Identification and characterization of two quiescent porin genes, nmpC and ompN, in Escherichia coli BE

Abstract

The genomic DNA of the BE strain of Escherichia coli has been scrutinized to detect porin genes that have not been identified so far. Southern blot analysis yielded two DNA segments which proved highly homologous to, yet distinct from, the ompC, ompF, and phoE porin genes. The two genes were cloned and sequenced. One of them, designated ompN, encodes a porin which, due to low levels of expression, has eluded prior identification. The functional properties (single-channel conductance) of the OmpN porin, purified to homogeneity, closely resemble those of the OmpC porin from E. coli K-12. The second DNA fragment detected corresponds to the nmpC gene, which, due to an insertion of an IS1 element in its coding region, is not expressed in E. coli BE.

FIG. 1

Southern blot analysis. Chromosomal DNAs were purified from E. coli CE1249 (lane a), BL21(DE3) (lane b), and BZB1107 (lane c), respectively, and digested with EcoRV. After fractionation of the DNA fragments by agarose gel electrophoresis, they were blotted onto a Hybond-N membrane and probed with [α-³²P]dATP-labeled ompF-specific DNA. With strain BL21(DE3), two EcoRV fragments appeared in positions corresponding to ∼3.1 and 2.6 kb and are labeled by arrows as fragments I and II. Fragments corresponding to ompF (asterisk) and ompC (circle) are indicated. HindIII-digested λ DNA is shown in lane M.

FIG. 2

Comparison of the predicted amino acid sequence of OmpN with sequences of various known E. coli porins. Sequences underlined below each block correspond to the β strands in the three-dimensional structures of OmpF and PhoE (6). A highly conserved porin-specific sequence motif, PEFGGD (14), in loop L3, and five conserved charged residues (R37, R75, D106, E110, and R126) which form a strong transversal electrostatic field in the channel interior are shown in bold. The additional amino acid residues, present in the predicted surface-exposed loop L7 in OmpN, are shown in bold italics. The overall similarity of OmpN is highest to OmpC (65% identical residues), followed by PhoE (62% identical residues) and OmpF (58% identical residues).

FIG. 3

SDS-PAGE analysis of E. coli porins. (A) Overexpression of OmpN porin in E. coli BL21(DE3)omp8. Membrane pellets were extracted with 3% octyl-POE as described in Materials and Methods. Protein samples (from 15 ml of culture) were applied to the gel without (−) or with (+) heat treatment for 10 min at 95°C in sample buffer. (B) Electrophoretic mobilities of purified porins (indicated at the top), which were used for functional analyses, represent monomers (high mobility) and trimers (low mobility). They were purified from E. coli outer membranes as described in the text. The protein samples (3 to 5 μg) were heated as described above. Lane M, molecular weight standard.