The porin oprE gene of Pseudomonas aeruginosa PAO1 was isolated. Its nucleotide sequence indicated that the structural gene of 1383 nucleotide residues encodes a precursor consisting of 460 amino acid residues with a signal peptide of 29 amino acid residues, which was confirmed by the N-terminal 23-amino-acid sequence and the reaction with anti-OprE polyclonal antiserum. Anaerobiosis induced OprE production at the transcription level. The transcription start site was determined to be 40 nucleotides upstream from the ATG initiation codon. The control region contained an appropriately situated E sigma 54 recognition site and the putative second half of an ANR box. The amino acid sequence of OprE had some clusters of sequence homologous with that of OprD of P. aeruginosa, which might be responsible for the outer membrane permeability of imipenem and basic amino acids.
Porin-deficient mutants of Pseudomonas aeruginosa PAO1 were selected by isolating latamoxef-resistant mutants following chemical mutagenesis. Highly latamoxef-resistant mutants had alterations in both the outer membrane proteins and penicillin-binding protein 3, a lethal target of latamoxef. Both of these alterations may be essential for cells to acquire high resistance to latamoxef. Many of the latamoxef-resistant mutants were also resistant to new quinolones and chloramphenicol. Resistance to these compounds was simultaneously co-transduced from one mutant into strain PAO1 when selection was made for transduction of ofloxacin resistance. Study of these transductants indicated that decreased amounts of outer membrane proteins C, D2, E1, and E2 lowered the outer membrane permeability and resulted in resistance. Three of these proteins were apparently identical to proteins previously reported to function as small pores. The results suggested that at least one of the four proteins was functioning as a porin for the antibacterial agents studied.
An eschericia coli K-12 mutant highly resistant to moxalactam but only slightly resistant to other beta-lactam antibiotics was obtained by mutagen treatment. The affinity of moxalactam for its target penicillin-binding proteins was unchanged, as was the level of beta-lactamase activity. The penetration of [14C] moxalactam, however, was markedly reduced in the mutant. Electrophoretic analysis revealed alterations of the outer membrane proteins. A reduction in the amount of one of the pore-forming proteins (porins) was especially noteworthy. These data suggest that moxalactam resistance is the result of an alteration in the outer membrane structure.
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