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

Abstract

Ectoine has osmoprotective effects on Sinorhizobium meliloti that differ from its effects in other bacteria. Ectoine does not accumulate in S. meliloti cells; instead, it is degraded. The products of the ehuABCD-eutABCDE operon were previously discovered to be responsible for the uptake and catabolism of ectoine in S. meliloti. However, the mechanism by which ectoine is involved in the regulation of the ehuABCD-eutABCDE operon remains unclear. The ehuR gene, which is upstream of and oriented in the same direction as the ehuABCD-eutABCDE operon, encodes a member of the MocR/GntR family of transcriptional regulators. Quantitative reverse transcription-PCR and promoter-lacZ reporter fusion experiments revealed that EhuR represses transcription of the ehuABCD-eutABCDE operon, but this repression is inhibited in the presence of ectoine. Electrophoretic mobility shift assays and DNase I footprinting assays revealed that EhuR bound specifically to the DNA regions overlapping the Ϫ35 region of the ehuA promoter and the ϩ1 region of the ehuR promoter. Surface plasmon resonance assays further demonstrated direct interactions between EhuR and the two promoters, although EhuR was found to have higher affinity for the ehuA promoter than for the ehuR promoter. In vitro, DNA binding by EhuR could be directly inhibited by a degradation product of ectoine. Our work demonstrates that EhuR is an important negative transcriptional regulator involved in the regulation of ectoine uptake and catabolism and is likely regulated by one or more end products of ectoine catabolism.IMPORTANCE Sinorhizobium meliloti is an important soil bacterium that displays symbiotic interactions with legume hosts. Ectoine serves as a key osmoprotectant for S. meliloti. However, ectoine does not accumulate in the cells; rather, it is degraded. In this study, we characterized the transcriptional regulation of the operon responsible for ectoine uptake and catabolism in S. meliloti. We identified and characterized the transcription repressor EhuR, which is the first MocR/GntR family member found to be involved in the regulation of compatible solute uptake and catabolism. More importantly, we demonstrated for the first time that an ectoine catabolic end product could modulate EhuR DNA-binding activity. Therefore, this work provides new insights into the unique mechanism of ectoine-induced osmoprotection in S. meliloti.KEYWORDS Sinorhizobium meliloti, EhuR, MocR, ectoine, transcriptional regulator S inorhizobium meliloti is a soil bacterium that can be found either free-living or on the roots of leguminous plants. The ability to adapt to changes in the osmolality of the external soil environment is of vital importance for the growth and survival of soil bacteria, and ectoine serves as a key osmoprotectant for S. meliloti (1). Unlike many other bacteria, however, S. meliloti does not produce ectoine. Therefore, other soil

Transcriptional regulation of ectoine catabolism in response to multiple metabolic and environmental cues

“…We, and others, have recently shown that EnuR (= EhuR) serves as a repressor for ectoine catabolism (Schulz et al ., ; Yu et al ., ). Therefore, it came somewhat as a surprise that the uehA‐lacZ reporter fusion present on the enuR + plasmid pBAS20 exhibited higher induction levels in response to ectoines in comparison with plasmid pBAS21 that lacks an intact enuR regulatory gene (Fig.…”

“…C). This is consistent with the notion that EnuR acts genetically as a repressor (Schulz et al ., ; Yu et al ., ). Induction of the reporter fusion by either ectoine or hydroxyectoine in a chromosomal enuR deletion strain was less prominent, but was not completely eliminated (Fig.…”

“…Another intermediate of ectoine catabolism is L‐2,4‐diaminobutyric acid (DABA) (Fig. ) which has been suggested to function as an inducer for the EnuR orthologue EhuR from S. meliloti (Yu et al ., ). We found that DABA is also recognized by the R. pomeroyi EnuR protein and determined a K d of ligand binding of 457 ± 1 µM via MST (Fig.…”

The MocR‐like transcription factors: pyridoxal 5′‐phosphate‐dependent regulators of bacterial metabolism

“…Ectoine, aspartate and PLP exhibited no effects on the binding of EhuR to DNA probes. In contrast, DABA (a degradation product in the ectoine catabolism pathway) alone can inhibit the binding of EhuR to both promoters in a concentration‐dependent manner, with a slightly stronger inhibitory effect on the binding to ehuA promoter .…”

“…meliloti, a soil bacterium that can be found either free‐living or on the roots of leguminous plants, the ehuABCD‐eutABCDE operon is involved in ectoine uptake and catabolism . A mocR gene ( ehuR ; http://www.ncbi.nlm.nih.gov/protein/SMB_20426) is located upstream of this operon and oriented in the same direction . Although ehuR and ehuABCD‐eutABCDE form a polycistronic transcriptional unit, ehuABCD‐eutABCDE has its own promoter.…”