Positive charges promote the recognition of proteins by the chaperone SlyD from Escherichia coli.

SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at its C-terminus that binds nickel and provides it for nickel-enzyme biogenesis. SlyD has been found to bind signal peptides of proteins that are translocated by the Tat pathway, a system for the transport of folded proteins across membranes. Using peptide arrays to analyze these signal peptide interactions, we found that SlyD interacted only with positively charged peptides, with a preference for arginines over lysines, and large hydrophobic residues enhanced binding. Especially a twin-arginine motif was recognized, a pair of highly conserved arginines adjacent to a stretch of hydrophobic residues. Using isothermal titration calorimetry (ITC) with purified SlyD and a signal peptide-containing model Tat substrate, we could show that the wild type twin-arginine signal peptide was bound with higher affinity than an RR>KK mutated variant, confirming that positive charges are recognized by SlyD, with a preference of arginines over lysines. The specific role of negative charges of the chaperone domain surface and of hydrophobic residues in the chaperone active site was further analyzed by ITC of mutated SlyD variants. Our data show that the supposed key hydrophobic residues of the active site are indeed crucial for binding, and that binding is influenced by negative charges on the chaperone domain. Recognition of positive charges is likely achieved by a large negatively charged surface region of the chaperone domain, which is highly conserved although individual positions are variable.

A larger TatBC complex associates with TatA clusters for transport of folded proteins across the bacterial cytoplasmic membrane.

The twin-arginine translocation (Tat) system transports folded proteins across energized biological membranes in bacteria, plastids, and plant mitochondria. In Escherichia coli, the three membrane proteins TatA, TatB and TatC associate to enable Tat transport. While TatB and TatC together form complexes that bind Tat-dependently transported proteins, the TatA component is responsible for the permeabilization of the membrane during transport. With wild type Tat systems, the TatB- and TatC-containing Tat complexes TC1 and TC2 can be differentiated. Their TatA content has not been resolved, nor could they be assigned to any step of the translocation mechanism. It is therefore a key question of current Tat research to understand how TatA associates with Tat systems during transport. By analyzing affinity-purified Tat complexes with mutations in TatC that selectively enrich either TC1 or TC2, we now for the first time demonstrate that both Tat complexes associate with TatA, but the larger TC2 recruits significantly more TatA than the smaller TC1. Most TatA co-purified as multimeric clusters. Using site-specific photo cross-linking, we could detect TatA-TatC interactions only near TatC transmembrane helices 5 and 6. Substrate-binding did not change the interacting positions but affected the stability of the interaction, pointing to a substrate-induced conformational transition. Together, our findings indicate that TatA clusters associate with TatBC without being integrated into the complex by major rearrangements. The increased TatA affinity of the larger Tat complex TC2 suggests that functional assembly is advanced in this complex.

Specialized biopolymers: versatile tools for microbial resilience.

Bacteria produce a wide range of specialized biopolymers that can be classified into polysaccharides, polyamides, and polyesters and are considered to fulfill storage functions. In this review, we highlight recent developments in the field linking metabolism of biopolymers to stress and signaling physiology of the producers and demonstrating that biopolymers contribute to bacterial stress resistance and shape structure and composition of microenvironments. While specialized biopolymers are currently the focus of much attention in biotechnology as innovative and biodegradable materials, our understanding about the regulation and functions of these valuable compounds for the producers, microbial communities, and our environment is still very limited. Addressing open questions about signals, mechanisms, and functions in the area of biopolymers harbors potential for exciting discoveries with high relevance for biotechnology and fundamental research.

The novel chloroplast glucose transporter pGlcT2 affects adaptation to extended light periods.

