Evaluation of a nisin-based germicidal formulation on teat skin of live cows.

A purified preparation of the nontoxic antimicrobial peptide, nisin (AMBICIN N), was used in the formulation of a germicidal sanitizer suitable for use on cow teats. The germicidal activity of the formulation against mastitis pathogens was measured on teat skin of live cows. The nisin-based formulation gave a mean log reduction of 3.90 against Staphylococcus aureus and 4.22 log reduction against Escherichia coli after exposure for 1 min to the germicide. This activity was comparable with that exhibited by a 1% iodophor teat dip but was significantly greater than that exhibited by the .1 and .5% iodophors and by the .5% chlorhexidine digluconate teat dips. The nisin-based formulation showed little or no potential for skin irritation after multiple application to skin, but iodophor and chlorhexidine digluconate teat dips showed significant potential for skin irritation in comparable studies.

Escherichia coli carbamoyl-phosphate synthetase: domains of glutaminase and synthetase subunit interaction.

Three catalytic domains of the Escherichia coli carbamoyl-phosphate synthetase (EC 6.3.5.5) have been identified in previous studies. These include the glutamine amide-N transfer domain in the carboxyl-terminal half of the glutaminase component and at least two adenine nucleotide binding sites in the synthetase component. To delineate the domains involved in subunit interactions, we have examined the effects of deletions and point mutations in the glutaminase and synthetase subunits on formation of the alpha beta holoenzyme. Deletion of the amino-terminal third of the glutaminase subunit abolishes interactions with the synthetase subunit, suggesting that this domain functions to stabilize the complex. Two subunit binding domains have been identified in the synthetase subunit. They are homologous to one another and are located in the amino-terminal and central regions of the synthetase component. These domains are adjacent to regions of the synthetase previously proposed to be involved in ATP binding and, possibly, activation of CO2. The new data enlarge the definition of the structural and functional domains in the two interdependent components of carbamoyl-phosphate synthetase.

In vivo synthesis of carbamyl phosphate from NH3 by the large subunit of Escherichia coli carbamyl phosphate synthetase.

The cloned carAB operon of Escherichia coli coding for the small and large subunits of carbamyl phosphate synthetase has been used to construct a recombinant plasmid with a 4.16 kilobase ClaI fragment of the car operon that lacks the major promoters, P1 and P2. The plasmid, pHN12, carries a functional carB gene. A mutant E. coli strain lacking both subunits of carbamyl phosphate synthetase when transformed with pHN12 overproduces the large subunit by 200-fold (8-10% of the cellular protein). The elevated levels of the large subunit enable the transformed cells to utilize NH3 but not glutamine as nitrogen donor for carbamyl phosphate synthesis. The large subunit has been purified from the overexpressing strain. The purified native large subunit is capable of synthesizing carbamyl phosphate from ammonia, HCO-3, and ATP. The kinetic properties of the large subunit compared with the holoenzyme indicate that the Michaelis constants of the large subunit for HCO-3 and ATP are modulated by its association with the small glutamine binding subunit.

Catalytic domains of carbamyl phosphate synthetase. Glutamine-hydrolyzing site of Escherichia coli carbamyl phosphate synthetase.

We present evidence that cysteine 269 of the small subunit of Escherichia coli carbamyl phosphate synthetase is essential for the hydrolysis of glutamine. When cysteine 269 is replaced with glycine or with serine by site-directed mutagenesis of the carA gene, the resulting enzymes are unable to catalyze carbamyl phosphate synthesis with glutamine as nitrogen donor. Even though the glycine 269, and particularly the serine 269 enzyme bind significant amounts of glutamine, neither glycine 269 nor serine 269 can hydrolyze glutamine. The mutations at cysteine 269 do not affect carbamyl phosphate synthesis with NH3 as substrate. The NH3-dependent activity of the mutant enzymes was equal to that of wild-type. Measurements of Km indicate that the enzyme uses unionized NH3 rather than ammonium ion as substrate. The apparent Km for NH3 of the wild-type enzyme is calculated to be about 5 mM, independent of pH. The substitution of cysteine 269 with glycine or with serine results in a decrease of the apparent Km value for NH3 from 5 mM with the wild-type to 3.9 mM with the glycine, and 2.9 mM with the serine enzyme. Neither the glycine nor the serine mutation at position 269 affects the ability of the enzyme to catalyze ATP synthesis from ADP and carbamyl phosphate. Allosteric properties of the large subunit are also unaffected. However, substitution of cysteine 269 with glycine or with serine causes an 8- and 18-fold stimulation of HCO-3 -dependent ATPase activity, respectively. The increase in ATPase activity and the decrease in apparent Km for NH3 provide additional evidence for an interaction of the glutamine binding domain of the small subunit with one of the two known ATP sites of the large subunit.