Null mutations in a Nudix gene, ygdP, implicate an alarmone response in a novel suppression of hybrid jamming

Induction of the toxic LamB-LacZ protein fusion, Hyb42-1, leads to a lethal generalized protein export defect. The prlF1 suppressor causes hyperactivation of the cytoplasmic Lon protease and relieves the inducer sensitivity of Hyb42-1. Since prlF1 does not cause a detectable change in the stability or level of the hybrid protein, we conducted a suppressor screen, seeking factors genetically downstream of lon with prlF1-like phenotypes. Two independent insertions in the ygdP open reading frame relieve the toxicity of the fusion protein and share two additional properties with prlF1: cold sensitivity and the ability to suppress the temperature sensitivity of a degP null mutation. Despite these similarities, ygdP does not appear to act in the same genetic pathway as prlF1 and lon, suggesting a fundamental link between the phenotypes. We speculate that the common properties of the suppressors relate to secretion defects. The ygdP gene (also known as nudH) has been shown to encode a Nudix protein that acts as a dinucleotide oligophosphate (alarmone) hydrolase. Our results suggest that loss of ygdP function leads to the induction of an alarmone-mediated response that affects secretion. Using an epitope-tagged ygdP construct, we present evidence that this response is sensitive to secretion-related stress and is regulated by differential proteolysis of YgdP in a self-limiting manner.     

Beta-galactosidase is inactivated by intermolecular disulfide bonds and is toxic when secreted to the periplasm of Escherichia coli.

The wild-type LamB-LacZ hybrid protein inhibits the export machinery upon induction when assayed by biochemical and genetic techniques, a phenotype referred to as hybrid protein jamming. This hybrid protein also renders cells sensitive to growth in the presence of the inducer maltose, presumably because of the jamming. We constructed a new version of this fusion by adding alkaline phosphatase, encoded by phoA, to the C terminus of the LamB-LacZ hybrid protein. This tripartite protein, LamB-LacZ-PhoA, is as toxic to cells as the hybrid LamB-LacZ; however, it does not jam at temperatures greater than 33 degrees C. Extreme C-terminal sequences of LacZ function as a critical folding domain and are therefore responsible for stabilizing the LacZ structure. To determine if this region of LacZ is important for jamming, we recombined a late nonsense mutation (X90) onto the hybrid construct. We found the toxicity of this new hybrid, LamB-LacZX90, to be nearly identical to that of the full-length protein, but it also does not jam the secretion machinery. This suggests that jamming is caused by LacZ folding. We found no inhibition of secretion in the tripartite and X90 fusion strains at 37 degrees C, suggesting that the toxicity of the new fusions is novel. Under these conditions, the tripartite and X90 fusion proteins form disulfide-bonded aggregates with high molecular weights in the periplasm. Accordingly, we believe that LacZ disrupts some essential function(s) in the periplasm.     

Evaluation of bottlenecks in proinsulin secretion by Escherichia coli

This work evaluates three potential bottlenecks in recombinant human proinsulin secretion by Escherichia coli: protein stability, secretion capacity and the effect of molecular size on secretion efficiency. A maximum secretion level of 7.2 mg g(-1) dry cell weight was obtained in the periplasm of E. coli JM109(DE3) host cells. This value probably represents an upper limit in the transport capacity of E. coli cells secreting ZZ-proinsulin and similar proteins with the protein A signal peptide. A selective deletion study was performed in the fusion partner and no effect of the molecular size (17-24 kDa) was detected on secretion efficiency. The protective effect against proteolysis provided by the ZZ domain was thoroughly demonstrated in the periplasm of E. coli and it was also shown that a single Z domain is able to provide the same protection level without compromising the downstream processing. The use of this shorter fusion partner enables a 1.6-fold increase in the recovery of the target protein after cleavage of the affinity handle.     

