FEMS Microbiology Letters 23 (1984) 309-311 309 Published by Elsevier FEM01823 Assay for acetyl coenzyme A" alkylhydroxylamine N-acetyltransferase in Klebsiella pneumoniae ATCC25304 (Aerobactin biosynthesis; acetyltransferase) J. Kusters and H. Diekmann Institut ff~r Mikrobiologie, Universiti~tHannover, D- 3000 Hannover 1, F.R.G. Received 12 April 1984 Accepted 13 April 1984 1. SUMMARY An alkylhydroxylamine N-acetyltransferase was partially purified from extracts of Kelbsiella pneu- moniae. N6-Hydroxylysine, NS-hydroxyornithine and N-methylhydroxylamine were acetylated with decreasing velocity. N6-Acetyl-L-lysine showed noncompetitive inhibition while L-ornithine, L- lysine, their a-acetyl derivatives and NS-hydroxy - lysine were without effect at 0.1 M. A sensitive assay using t4C-labelled acetyl coenzyme A was developed. 2. INTRODUCTION The identification of the ColV plasmid coded virulence factor [1] as aerobactin [2] has renewed the interest in the biosynthesis of this siderophore [3]. From the work of Parniak et al. [4] it is evident that the biosynthetic pathway starts from lysine which is hydroxylated to N6-hydroxylysine. The acetylation to the monohydroxamic acid N 6- acetyl-N6-hydroxylysine is analogous to the for- mation of NS-acetyl-NS-hydroxyornithine studied by Ong and Emery in Ustilago sphaerogena [5], by Plattner in Rhodotorula glutinis and by Kusters in Aspergillus quadricinctus in our laboratory (unpub- lished). We have studied the occurrence and some prop- erties of N-acetyltransferase activity in extracts from Klebsiella pneumoniae, a producer of aero- bactin [6], and have developed an assay procedure which may be used in testing non-producing mutants. 3. MATERIALS AND METHODS 3.1. Cultivation K. pneumoniae (Schroeter) Trevisan ATCC25 304 (F. Gibson N(~W) was grown at 37°C in 5 1 of sucrose medium, pH 7.0 containing 0.1 g. 1 1 L- arginine, instead of 1.5 g.1-1 as described by Mullis et al. [7]. The medium contained 0.8 /~M Fe 3+ as determined with bathophenanthroline [8]. Seed cultures were prepared by inoculation of 200 ml nutrient broth (Difco, pH 7.2) from a 24-h-old nutrient agar slant and incubating for 12 h at 37 °C and 140 rev./min on a rotatory shaker. 50 ml of this culture were used to inoculate 5 1 medium in a glass fermenter model MF-14 (New Brunswick Scientific Co.). After incubation for 10 h at 200 rev./min and 2 1 air per min, cells were 0378-1097/84/$03.00 © 1984 Federation of European Microbiological Societies Downloaded from https://academic.oup.com/femsle/article-abstract/23/2-3/309/479312 by guest on 26 May 2020