Intracellular sugar compartmentation is critical in plant development and acclimation to challenging environmental conditions. Sugar transport proteins are present in plasma membranes and in membranes of organelles such as vacuoles, the Golgi apparatus, and plastids. However, there may exist other transport proteins with uncharacterized roles in sugar compartmentation. Here we report one such novel transporter of the Monosaccharide Transporter Family, the closest phylogenetic homolog of which is the chloroplast-localized glucose transporter pGlcT and that we therefore term plastidic glucose transporter 2 (pGlcT2). We show, using gene-complemented glucose uptake deficiency of an Escherichia coli ptsG/manXYZ mutant strain and biochemical characterization, that this protein specifically facilitates glucose transport, whereas other sugars do not serve as substrates. In addition, we demonstrate pGlcT2-GFP localized to the chloroplast envelope and that pGlcT2 is mainly produced in seedlings and in the rosette center of mature Arabidopsis plants. Therefore, in conjunction with molecular and metabolic data, we propose pGlcT2 acts as a glucose importer that can limit cytosolic glucose availability in developing pGlcT2-overexpressing seedlings. Finally, we show both overexpression and deletion of pGlcT2 resulted in impaired growth efficiency under long day and continuous light conditions, suggesting pGlcT2 contributes to a release of glucose derived from starch mobilization late in the light phase. Together, these data indicate the facilitator pGlcT2 changes the direction in which it transports glucose during plant development and suggest the activity of pGlcT2 must be controlled spatially and temporarily in order to prevent developmental defects during adaptation to periods of extended light.

Occurrence and potential mechanism of holin-mediated non-lytic protein translocation in bacteria.

Holins are generally believed to generate large membrane lesions that permit the passage of endolysins across the cytoplasmic membrane of prokaryotes, ultimately resulting in cell wall degradation and cell lysis. However, there are more and more examples known for non-lytic holin-dependent secretion of proteins by bacteria, indicating that holins somehow can transport proteins without causing large membrane lesions. Phage-derived holins can be used for a non-lytic endolysin translocation to permeabilize the cell wall for the passage of secreted proteins. In addition, clostridia, which do not possess the Tat pathway for transport of folded proteins, most likely employ non-lytic holin-mediated transport also for secretion of toxins and bacteriocins that are incompatible with the general Sec pathway. The mechanism for non-lytic holin-mediated transport is unknown, but the recent finding that the small holin TpeE mediates a non-lytic toxin secretion in Clostridium perfringens opened new perspectives. TpeE contains only one short transmembrane helix that is followed by an amphipathic helix, which is reminiscent of TatA, the membrane-permeabilizing component of the Tat translocon for folded proteins. Here we review the known cases of non-lytic holin-mediated transport and then focus on the structural and functional comparison of TatA and TpeE, resulting in a mechanistic model for holin-mediated transport. This model is strongly supported by a so far not recognized naturally occurring holin-endolysin fusion protein.

TatA and TatB generate a hydrophobic mismatch important for the function and assembly of the Tat translocon in Escherichia coli.

The twin-arginine translocation (Tat) system serves to translocate folded proteins across energy-transducing membranes in bacteria, archaea, plastids, and some mitochondria. In Escherichia coli, TatA, TatB, and TatC constitute functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) followed by an amphipathic helix. The TMHs of TatA and TatB generate a hydrophobic mismatch with the membrane, as the helices comprise only 12 consecutive hydrophobic residues; however, the purpose of this mismatch is unclear. Here, we shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both and analyzed effects on translocon function and assembly. We found the WT length helices functioned best, but some variation was clearly tolerated. Defects in function were exacerbated by simultaneous mutations in TatA and TatB, indicating partial compensation of mutations in each by the other. Furthermore, length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residue-length is important but not essential for this interaction and translocon assembly. To also address potential effects of helix length on TatA interactions, we characterized these interactions by molecular dynamics simulations, after having characterized the TatA assemblies by metal-tagging transmission electron microscopy. In these simulations, we found that interacting short TMHs of larger TatA assemblies were thinning the membrane and-together with laterally-aligned tilted amphipathic helices-generated a deep V-shaped membrane groove. We propose the 12 consecutive hydrophobic residues may thus serve to destabilize the membrane during Tat transport, and their conservation could represent a delicate compromise between functionality and minimization of proton leakage.

PvdM of fluorescent pseudomonads is required for the oxidation of ferribactin by PvdP in periplasmic pyoverdine maturation.

