Kikuo Tsukamoto

Cloning and sequencing of an Na+/H+ antiporter gene from the marine bacterium Vibrio alginolyticus

A gene has been cloned from a DNA library from the marine bacterium Vibrio alginolyticus that functionally complements a mutant strain of Escherichia coli, NM81, defective in an Na+/H+ antiporter (NhaA). The cloned Vibrio gene restored NM81 to grow in a medium containing 0.5 M NaCl at pH 7.5 and concomitantly led to an increase in Na+/H+ antiport activity. The nucleotide sequence of the cloned fragment revealed an open reading frame, which encodes a protein with a predicted 383 amino acid sequence and molecular mass of 40,400 Da. The hydropathy profile is characteristic of a membrane protein with 11 membrane spanning regions. The deduced amino acid sequence is 58% identical with E. coli NhaA.

Function of the conserved triad residues in the class C β‐lactamase from Citrobacter freundii GN346

The conserved KTG triad in the class C β‐lactamase from Citrobacter freundii GN346 was examined as to its function by means of site‐directed mutagenesis. The following conversions were performed; Lys‐315 to arginine, alanine or glutamic acid, Thr‐316 to valine, and Gly‐317 to alanine, proline or isoleucine. The resultant mutant enzymes revealed that a basic amino acid at position 315 and a small uncharged residue at position 317 are essential for the enzyme activity, but a hydroxyl group at residue 316 is not required for the enzymatic catalysis. The kinetic properties of the purified Arg‐315 and Val‐316 enzymes provided information on the function of these residues.

Substitution of aspartic acid‐217 of Citrobacter freundii cephalosporinase and properties of the mutant enzymes

On the assumption that Asp‐217 of a Citrobacter freundii cephalosporinase forms a salt‐bridge with the conserved Lys‐67, Asp‐217 was changed to glutamic acid, threonine or lysine. The mutant enzymes retained about the same level of activity as that of the wild‐type enzyme, and the participation of Asp‐217 in the salt‐bridge was ruled out. However, the mutations resulted in an increase in hydrolytic activity toward oxyimino‐cephalosporins such as cefuroxime, cefmenoxime and ceftazidime, suggesting a possible mechanism of the bacterial resistance to the novel β‐lactams by a single mutation in cephalosporinases.

Carbenicillin‐Hydrolyzing Penicillinases of Proteus mirabilis and the PSE‐Type Penicillinases of Pseudomonas aeruginosa

Three carbenicillin‐hydrolyzing penicillinases were found in Proteus mirabilis strains, N‐3, N‐29, and GN79. The former two strains were isolated in 1978, but strain GN79 was one of our stock cultures isolated in 1965. These penicillinases closely resembled each other, and the PSE‐1 and PSE‐4 enzymes produced by Pseudomonas aeruginosa, in their substrate profiles and kinetic properties for hydrolyzing various β‐lactams. However, differences were found in their molecular weights and isoelectric points which ranged from 22,000 to 27,000 and from 6.0 to 6.9, respectively. The antiserum against the purified penicillinase of N‐29 cross‐reacted with the enzyme of N‐3 and inhibited its activity by more than 80%. The antiserum also reacted with the PSE‐1 and PSE‐4 enzymes. The antiserum did not react with the penicillinase from strain GN79 and the PSE‐2 and PSE‐3 enzymes of P. aeruginosa. Enzyme production in N‐3 and N‐29 was mediated by R plasmids.

A survey of a functional amino acid of class Cβ-lactamase corresponding to Glu166 of class A β-lactamases

The class C β‐lactamase of Cltrobacter freundii GN346 is a typical cephalosporinase comprising 361 amino acids. The aspartic acid at position 217 and glutamic acid at position 219 in this β‐lactamase were, respectively, previously shown not to be the counterpart of Glu166 (ABL166) in class A β‐lactamases, even though sequence alignment of class A and C enzymes strongly suggested this possibility [(1990) FEBS Lett. 264, 211‐214; (1990) J. Bacteriol. 172, 4348‐4351]. We tried again to assign candidates for the counterpart of Glu166 through sequence alignment based on other criteria, the glutamic acids at positions 195 and 205 in the class C β‐lactamase being selected. To investigate this possibility, these two glutamic acids were changed to glutamine, lysine or alanine, respectively. All the mutant enzymes showed more than 50% of the activity of the wild‐type enzyme, indicating that the possibility was ruled out. These results strongly suggested the possibility that the class C β‐lactamase lacks a functional acidic residue corresponding to Glu166 in class A enzymes.