Tetsuo Sawai

Amino Acid Substitutions in a Variant of IMP-1 Metallo-β-Lactamase

ABSTRACT In the course of surveying for the carbapenem-hydrolyzing metallo-β-lactamase gene blaIMP in pathogenic bacteria by the PCR method, we detected a gene encoding a variant metallo-β-lactamase, designated IMP-3, which differed from IMP-1 by having low hydrolyzing activity for penicillins and carbapenems. PCR product direct sequencing of a 2.2-kb segment revealed that the geneblaIMP-3 was located on a cassette inserted within a class I integron in the pMS390 plasmid. The 741-bp nucleotide sequence of blaIMP-3 was identical to that ofblaIMP-1, except for seven base substitutions. Among these were two, at nucleotide positions 314 and 640, which caused amino acid alterations. Hybrid bla genes were constructed from blaIMP-3 andblaIMP-1 by recombinant DNA techniques, and β-lactamases encoded by these genes were compared with those of the parents IMP-3 and IMP-1 under the same experimental conditions. The kinetic parameters indicated that the inefficient hydrolysis of benzylpenicillin, ampicillin, imipenem, and ceftazidime by IMP-3 was due to the substitution of glycine for serine at amino acid residue 196 in the mature enzyme. This alteration corresponded to the presence of guanine instead of an adenine at nucleotide position 640 of theblaIMP-3 gene. This indicated that extension of the substrate profile in the metallo-β-lactamase IMP-1 compared to IMP-3 is the result of a one-step single-base mutation, suggesting that the gene blaIMP-3 is an ancestor ofblaIMP-1.

Energetics of tetracycline efflux system encoded by Tn10 in Escherichia coli

Tritiated tetracycline was actively accumulated in inverted membrane vesicles prepared from Escherichia coli W3104rif, which has a transposon, Tn10, on the plasmid, R388, by means of a protonmotive force when NADH was added as an energy source. The tetracycline accumulation was reduced to about one‐half the full value on the addition of a cation/proton‐exchange ionophore, nigericin. In contrast, remarkable stimulation of the tetracycline accumulation was observed with a K+‐specific ionophore, valinomycin. The accumulation of [3H]tetracycline could also be driven by an artificially imposed interior‐acidic pH gradient (ΔpH), but not, however, by an artificially imposed interior‐positive membrane potential (Δψ). These results strongly indicate that the plasmid‐encoded tetracycline transport was mainly due to an electrically neutral proton/tetracycline antiport system.

Iodometric Assay Method for Beta-Lactamase with Various Beta-Lactam Antibiotics as Substrates

The rapid fixed-time assay for penicillinase was modified for measuring β-lactamase activity with twelve substrates, i.e., benzylpenicillin, ampicillin, cloxacillin, methicillin, carbenicillin, cefazolin, cephalothin, cephaloglycin, cephalexin, cephalosporin C, 7-aminocephalosporanic acid, and cefoxitin. The method depends upon the reduction of iodine by the hydrolyzed substrate. Determined experimentally, 1 mol of hydrolyzed penicillins consumed 3.4 to 4.0 mol of iodine (I2). Iodine consumption of hydrolyzed cephalosporins varied widely from 1.7 for cephalothin to 3.7 for cefazolin. The method is useful for routine assay of β-lactamase activity with various substrates.

A novel glycylcycline, 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline, is neither transported nor recognized by the transposon Tn10-encoded metal-tetracycline/H+ antiporter.

A novel tetracycline derivative, DMG-DMDOT [9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline] , is one of the glycylcyclines which have a broad antibacterial spectrum, including many tetracyclineresistant bacteria (R.T. Testa, P.J. Petersen, N.V. Jacobus, P.-E. Sum, V.J. Lee, and F.P. Tally, Antimicrob. Agents Chemother. 37:2270-2277, 1993). The mechanism by which DMG-DMDOT overcomes efflux-based tetracycline resistance was investigated. Tetracycline-resistant Escherichia coli cells carrying an R plasmid encoding the tet(B) gene, which encodes the typical tetracycline efflux pump [TetA(B)] of gram-negative bacteria, were as susceptible to DMG-DMDOT as was the tetracycline-susceptible host. When mid-log-phase cells carrying the tet(B) gene were incubated with a subbactericidal concentration of DMG-DMDOT (0.5 micrograms/ml) for 2 h, a significant amount of the TetA(B) protein was detected in the cell membrane by Western blotting (immunoblotting) with an anti-carboxyl-terminal antibody, similar to the case in which tetracycline was used as the inducer, indicating that the tet repressor, TetR, can recognize DMG-DMDOT as an efficient inducer. Everted membrane vesicles prepared from cells producing the TetA(B) protein showed absolutely no transport activity for DMG-DMDOT. Furthermore, the presence of excess DMG-DMDOT had no effect on the tetracycline transport activity of the everted vesicles, indicating that DMG-DMDOT is not recognized as a substrate by the TetA(B) protein.

Stoichiometry of metal-tetracycline/H+ antiport mediated by transposon Tn10-encoded tetracycline resistance protein in Escherichia coli

The tetracycline resistance protein (TetA) endoded by transposon Tn10 mediates the efflux of divalent cation‐tetracycline chelating complexes [Yamaguchi, A., Udagawa, T. and Sawai, T. (1990) J. Biol. Chem. 265, 4809–4813]. It was confirmed that protons were antiported with the complexes through an electrically‐neutral process because the antiport consumed ΔpH but not Δψ. The quantitative relationship between ΔpH and ΔpTC determined by a flow‐dialysis method clearly indicated a 1:1 stoichiometry of the monocationic metal‐tetracycline/H+ exchange.

Delta pH-dependent accumulation of tetracycline in Escherichia coli.

