Role of the ompT Mutation in Stimulated Decrease in Colony-Forming Ability Due to Intracellular Protein Aggregate Formation in Escherichia coli Strain BL21

Recently we found that the cells of Escherichia coli strain BL21 producing a fusion protein, GST-Sup35NM, show a much more rapid decrease in colony-forming ability in the stationary phase than control cells. In this study, it was found that an extract of the cells producing GST-Sup35NM forms fibrous protein polymers containing GST-Sup35NM. In the course of the study, we realized that strain BL21 carried the ompT mutation. We suspected that the deficiency in OmpT protease was responsible for the observed phenotype. To test this, we introduced the wild-type ompT gene into strain BL21, and found that the transformed cells recovered the wild-type phenotype. We concluded that OmpT protease, though known to localize on the cell surface, is involved in protein quality control within the cell.     

Production of a polymer-forming fusion protein in Escerichia coli strain BL21

In the course of studying [PSI(+)], a yeast prion, we found inadvertently that Escherichia coli strain BL21 overproducing a fusion protein, in which the prion-domain of Sup35 was connected to the C terminus of glutathione S-transferase, grew normally to the stationary phase and rapidly decreased in colony-forming ability thereafter. Evidence indicated that protein polymers consisting mainly of the fusion protein GST-Sup35NM (about 70% of the mass) and its N-terminal fragments were formed in extract prepared from the cells producing GST-Sup35NM. It was further found that cells of strain BL21 accumulated the protein polymers during prolonged cultivation. Based on these results, we contend that the initially observed defect in colony forming ability is the direct or indirect consequence of intracellular formation and accumulation of the protein polymers.     

Effects of mutations in yeast prion [PSI+] on amyloid toxicity manifested in Escherichia coli strain BL21

We previously showed that over production of a fusion protein in which the prion domain of Saccharomyces cerevisiae [PSI+] is connected to glutathione S-transferase (GST-Sup35NM) causes a marked decrease in the colony forming ability of Escherichia coli strain BL21 after reaching stationary phase. Evidence indicated that the observed toxicity was attributable to intracellular formation of fibrous aggregates of GST-Sup35NM. In this report, we describe the isolation of plasmids that encode mutant forms of GST-Sup35NM which do not confer the toxicity to E. coli strain BL21. Each of the four spontaneous mutant-forms of GST-Sup35NM obtained revealed amino acid substitutions. One substitution was located in the N domain, and the others in the M domain. Congo red binding assay indicated that none of these mutant protein underwent conformational alteration in vitro. From these results, we conclude that the M domain, in collaboration with the N domain, plays an essential role in aggregation of Sup35NM. In addition, our data demonstrate the usefulness of the E. coli expression system in studying aggregate-forming proteins.     

Effects of mutations in yeast prion [PSI+] on amyloid toxicity manifested in Escherichia coli strain BL21

We previously showed that over production of a fusion protein in which the prion domain of Saccharomyces cerevisiae [PSI⁺] is connected to glutathione S-transferase (GST-Sup35NM) causes a marked decrease in the colony forming ability of Escherichia coli strain BL21 after reaching stationary phase. Evidence indicated that the observed toxicity was attributable to intracellular formation of fibrous aggregates of GST-Sup35NM. In this report, we describe the isolation of plasmids that encode mutant forms of GST-Sup35NM which do not confer the toxicity to E. coli strain BL21. Each of the four spontaneous mutant-forms of GST-Sup35NM obtained revealed amino acid substitutions. One substitution was located in the N domain, and the others in the M domain. Congo red binding assay indicated that none of these mutant proteins underwent conformational alteration in vitro. From these results, we conclude that the M domain, in collaboration with the N domain, plays an essential role in aggregation of Sup35NM. In addition, our data demonstrate the usefulness of the E. coli expression system in studying aggregate-forming proteins.Key words: [PSI⁺], yeast prion, protein aggregation, mutant selection, predication of protein secondary structure     

ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification.

Bacteriophage T7 RNA polymerase is stable in Escherichia coli but very susceptible to cleavage by at least one endoprotease after cell lysis. The major source of this endoprotease activity was found to be localized to the outer membrane of the cell. A rapid whole-cell assay was developed to screen different strains for the presence of this proteolytic activity. Using this assay, we identified some common laboratory strains that totally lack the protease. Genetic and Southern analyses of these null strains allowed us to conclude that the protease that cleaves T7 RNA polymerase is OmpT (formerly termed protein a), a known outer membrane endoprotease, and that the null phenotype results from deletion of the OmpT structural gene. A recombinant plasmid carrying the ompT gene enables these deletion strains to synthesize OmpT and converts them to a protease-positive phenotype. The plasmid led to overproduction of OmpT protein and protease activity in the E. coli K-12 and B strains we used, but only weak expression in the E. coli C strain, C1757. This strain-dependent difference in ompT expression was investigated with respect to the known influence of envZ on OmpT synthesis. A small deletion in the ompT region of the plasmid greatly diminishes the amount of OmpT protein and plasmid-encoded protease present in outer membranes. Use of ompT deletion strains for production of T7 RNA polymerase from the cloned gene has made purification of intact T7 RNA polymerase routine. Such strains may be useful for purification of other proteins expressed in E. coli.     

Prevalence of ompT among Escherichia coli isolates of human origin

OmpT is a protease associated with the outer membrane of Escherichia coli and possesses a high degree of homology to the plasminogen activator, Pla, of Yersinia pestis. We show here that OmpT from intact cells can indeed activate plasminogen. Clinical specimens of E. coli were examined for protease activity and for the ompT gene. Few isolates (12%) were found to be positive for OmpT activity, whereas most (77%) carried the ompT gene and expressed the cloned protease gene. In this report we present evidence suggesting that the surface architecture of E. coli influences the activity of OmpT and that OmpT may be indicative of the pathogenic potential of the organism.     

Artifactual processing of penicillin-binding proteins 7 and 1b by the OmpT protease of Escherichia coli.

Penicillin-binding proteins (PBPs) were visualized in strains of Escherichia coli that carried mutations in one or more of the following protease genes: tsp, degP, ptr, and ompT. In the absence of a functional ompT gene, PBPs 1b alpha and 7 were not processed to the shortened forms 1b beta and 8, respectively. Cleavage of PBPs 1b alpha and 7 could be restored by introduction of a plasmid carrying the wild-type ompT gene. These PBPs were processed only after cell lysis or after membrane perturbation of whole cells by freeze-thaw, suggesting that the cleavage was a nonspecific artifact due to contact with OmpT, an outer membrane protease, and that such processing was not biologically significant in vivo. The degradation of other PBPs during purification or storage may also be effected by OmpT.     

Proteolysis of bacteriophage phi X174 prohead accessory protein gpB by Escherichia coli OmpT protease is not essential for phage maturation in vivo.

To examine whether cleavage of the phi X174 prohead accessory protein, gpB, by the OmpT protease is required for phage development in vivo, a phage mutant lacking the OmpT cleavage site and an Escherichia coli C delta ompT strain were constructed. The results of burst size experiments suggest that neither the cleavage site nor the OmpT protein is required for phi X174 development.     

大肠杆菌外膜蛋白酶T及其突变体的表达、复性及生物活性分析

外膜蛋白酶T(Outer-membrane protease T,OmpT)是定位于大肠杆菌外膜,具有高度底物特异性的蛋白水解酶。本文旨在建立克隆表达膜蛋白OmpT和体外复性的方法,考察其蛋白酶活性。首先以大肠杆菌基因组DNA为模板,PCR扩增ompT基因,连接至pET28a(pET-ompT),引入点突变Asp85Ala,构建表达质粒pET-ompT85。然后将两种重组质粒转化入BL21(DE3),均以包涵体形式大量表达。纯化后的蛋白经稀释法复性,并加入粗制脂多糖(Lipopolysaccharide,LPS)恢复蛋白酶活性。通过SDS-PAGE、鱼精蛋白水解试验及生长曲线观察表明,重组蛋白OmpT在体外能水解抗菌肽鱼精蛋白和兔肌肉肌酸激酶,而OmpT突变体则无上述功能。上述结果表明本文获得了具有蛋白水解酶功能的重组蛋白OmpT,该蛋白在体外可保护大肠杆菌抵抗鱼精蛋白的杀菌作用。     

In vivo degradation of secreted fusion proteins by the Escherichia coli outer membrane protease OmpT.

