Affiliation:
1. Laboratoire de Biologie Végétale et Microbiologie, CNRS FRE 2294, Faculté des Sciences, Université de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cédex, France
Abstract
ABSTRACT
Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb
Sinorhizobium meliloti
DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of
Escherichia coli
(34%) and to the glycine betaine transporter OpuD of
Bacillus subtilis
(30%). Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids). After heterologous expression of
betS
in
E. coli
mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock. Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport. Kinetic analysis revealed that BetS has a high affinity for betaines, with
K
m
s of 16 ± 2 μM and 56 ± 6 μM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl. BetS activity appears to be Na
+
driven. In an
S. meliloti betS
mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress. β-Galactosidase activities of a
betS-lacZ
strain grown in various conditions showed that
betS
is constitutively expressed. Osmotic upshock experiments performed with wild-type and
betS
mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation. Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by
S. meliloti
subjected to osmotic upshock.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
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