Involvement of EupR, a response regulator of the NarL/FixJ family, in the control of the uptake of the compatible solutes ectoines by the halophilic bacterium Chromohalobacter salexigens

Abstract

Background: Osmosensing and associated signal transduction pathways have not yet been described in obligately halophilic bacteria. Chromohalobacter salexigens is a halophilic bacterium with a broad range of salt tolerance. In response to osmotic stress, it synthesizes and accumulates large amounts of the compatible solutes ectoine and hydroxyectoine. In a previous work, we showed that ectoines can be also accumulated upon transport from the external medium, and that they can be used as carbon sources at optimal, but not at low salinity. This was related to an insufficient ectoine(s) transport under these conditions.

Results: A C. salexigens Tn1732-induced mutant (CHR95) showed a delayed growth with glucose at low and optimal salinities, could not grow at high salinity, and was able to use ectoines as carbon sources at low salinity. CHR95 was affected in the transport and/or metabolism of glucose, and showed a deregulated ectoine uptake at any salinity, but it was not affected in ectoine metabolism. Transposon insertion in CHR95 caused deletion of three genes, Csal0865-Csal0867: acs, encoding an acetyl-CoA synthase, mntR, encoding a transcriptional regulator of the DtxR/MntR family, and eupR, encoding a putative two-component response regulator with a LuxR_C-like DNA-binding helix-turn-helix domain. A single mntR mutant was sensitive to manganese, suggesting that mntR encodes a manganese-dependent transcriptional regulator. Deletion of eupR led to salt-sensitivity and enabled the mutant strain to use ectoines as carbon source at low salinity. Domain analysis included EupR as a member of the NarL/FixJ family of two component response regulators. Finally, the protein encoded by Csal869, located three genes downstream of eupR was suggested to be the cognate histidine kinase of EupR. This protein was predicted to be a hybrid histidine kinase with one transmembrane and one cytoplasmic sensor domain.

Conclusions: This work represents the first example of the involvement of a two-component response regulator in the osmoadaptation of a true halophilic bacterium. Our results pave the way to the elucidation of the signal transduction pathway involved in the control of ectoine transport in C. salexigens.

Figure 1

C. salexigens CHR95 can use ectoine as the sole carbon source at low salinity. Wild type (solid symbols) and CHR95 (open symbols) strains were grown at 37°C in M63 minimal medium with 20 mM glucose, 20 mM ectoine, or 20 mM hydroxyectoine and 0.6 (A), 0.75 (B), 1.5 (C) and 2.5 (D) M NaCl. Values shown are the mean of two replicas of each conditions in three independent experiment ± SD (standard deviation)

Figure 2

C. salexigens CHR95 is affected in the transport and metabolism of glucose. Cells grown in M63 with 1.5 M NaCl up to exponential phase were centrifuged, resuspended in the same medium to an OD600 of c. 0.6, supplemented with 100 μM of [¹⁴C]-glucose. After different times of incubation at 37°C, the glucose remaining in the supernatant (S) and cytoplasmatic solutes synthesized from ectoine, present in the ethanol insoluble (EIF) and soluble (ESF) fractions, respectively, were determined as described in Methods. The data are the averages of three different replicates ± SD (standard deviation).

Figure 3

C. salexigens CHR95 shows a deregulated ectoine uptake. The wild-type strain and the mutant CHR95 (ΔacseupRmntR::Tn1732) were grown in glucose M63 minimal medium containing the indicated concentration of NaCl. The measurement of 40 [¹⁴C]-ectoine uptake rates (vi, expressed as nmol min^-1 OD^-1 units) was performed as described in Methods. Experiments were repeated twice, and the data correspond to mean values.

Figure 4

C. salexigens CHR95 is not affected in the metabolism of ectoine. Cells grown in M63 with 1.5 M NaCl up to exponential phase were centrifuged, resuspended in the same medium to an OD₆₀₀ of ca. 0.6, supplemented with 87 μM of [¹⁴C]-ectoine and incubated with and without 20 mM of glucose. After 2 h incubation at 37°C, CO₂ production from ectoine (A) and macromolecules (EIF, B) and cytoplasmatic solutes (ESF, C) synthesized from ectoine, present in the ethanol insoluble and soluble fractions, respectively, were determined as described in Methods. The data are the averages of three different replicates ± SD (standard deviation).

Figure 5

Genetic organization of the C. salexigens eupR region and constructions derived from it. (A) C. salexigens genomic region containing eupR and Csal869, encoding its putative cognate histidine kinase, the mntH-mntR genes related to manganese transport, and the acs gene encoding a putative acetyl-CoA synthase. Promoters are indicated by angled arrows. The transcriptional terminator downstream of eupR is shown as a lollipop. (B) The same genomic region in C. salexigens CHR95. The insertion of Tn1732 deleted acs, eupR and mntR. (C) Generation of the eupR strain. eupR was inactivated by the insertion of an Ωaac cassette, which carries resistance genes for geneticin and gentamicin, into its unique site HpaI site (H). (D) Generation of the mntR strain. mntR was inactivated by the insertion of an Ω cassette, which carries resistance genes for streptomycin and spectinomycin, into its unique site HpaI site (H).

Figure 6

C. salexigens MntR is involved in the control of manganese uptake. 100 μL of overnight cultures of the wild type, CHR95 (ΔacseupRmntR::Tn1732) and CHR 161 (mntR::Ω) were placed on SW2 plates with 0.5 mM MnCl₂ and growth was observed after incubation at 37°C for 48 h.

Figure 7

C. salexigens EupR is involved in the control of ectoine uptake. Wild type strain (squares), CHR161 mutant (mntR::Ω) (triangles) and CHR183 mutant (eupR::Ωaac) (circles) were grown at 37°C in M63 medium with 20 mM ectoine (black markers) or 20 mM hydroxyectoine (white markers) and 0.6 (A), 0.75 (B) or 1.5 (C) M NaCl. Values shown are the mean of two replicas of each condition in three independent experiment ± SD (standard deviation)

Figure 8

In silico analysis of EupR and its putative cognate histidine kinase. (A) EupR is a two-component response regulator of the NarL/FixJ family of proteins. Neighbor-Joining tree based on proteins with a common LuxR_C-like conserved domain. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons. Bootstrap probabilities (as percentage) were determined from 1000 resamplings. Domain architecture of each group is represented at the side of the tree. The figure is based on the graphical output of the SMART web interface at http://smart.embl-heidelberg.de, with modifications. Sizes and positions of conserved domains are indicated by the labeled symbols. (B) Domain architecture of the EupR cognate histidine kinase. The figure is based on the graphical output of the SMART web interface at http://smart.embl-heidelberg.de, with modifications. Positions of conserved domains are indicated by symbols.