The Escherichia coli outer membrane protease OmpT (protease VII) has been shown to degrade several proteins in vitro, but its function in vivo is uncertain. We demonstrate that OmpT participates in the degradation of a fusion protein secreted into the periplasmic space. A strain with mutations in degP (K.L. Strauch and J. Beckwith, Proc. Natl. Acad. Sci. USA 85:1576-1580, 1988) and ompT exhibits a cumulative decrease in protein degradation and should be useful for the expression of proteolytically sensitive secreted proteins.     

Autodisplay: one-component system for efficient surface display and release of soluble recombinant proteins from Escherichia coli.

The immunoglobulin A protease family of secreted proteins are derived from self-translocating polyprotein precursors which contain C-terminal domains promoting the translocation of the N-terminally attached passenger domains across gram-negative bacterial outer membranes. Computer predictions identified the C-terminal domain of the Escherichia coli adhesin involved in diffuse adherence (AIDA-I) as a member of the autotransporter family. A model of the beta-barrel structure, proposed to be responsible for outer membrane translocation, served as a basis for the construction of fusion proteins containing heterologous passengers. Autotransporter-mediated surface display (autodisplay) was investigated for the cholera toxin B subunit and the peptide antigen tag PEYFK. Up to 5% of total cellular protein was detectable in the outer membrane as passenger autotransporter fusion protein synthesized under control of the constitutive P(TK) promoter. Efficient presentation of the passenger domains was demonstrated in the outer membrane protease T-deficient (ompT) strain E. coli UT5600 and the ompT dsbA double mutant JK321. Surface exposure was ascertained by enzyme-linked immunosorbent assay, immunofluorescence microscopy, and immunogold electron microscopy using antisera specific for the passenger domains. In strain UT2300 (ompT+), the passenger domains were released from the cell surface by the OmpT protease at a novel specific cleavage site, R / V. Autodisplay represents a useful tool for future protein translocation studies with interesting biotechnological possibilities.     

New outer membrane-associated protease of Escherichia coli K-12.

The gene for a new outer membrane-associated protease, designated OmpP, of Escherichia coli has been cloned and sequenced. The gene encodes a 315-residue precursor protein possessing a 23-residue signal sequence. Including conservative substitutions and omitting the signal peptides, OmpP is 87% identical to the outer membrane protease OmpT. OmpP possessed the same enzymatic activity as OmpT. Immuno-electron microscopy demonstrated the exposure of the protein at the cell surface. Digestion of intact cells with proteinase K removed 155 N-terminal residues of OmpP, while the C-terminal half remained protected. It is possible that much of this N-terminal part is cell surface exposed and carries the enzymatic activity. Synthesis of OmpP was found to be thermoregulated, as is the expression of ompT (i.e., there is a low rate of synthesis at low temperatures) and, in addition, was found to be controlled by the cyclic AMP system.     

