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The Tat pathway transports folded proteins across the bacterial cytoplasmic membrane and is a major route of protein export in the Streptomyces genus of bacteria. In this study, we have examined the localization of Tat components in the model organism Streptomyces coelicolor by constructing enhanced green fluorescent protein (eGFP) and mCherry fusions with the TatA, TatB, and TatC proteins. All three components colocalized dynamically in the vegetative hyphae, with foci of each tagged protein being prominent at the tips of emerging germ tubes and of the vegetative hyphae, suggesting that this may be a primary site of Tat secretion. Time-lapse imaging revealed that localization of the Tat components was highly dynamic during tip growth and again demonstrated a strong preference for apical sites in growing hyphae. During aerial hypha formation, TatA-eGFP and TatB-eGFP fusions relocalized to prespore compartments, indicating repositioning of Tat components during the Streptomyces life cycle.

The yjeE , yeaZ , and ygjD genes are highly conserved in the genomes of eubacteria, and ygjD orthologs are also found throughout the Archaea and eukaryotes. In this study, we have constructed conditional expression strains for each of these genes in the model organism Escherichia coli K12. We show that each gene is essential for the viability of E. coli under laboratory growth conditions. Growth of the conditional strains under nonpermissive conditions results in dramatic changes in cell ultrastructure. Deliberate repression of the expression of yeaZ results in cells with highly condensed nucleoids, while repression of yjeE and ygjD expression results in at least a proportion of very enlarged cells with an unusual peripheral distribution of DNA. Each of the three conditional expression strains can be complemented by multicopy clones harboring the rstA gene, which encodes a two-component-system response regulator, strongly suggesting that these proteins are involved in the same essential cellular pathway. The results of bacterial two-hybrid experiments show that YeaZ can interact with both YjeE and YgjD but that YgjD is the preferred interaction partner. The results of in vitro experiments indicate that YeaZ mediates the proteolysis of YgjD, suggesting that YeaZ and YjeE act as regulators to control the activity of this protein. Our results are consistent with these proteins forming a link between DNA metabolism and cell division.

We have functionally produced the outer membrane cytochrome OmcA from Shewanella oneidensis in Escherichia coli . Substrate accessibility experiments indicate that OmcA is surface exposed in an E. coli B strain but not in a K-12 strain. We show that a functional type II secretion system is required for surface localization.