Fluorescent pseudomonads such as Pseudomonas aeruginosa or Pseudomonas fluorescens produce pyoverdine siderophores that ensure iron-supply in iron-limited environments. After its synthesis in the cytoplasm, the nonfluorescent pyoverdine precursor ferribactin is exported into the periplasm, where the enzymes PvdQ, PvdP, PvdO, PvdN, and PtaA are responsible for fluorophore maturation and tailoring steps. While the roles of all these enzymes are clear, little is known about the role of PvdM, a human renal dipeptidase-related protein that is predicted to be periplasmic and that is essential for pyoverdine biogenesis. Here, we reveal the subcellular localization and functional role of PvdM. Using the model organism P. fluorescens, we show that PvdM is anchored to the periplasmic side of the cytoplasmic membrane, where it is indispensable for the activity of the tyrosinase PvdP. While PvdM does not share the metallopeptidase function of renal dipeptidase, it still has the corresponding peptide-binding site. The substrate of PvdP, deacylated ferribactin, is secreted by a ΔpvdM mutant strain, indicating that PvdM prevents loss of this periplasmic biosynthesis intermediate into the medium by ensuring the efficient transfer of ferribactin to PvdP in vivo. We propose that PvdM belongs to a new dipeptidase-related protein subfamily with inactivated Zn2+ coordination sites, members of which are usually genetically linked to TonB-dependent uptake systems and often associated with periplasmic FAD-dependent oxidoreductases related to d-amino acid oxidases. We suggest that these proteins are necessary for selective binding, exposure, or transfer of specific d- and l-amino acid-containing peptides and other periplasmic biomolecules in manifold pathways.

The Phage T4 Antiholin RI Has a Cleavable Signal Peptide, Not a SAR Domain.

Holin/endolysin-mediated lysis of phage T4 of Escherichia coli is tightly regulated by the antiholins RI and RIII. While regulation by the cytoplasmic RIII plays a minor role, the periplasmic antiholin RI binds tightly to the holin T and is believed to directly sense periplasmic phage DNA from superinfections as a trigger for the inhibition of lysis. RI has been reported to contain a non-cleavable signal peptide that anchors the protein to the membrane. Lysis is believed to be induced at some stage by a membrane depolarization that causes a release of RI into the periplasm without cleavage of the signal anchor. For the current model of phage lysis induction, it is thus a fundamental assumption that the N-terminal trans-membrane domain (TMD) of RI is such a signal anchor release (SAR) domain. Here we show that, in contrast to previous reports, this domain of RI is a cleavable signal peptide. RI is processed and released into the periplasm as a mature protein, and inactivation of its signal peptidase cleavage site blocks processing and membrane release. The signal peptide of RI can also mediate the normal translocation of a well-characterized Sec substrate, PhoA, into the periplasm. This simplifies the current view of phage lysis regulation and suggests a fundamentally different interpretation of the recently published structure of the soluble domains of the RI-T complex.

An unusual overrepresentation of genetic factors related to iron homeostasis in the genome of the fluorescent Pseudomonas sp. ABC1.

Members of the genus Pseudomonas inhabit diverse environments, such as soil, water, plants and humans. The variability of habitats is reflected in the diversity of the structure and composition of their genomes. This cosmopolitan bacterial genus includes species of biotechnological, medical and environmental importance. In this study, we report on the most relevant genomic characteristics of Pseudomonas sp. strain ABC1, a siderophore-producing fluorescent strain recently isolated from soil. Phylogenomic analyses revealed that this strain corresponds to a novel species forming a sister clade of the recently proposed Pseudomonas kirkiae. The genomic information reveals an overrepresented repertoire of mechanisms to hoard iron when compared to related strains, including a high representation of fecI-fecR family genes related to iron regulation and acquisition. The genome of the Pseudomonas sp. ABC1 contains the genes for non-ribosomal peptide synthetases (NRPSs) of a novel putative Azotobacter-related pyoverdine-type siderophore, a yersiniabactin-type siderophore and an antimicrobial betalactone; the last two are found only in a limited number of Pseudomonas genomes. Strain ABC1 can produce siderophores in a low-cost medium, and the supernatants from cultures of this strain promote plant growth, highlighting their biotechnological potential as a sustainable industrial microorganism.

