FEMS Microbiology Letters 58 (1989) 229-232 229 Published by Elsevier FEM 03505 Nitrogen-limited behaviour of micro-organisms growing in the presence of large concentrations of ammonium ions Ed T. Buurman, M. Joost Teixeira de Mattos and Oense M. Neijssel Department of Microbiology, Biotechnology Centre, University of A msterdam, Amsterdam, The Netherlands Received 24 November 1988 Accepted 26 November 1988 Key words: Klebsiella pneumoniae; Ammonia assimilation; Glutamate dehydrogenase; Glutamate synthase; pH; Chemostat culture 1. SUMMARY Cells of Klebsiella pneumoniae NCTC 418 grown at low culture pH values (4.5-5) in a glu- cose-limited chemostat culture contained elevated levels of glutamate synthase (EC 2.6.1.53). This can be taken as an indication that these cells show the physiology of nitrogen-limited cells, in spite of the fact that high concentrations (about 80 mM) of ammonium ions were present in the culture extracellular fluids. This phenomenon can be ex- plained by the rapid diffusion of ammonia (NH3) through the cell membrane, leading to very low cytoplasmic ammonium (NH~-) and NH 3 levels in cells that possess an almost neutral cytoplasmic pH value, but are growing at low culture pH values. 2. INTRODUCTION Ammonium salts are frequently used as a nitrogen source in microbiological media. An in- Correspondence to: Dr. O.M. Neijssel, Department of Microbi- ology, Biotechnology Centre, University of Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands. teresting problem is how ammonia (NH3) or am- monium ions (NH~-) enter the cell. Kleiner [1] estimated that the permeability coefficient for NH 3 through the cell membrane of KlebsieUa pneumo- niae is high (2 × 10 3 cm. s-l), which indicates that this compound will diffuse rapidly through the cell membrane. On the other hand it has been shown that micro-organisms contain NH~- trans- port systems which are derepressed when the medium contains nitrogen sources other than am- monium salts (for review, see [21). In addition, there is uncertainty about which chemical species (NH 3 or NH4~) is the substrate for the two main assimilatory enzymes: glutamate dehydrogenase and glutamine synthetase [3]. If NH 3 can indeed diffuse very quickly through the cell membrane, this will lead to serious conse- quences when neutrophilic organisms such as K. pneumoniae or Escherichia coli, possessing a cyto- plasmic pH value of 7.5-8 [4], are growing at low medium pH values. Since the NH 3 gradient across the membrane will be close to zero and the ex- tracellular NH 3 concentration will be vanishingly small at such low extracellular pH values, the intracellular NH 3 and NH~- concentrations can- not be large. Thus, on the basis of this hypothesis one could predict that when Klebsiella pneumoniae is growing in a nominally glucose-limited chemo- 0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies