Saburo Yamagishi

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.

Physical and functional mapping of Tn2603, a transposon encoding ampicillin, streptomycin, sulfonamide, and mercury resistance.

SummaryA map of cleavage sites for restriction endonucleases EcoR1, BamHI, HindIII, and SalI on Tn2603, a transposon encoding resistance to ampicillin, streptomycin, sulfonamide, and mercury, was constructed by an analysis of restriction cleavage patterns of plasmid pMK1.:: Tn2603 and its deletion derivative. By cloning the fragments generated from pMK1::Tn2603 with these restriction endonucleases to a pACYC184 plasmid vehicle, the regions necessary for expression of resistance were located on the restriction cleavage map of Tn2603. Ampicillin, streptomycin, and sulfonamide-resistance genes were mapped in a cluster on the region between the center and the right and the mercury-resistance gene was located to the left of the map. The final functional map of Tn2603 was compared with those of Tn4 and Tn21 and the evolutional relationships between them were discussed.

Inducible oxacillin-hydrolyzing penicillinase in Aeromonas hydrophila isolated from fish.

An inducible penicillinase was shown to be present in a strain of Aeromonas hydrophila subsp. hydrophila isolated from freshwater fish. Enzyme induction was observed with benzylpenicillin or 6-aminopenicillanic acid, and the enzyme was cell bound. The penicillinase was purified 50-fold from a crude cell extract. The molecular weight was estimated to be 23,000 by gel filtration. The pH and temperature optima for the enzyme activity were 8.0 and 35°C, respectively. The penicillinase showed a unique substrate profile by hydrolyzing oxacillin about twice as rapidly as benzylpenicillin. The enzyme activity was weakly inhibited by sodium chloride but was not affected by p-chloromercuribenzoate. The property of penicillinase production by the A. hydrophila strain could not be transferred to Escherichia coli and also could not be eliminated from the bacteria by ethidium bromide treatment.

Gene Expression of Ampicillin Resistance Transposons, Tn2601 and Tn2602

To establish the mode of gene expression specified by transposon, we investigated the correlation among the homology of the DNA sequence, the extent of transposon‐specific transcription, the specific activity of penicillinase (PCase) per cell, and the transposition frequency by using two ampicillin resistance transposons (TnAs), Tn2601 and Tn2602. Although both the TnAs specify the so‐called type I PCase, Tn2602 always conferred 10–to 20‐fold higher PCase activity per cell than Tn2601 regardless of the kind of replicon carrying TnA. The transposition frequency of Tn2602 also was 8 to 50 times higher than that of Tn2601 in all combinations of donor and recipient plasmids examined. As a result, the transposability expressed by the TnA was thought to correlate with the productivity of PCase in the cell specified by the corresponding TnA. The level of TnA‐specific transcription of Tn2602 was noticeably higher than that of Tn2601, whereas the two TnAs shared a high degree (more than 90%) of DNA sequence homology. These results suggest that the difference in rates of transcription of the two transposons plays a key role in determining the difference in the productivity of PCase and the transposition‐protein(s) of Tn2601 and Tn2602.

Thermolabile Repression of Cephalosporinase Synthesis in Citrobacter freundii

An unusual regulatory system of cephalosporinase synthesis in Citrobacter freundii has been found. When the bacteria are grown at 20 C, the cephalosporinase is synthesized as a typical inducible enzyme and benzylpenicillin acts as an effective inducer. The enzyme, however, is synthesized in the absence of the inducer at growth temperatures above 25 C. When the growth temperature is shifted from 20 C to 37 C, the induction of enzyme synthesis is observed after about one half of the organism doubling time, but it does not occur in the presence of chloramphenicol. The reverse control mutants, the enzyme constitutive synthesis of which is markedly depressed by benzylpenicillin, were isolated from the C. freundii wild strain. The possibility that the enzyme synthesis is governed by a regulatory system analogous to the its mutant of the lac operon in Escherichia coli was suggested.

Restriction Endonuclease Cleavage Maps of the Ampicillin Transposons Tn2601 and Tn2602

This paper reports the cleavage maps of ampicillin transposons Tn2601 and Tn2602, for restriction endonucleases BamHI, PvuII, AvaI, HincII, and HaeII. Both of the transposons are very similar to the well‐known ampicillin transposon Tn3 in size, endonuclease cleavage sites, and possession of a short inverted repeat sequence at both ends. A slight difference in the cleavage pattern among these three transposons was observed in the region around the BamHI site which was assumed to be a part of the repressor gene for transposition.