The respiratory formate dehydrogenase N (FDH-N) enzyme of Escherichia coli is a seleno-molybdoenzyme that is synthesized when the bacterium grows anaerobically with nitrate as exogenous electron acceptor. FDH-N can comprise up to 10% of the total membrane protein (9). Together with nitrate reductase-A, it forms a respiratory chain transferring electrons from formate to nitrate and results in the generation of a protonmotive force (10). FDH-N has a number of cofactors, including bis-molybdopterin guanine dinucleotide cofactor, selenocysteine, and a single [4Fe-4S] cluster. Consequently, synthesis of this enzyme requires careful control. Transcription of both the fdnGHI operon, which encodes FDH-N, and narGHJI, which encodes nitrate reductase-A, is coordinately controlled. Expression of both operons is maximal anaerobically in the presence of nitrate and is controlled by the transcription factors Fnr and NarL (8). The high-resolution X-ray structure of FDH-N has revealed that it adopts an (αβγ)3 “trimer-of-trimers ” architecture, with the active site of the enzyme located in the periplasm (13). FDH-N is translocated across the membrane by the twin arginine translocation (Tat) pathway (21). The Tat translocase is dedicated to the transport of prefolded proteins, which bear an N-terminal signal peptide with the conserved S/T-R-R-x-F-L-K twin arginine motif (2, 3). Translocation of FDH-N is mediated by virtue of a Tat signal peptide on the FdnG subunit. Stanley et al. have shown previously that the FdnG signal peptide is able to mediate export of the reporter proteins β-lactamase and chloramphenicol acetyltransferase to the periplasm in a Tat-dependent fashion (24). During the course of these fusion studies we noted that point mutations in the first or second arginine codons of the twin arginine motif resulted in a dramatic overproduction of the fusion protein. The mRNA specifying the first 17 codons of the fdnG gene is predicted to fold into a stable stem-loop structure, and we demonstrate here that this stem-loop mediates translational control of FDH-N synthesis. As shown in Table ​Table1,1, substitution of Arg5, either conservatively for Lys or nonconservatively for Ser, resulted in a marked increase (up to 60-fold) in fusion protein synthesis, regardless of the nature of the reporter protein. In contrast, substitution of Lys10 for Glu had no significant effect on levels of fusion protein (Table ​(Table1).1). No significant differences in fusion protein synthesis were noted in a tat mutant background, suggesting that these observations were not directly related to operation of the Tat pathway. When the mfold program (25) was used, the mRNA covering the start codon and signal peptide-coding region of fdnG could be folded into a stem-loop structure (Fig. ​(Fig.1B).1B). The ΔG value (at 37°C and physiological pH) associated with this structure was calculated to be −12.6 kCal/mol, suggesting that the folded mRNA would be relatively stable. Such a folded mRNA structure would be consistent with the results seen in Table ​Table1,1, since mutations at codon 5, which fall within one arm of the predicted stem, would be expected to disrupt the fold, whereas the mutation at codon 10, which is located within the putative loop region, would not be expected to disrupt the structure. FIG. 1. The mRNA specifying the fdnG signal peptide coding region can be folded into a stem-loop structure. The position of the initiation codon is indicated in bold type; the Shine-Dalgarno ribosome-binding sequence is underlined. Numbering is shown relative ... TABLE 1. Total cellular activity of reporter proteins fused to the wild-type or amino acid-substituted FdnG signal sequencea To test whether a hairpin in the fdnG mRNA was controlling expression at a translational level, we constructed a number of additional mutations in codons 3 to 14 of fdnG. As shown in Fig. ​Fig.2A,2A, substitutions that were predicted to severely disrupt the mRNA fold (R5S [CGC→AGC], R5K [CGC→AAG], R5Rhigh [CGC→CGT], and R5Rlow [CGC→AGG], where the natural Arg codon was replaced with Arg codons of higher and lower usage) (18) led to significant upregulation of β-galactosidase activity. Mutations in codon 6 that were predicted to have more modest effects on the mRNA secondary structure gave less-dramatic increases in β-galactosidase activity. As predicted by the model, substitutions of codons that fell within the putative loop region (Fig. ​(Fig.2B)2B) did not have significant effects on the activity of β-galactosidase, with the exception of the I11V mutation, which resulted in a marked decrease in the activity of β-galactosidase. Interestingly, this substitution is predicted to result in a significant increase in the stability of the stem-loop (from −12.6 to −16.4 kCal/mol) due to the formation of an extra base pair at the top of the stem. The results of substitutions in the putative second arm of the stem (Fig. ​(Fig.2C)2C) were also consistent with the premise that a stem-loop structure was controlling translation of fdnG-lacZ. A plot of ΔG value for the structures associated with the wild-type sequence and each of the mutations against the observed β-galactosidase activity (Fig. ​(Fig.2E)2E) shows a linear relationship. Indeed, a straight line can be drawn through the points with a correlation factor (R2) of 0.82, providing strong support for the model. As a final test, we looked at the effect of introducing a compensatory mutation into the second arm of the stem, to restore base pairing, with a mutation in the first arm of the stem that disrupts the stem-loop structure. When introduced singly, both the AGA→AAG mutations at codon 6 and the ATC→ACT mutations at codon 11 led to increased β-galactosidase activity (Fig. ​(Fig.2D).2D). However, when these two sets of mutations were combined, β-galactosidase activity was restored to the wild-type level, indicating that the two mutations had a compensatory effect. FIG. 2. The effects of mutations in the stem and loop regions of the fdnG mRNA on expression of fdnG-lacZ. Constructs were transformed into strain MC4100-P (4), which carries the pcnB allele, providing more careful control of plasmid copy number (14, 15). (A) ... In addition to the substantial transcriptional regulation already reported (1), our findings strongly suggest that a further, possibly up to 10-fold, level of control of fdnGHI expression was exerted at the translational level. To confirm the physiological relevance of these findings, we used published methods (11) to introduce two sets of mutations (R5Rhigh [CGC→CGT] and R5Rlow [CGC→AGG]) in the fdnG gene in the chromosome. In each case we made a substitution that retained an Arg codon at position 5 so that the cellular location of FDH-N would not be compromised. To circumvent possible translational errors associated with use of rare arginine codons, such as AGG, in E. coli (see, e.g., reference 5), we additionally introduced into each strain plasmid pUt-AGA/AGG, which carries the genes encoding tRNAArgUCU and tRNAArgUCC (22). In the presence of this plasmid, but not in its absence, we saw an approximate fourfold increase in FDH-N activity from the strain with the R5Rlow chromosomal replacement of fdnG (Fig. ​(Fig.3A)3A) and a corresponding overproduction of the FdnG polypeptide (Fig. ​(Fig.3C).3C). This result strongly suggests that the translational control of fdnG expression is of physiological significance, since we have clearly demonstrated that the cell has the capacity to synthesize, assemble, and export fourfold-higher levels of FDH-N. Interestingly, as shown in Fig. ​Fig.3B3B the fourfold overproduction of FDH-N activity was associated with a marked increase in NAR activity. This suggests that there might be a further level of coordinate control of FDH-N and NAR synthesis not previously observed. FIG. 3. The stem-loop structure regulates FDH-N activity in vivo. FDH-N (A) and NAR (B) enzyme assays were performed with crude cell extracts derived from strains MC4100, NRS9 (as MC4100; FdnG R5R [CGC→CGT]), and CMP1 (as MC4100; FdnG R5R [CGC→AGG]) ... In conclusion, we have demonstrated that there is control of FDH-N synthesis at the translational level. Interestingly, the stem-loop structure seen here for the E. coli mRNA is also predicted to be conserved in the fdnG coding regions of several other bacteria, including Salmonella enterica serovar Typhimurium, Haemophilus influenzae, and Pseudomonas aeruginosa. Notably, however, the stem-loop structure is apparently not conserved over the similar coding region of E. coli fdoG. While the mechanism underlying modulation of FDH-N translation is presently unclear, it may potentially serve as a quality control feature ensuring that only the fully assembled complex is transported to the periplasm.