A tunable anthranilate-inducible gene expression system for Pseudomonas species.

Pseudomonads are among the most common bacteria in soils, limnic ecosystems, and human, animal, or plant host environments, including intensively studied species such as Pseudomonas aeruginosa, P. putida, or P. fluorescens. Various gene expression systems are established for some species, but there is still a need for a simple system that is suitable for a wide range of pseudomonads and that can be used for physiological applications, i.e., with a tuning capacity at lower expression levels. Here, we report the establishment of the anthranilate-dependent PantA promoter for tunable gene expression in pseudomonads. During studies on P. fluorescens, we constructed an anthranilate-inducible AntR/PantA-based expression system, named pUCP20-ANT, and used GFP as reporter to analyze gene expression. This system was compared with the rhamnose-inducible RhaSR/PrhaB-based expression system in an otherwise identical vector background. While the rhamnose-inducible system did not respond to lower inducer concentrations and always reached high levels over time when induced, expression levels of the pUCP20-ANT system could be adjusted to a range of distinct lower or higher levels by variation of anthranilate concentrations in the medium. Importantly, the anthranilate-inducible expression system worked also in strains of P. aeruginosa and P. putida and therefore will be most likely useful for physiological and biotechnological purposes in a wide range of pseudomonads. KEY POINTS: • We established an anthranilate-inducible gene expression system for pseudomonads. • This system permits tuning of gene expression in a wide range of pseudomonads. • It will be very useful for physiological and biotechnological applications.

A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli.

The twin-arginine translocation (Tat) system transports folded proteins across membranes of prokaryotes, plant plastids, and some mitochondria. According to blue-native polyacrylamide gel electrophoresis after solubilization with digitonin, distinct interactions between the components TatA, TatB, and TatC result in two major TatBC-containing complexes in Escherichia coli that can bind protein substrates. We now report the first detection of a TatABC complex that likely represents the state at which transport occurs. This complex was initially found when the photo cross-linking amino acid p-benzoyl-l-phenylalanine (Bpa) was introduced at position I50 on the periplasmic side of the first trans-membrane domain of TatC. Cross-linking of TatCI50Bpa resulted in TatC-TatC-cross-links, indicating a close proximity to neighboring TatC in the complex. However, the new complex was not caused by cross-links but rather by non-covalent side chain interactions, as it was also detectable without UV-cross-linking or with an I50Y exchange. The new complex did not contain any detectable substrate. It was slightly upshifted relative to previously reported substrate-containing TatABC complexes. In the absence of TatA, an inactive TatBCI50Bpa complex was formed of the size of wild-type substrate-containing TatABC complexes, suggesting that TatB occupies TatA-binding sites at TatCI50Bpa. When substrate binding was abolished by point mutations, this TatBCI50Bpa complex shifted analogously to active TatABCI50Bpa complexes, indicating that a defect substrate-binding site further enhances TatB association to TatA-binding sites. Only TatA could shift the complex with an intact substrate-binding site, which explains the TatA requirement for substrate transport by TatABC systems.

Evidence for an Adaptation of a Phage-Derived Holin/Endolysin System to Toxin Transport in Clostridioides difficile.

