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PMID: 8752321 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Three transport systems for the osmoprotectant glycine betaine operate in Bacillus subtilis: characterization of OpuD.

Journal of bacteriology ·Vol. 178 ·No. 17 ·1996-09-00 ·Pages 5071-9

Kappes RM, Kempf B, Bremer E

Abstract

The accumulation of the osmoprotectant glycine betaine from exogenous sources provides a high degree of osmotic tolerance to Bacillus subtilis. We have identified, through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, a new glycine betaine transport system from B. subtilis. The DNA sequence of a 2,310-bp segment of the cloned region revealed a single gene (opuD) whose product (OpuD) was essential for glycine betaine uptake and osmoprotection in E. coli. The opuD gene encodes a hydrophobic 56.13-kDa protein (512 amino acid residues). OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporter CaiT from E. coli and a BetT-like protein from Haemophilus influenzae. These membrane proteins form a family of transporters involved in the uptake of trimethylammonium compounds. The OpuD-mediated glycine betaine transport activity in B. subtilis is controlled by the environmental osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine betaine uptake activity. An opuD mutant was constructed by marker replacement, and the OpuD-mediated glycine betaine uptake activity was compared with that of the previously identified multicomponent OpuA and OpuC (ProU) glycine betaine uptake systems. In addition, a set of mutants was constructed, each of which synthesized only one of the three glycine betaine uptake systems. These mutants were used to determine the kinetic parameters for glycine betaine transport through OpuA, OpuC, and OpuD. Each of these uptake systems shows high substrate affinity, with Km values in the low micromolar range, which should allow B. subtilis to efficiently acquire the osmoprotectant from the environment. The systems differed in their contribution to the overall glycine betaine accumulation and osmoprotection. A triple opuA, opuC, and opuD mutant strain was isolated, and it showed no glycine betaine uptake activity, demonstrating that three transport systems for this osmoprotectant operate in B. subtilis.

MeSH Terms
Amino Acid Sequence Bacillus subtilis/genetics,metabolism Bacterial Proteins/genetics,metabolism Base Sequence Betaine/metabolism Biological Transport Carrier Proteins/genetics,metabolism Cloning, Molecular DNA Transposable Elements DNA, Bacterial Kinetics Membrane Transport Proteins Molecular Sequence Data Mutation Osmolar Concentration Plasmids Quaternary Ammonium Compounds Sequence Homology, Amino Acid
Chemicals
Bacterial Proteins Carrier Proteins DNA Transposable Elements DNA, Bacterial Membrane Transport Proteins OpuD protein, Bacillus subtilis Quaternary Ammonium Compounds Betaine
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Kappes R M
Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
Kempf B
Bremer E
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.178.17.5071-5079.1996
Published
1996-09-00
Pages
5071-9
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC178300
Subset
IM
Databases
GENBANK
AF008220, U50082
External Links
PubMed source DOI: 10.1128/jb.178.17.5071-5079.1996 PMC Full Text View journal details
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