A novel fusion protein system for the production of native human pepsinogen in the bacterial periplasm

Human pepsinogen is the secreted inactive precursor of pepsin. Under the acidic conditions present in the stomach it is autocatalytically cleaved into the active protease. Pepsinogen contains three consecutive disulfides, and was used here as a model protein to investigate the production of aspartic proteases in the Escherichia coli periplasm. Various N-terminal translocation signals were applied and several different expression vectors were tested. After fusion to pelB, dsbA or ompT signal peptides no recombinant product could be obtained in the periplasm using the T7 promoter. As a new approach, human pepsinogen was fused to E. coli ecotin (E. coli trypsin inhibitor), which is a periplasmic homodimeric protein of 142 amino acids per monomer containing one disulfide bridge. The fusion protein was expressed in pTrc99a. After induction, the ecotin-pepsinogen fusion protein was translocated into the periplasm and the ecotin signal peptide was cleaved. Upon acid treatment, the fusion protein was converted into pepsin, indicating that pepsinogen was produced in its native form. In shake flasks experiments, the amount of active fusion protein present in the periplasm was 100 microg per litre OD 1, corresponding to 70 microg pepsinogen. After large scale cultivation, the fusion protein was isolated from the periplasmic extract. It was purified to homogeneity with a yield of 20%. The purified protein was native. Acid-induced activation of the fusion protein proceeded very fast. As soon as pepsin was present, the ecotin part of the fusion protein was rapidly digested, followed by a further activation of pepsinogen.     

Membrane topology of penicillin-binding protein 3 of Escherichia coli

The beta-lactamase fusion vector, pJBS633, has been used to analyse the organization of penicillin-binding protein 3 (PBP3) in the cytoplasmic membrane of Escherichia coli. The fusion junctions in 84 in-frame fusions of the coding region of mature TEM beta-lactamase to random positions within the PBP3 gene were determined. Fusions of beta-lactamase to 61 different positions in PBP3 were obtained. Fusions to positions within the first 31 residues of PBP3 resulted in enzymatically active fusion proteins which could not protect single cells of E. coli from killing by ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were not translocated to the periplasm. However, all fusions that contained greater than or equal to 36 residues of PBP3 provided single cells of E. coli with substantial levels of resistance to ampicillin, indicating that the beta-lactamase moieties of these fusion proteins were translocated to the periplasm. PBP3 therefore appeared to have a simple membrane topology with residues 36 to the carboxy-terminus exposed on the periplasmic side of the cytoplasmic membrane. This topology was confirmed by showing that PBP3 was protected from proteolytic digestion at the cytoplasmic side of the inner membrane but was completely digested by proteolytic attack from the periplasmic side. PBP3 was only inserted in the cytoplasmic membrane at its amino terminus since replacement of its putative lipoprotein signal peptide with a normal signal peptide resulted in a water-soluble, periplasmic form of the enzyme. The periplasmic form of PBP3 retained its penicillin-binding activity and appeared to be truly water-soluble since it fractionated, in the absence of detergents, with the expected molecular weight on Sephadex G-100 and was not retarded by hydrophobic interaction chromatography on Phenyl-Superose.     

Discrimination between SRP- and SecA/SecB-dependent substrates involves selective recognition of nascent chains by SRP and trigger factor

Besides SecA and SecB, Escherichia coli cells possess a signal recognition particle (SRP) to target exported proteins to the SecY translocon. Using chemical and site-specific cross-linking in vitro, we show that SRP recognizes the first signal anchor sequence of a polytopic membrane protein (MtlA) resulting in cotranslational targeting of MtlA to SecY and phospholipids of the plasma membrane. In contrast, a possible interaction of SRP with the secretory protein pOmpA is prevented by the association of trigger factor with nascent pOmpA. Trigger factor also prevents SecA from binding to the first 125 amino acids of pOmpA when they are still associated with the ribosome. Under no experimental conditions was SecA found to interact with MtlA. Likewise, virtually no binding of trigger factor to ribosome-bound MtlA occurs even in the complete absence of SRP. Collectively, our results indicate that at the stage of nascent polypeptides, polytopic membrane proteins are selected by SRP for co-translational membrane targeting, whereas secretory proteins are directed into the SecA/SecB-mediated post-translational targeting pathway by means of their preferential recognition by trigger factor.     

A method for the evaluation of the efficiency of signal sequences for secretion and correct N-terminal processing of human parathyroid hormone produced in Escherichia coli.

Expression plasmids have been constructed for evaluation of different signal sequences for secretion and correct amino terminal processing of foreign proteins expressed in Escherichia coli. cDNA representing the N-terminal region (1-37) of human parathyroid hormone was inserted between DNA coding for two different forms of the signal sequence and two IgG binding domains (ZZ) derived from Staphylococcal protein A. The expression products were secreted to the periplasm and even to the growth medium and were easily purified by affinity chromatography using the ZZ part as a specific handle. Further analyses showed that the expression products were correctly processed to the mature protein hPTH(1-37)ZZ in a construct where the wild type signal sequence of Staphylococcus protein A was used. When a mutated signal sequence which lacks the normal cleavage site was employed, the fusion protein was not cleaved. Since signal sequences seem to be processed in the correct way in this system, we conclude that the general design of this type of expression vector is well suited for studying the N-terminal processing and secretion of heterologous proteins in E. coli.     