The pathogenicity locus (PaLoc) of Clostridioides difficile usually comprises five genes (tcdR, tcdB, tcdE, tcdA, tcdC). While the proteins TcdA and TcdB represent the main toxins of this pathogen, TcdR and TcdC are involved in the regulation of their production. TcdE is a holin family protein, members of which are usually involved in the transport of cell wall-degrading enzymes (endolysins) for phage-induced lysis. In the past, TcdE has been shown to contribute to the release of TcdA and TcdB, but it is unclear whether it mediates a specific transport or rather a lysis of cells. TcdE of C. difficile strains analyzed so far can be produced in three isoforms that are initiated from distinct N-terminal ATG codons. When produced in Escherichia coli, we found that the longest TcdE isoform had a moderate effect on cell growth, whereas the shortest isoform strongly induced lysis. The effect of the longest isoform was inhibitory for cell lysis, implying a regulatory function of the N-terminal 24 residues. We analyzed the PaLoc sequence of 44 C. difficile isolates and found that four of these apparently encode only the short TcdE isoforms, and the most closely related holins from C. difficile phages only possess one of these initiation codons, indicating that an N-terminal extension of TcdE evolved in C. difficile. All PaLoc sequences comprised also a conserved gene encoding a short fragment of an endolysin remnant of a phage holin/endolysin pair. We could produce this peptide, which we named TcdL, and demonstrated by bacterial two-hybrid analysis a self-interaction and an interaction with TcdB that might serve to mediate TcdE-dependent transport.

The biosynthesis of pyoverdines.

Pyoverdines are fluorescent siderophores of pseudomonads that play important roles for growth under iron-limiting conditions. The production of pyoverdines by fluorescent pseudomonads permits their colonization of hosts ranging from humans to plants. Prominent examples include pathogenic or non-pathogenic species such as Pseudomonas aeruginosa, P. putida, P. syringae, or P. fluorescens. Many distinct pyoverdines have been identified, all of which have a dihydroxyquinoline fluorophore in common, derived from oxidative cyclizations of non-ribosomal peptides. These serve as precursor of pyoverdines and are commonly known as ferribactins. Ferribactins of distinct species or even strains often differ in their sequence, resulting in a large variety of pyoverdines. However, synthesis of all ferribactins begins with an L-Glu/D-Tyr/L-Dab sequence, and the fluorophore is generated from the D-Tyr/L-Dab residues. In addition, the initial L-Glu residue is modified to various acids and amides that are responsible for the range of distinguishable pyoverdines in individual strains. While ferribactin synthesis is a cytoplasmic process, the maturation to the fluorescent pyoverdine as well as the tailoring of the initial glutamate are exclusively periplasmic processes that have been a mystery until recently. Here we review the current knowledge of pyoverdine biosynthesis with a focus on the recent advancements regarding the periplasmic maturation and tailoring reactions.

Evidence for a second regulatory binding site on PspF that is occupied by the C-terminal domain of PspA.

PspA is a key component of the bacterial Psp membrane-stress response system. The biochemical and functional characterization of PspA is impeded by its oligomerization and aggregation properties. It was recently possible to solve the coiled coil structure of a completely soluble PspA fragment, PspA(1-144), that associates with the σ54 enhancer binding protein PspF at its W56-loop and thereby down-regulates the Psp response. We now found that the C-terminal part of PspA, PspA(145-222), also interacts with PspF and inhibits its activity in the absence of full-length PspA. Surprisingly, PspA(145-222) effects changed completely in the presence of full-length PspA, as promoter activity was triggered instead of being inhibited under this condition. PspA(145-222) thus interfered with the inhibitory effect of full-length PspA on PspF, most likely by interacting with full-length PspA that remained bound to PspF. In support of this view, a comprehensive bacterial-2-hybrid screen as well as co-purification analyses indicated a self-interaction of PspA(145-222) and an interaction with full-length PspA. This is the first direct demonstration of PspA/PspA and PspA/PspF interactions in vivo that are mediated by the C-terminus of PspA. The data indicate that regulatory binding sites on PspF do not only exist for the N-terminal coiled coil domain but also for the C-terminal domain of PspA. The inhibition of PspF by PspA-(145-222) was reduced upon membrane stress, whereas the inhibition of PspF by PspA(1-144) did not respond to membrane stress. We therefore propose that the C-terminal domain of PspA is crucial for the regulation of PspF in response to Psp system stimuli.

The TatA component of the twin-arginine translocation system locally weakens the cytoplasmic membrane of Escherichia coli upon protein substrate binding.