Overexpression of either heterologous or homologous proteins that are routed to the periplasm via the twin-arginine translocation (Tat) pathway results in a block of export and concomitant accumulation of the respective protein precursor in the cytoplasm. Screening of a plasmid-encoded genomic library for mutants that confer enhanced export of a TorA signal sequence (ssTorA)-GFP-SsrA fusion protein, and thus result in higher cell fluorescence, yielded the pspA gene encoding phage shock protein A. Coexpression of pspA relieved the secretion block observed with ssTorA-GFP-SsrA or upon overexpression of the native Tat proteins SufI and CueO. A similar effect was observed with the Synechocystis sp. strain PCC6803 PspA homologue, VIPP1, indicating that the role of PspA in Tat export may be phylogenetically conserved. Mutations in Tat components that completely abolish export result in a marked induction of PspA protein synthesis, consistent with its proposed role in enhancing protein translocation via Tat.

The TatA and TatB proteins are essential components of the twin arginine protein translocation pathway in Escherichia coli . C-terminal truncation analysis of the TatA protein revealed that a plasmid-expressed TatA protein shortened by 40 amino acids is still fully competent to support protein translocation. Similar truncation analysis of TatB indicated that the final 30 residues of TatB are dispensable for function. Further deletion experiments with TatB indicated that removal of even 70 residues from its C terminus still allowed significant transport. These results imply that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity. A chimeric protein comprising the N-terminal region of TatA fused to the amphipathic and C-terminal domains of TatB supports a low level of Tat activity in a strain in which the wild-type copy of either tatA or tatB (but not both) is deleted.

The transcription start sites for the tatABCD and tatE loci, encoding components of the Tat (twin-arginine translocase) protein export pathway, have been identified. Expression studies indicate that the tatABCD and tatE transcription units are expressed constitutively. Translational fusion experiments suggest that TatA is synthesized at a much higher level than the other Tat proteins.

