Isolation of the putP gene of Corynebacterium glutamicum and characterization of a low-affinity uptake system for compatible solutes.
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.
Peter
H
H
Institut für Biotechnologie 1, Forschungszentrum Jülich, Postfach 1913, D-52425 Jülich, Germany.
Bader
A
A
Burkovski
A
A
Lambert
C
C
Krämer
R
R
D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral; D001426:Bacterial Proteins; D001622:Betaine / Q000378:metabolism; D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D003352:Corynebacterium / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D015698:Genomic Library; D026901:Membrane Transport Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011392:Proline / Q000378:metabolism*; D014176:Protein Biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D027981:Symporters; D014882:Water-Electrolyte Balance / Q000235:genetics
D026921:Amino Acid Transport Systems, Neutral; D001426:Bacterial Proteins; C101652:BetP protein, Corynebacterium glutamicum; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D027981:Symporters; D001622:Betaine; C033082:proline transporter; D011392:Proline