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.     

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.     

Comparison of Escherichia coli K-12 outer membrane protease OmpT and Salmonella typhimurium E protein.

The predicted amino acid sequence of OmpT, an Escherichia coli outer membrane protease, was found to be highly homologous to that predicted for the pgtE gene product of Salmonella typhimurium. In this paper, it is shown that pgtE codes for a protein functionally homologous to OmpT as judged by its ability to proteolyze T7 RNA polymerase and to localize in the outer membrane of E. coli.     

Overproduction and purification of the omega subunit of Escherichia coli RNA polymerase

This paper reports the construction of plasmids which direct the overproduction of the omega subunit of Escherichia coli RNA polymerase and the subsequent purification of omega. Useful overproduction is achieved only if the natural ribosomal binding site region of rpoZ is replaced with the ribosomal binding site region of bacteriophage T7 gene 10. Overproduction is directed by T7 RNA polymerase which is provided on a separate plasmid. omega is purified by three column steps either from the insoluble inclusion body fraction or from the soluble fractions of lysates. The final yield is approximately 2 mg omega per 10 g cells wet wt. Additionally, we found that recombinant omega is readily cleaved by an endogenous protease. Sequence analysis of the most prevalent proteolytic fragment suggested that the protease responsible was the product of the ompT gene. Cleavage of omega is greatly reduced in ompT- strains.     

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。     

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.     

The plasmid F OmpP protease, a homologue of OmpT, as a potential obstacle to E. coli-based protein production

OmpT, an outer membrane-localized protease of Escherichia coli, cleaves a number of exogenous and endogenous proteins during their purification. SecY, an endogenous membrane protein, is a target of this artificial proteolysis in vitro. Here we report that SecY cleavage occurs even in cell extracts from ompT-disrupted cells, if they carry an F plasmid derivative. A gene, ompP, on the F plasmid was shown to be responsible for this proteolysis. These results indicate that the absence of an F-like plasmid should be checked when choosing a host strain for E. coli-based protein production.     

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.     

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.     

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.     

Characterization of degQ and degS, Escherichia coli genes encoding homologs of the DegP protease.

The degQ and degS genes of Escherichia coli encode proteins of 455 and 355 residues, respectively, which are homologs of the DegP protease. The purified DegQ protein has the properties of a serine endoprotease and is processed by the removal of a 27-residue amino-terminal signal sequence. A plasmid expressing degQ rescues the temperature-sensitive phenotype of a strain bearing the degP41 deletion, implying that DegQ, like DegP, functions as a periplasmic protease in vivo. Deletions in the degQ gene cause no obvious growth defect, while those in the degS gene result in a small-colony phenotype. The latter phenotype is rescued by a plasmid expressing the degS gene but not by plasmids expressing the degQ or degP genes. This result and the inability of a plasmid expressing degS to rescue the temperature-sensitive degP41 phenotype indicate that the DegS protein is functionally different from the DegQ and DegP proteins.     

Phage T7 RNA-polymerase: gene cloning and its structure

Gene 1.0 to T7 phage coding for a phage-specific RNA polymerase has been cloned in a plasmid vector. One of the clones obtained, viz. E. coli K12 HB101 (pSK-T7-1.0a), produced an active T7 RNA polymerase as revealed by the fact that its extract stimulated RNA synthesis on the T7 DNA template in the presence of rifampicin. Analysis of gene 1.0 fragments isolated from pSK-T7-1.0a plasmid made it possible to refine the sequence of this gene which has been recently published by the authors of the present paper and, independently, by other workers. Another plasmid, pSK-T7-1.0b, did not differ from pSK-T7-1.0a in the restriction map. However, it failed to induce production of a rifampicin-insensitive RNA polymerase. Sequencing revealed a deletion of one T residue in gene 1.0 present in this plasmid.     

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.     

“Host Shutoff” Function of Bacteriophage T7: Involvement of T7 Gene 2 and Gene 0.7 in the Inactivation of Escherichia coli RNA Polymerase

The "host shutoff" function of bacteriophage T7 involves an inactivation of the host Escherichia coli RNA polymerase by an inhibitor protein bound to the enzyme. When this inhibitor protein, termed I protein, was removed from the inactive RNA polymerase complex prepared from T7-infected cells by glycerol gradient centrifugation in the presence of 1 M KCl, the enzyme recovered its activity equivalent to about 70 to 80% of the activity of the enzyme from uninfected cells. Analysis of the activity of E. coli RNA polymerase from E. coli cells infected with various T7 mutant phages indicated that the T7 gene 2 codes for the inhibitor I protein. The activity of E. coli RNA polymerase from gene 2 mutant phage-infected cells, which was about 70% of that from uninfected cells, did not increase after glycerol gradient centrifugation in the presence of 1 M KCl, indicating that the salt-removable inhibitor was not present with the enzyme. It was found that the reduction in E. coli RNA polymerase activity in cells infected with T7(+) or gene 2 mutant phage, i.e., about 70% of the activity of the enzyme compared to that from uninfected cells after glycerol gradient centrifugation in the presence of 1 M KCl, results from the function of T7 gene 0.7. E. coli RNA polymerase from gene 0.7 mutant phage-infected cells was inactive but recovered a full activity equivalent to that from uninfected cells after removal of the inhibitor I protein with 1 M KCl. E. coli RNA polymerase from the cells infected with newly constructed mutant phages having mutations in both gene 2 and gene 0.7 retained the full activity equivalent to that from uninfected cells with or without treatment of the enzyme with 1 M KCl. From these results, we conclude that both gene 2 and gene 0.7 of T7 are involved in accomplishing complete shutoff of the host E. coli RNA polymerase activity in T7 infection.     

Identification and network of outer membrane proteins regulating streptomysin resistance in Escherichia coli

Bacterial Outer membrane (OM) proteins involved in antibiotic resistance have been reported. However, little is known about the OM proteins and their interaction network regulating streptomycin (SM) resistance. In the present study, a subproteomic approach was utilized to characterize OM proteins of Escherichia coli with SM resistance. TolC, OmpT and LamB were found to be up-regulated, and FadL, OmpW and a location-unknown protein Dps were down-regulated in the SM-resistant E. coli strain. These changes at the level of protein expression were validated using Western blotting. The possible roles of the altered proteins involved in the SM resistance were investigated using genetic modified strains with the deletion of these altered genes. It is found that decreased and elevated minimum inhibitory concentrations and survival capabilities of the gene deleted strains and their resistant strains, Delta tolC, Delta ompT, Delta dps, Delta tolC-R, Delta ompT-R, Delta dps-R and Delta fadL-R, were correlated with the changes of TolC, OmpT, Dps and FadL at the protein expression levels detected by 2-DE gels, respectively. The results may suggest that these proteins are the key OM proteins and play important roles in the regulation of SM resistance in E. coli. Furthermore, an interaction network of altered OM proteins involved in the SM resistance was proposed in this report. Of the six altered proteins, TolC may play a central role in the network. These findings may provide novel insights into mechanisms of SM resistance in E. coli.     

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.