The twin-arginine translocation (Tat) system that comprises the TatA, TatB, and TatC components transports folded proteins across energized membranes of prokaryotes and plant plastids. It is not known, however, how the transport of this protein cargo is achieved. Favored models suggest that the TatA component supports transport by weakening the membrane upon full translocon assembly. Using Escherichia coli as a model organism, we now demonstrate in vivo that the N terminus of TatA can indeed destabilize the membrane, resulting in a lowered membrane energization in growing cells. We found that in full-length TatA, this effect is counterbalanced by its amphipathic helix. Consistent with these observations, the TatA N terminus induced proton leakage in vitro, indicating membrane destabilization. Fluorescence quenching data revealed that substrate binding causes the TatA hinge region and the N-terminal part of the TatA amphipathic helix to move toward the membrane surface. In the presence of TatBC, substrate binding also reduced the exposure of a specific region in the amphipathic helix, indicating a participation of TatBC. Of note, the substrate-induced reorientation of the TatA amphipathic helix correlated with detectable membrane weakening. We therefore propose a two-state model in which membrane-destabilizing effects of the short TatA membrane anchor are compensated by the membrane-immersed N-terminal part of the amphipathic helix in a resting state. We conclude that substrate binding to TatABC complexes switches the position of the amphipathic helix, which locally weakens the membrane on demand to allow substrate translocation across the membrane.

PvdO is required for the oxidation of dihydropyoverdine as the last step of fluorophore formation in Pseudomonas fluorescens.

Pyoverdines are important siderophores that guarantee iron supply to important pathogenic and non-pathogenic pseudomonads in host habitats. A key characteristic of all pyoverdines is the fluorescent dihydroxyquinoline group that contributes two ligands to the iron complexes. Pyoverdines are derived from the non-ribosomally synthesized peptide ferribactin, and their fluorophore is generated by periplasmic oxidation and cyclization reactions of d-tyrosine and l-diaminobutyric acid. The formation of the fluorophore is known to be driven by the periplasmic tyrosinase PvdP. Here we report that the putative periplasmic oxidoreductase PvdO of Pseudomonas fluorescens A506 is required for the final oxidation of dihydropyoverdine to pyoverdine, which completes the fluorophore. The pvdO deletion mutant accumulates dihydropyoverdine, and this phenotype is fully complemented by recombinant PvdO. The autoxidation of dihydropyoverdine at alkaline pH and the presence of high copper concentrations can mask this phenotype. Mutagenesis of conserved residues with potential catalytic function identified Glu-260 as an essential residue whose mutation abolished function without affecting stability or transport. Glu-260 of PvdO is at the exact position of the active-site cysteine in the structurally related formylglycine-generating enzyme. Evolution thus used the same protein fold for two distinct functionalities. As purified PvdO was inactive, additional factors are required for catalysis.

The periplasmic transaminase PtaA of Pseudomonas fluorescens converts the glutamic acid residue at the pyoverdine fluorophore to α-ketoglutaric acid.

The periplasmic conversion of ferribactin to pyoverdine is essential for siderophore biogenesis in fluorescent pseudomonads, such as pathogenic Pseudomonas aeruginosa or plant growth-promoting Pseudomonas fluorescens The non-ribosomal peptide ferribactin undergoes cyclizations and oxidations that result in the fluorophore, and a strictly conserved fluorophore-bound glutamic acid residue is converted to a range of variants, including succinamide, succinic acid, and α-ketoglutaric acid residues. We recently discovered that the pyridoxal phosphate-containing enzyme PvdN is responsible for the generation of the succinamide, which can be hydrolyzed to succinic acid. Based on this, a distinct unknown enzyme was postulated to be responsible for the conversion of the glutamic acid to α-ketoglutaric acid. Here we report the identification and characterization of this enzyme in P. fluorescens strain A506. In silico analyses indicated a periplasmic transaminase in fluorescent pseudomonads and other proteobacteria that we termed PtaA for "periplasmic transaminase A" An in-frame-deleted ptaA mutant selectively lacked the α-ketoglutaric acid form of pyoverdine, and recombinant PtaA complemented this phenotype. The ptaA/pvdN double mutant produced exclusively the glutamic acid form of pyoverdine. PtaA is homodimeric and contains a pyridoxal phosphate cofactor. Mutation of the active-site lysine abolished PtaA activity and affected folding as well as Tat-dependent transport of the enzyme. In pseudomonads, the occurrence of ptaA correlates with the occurrence of α-ketoglutaric acid forms of pyoverdines. As this enzyme is not restricted to pyoverdine-producing bacteria, its catalysis of periplasmic transaminations is most likely a general tool for specific biosynthetic pathways.

