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

Abstract

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 ...

A PhoP-Regulated Outer Membrane Protease of Salmonella enterica Serovar Typhimurium Promotes Resistance to Alpha-Helical Antimicrobial Peptides

“…E. coli contains two highly homologous genes encoding OmpT-like proteases: ompT on the chromosome (15) and ompP (25) on plasmid F (29). OmpP and OmpT have 87% sequence identity (25). A pgtE-specific probe hybridized to a single Salmonella genomic fragment on a Southern blot (data not shown).…”

VirK Is a Periplasmic Protein Required for Efficient Secretion of Plasmid-Encoded Toxin from Enteroaggregative Escherichia coli

“…2). Periplasmic fractions were generated from E. coli HB101 and UT5600, a strain which expresses high levels of periplasmic proteins but lacks the OmpT and OmpP proteases (16). The periplasmic proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane.…”

Substrate Specificity of the Escherichia coli Outer Membrane Protease OmpP

“…Twenty-eight of 33 fifth-round peptides contained pairs of Arg residues. Kaufmann et al (10), who first purified OmpP, reported that it cleaves primarily between consecutive basic amino acid residues in a manner not unlike that of its homologue, OmpT. Therefore, pairs of Arg/Lys residues were assigned to the P1 and P1Ј positions (Fig.…”

“…Early evidence suggested that its synthesis is regulated by phosphate and the growth temperature (10,21) and that it plays a role in the catabolism of peptides under nitrogen-limited conditions (1). The enzyme was first isolated by Henning and coworkers (10), who showed that it exhibits 71% amino acid identity with OmpT, with which it also shares a strong preference for cleavage of polypeptide substrates between pairs of basic amino acids. OmpP is stable over a broad pH range (pH 6.4 to pH 9.2) and at temperatures up to 75°C but is inactivated by Mg 2ϩ , Mn 2ϩ , Co 2ϩ , or Ca 2ϩ ions (30).…”