The superoxide dismutase SodA is targeted to the periplasm in a SecA-dependent manner by a novel mechanism

The manganese/iron-type superoxide dismutase (SodA) of Rhizobium leguminosarum bv. viciae 3841 is exported to the periplasm of R. l. bv. viciae and Escherichia coli. However, it does not possess a hydrophobic cleaved N-terminal signal peptide typically present in soluble proteins exported by the Sec-dependent (Sec) pathway or the twin-arginine translocation (TAT) pathway. A tatC mutant of R. l. bv. viciae exported SodA to the periplasm, ruling out export of SodA as a complex with a TAT substrate as a chaperone. The export of SodA was unaffected in a secB mutant of E. coli, but its export from R. l. bv. viciae was inhibited by azide, an inhibitor of SecA ATPase activity. A temperature-sensitive secA mutant of E. coli was strongly reduced for SodA export. The 10 N-terminal amino acid residues of SodA were sufficient to target the reporter protein alkaline phosphatase to the periplasm. Our results demonstrate the export of a protein lacking a classical signal peptide to the periplasm by a SecA-dependent, but SecB-independent targeting mechanism. Export of the R. l. bv. viciae SodA to the periplasm was not limited to the genus Rhizobium, but was also observed in other proteobacteria.     

Chaperone-mediated folding and maturation of the penicillin acylase precursor in the cytoplasm of Escherichia coli

Expression of the leaderless pac gene (LL pac), which lacks the coding region for the signal peptide of penicillin acylase (PAC), in Escherichia coli was conducted. It was demonstrated that the PAC precursor, proPAC, can be produced and even processed to form mature PAC in the cytoplasm, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E. coli. The outcome of proPAC folding and PAC maturation could be affected by several factors, such as inducer type, proPAC formation rate, and chaperone availability. Misfolding of proPAC in the cytoplasm could be partially resolved through the coexpression of cytoplasmic chaperones, such as trigger factor, GroEL/ES, or DnaK/J-GrpE. The three chaperones tested showed different extents of the effect on proPAC solublization and PAC maturation, and trigger factor had the most prominent one. However, the chaperone-mediated solublization of proPAC did not guarantee its maturation, which is usually limited by the first autoproteolytic step. It was observed that arabinose could act as an effective inducer for the induction of LL pac expression regulated by the lac-derived promoter system of trc. In addition, PAC maturation could be highly facilitated by arabinose supplementation and coexpression of trigger factor, suggesting that the coordination of chaperone systems with proper culture conditions could dramatically impact recombinant protein production. This study suggests that folding/misfolding of proPAC could be a major step limiting the overproduction of PAC in E. coli and that the problem could be resolved through the search for appropriate chaperones for coexpression. It also demonstrates the analogy in the issues of proPAC misfolding as well as the expression bottleneck occurring in the cytoplasm (i.e., LL pac expression) and those occurring in the periplasm (i.e., wild-type pac expression).     

Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli

The twin-arginine translocation (Tat) system targets cofactor-containing proteins across the Escherichia coli cytoplasmic membrane via distinct signal peptides bearing a twin-arginine motif. In this study, we have analysed the mechanism and capabilities of the E. coli Tat system using green fluorescent protein (GFP) fused to the twin-arginine signal peptide of TMAO reductase (TorA). Fractionation studies and fluorescence measurements demonstrate that GFP is exported to the periplasm where it is fully active. Export is almost totally blocked in tat deletion mutants, indicating that the observed export in wild-type cells occurs predominantly, if not exclusively, by the Tat pathway. Imaging studies reveal a halo of fluorescence in wild-type cells corresponding to the exported periplasmic form; the GFP is distributed uniformly throughout the cytoplasm in a tat mutant. Because previous work has shown GFP to be incapable of folding in the periplasm, we propose that GFP is exported in a fully folded, active state. These data also show for the first time that heterologous proteins can be exported in an active form by the Tat pathway.     

