Outer membrane protein 3B of Escherichia coli K-12: Effects of growth temperature on the amount of the protein and further characterization on acrylamide gels

Manning, Paul A.

doi:10.1016/0378-1097(77)90072-6

Cited by 14 publications

(16 citation statements)

References 8 publications

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“…Bands b and c represent protein I of Henning et al (1973), while band d is the heatmodifiable protein, described as protein 11" by Garten et al (1975). A band moving like protein a, Schnaitman's protein 3B (Manning & Reeves, 1977), was seen in the profiles of strain J5 and serotypes 0 1 11 K58, 0 4 K2 and 075 K-. The protein b band was usually rather faint, though visible in all strains when OM preparations were applied to the gels in small amounts.…”

Section: Results a N D Discussionmentioning

confidence: 99%

Antigenic Cross-reactivity of Major Outer Membrane Proteins in Enterobacteriaceae Species

Hofstra¹,

Dankert²

1979

Journal of General Microbiology

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293The protein constituents in the outer membrane (OM) of several serotypes of Escherichia coli and some other Enterobacteriaceae cross-reacted antigenically. Solubilized OM preparations of these bacteria were applied in interfacial precipitin tests to antisera elicited in rabbits against whole bacterial cells, absorbed with their appropriate lipopolysaccharide before testing. The resulting immunecomplexes were analysed on polyacrylamide gels. Protein profiles of the imrnunoprecipitates showed a considerable antigenic cross-reactivity of outer membrane proteins between most E. coli serotypes. Cross-reactivity, though substantially lower, was also found with OM from three other Enterobacteriaceae species, but was not detectable with Pseudomonas aeruginosa OM. When OM preparations were solubilized at room temperature, the peptidoglycan-bound proteins in the molecular weight range 37000 to 41000 predominated in the protein profiles of the immunecomplexes. In profiles of immunecomplexes obtained with boiled OM preparations, a heat-modifiable protein (mol. wt 33000) predominated. The major OM proteins of the Gram-negative bacterium may therefore play a role as common surface antigens of the family of Enterobacteriaceae.

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Section: Results a N D Discussionmentioning

confidence: 99%

Antigenic Cross-reactivity of Major Outer Membrane Proteins in Enterobacteriaceae Species

Hofstra¹,

Dankert²

1979

Journal of General Microbiology

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“…OmpT is synthesized as a 42-kDa proprotein that is processed in the membrane to the mature 40-kDa form (14). Since its synthesis is reduced significantly in cells grown at or below 32°C, the structural gene is designated ompT in reference to the temperaturedependent appearance of its product (28). However, when we tried to purify T7 RNA polymerase from an ompT+ strain grown at 30°C, we still had problems with proteolysis, presumably because some OmpT protein still was present.…”

Section: Discussionmentioning

confidence: 99%

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

1988

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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.The DNA-dependent RNA polymerase of bacteriophage T7 is a single-chain enzyme with a molecular weight close to 100,000 that has stringent specificity for its own promoters (7,33,45). The gene specifying T7 RNA polymerase, T7 gene 1, has been cloned, and the protein has been successfully expressed at high levels in Escherichia coli (8, 49). As one of the simplest RNA polymerases known, it is an attractive candidate for attempts at crystallization, both alone and in association with its promoter sequence, and for specific biochemical and mutational analysis to map functional regions.Expression of cloned gene in E. coli is very efficient, and high levels of intact enzyme rapidly accumulate in the cells. However, during purification the protein becomes accessible to an endoprotease that introduces one or more nicks into the polypeptide chain (8,49 devoid of protease activity would be one way to circumvent this problem. As a first step towards isolating mutants defective in the endoprotease, we decided to characterize the enzyme's cellular location. From these studies we were able to devise an assay that allowed for rapid screening of the activity. When various laboratory strains of E. coli were tested for their ability to cleave T7 RNA polymerase, some strains were identified that appeared to lack protease activity.In this paper, we report the results of these studies and show that the protease that nicks T7 RNA polymerase is the product of the ompT gene, which encodes a previo...

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“…Synthesis of OmpT is thermoregulated (29) above, OmpP (and the 45-kDa protein shown in Fig. 2) was absent in cells grown in the presence of glucose.…”

Section: Figmentioning

confidence: 99%

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

1994

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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 T-even-type Escherichia coli phage Ox2 (20) (Dianova, Hamburg [16]). Strain Y1090 was lysogenized with one of these, Agtll-15. The lysogen was thermoinduced, and the phage was precipitated with polyethylene glycol (57) and then centrifuged through a CsCl step gradient (43). The 4.5-kb EcoRI insert of this phage was cloned into pUC18, yielding pAK1. The ompP gene was present on a 2.2-kb BglII fragment of this insert (see Results), which was cloned into the BamHI site of pUC18, resulting in pAK1-5. DNA sequencing (44) was performed using plasmids which were purified by CsCl density gradient centrifugation. Sequencing was started with a 17-mer sequencing primer and a 24-mer reverse sequencing primer (New England Biolabs). The sequence from bp 1 to 1020 was obtained from subclones by using restriction sites for AccI (bp 1), BglII (bp 50), HindIII (bp 561 and 610), and HinclI (bp 334 and 777; for the positions, see Fig. 4). For further sequencing, new primers annealing within the cloned chromosomal fragment were obtained from Appligene (Heidelberg, Germany [also shown in Fig. 4]). This strategy left a few uncertainties and gaps in the complementary strand upstream from bp 900 and 1400. A site for EclXI starts at bp 1474 (CGGCCG), and an EcoRI site exists in the vector (see Fig. 3). A deletion was obtained by restricting pAK1-5 with the two enzymes, followed by filling-in with the Klenow fragment of DNA polymerase I and religation. The resulting DNA fragment was sequenced by using the reverse sequencing primer, covering the sequence from bp 1450 to 1100. For the generation of two other deletions, pAK1-5 was opened with EclXI, digested for various time intervals with exonuclease III, and made blunt-ended with nuclease SI. After cleavage with EcoRI, the DNA was made blunt ended as above and religated. Two deletions were recovered; one started at bp 1066 and the other started at position 903. Both were sequenced with the reverse primer, providing the sequence from bp 1050 to 650. With these deletions, the sequence was completed for both strands from bp 334 to 1675. Southern blots were probed by ...

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Outer membrane protein 3B of Escherichia coli K-12: Effects of growth temperature on the amount of the protein and further characterization on acrylamide gels

Cited by 14 publications

References 8 publications

Antigenic Cross-reactivity of Major Outer Membrane Proteins in Enterobacteriaceae Species

Antigenic Cross-reactivity of Major Outer Membrane Proteins in Enterobacteriaceae Species

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

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

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