Identification of a third osmoprotectant transport system, the osmU system, in Salmonella enterica

Abstract

The growth of Salmonella enterica serovar Typhimurium mutants lacking the ProP and ProU osmoprotectant transport systems is stimulated by glycine betaine in high-osmolarity media, suggesting that this organism has an additional osmoprotectant transport system. Bioinformatic analysis revealed that the genome of this organism contains a hitherto-unidentified operon, designated osmU, consisting of four genes whose products show high similarity to ABC-type transport systems for osmoprotectants in other bacteria. The osmU operon was inactivated by a site-directed deletion, which abolished the ability of glycine betaine to alleviate the inhibitory effect of high osmolarity and eliminated the accumulation of [(14)C]glycine betaine and [(14)C]choline-O-sulfate in high-osmolarity media in a strain lacking the ProP and ProU systems. Although the OsmU system can take up glycine betaine and choline-O-sulfate, these two osmoprotectants are recognized at low affinity by this transporter, suggesting that there might be more efficient substrates that are yet to be discovered. The transcription of osmU is induced 23-fold by osmotic stress (0.3 M NaCl). The osmU operon is present in the genomes of a number of Enterobacteriaceae, and orthologs of the OsmU system can be recognized in a wide variety of Bacteria and Archaea. The structure of the periplasmic binding protein component of this transporter, OsmX, was modeled on the crystallographic structure of the glycine betaine-binding protein ProX of Archaeoglobus fulgidus; the resultant model indicated that the amino acids that constitute substrate-binding site, including an "aromatic cage" made up of four tyrosines, are conserved between these two proteins.

Fig 1

Osmoprotectants used in the present study.

Fig 2

Glycine betaine can alleviate the inhibitory effects of high osmolarity in a ProP⁻ ProU⁻ OsmU⁺ strain. (A and B) The effect of glycine betaine in liquid M63 glucose plus 0.65 M NaCl was determined as described in Materials and Methods. Open symbols, strains grown in the absence glycine betaine; closed symbols, strains grown with of 1 mM glycine betaine (GB). Results for strains TL1 (wild type; ProP⁺ ProU⁺ OsmU⁺), TL188 (ProP⁺ ProU⁻ OsmU⁺), and TL3463 (ProP⁻ ProU⁺ OsmU⁺) (A) and strain TL3465 (ProP⁻ ProU⁻ OsmU⁺) (B) are shown. (C) Osmoprotective effect of glycine betaine on solid medium containing M63 glucose plus 0.65 M NaCl (left petri dish) or with 1 mM glycine betaine (right petri dish). Cells from single colonies grown on M63 glucose plates were streaked onto these plates, followed by incubation at 37°C for 4 days. The test strains were as follows (clockwise from the top): TL1 (ProP⁺ ProU⁺ OsmU⁺), TL3465 (ProP⁻ ProU⁻ OsmU⁺), and TL4099 (ProP⁻ ProU⁻ OsmU⁻).

Fig 3

Deletion of the osmU operon eliminates the accumulation of glycine betaine as an osmoprotectant in a ProP⁻ ProU⁻ strain. The effect of glycine betaine on the growth of strains was determined in M63 glucose plus 0.65 M NaCl, as described in Materials and Methods. Open and closed symbols: strains grown in the absence or presence of 1 mM glycine betaine, respectively. (A) Strain TL4099 (ProP⁻ ProU⁻ OsmU⁻); (B) strains TL4093 (ProP⁺ ProU⁺ OsmU⁻), TL4095 (ProP⁺ ProU⁻ OsmU⁻), and TL4097 (ProP⁻ ProU⁺ OsmU ⁻).

Fig 4

The osmU operon is induced by osmotic stress. Strain TL1 was grown in MOPS medium plus glucose with 0 M or 0.3 M NaCl, and the osmU and gnd transcript levels were determined by qRT-PCR as described in Materials and Methods. The data are shown as the ratios of osmU and gnd mRNAs, with the ratio for the cells grown with 0 M NaCl set to 1. For both media, the results are the averages of results obtained with four independent mid-exponential phase cultures. In a two-sample, one-tailed t test, the difference in the ratios of osmU/gnd expression at 0.3 M NaCl and 0 M NaCl was significant (P < 0.01).

Fig 5

Conservation of orthologous genes flanking the osmU operon in Enterobacteriaceae. The synteny of genes was analyzed and displayed using the SEED database and algorithm (http://pubseed.theseed.org/seedviewer.cgi) (43). Sets of orthologous genes that are conserved around the osmU operon are shown in the same colors, with the direction of transcription indicated by the arrows; genes that are gray are not conserved across these organisms. Conserved orthologs encode the proteins as follows: dmsBCD, three subunits of dimethylsulfate reductase; clcB, Cl⁻ channel; bioD, dethiobiotin synthetase; mlc, maltose regulon repressor; ynfL, putative LysR family repressor; ynfM, major superfamily transporter; mldR, multidrug resistance protein B; hypP, hypothetical protein. The organisms shown are S. Typhimurium, Escherichia fergusonii B253, Citrobacter rodentium ICC168, Enterobacter cloacae subsp. cloacae NTC9394, Cronobacter turicensis, Pantoea sp. strain At-9b, Erwinia billingiae Eb661, Serratia marcescens DB11, and Chromobacterium violaceum ATCC 12472.

Fig 6

Phylogenetic relationship of the S. Typhimurium OsmX protein to YehZ and the extracellular binding protein components of other osmoprotectant transport systems whose three-dimensional structure has been determined. The phylogenetic tree of the protein sequences (including the signal peptides) was constructed with the PhyML method and displayed by the web service Phylogeny.fr (http://www.phylogeny.fr), using the “advanced mode, bootstrapping procedure” (100 bootstraps), without the Gblocks program (7, 17, 18, 22). The protein sequences (database accession codes) are as follows: OsmX, S. Typhimurium (GI:16764838); YehZ, S. Typhimurium (GI:16765495); ProX, A. fulgidus DSM 4304 (GI:11498587); OpuCC, B. subtilis subsp. subtilis strain 168 (GI:16080434); OpuBC, B. subtilis subsp. subtilis strain 168 (GI:16080424); OpuCC, S. aureus subsp. aureus Mu50 (GI:15925436); OpuAC, L. lactis (GI:296278460); OpuAC, B. subtilis subsp. subtilis strain 168 (GI:16077369); ChoX, S. meliloti 1021 (GI:15966152), and ProX, E. coli strain K-12 substrain MG1655 (GI:16130593).

Fig 7

S. enterica OsmX modeled on the glycine betaine-bound conformation of A. fulgidus ProX. A surface slab view is shown for ProX (A) and the modeled OsmX (B) bound to glycine betaine (yellow). Residues within 5 Å of the bound substrate are indicated in cyan. Figure was constructed with PyMOL (www.pymol.org) as described in Materials and Methods. The amino acids are numbered starting from the first residue of the mature protein for ProX (49) and starting from the N-terminal methionine of the predicted full-length protein, including the signal sequence, for OsmX.