Secretion of recombinant proteins via the chaperone/usher pathway in Escherichia coli

F1 antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in Escherichia coli. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1beta (hIL-1beta), and mature Caf1, the processed product (hIL-1beta:Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M chaperone led to the accumulation of soluble hIL-1beta:Caf1 in the periplasm. Soluble hIL-1beta:Caf1 reacted with monoclonal antibodies directed against structural epitopes of hIL-1beta. The results indicate that Caf1M-induced release of hIL-1beta:Caf1 from the inner membrane promotes folding of the hIL-1beta domain. Similar results were obtained with the fusion of Caf1 to hIL-1beta receptor antagonist or to human granulocyte-macrophage colony-stimulating factor. Following coexpression of the hIL-1beta:Caf1 precursor with both the Caf1M chaperone and Caf1A outer membrane protein, hIL-1beta:Caf1 could be detected on the cell surface of E. coli. These results demonstrate for the first time the potential application of the chaperone/usher secretion pathway in the transport of subunits with large heterogeneous N-terminal fusions. This represents a novel means for the delivery of correctly folded heterologous proteins to the periplasm and cell surface as either polymers or cleavable monomeric domains.     

Genetic toggling of alkaline phosphatase folding reveals signal peptides for all major modes of transport across the inner membrane of bacteria

Prediction of export pathway specificity in prokaryotes is a challenging endeavor due to the similar overall architecture of N-terminal signal peptides for the Sec-, SRP- (signal recognition particle), and Tat (twin arginine translocation)-dependent pathways. Thus, we sought to create a facile experimental strategy for unbiased discovery of pathway specificity conferred by N-terminal signals. Using a limited collection of Escherichia coli strains that allow protein oxidation in the cytoplasm or, conversely, disable protein oxidation in the periplasm, we were able to discriminate the specific mode of export for PhoA (alkaline phosphatase) fusions to signal peptides for all of the major modes of transport across the inner membrane (Sec, SRP, or Tat). Based on these findings, we developed a mini-Tn5 phoA approach to isolate pathway-specific export signals from libraries of random fusions between exported proteins and the phoA gene. Interestingly, we observed that reduced PhoA was exported in a Tat-independent manner when targeted for Tat export in the absence of the essential translocon component TatC. This suggests that initial docking to TatC serves as a key specificity determinant for Tat-specific routing of PhoA, and in its absence, substrates can be rerouted to the Sec pathway, provided they remain compatible with the Sec export mechanism. Finally, the utility of our approach was demonstrated by experimental verification that four secreted proteins from Mycobacterium tuberculosis carrying putative Tat signals are bona fide Tat substrates and thus represent potential Tat-dependent virulence factors in this important human pathogen.     

Periplasmic production of native human proinsulin as a fusion to E. coli ecotin

Native proinsulin belongs to the class of the difficult-to-express proteins in Escherichia coli. Problems mainly arise due to its small size, a high proteolytic decay, and the necessity to form a native disulfide pattern. In the present study, human proinsulin was produced in the periplasm of E. coli as a fusion to ecotin, which is a small periplasmic protein of 16 kDa encoded by the host, containing one disulfide bond. The fusion protein was secreted to the periplasm and native proinsulin was determined by ELISA. Cultivation parameters were studied in parallel batch mode fermentations using E. coli BL21(DE3)Gold as a host. After improvement of fed-batch high density fermentation conditions, 153 mg fusion protein corresponding to 51.5mg native proinsulin was obtained per L. Proteins were extracted from the periplasm by osmotic shock treatment. The fusion protein was purified in one step by ecotin affinity chromatography on immobilized trypsinogen. After thrombin cleavage of the fusion protein, the products were separated by Ni-NTA chromatography. Proinsulin was quantified by ELISA and characterized by mass spectrometry. To evaluate the influence of periplasmic proteases, the amount of ecotin-proinsulin was determined in E. coli BL21(DE3)Gold and in a periplasmic protease deficient strain, E. coli SF120.     

Cytoplasmic overexpression, folding, and processing of penicillin acylase precursor in Escherichia coli

