Isolation of the putP gene of Corynebacterium glutamicum and characterization of a low-affinity uptake system for compat
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© Springer-Verlag 1997
O R I G I N A L PA P E R
Heidi Peter · Andrea Bader · Andreas Burkovski · Camille Lambert · Reinhard Krämer
Isolation of the putP gene of Corynebacterium glutamicum and characterization of a low-affinity uptake system for compatible solutes Received: 27 February 1997 / Accepted: 24 April 1997
Abstract Corynebacterium glutamicum accumulates the compatible solutes proline, glycine betaine, and ectoine under conditions of high osmolality. Uptake of proline is mediated by both a high-affinity and a low-affinity secondary transport system. The low-affinity uptake system also accepts glycine betaine and ectoine as substrates. In the present study, the gene encoding the high-affinity proline uptake system PutP was isolated by heterologous complementation of Escherichia coli mutant strain WG389, which lacks the transport systems BetT, PutP, ProP, and ProU and is unable to synthesize proline and glycine betaine. This gene (putP) encodes a protein of 524 amino acids that shares identity with the proline transport systems PutP of E. coli, Staphylococcus aureus, Salmonella typhimurium, Haemophilus influenzae, and Klebsiella pneumoniae. Functional studies of PutP synthesized in E. coli mutant strain MKH13, which also lacks the transport systems for compatible solutes and is unable to synthesize glycine betaine, revealed that this carrier system is not regulated by the external osmolality on the level of activity. Km values of 7.6 mM for proline and 1.3 mM for sodium as cotransported ion were determined. Deletion of the putP gene allowed the functional characterization of another proline uptake system with low affinity. Key words Corynebacterium glutamicum · PutP · Proline transport · Compatible solutes · Osmoregulation Abbreviations PMBN Polymyxin B nonapeptide · CCCP Carbonyl cyanide m-chlorophenylhydrazone · IPTG Isopropyl-β-D-thiogalactopyranoside
H. Peter · A. Bader · A. Burkovski · C. Lambert · R. Krämer (Y) Institut für Biotechnologie 1, Forschungszentrum Jülich, Postfach 1913, D-52425 Jülich, Germany Tel. +49-2461-61-5515; Fax +49-2461-61-2710 e-mail: [email protected]
Introduction The gram-positive bacterium Corynebacterium glutamicum is used in industrial fermentations for the production of amino acids such as glutamate, lysine, and isoleucine. During the fermentation process, the osmotic conditions change significantly. Also in its natural habitat, the soil, C. glutamicum requires effective adaptation mechanisms in order to prevent cell lysis or dehydration as a consequence of frequently changing osmotic conditions in its surroundings. A strategy of bacteria to overcome hyperosmotic stress is the accumulation of osmoprotective solutes such as glycine betaine and proline [for reviews, see Csonka (1989) and Csonka and Hanson (1991)]. Proline uptake of the well-studied gram-negative organisms Escherichia coli and Salmonella typhimurium is mediated by three transport systems: ProU, ProP, and PutP (Wood 1988; Booth and Higgins 1990; Lucht and Bremer 1994). ProU and ProP accept a broad rang
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