The TatC protein is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. It is a polytopic membrane protein that forms a complex with TatB, together acting as the receptor for Tat substrates. In this study we have constructed 57 individual cysteine substitutions throughout the protein. Each of the substitutions resulted in a TatC protein that was competent to support Tat-dependent protein translocation. Accessibility studies with membrane-permeant and -impermeant thiol-reactive reagents demonstrated that TatC has six transmembrane helices, rather than the four suggested by a previous study (K. Gouffi, C.-L. Santini, and L.-F. Wu, FEBS Lett. 525:65-70, 2002). Disulfide cross-linking experiments with TatC proteins containing single cysteine residues showed that each transmembrane domain of TatC was able to interact with the same domain from a neighboring TatC protein. Surprisingly, only three of these cysteine variants retained the ability to cross-link at low temperatures. These results are consistent with the likelihood that most of the disulfide cross-links are between TatC proteins in separate TatBC complexes, suggesting that TatC is located on the periphery of the complex.

The Tat system is a recently discovered protein export pathway that serves to translocate folded proteins, often containing redox cofactors, across the bacterial cytoplasmic membrane. Here we report that tat strains are associated with a mutant cell septation phenotype, where chains of up to 10 cells are evident. Mutant strains are also hypersensitive to hydrophobic drugs and to lysis by lysozyme in the absence of EDTA, and they leak periplasmic enzymes, characteristics that are consistent with an outer membrane defect. Both phenotypes are similar to those displayed by strains carrying point mutations in the lpxC (envA) gene. The phenotype was not replicated by mutations affecting synthesis and/or activity of all known or predicted Tat substrates. Approximately 20% of all proteins synthesized by Escherichia coli are predicted to be located outside the cytoplasmic compartment. Most proteins destined for export are synthesized with N-terminal signal sequences that direct translocation by the general secretory (Sec) apparatus (17, 21). Secdependent signal sequences lack sequence similarity but have similar overall physical-chemical properties (32). A subset of periplasmic and periplasmically oriented proteins are exported by a mechanism distinct from the Sec pathway (3, 25, 26, 34). Such proteins are synthesized with, or in some cases associate with partner proteins synthesized with, signal sequences harboring the (S/T)RRxFLK “twin-arginine” motif (2). These distinctive signal sequences target substrate proteins to the twin-arginine signal peptide-dependent protein translocase (Tat translocase) which is structurally and mechanistically related to the DpH-dependent protein translocase of plant thylakoid membranes (8, 29). Many Tat-dependent substrate proteins bind redox cofactors and are involved in bacterial energy metabolism. Cofactor acquisition is a cytoplasmic event and is a prerequisite for export, suggesting that proteins are translocated by the Tat apparatus in a folded conformation (22, 25). Minimally the E. coli Tat translocase comprises four probable membrane proteins, encoded by the tatA, tatB, tatC, and tatE genes. The tatA, tatB, and tatC genes are cotranscribed with a fourth gene, tatD, encoding a soluble protein with no discernible role in Tat-dependent protein export (35). TatA, TatB, and TatE are sequence-related proteins, each of which is predicted to comprise a single N-terminal transmembrane a-helix, immediately followed by an amphipathic a-helix at the cytoplasmic side of the membrane (7, 26). TatA and TatE are more than 50% identical at the amino acid level and have overlapping functions in Tat-dependent protein export. Thus, comutation of both tatA and tatE is necessary to observe a complete block in export by the Tat pathway (26). TatB, which is more divergently related to TatA/E (approximately 25% amino acid identity), is a distinct and essential component of the Tat apparatus (27). The tatC gene encodes a highly hydrophobic protein, predicted to contain six membrane-spanning a-helices, which is critical for Tat function (5). In each case, a block in Tat-dependent protein export results in the mislocalization of Tat-dependent substrate proteins to the cytoplasmic compartment. Tat mutant strains are unable to grow anaerobically with either dimethyl sulfoxide or trimethylamine-Noxide as sole terminal electron acceptor, reflecting the failure to correctly localize the terminal reductases. The mutant strains otherwise display no discernible growth phenotype. In this paper, we report that strains lacking genes encoding essential Tat components form chains of up to 10 cells, which appear to be defective in cell separation. Electron microscopy reveals that the phenotype is similar to that of an lpxC (envA) mutant, which has a defect in the synthesis of outer membrane lipid A. Consistent with this, tat mutant strains are hypersensitive to hydrophobic drugs and to lysozyme-induced lysis in the absence of EDTA. We show that this phenotype is not due to the mislocalization of any single Tat substrate protein.