Penicillin acylase (PAC) precursor, proPAC, was overproduced in a soluble or insoluble form in the cytoplasm of Escherichia coli through the expression of the leader-less pac gene (ll-pac) devoid of the coding region for the signal peptide of PAC. Also, a portion of the overexpressed proPAC was further processed to form mature PAC, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E. coli. The cultivation performance for ll-pac expression was limited by several factors, including (1) misfolding of proPAC, resulting in the aggregation of insoluble proPAC as inclusion bodies, (2) intracellular proteolysis, leading to the degradation of the overexpressed gene products, and (3) inefficient PAC maturation, limiting the formation of active PAC. The effect of coexpression of various cytoplasmic chaperones, including trigger factor, GroEL/ES, DnaK/J-GrpE, and their combinations, on ll-pac expression was investigated. Intracellular proteolysis of the overexpressed gene products could be prevented by coexpression of GroEL/ES. On the other hand, coexpression of trigger factor appeared to be able to facilitate the folding of soluble proPAC and to improve PAC maturation. The roles of trigger factor and GroEL/ES could be coordinated to significantly improve ll-pac expression performance. DnaK/J-GrpE had an effect for solublization of proPAC and perhaps, similar to trigger factor, for improving PAC maturation. The ll-pac expression performance was also significantly improved through the simultaneous coexpression of DnaK/J-GrpE and GroEL/ES. The results of the study suggest that the folding and/or processing of proPAC could be a major issue limiting the overproduction of PAC in E. coli and the bottleneck could be eliminated through the coexpression of appropriate chaperone(s).     

Screening for synthetic lethal mutants in Escherichia coli and identification of EnvC (YibP) as a periplasmic septal ring factor with murein hydrolase activity

Bacterial cytokinesis is driven by the septal ring apparatus, the assembly of which in Escherichia coli is directed to mid-cell by the Min system. Despite suffering aberrant divisions at the poles, cells lacking the minCDE operon (Min(-)) have an almost normal growth rate. We developed a generally applicable screening method for synthetic lethality in E. coli, and used it to select for transposon mutations (slm) that are synthetically lethal (or sick) in combination with DeltaminCDE. One of the slm insertions mapped to envC (yibP), proposed to encode a lysostaphin-like, metallo-endopeptidase that is exported to the periplasm by the general secretory (Sec) pathway. Min(-) EnvC(-) cells showed a severe division defect, supporting a role for EnvC in septal ring function. Accordingly, we show that an EnvC-green fluorescent protein fusion, when directed to the periplasm via the twin-arginine export system, is both functional and part of the septal ring apparatus. Using an in-gel assay, we also present evidence that EnvC possesses murein hydrolytic activity. Our results suggest that EnvC plays a direct role in septal murein cleavage to allow outer membrane constriction and daughter cell separation. By uncovering genetic interactions, the synthetic lethal screen described here provides an attractive new tool for studying gene function in E. coli.     

DmsD is required for the biogenesis of DMSO reductase in Escherichia coli but not for the interaction of the DmsA signal peptide with the Tat apparatus

The DmsD protein is essential for the biogenesis of DMSO reductase in Escherichia coli, and binds the signal peptide of the DmsA subunit, a Tat substrate. This suggests a role as a guidance factor to target pre-DmsA to the translocase. Here, we have analysed the export of fusion proteins in which the DmsA and TorA signal peptides are fused to green fluorescent protein. Both chimeras are efficiently exported to the periplasm in wild-type E. coli cells and we show that their export efficiencies are essentially identical in a mutant lacking DmsD. An authentic Tat substrate, TMAO reductase, is also efficiently exported in the dmsD mutant. The data indicate that DmsD carries out a critical role in DMSO reductase biogenesis/assembly but is not required for the functioning of the DmsA signal peptide.     

Molecular aspects of complement-mediated bacterial killing. Periplasmic conversion of C9 from a protoxin to a toxin

As part of the membrane attack complex complement protein C9 is responsible for direct killing of bacteria. Here we show that in the periplasmic space of an Escherichia coli cell C9 is converted from a protoxin to a toxin by periplasmic conditions missing in spheroplasts. This conversion is independent of the pathway by which C9 enters the periplasm. Both, C9 shocked into the periplasm and plasmid-expressed C9 targeted to the periplasm via a signal sequence are toxic. Toxicity requires disulfide-linked C9 because export into the periplasm of cells defective in disulfide bond synthesis (dsbA and dsbB mutants) is not toxic unless N-acetylcysteine is added externally to promote cystines. A N-terminal fragment, C9[1-144], is not toxic nor is cytoplasmically expressed C9, even in trxB mutants that are able to form disulfide bonds in the cytoplasm. Importantly, expression of full-length C9 in complement-resistant cells has no effect on their viability. Expression and translocation into the periplasm may provide a novel model to identify molecular mechanisms of other bactericidal disulfide-linked proteins and to investigate the nature of bacterial complement resistance.     

A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

The prlA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose-binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several prlA mutants. The export efficiency can be substantial; in a strain carrying the prlA4 allele, 30% of signal-sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal-sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all.