Tat transport in Escherichia coli requires zwitterionic phosphatidylethanolamine but no specific negatively charged phospholipid.

Translocation of folded proteins by the Tat system of Escherichia coli is believed to rely on the presence of phosphatidylethanolamine (PE) and the negatively charged phospholipids cardiolipin (CL) and phosphatidylglycerol (PG). Here, we show that while PE is indeed essential for activity, the Tat system is fully functional in a clsA/clsB/clsC deletion strain lacking CL, and in a pgsA deletion strain lacking both PG and CL during aerobic growth on complex media. In contrast to early studies that relied on strains with reduced lipid levels, this study therefore demonstrates that PG and CL are dispensable for Tat transport. The lack of these lipids may be compensated by other anionic phospholipids such as phosphatidic acid, CDP-diacylglycerol or N-acyl-PE.

PvdN Enzyme Catalyzes a Periplasmic Pyoverdine Modification.

Pyoverdines are high affinity siderophores produced by a broad range of pseudomonads to enhance growth under iron deficiency. They are especially relevant for pathogenic and mutualistic strains that inhabit iron-limited environments. Pyoverdines are generated from non-ribosomally synthesized highly modified peptides. They all contain an aromatic chromophore that is formed in the periplasm by intramolecular cyclization steps. Although the cytoplasmic peptide synthesis and side-chain modifications are well characterized, the periplasmic maturation steps are far from understood. Out of five periplasmic enzymes, PvdM, PvdN, PvdO, PvdP, and PvdQ, functions have been attributed only to PvdP and PvdQ. The other three enzymes are also regarded as essential for siderophore biosynthesis. The structure of PvdN has been solved recently, but no function could be assigned. Here we present the first in-frame deletion of the PvdN-encoding gene. Unexpectedly, PvdN turned out to be required for a specific modification of pyoverdine, whereas the overall amount of fluorescent pyoverdines was not altered by the mutation. The mutant strain grew normally under iron-limiting conditions. Mass spectrometry identified the PvdN-dependent modification as a transformation of the N-terminal glutamic acid to a succinamide. We postulate a pathway for this transformation catalyzed by the enzyme PvdN, which is most likely functional in the case of all pyoverdines.

The Tat Substrate CueO Is Transported in an Incomplete Folding State.

In Escherichia coli, cytoplasmic copper ions are toxic to cells even at the lowest concentrations. As a defense strategy, the cuprous oxidase CueO is secreted into the periplasm to oxidize the more membrane-permeable and toxic Cu(I) before it can enter the cytoplasm. CueO itself is a multicopper oxidase that requires copper for activity. Because it is transported by the twin-arginine translocation (Tat) pathway, which transports folded proteins, a requirement for cofactor assembly before translocation has been discussed. Here we show that CueO is transported as an apo-protein. Periplasmic CueO was readily activated by the addition of copper ions in vitro or under copper stress conditions in vivo Cytoplasmic CueO did not contain copper, even under copper stress conditions. In vitro Tat transport proved that the cofactor assembly was not required for functional Tat transport of CueO. Due to the post-translocational activation of CueO, this enzyme contributes to copper resistance not only by its cuprous oxidase activity but also by chelation of copper ions before they can enter the cytoplasm. Apo-CueO was indistinguishable from holo-CueO in terms of secondary structural elements. Importantly, the binding of copper to apo-CueO greatly stabilized the protein, indicating a transformation from an open or flexible domain arrangement with accessible copper sites to a closed structure with deeply buried copper ions. CueO is thus the first example for a natural Tat substrate of such incomplete folding state. The Tat system may need to transport flexibly folded proteins in any case when cofactor assembly or quaternary structure formation occurs after transport.