Ectoine-Induced Proteins in Sinorhizobium meliloti Include an Ectoine ABC-Type Transporter Involved in Osmoprotection and Ectoine Catabolism

Abstract

To understand the mechanisms of ectoine-induced osmoprotection in Sinorhizobium meliloti, a proteomic examination of S. meliloti cells grown in minimal medium supplemented with ectoine was undertaken. This revealed the induction of 10 proteins. The protein products of eight genes were identified by using matrixassisted laser desorption ionization-time-of-flight mass spectrometry. Five of these genes, with four other genes whose products were not detected on two-dimensional gels, belong to the same gene cluster, which is localized on the pSymB megaplasmid. Four of the nine genes encode the characteristic components of an ATP-binding cassette transporter that was named ehu, for ectoine/hydroxyectoine uptake. This transporter was encoded by four genes (ehuA, ehuB, ehuC, and ehuD) that formed an operon with another gene cluster that contains five genes, named eutABCDE for ectoine utilization. On the basis of sequence homologies, eutABCDE encode enzymes with putative and hypothetical functions in ectoine catabolism. Analysis of the properties of ehuA and eutA mutants suggests that S. meliloti possesses at least one additional ectoine catabolic pathway as well as a lower-affinity transport system for ectoine and hydroxyectoine. The expression of ehuB, as determined by measurements of UidA activity, was shown to be induced by ectoine and hydroxyectoine but not by glycine betaine or by high osmolality.

RpoE2 of Sinorhizobium meliloti is necessary for trehalose synthesis and growth in hyperosmotic media

“…]; this value is similar to the level found in strain 102F34 in the same experimental conditions (Gouffi et al, 1999;Jebbar et al, 2005;Talibart et al, 1994). Trehalose was not detected in the rpoE2 mutant in the same experimental conditions.…”

“…In contrast, in the absence of osmoprotectants, or in the presence of nonaccumulated osmoprotectants, osmoprotection is achieved by the synthesis and accumulation of glutamate, NAGGN and trehalose (Gouffi et al, 1999;Jebbar et al, 2005). The growth of the rpoE2 mutant is affected in hyperosmotic medium deprived of osmoprotectants or containing nonaccumulated osmoprotectants, suggesting that synthesis of glutamate, NAGGN or trehalose is affected.…”

The Compatible-Solute-Binding Protein OpuAC from Bacillus subtilis : Ligand Binding, Site-Directed Mutagenesis, and Crystallographic Studies

“…This result is surprising, since the site-directed change of the Trp residues to either Phe or Tyr residues within the E. coli ProX ligand-binding site has essentially no influence on ligand binding (40). Furthermore, mutational analysis of the aromatic residues within the binding site of the ectoine/hydroxyectoine binding protein EhuB from Sinorhizobium meliloti (14,21) (14). Replacements of these aromatic residues by Trp, the amino acid that has the strongest electronegative potential and is hence best suited for cation-interactions (11,31), created superbinding variants of EhuB that bind both ectoine and hydroxyectoine with K d values in a low nM range (14).…”

Negative Regulation of Ectoine Uptake and Catabolism in Sinorhizobium meliloti: Characterization of the EhuR Gene

“…bacteria presumably serve as potential sources of ectoine for S. meliloti (1). In addition, ectoine does not accumulate within S. meliloti cells; instead, it is degraded.…”

“…In addition, ectoine does not accumulate within S. meliloti cells; instead, it is degraded. It has been demonstrated that the ehuABCD-eutABCDE operon is involved in ectoine uptake and catabolism in S. meliloti, and an ABC transporter encoded by four genes (ehuA, ehuB, ehuC, and ehuD) has been reported to be responsible for ectoine uptake in this species (1). The ehuABCD genes form an operon with another cluster of putative ectoinecatabolic genes, previously named eutA, eutB, eutC, eutD, and eutE.…”