The effects of ionophores on tetracycline accumulation in Escherichia coli cells were investigated in the presence of polymyxin B nonapeptide. Accumulation was inhibited by nigericin but not by valinomycin. Tetracycline accumulation was stimulated by decreasing the pH of the medium and inhibited by the addition of magnesium ions. These results indicated that tetracycline enters cells through diffusion as a protonated form (TH2) and is accumulated as a membrane-impermeable magnesium-tetracycline chelate complex (THMg+). This noncarrier diffusion hypothesis was confirmed by the fact that tetracycline accumulated in protein-free liposomes through an artificially imposed pH difference.

Functional Analysis of the Active Site of a Metallo-β-Lactamase Proliferating in Japan

ABSTRACT An R-plasmid-mediated metallo-β-lactamase was found inKlebsiella pneumoniae DK4 isolated in Japan in 1991. The nucleotide sequence of its structural gene revealed that the β-lactamase termed DK4 was identical to the IMP-1 metallo-β-lactamase which was mediated by a chromosomal gene ofSerratia marcescens TN9106 isolated in Japan in 1991 (E. Osano et al., Antimicrob. Agents Chemother. 38:71–78, 1994). The dose effect of DK4 β-lactamase production on the resistance levels indicated a significant contribution of the enzyme to bacterial resistance to all the β-lactams except monobactams. The enzymatic characteristics of the DK4 β-lactamase and its kinetic parameters for nine β-lactams were examined. The DK4 β-lactamase was confirmed to contain 2 mol of zinc per mol of enzyme protein. The apoenzyme that lacked the two zincs was structurally unstable, and the activities of only 30% of the apoenzyme molecules could be restored by the addition of 1 mM zinc sulfate. The substitution of five conserved histidines (His28, His86, His88, His149, His210) and a cysteine (Cys168) for an alanine indicated that His86, His88, and His149 served as ligands to one of the zincs and that Cys168 played a role as a ligand to the second zinc. Both zinc molecules contribute to the enzymatic process. Mutant enzymes that lack only one of these retained some activity. Additionally, a conserved aspartic acid at position 90 was replaced by asparagine. This mutant enzyme showed an approximately 1,000 times lower kcat value for cephalothin than that of the wild-type enzyme but retained the two zincs even after dialysis against zinc-free buffer. The observed effect of pH on the activity suggested that Asp90 functions as a general base in the enzymatic process.

Orientation of the carboxyl terminus of the transposon Tn10-encoded tetracycline resistance protein in Escherichia coli

A site‐directed antibody was generated against a synthetic polypeptide corresponding to the 14 amino acid residues of the carboxyl terminus of the Tn10 TetA protein. The antibody reacted preferentially with inside‐out vesicles, rather than right‐side‐out vesicles, prepared from Escherichia coli cells harboring transposon Tn10. When inside‐out vesicles were treated with trypsin, the TetA protein was completely digested in the vicinity of the carboxyl terminus, as judged on immunoblot analysis using the antibody. In contrast, when right‐side‐out vesicles were treated with trypsin, the TetA protein was hardly digested. These results indicate that the carboxyl terminus of TetA is exposed to the cytoplasmic side of the membrane.

Identification of porins in outer membrane of Proteus, Morganella, and Providencia spp. and their role in outer membrane permeation of beta-lactams.

Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia rettgeri, and Providencia alcalifaciens, which were once classified into the same genus, Proteus, were studied. Cefoxitin-resistant mutants from these species were isolated, and it was confirmed that the resistance was attributed to the lack of an outer membrane protein, resulting in a significant decrease in the penetration of hydrophilic cephalosporins through the outer membrane. Comparison of the mutant strains with their parental strains in the diffusion rates of six monoanionic cephalosporins, a zwitterionic cephalosporin (cephaloridine), and a divalent anionic cephalosporin (cephalosporin C) suggested that each species had only one kind of porin protein, with molecular weights of 40,000 (Proteus mirabilis) or 37,000 (the other four species) and that the porins formed channels with cation selectivity, except for Proteus vulgaris. Porin proteins were purified from all the bacterial species except Providencia alcalifaciens, and the radius of the pores formed by the purified porins was estimated by the use of the liposome swelling assay. The pore radii were estimated to be approximately 0.59 nm (Proteus mirabilis), 0.63 nm (Proteus vulgaris), 0.58 nm (Providencia rettgeri), and 0.60 nm (M. morganii), similar to the size of the pore radius of Escherichia coli porins. Images

The tetracycline efflux protein encoded by the tet(K) gene from Staphylococcus aureus is a metal-tetracycline/H+ antiporter

The tet(K) gene from Staphylococcus aureus was highly expressed in Escherichia coli by an alteration of its initiation codon from TTG to ATG and its ribosome‐binding sequence from GAGG to GGAGG [Noguchi, N. et al. (1994) Biol. Pharm. Bull. 17, 352–355]. The inverted membrane vesicles prepared from the tet(K)‐expressing cells showed respiration‐dependent [3H]tetracycline transport comparable to the vesicles from the tet(B)‐expressing cells. The affinity of Tet(K) vesicles to tetracycline was the same as that of Tet(B) vesicles, whereas the former V max value was about 60% of the latter one. Contrary to Tet(B) vesicles, Tet(K) vesicles showed no significant minocycline uptake, which was consistent with the low minocycline resistance of the Tet(K)‐producing cells. The tetracycline transport mediated by Tet(K) vesicles was coupled with proton transport and the translocation of 60Co2+ ions as well as in Tet(B) vesicles. This observation indicates that the class K tetracycline resistance determinant from Gram‐positive bacteria also encodes a metaltetracycline/H+ antiporter that is functionally similar to that encoded by tet(B), although there is a considerable difference in the primary sequences and the putative topologies of these Tet proteins.