Antimicrobial properties of arginine- and lysine-rich histones and involvement of bacterial outer membrane protease T in their differential mode of actions

There is growing evidence of the antimicrobial properties of histones and histone-derived peptides; however, most of them are specific to lysine (Lys)-rich histones (H1, H2A, and H2B). In the present study, we focused on arginine (Arg)-rich histones (H3 and H4) and investigated their antimicrobial properties in comparison with those of histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against the bacterial outer membrane protease T (OmpT) gene-expressing Escherichia coli strain JCM5491 with calculated 50% growth inhibitory concentrations of 3.8, 10, and 12.7 μM, respectively. A lysate prepared from the JCM5491 cells was capable of strongly, moderately, and slightly fragmenting histones H2B, H3, and H4, respectively. While the lysate prepared from the cells of the ompT-deleted E. coli strain BL21(DE3) did not digest these histones, the ompT-transformed BL21(DE3), termed BL21/OmpT⁺, cell lysate digested the histones more strongly than the JCM5491 cell lysate. Laser confocal and scanning electron microscopic analyses demonstrated that while histone H2B penetrated the cell membrane of JCM5491 or BL21/OmpT⁺ cells, histones H3 and H4 remained on the cell surface and subsequently disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. The BL21(DE3) cells treated with each histone showed no bleb formation, but cell integrity was affected and the cell surface was corrugated. Consequently, it is suggested that OmpT is involved in the antimicrobial properties of Arg- and Lys-rich histones and that the modes of antimicrobial action of these histones are different.     

Substrate specificity of the Escherichia coli outer membrane protease OmpP

Escherichia coli OmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (k(cat)/K(m) = 3.0 x 10(6) M(-1)s(-1)). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1' sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3'. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1', site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F' episome results in increased resistance to the antimicrobial peptide protamine both in ompT mutants and in wild-type E. coli cells.     

Secretion-dependent proteolysis of heterologous protein by recombinant Escherichia coli is connected to an increased activity of the energy-generating dissimilatory pathway

The synthesis of a proteolytically unstable protein, originally designed for periplasmic export in recombinant Escherichia coli BL21(DE3), a strain naturally deficient for the ATP-dependent protease Lon (or La) and the outer membrane protease OmpT, is associated with a severe growth inhibition. This inhibition is not observed in BL21(DE3) synthesizing a closely related but proteolytically stable protein that is sequestered into inclusion bodies. It is shown that the growth inhibition is mainly caused by a slower cell division rate and a reduced growth yield and not by a general loss of cell division competence. Cells proceed with their normal growth characteristics when exposed again to conditions that do not sustain the expression of the heterologous gene. The performance of cells synthesizing either the stable or the degraded protein was also studied in high cell density cultures by employing a new method to calculate the actual specific growth rate, the biomass yield coefficient, and the dissimilated fraction of the carbon substrate in real-time. It is shown that the growth inhibition of cells synthesizing the proteolytically degraded protein is connected to an increased dissimilation of the carbon substrate resulting in a concomitant reduction of the growth rate and the biomass yield coefficient with respect to the carbon source. It is postulated that the increased dissimilation of the carbon substrate by lon-deficient Bl21(DE3) cells synthesizing the proteolytically unstable protein may result from a higher energy demand required for the in vivo degradation of this protein by ATP-dependent proteases different from the protease Lon.     

Automated protein backbone assignment using the projection-decomposition approach

Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease activity. In the implementation of the expression system we used freshly in-house prepared cell extract. Batch-mode cell-free expression with this setup was up to twofold more economical than continuous-exchange expression, with yields of 0.2-0.9 mg of purified protein per mL of reaction mixture. Native folding of the proteins thus obtained is documented with 2D [(15)N,(1)H]-HSQC NMR.     

OmpT expression and activity increase in response to recombinant chloramphenicol acetyltransferase overexpression and heat shock in E. coli

The activity of a 35 kDa protease increased in response to induced expression of chloramphenicol acetyltransferase (CAT) in E. coli. This protease was partially purified, extensively characterized, and identified via the use of zymogram gels as the outer membrane protease, OmpT. In experiments targeting the overlap of well-characterized stress responses, OmpT activity was found to increase in response to heat shock but was only minimally affected by amino acid limitation. The largest increase in activity was found after induction of CAT. OmpT expression levels also increased in response to induction of recombinant CAT overexpression and heat shock. This is the first report of increased activity and expression of an outer membrane protease during cytoplasmic overexpression of a recombinant protein.