Identification and characterization of a new porin gene of Klebsiella pneumoniae: its role in beta-lactam antibiotic resistance

Abstract

Klebsiella pneumoniae porin genes were analyzed to detect mutations accounting for the porin deficiency observed in many beta-lactam-resistant strains. PCR and Southern blot analysis revealed the existence of a third porin gene in addition to the OmpK36 and OmpK35 porin genes previously described. This new porin gene was designated ompK37 and is present in all of the clinical isolates tested. The OmpK37 porin gene was cloned, sequenced, and overexpressed in Escherichia coli. In contrast to that of the major porins, OmpK37 porin expression was only detectable by Western blot analysis in porin-deficient beta-lactam-resistant strains, suggesting strong down regulation under standard laboratory conditions. Functional characterization suggested a narrower pore for the OmpK37 porin than for K. pneumoniae porins OmpK36 and OmpK35. This correlated with the susceptibility to certain beta-lactam antibiotics, since a K. pneumoniae strain expressing porin OmpK37, but not porin OmpK36 or OmpK35, was less susceptible to beta-lactam antibiotics than the same strain expressing either porin OmpK36 or OmpK35.

FIG. 1

PCR and Southern blot analysis of the K. pneumoniae SD8 chromosome. (A) Resolution by agarose gel electrophoresis of PCR products obtained by amplification of internal sequences of porin genes from K. pneumoniae SD8 with primers U681 and L1316. The arrow indicates the PCR fragment corresponding to the OmpK37 porin gene. (B) Southern blot analysis of porin genes ompK36, ompK35, and ompK37 of strain SD8. Chromosomal DNA was digested with EcoRI, EcoRV, HindIII, and KpnI (from left to right in each panel) and hybridized with a probe specific for each gene. Molecular masses are indicated in kilobases on the left for panel A and on the right for panel B.

FIG. 2

Alignment of the OmpK37 sequence with selected sequences from other enterobacterial porins from E. coli (porins OmpC, OmpF, OmpN, PhoE, and NmpC) and S. typhi (OmpS1 and OmpS2). Porins were selected on the basis of their high degree of amino acid identity with OmpK37. Protein sequences were derived from nucleotide sequences. Secondary-structural motifs are those of the OmpF structure (8).

FIG. 3

SDS-PAGE analysis of OMPs isolated from strain CSUB10R and strain CSUB10R expressing different K. pneumoniae porins. Purified OMPs (5 μg) from strain CSUB10R and its derived clones carrying plasmids pQE1K, pQE3K, pQE7K, pQE31, pQE33, pSHA16K, and pSHA25K (form left to right, lanes 1, 2, 3, 4, 5, 6, 7, and 8, respectively) were resolved by SDS-PAGE, Coomassie blue stained, and analyzed by densitometry. The porin expressed by each clone is indicated at the top of each lane. Densitometric analysis results for the corresponding porin are indicated in arbitrary units at the bottom of each lane and are the mean ± the standard deviation of at least three independent experiments. Purified OmpK37 is shown in lane 9. ND, not detected.

FIG. 4

Western blot analysis of OmpK37 expression using monospecific antiserum. K. pneumoniae SD8, LB4, LB66, and CSUB10R were subjected to porin isolation methods, and the resulting materials were analyzed in lanes 2 to 5, respectively. Purified porins OmpK37, OmpK36, and OmpK35 were included as controls in lanes 1, 6, and 7, respectively. Molecular masses in kilodaltons are indicated on the left.