Biochemistry and regulation of a novel Escherichia coli K-12 porin protein, OmpG, which produces unusually large channels

Abstract

A novel porin, OmpG, is produced in response to a chromosomal mutation termed cog-192. Molecular characterization of cog-192 revealed that it is a large chromosomal deletion extending from the 3' end of pspA through to the 5' end of an open reading frame located immediately upstream of ompG. As a result of this 13.1-kb deletion, the expression of ompG was placed under the control of the pspA promoter. Characterization of OmpG revealed that it is quite different from other porins. Proteoliposome swelling assays showed that OmpG channels were much larger than those of the OmpF and OmpC porins, with an estimated limited diameter of about 2 nm. The channel lacked any obvious solute specificity. The folding model of OmpG suggests that it is the first 16-stranded beta-barrel porin that lacks the large external loop, L3, which constricts the channels of other nonspecific and specific porins. Consistent with the folding model, circular dichroism showed that OmpG contains largely a beta-sheet structure. In contrast to other Escherichia coli porins, there is no evidence that OmpG exists as stable oligomers. Although ompG DNA was present in all E. coli strains examined so far, its expression under laboratory conditions was seen only due to rare chromosomal mutations. Curiously, OmpG was constitutively expressed, albeit at low levels, in Salmonella, Shigella, and Pseudomonas species.

FIG. 1

Circular dichroism analysis of purified OmpG. Purification and measurement were carried out as described in Materials and Methods. The magnitude of mean residue ellipticity (about −5,000 deg² dmol⁻¹) is significantly smaller than that found with OmpA or P. aeruginosa OprF (−9,000 to −10,000 deg² dmol⁻¹ [38]), two outer membrane proteins that contain substantial α-helical domains. The spectrum also does not show the two sharp negative peaks at 208 and 220 nm, which are characteristic of α helices.

FIG. 2

Predicted folding pattern of OmpG. The OmpG amino acid sequence was analyzed by using computer programs based on the algorithms of Schirmer and Cowan (32) and Paul and Rosenbusch (27). Transmembrane β strands were predicted whenever a high value of hydrophobicity on alternate residues in a 10-residue stretch was found, and this prediction was strengthened by the presence of turn-promoting sequences on either side. Most of the transmembrane segments could be assigned unequivocally, but the presence of the 12th and 13th segments is less convincing, as the hydrophobicity of the alternate residues is not high.

FIG. 3

SDS-PAGE analysis of envelopes and detergent-solubilized OmpG. Lane 1, untreated envelopes obtained from a strain lacking OmpF, OmpC, LamB, and OmpA; lane 2, pellet after OG extraction; lane 3, supernatant of 0.5% OG extraction; lane 4, supernatant of 0.5 and 1% OG extraction; lane 5, same as lane 4 but dialyzed against 10 mM HEPES (pH 7.4) buffer; lane 6, unheated envelopes; lane 7, same as lane 4 but unheated; lane 8, same as lane 5 but unheated. Positions of denatured OmpG and heat-modifiable OmpG (OmpG*) are shown. Samples present in lanes 1 to 5 were heated by boiling for 5 min prior to SDS-PAGE analysis.

FIG. 4

Rates of penetration of various sugars through the OmpG channel. Proteoliposomes containing OmpG extract were made as described in Material and Methods, and portions of the suspension were diluted into iso-osmotic solutions of sugars to determine the rates of osmotic swelling of the vesicles. The rates were normalized to the swelling rate in l-arabinose, which was taken as 100%. The sugars used (and, in parentheses, their molecular weights) were l-arabinose (150), d-glucose (180), N-acetylglucosamine (221), 2,3-diacetamido-2,3-dideoxy-d-glucose (262), lactose (342), sucrose (342), and maltose (342). As a control, proteoliposomes containing OmpC extract were used.

FIG. 5

Immunoblot analysis of OmpG from envelopes of various gram-negative bacteria. Lanes 1 and 2 contain envelopes (1.5 μg of protein) from OmpG⁻ and OmpG⁺ E. coli strains, respectively; lanes 3 to 7 contain 0.75, 1.5, 3, 6, and 12 μg of total protein, respectively. Gels were blotted with polyclonal antibodies raised against E. coli OmpG.

FIG. 6

Molecular organization of the chromosomal region encompassing ompG and the psp operon. Ten uncharacterized genes (b1309 to 1317 and orf1) are present between ompG and the psp operon. These 10 ORFs as well as a significant portion of the psp operon are deleted (marked by dotted lines) in the cog-192 strain. The deletion aligns ompG with the pspA promoter; precise ends of the deletion are shown in a close-up diagram below. Double-headed arrows show DNA probes used in Southern and/or Northern blot analysis. Locations of various primers and of restriction enzyme sites are shown at the top. Abbreviations: B, BglII; C, ClaI; N, NcoI; and S, SalI.

FIG. 7

Northern blot analysis to detect ompG-specific transcripts. Total RNA (3 μg) obtained from DME553 (cog⁺; lanes 1 and 7), RAM123 (cog-192; lanes 2 and 8), RAM123 recA::Km^r (lane 3), RAM123 recA::Km^r/pBR322 (lane 4), RAM123 recA::Km^r/pBR322-pspFAB (lane 5), and RAM123 recA::Km^r/pBR322-pspA (lane 6) was probed with an ompG-specific probe. Molecular sizes of the two ompG-specific mRNA species are 1.4 and 2.0 kb.