced also with a trace metal whose aqueous and redox chemistry make it both relatively bio‐unavailable and strongly cytotoxic. Successful adaptation to this environmental context has provided fungi with an iron uptake strategy that has three features: it relies on redox cycling to enhance iron bio‐availability and reduce iron cytotoxicity; it includes both high‐ and low‐affinity pathways that are mechanistically distinct; and it is autoregulating so as to maintain intracellular iron homeostasis. Using Saccharomyces cerevisiae as a paradigm, this review summarizes current knowledge about the four pathways by which this yeast accumulates iron. These four pathways include: siderophore iron accumulation; high affinity iron uptake via an iron permease; and two lower affinity uptake pathways through relatively non‐specific divalent metal ion transporters. All of these four pathways are directly or indirectly dependent on the activity of metalloreductase activity expressed extracellularly on the plasma membrane. A variety of experimental and genomics data indicate that this resourcefulness is shared by many, if not most, fungi. On the other hand, while the autoregulation of iron metabolism in Baker's yeast is well‐understood, little is known about the apparent homeostatic mechanisms in these other yeasts and fungi. The integration of these multiple uptake mechanisms and their regulation into over‐all iron homeostasis in yeast concludes this brief review.
21471124
An Oxidase-Permease-based Iron Transport System in Schizosaccharomyces pombe and Its Expression in Saccharomyces cerevisiae*
Genetic studies have demonstrated that high affinity ferrous transport in Saccharomyces cerevisiae requires an oxidase (Fet3p) and a permease (Ftr1p). Using an iron-independent galactose-based expression system, we show that expression of these two genes can mediate high affinity ferrous iron transport, indicating that these two genes are not only necessary, but sufficient for high affinity iron transport. Schizosaccharomyces pombe also employ an oxidase-permease system for high affinity iron transport. The S. pombe genes, fio1+ (ferrous iron oxidase) and fip1+ (ferriferous permease), share significant similarity to FET3 and FTR1 from S. cerevisiae. Both fio1+ and fip1+ are transcriptionally regulated by iron need, and disruption of fio1+ results in a loss of high affinity iron transport. Expression of fio1+ alone in an S. cerevisiae fet3 disruption strain does not result in high affinity iron transport. This result indicates that the S. pombe ferroxidase, while functionally homologous to the S. cerevisiae ferroxidase, does not have enough similarity to interact with the S. cerevisiae permease. Simultaneous expression of both S. pombe genes, fio1+ and fip1+, in S. cerevisiae can reconstitute high affinity iron transport. These results demonstrate that the oxidase and permease are all that is required to reconstitute high affinity iron transport and suggest that such transport systems are found in other eukaryotes.
9610812
Bacteria capture iron from heme by keeping tetrapyrrol skeleton intact
Because heme is a major iron-containing molecule in vertebrates, the ability to use heme-bound iron is a determining factor in successful infection by bacterial pathogens. Until today, all known enzymes performing iron extraction from heme did so through the rupture of the tetrapyrrol skeleton. Here, we identified 2 Escherichia coli paralogs, YfeX and EfeB, without any previously known physiological functions. YfeX and EfeB promote iron extraction from heme preserving the tetrapyrrol ring intact. This novel enzymatic reaction corresponds to the deferrochelation of the heme. YfeX and EfeB are the sole proteins able to provide iron from exogenous heme sources to E. coli. YfeX is located in the cytoplasm. EfeB is periplasmic and enables iron extraction from heme in the periplasm and iron uptake in the absence of any heme permease. YfeX and EfeB are widespread and highly conserved in bacteria. We propose that their physiological function is to retrieve iron from heme.
39256890
Genome-Wide Transcriptional Profiling Analysis of Adaptation of Bacillus subtilis to High Salinity
ABSTRACT The gram-positive soil bacterium Bacillus subtilis often faces increases in the salinity in its natural habitats. A transcriptional profiling approach was utilized to investigate both the initial reaction to a sudden increase in salinity elicited by the addition of 0.4 M NaCl and the cellular adaptation reactions to prolonged growth at high salinity (1.2 M NaCl). Following salt shock, a sigB mutant displayed immediate and transient induction and repression of 75 and 51 genes, respectively. Continuous propagation of this strain in the presence of 1.2 M NaCl triggered the induction of 123 genes and led to the repression of 101 genes. In summary, our studies revealed (i) an immediate and transient induction of the SigW regulon following salt shock, (ii) a role of the DegS/DegU two-component system in sensing high salinity, (iii) a high-salinity-mediated iron limitation, and (iv) a repression of chemotaxis and motility genes by high salinity, causing severe impairment of the swarming capability of B. subtilis cells. Initial adaptation to salt shock and continuous growth at high salinity share only a limited set of induced and repressed genes. This finding strongly suggests that these two phases of adaptation require distinctively different physiological adaptation reactions by the B. subtilis cell. The large portion of genes with unassigned functions among the high-salinity-induced or -repressed genes demonstrates that major aspects of the cellular adaptation of B. subtilis to high salinity are unexplored so far.
20843204
Ferri‐bacillibactin uptake and hydrolysis in Bacillus subtilis
Upon iron limitation, Bacillus subtilis secretes the catecholic trilactone (2,3‐dihydroxybenzoate‐glycine‐threonine)3 siderophore bacillibactin (BB) for ferric iron scavenging. Here, we show that ferri‐BB uptake is mediated by the FeuABC transporter and that YuiI, a novel trilactone hydrolase, catalyses ferri‐BB hydrolysis leading to cytosolic iron release. Among several Fur‐regulated ABC transport mutants, only ΔfeuABC exhibited impaired growth during iron starvation. Quantification of intra‐ and extracellular (ferri)‐BB in iron‐depleted ΔfeuABC cultures revealed a fourfold increase of the extracellular siderophore concentration, confirming a blocked ferri‐BB uptake in the absence of FeuABC. Ferri‐BB was found to bind selectively to the periplasmic binding protein FeuA (Kd = 57 ± 1 nM), proving high‐affinity transport of the iron‐charged siderophore. During iron starvation, a ΔyuiI mutant displayed impaired growth and strong intracellular (30‐fold) and extracellular (6.5‐fold) (ferri)‐BB accumulation. Kinetic studies in vitro revealed that YuiI hydrolyses both BB and ferri‐BB. While BB hydrolysis led to strong accumulation of the tri‐ and dimeric reaction intermediates, ferri‐BB hydrolysis yielded exclusively the monomeric reaction product and occurred with a 25‐fold higher catalytic efficiency than BB single hydrolysis. Thus, ferri‐BB was the preferred substrate of the YuiI esterase whose gene locus was designated besA.