Conserved ABC Transport System Regulated by the General Stress Response Pathways of Alpha- and Gammaproteobacteria

Abstract

Brucella abortus σ^E1 is an EcfG family sigma factor that regulates the transcription of dozens of genes in response to diverse stress conditions and is required for maintenance of chronic infection in a mouse model. A putative ATP-binding cassette transporter operon, bab1_0223-bab1_0226, is among the most highly activated gene sets in the σ^E1 regulon. The proteins encoded by the operon resemble quaternary ammonium-compatible solute importers but are most similar in sequence to the broadly conserved YehZYXW system, which remains largely uncharacterized. Transcription of yehZYXW is activated by the general stress sigma factor σ^S in Enterobacteriaceae, which suggests a functional role for this transport system in bacterial stress response across the classes Alphaproteobacteria and Gammaproteobacteria We present evidence that B. abortus YehZYXW does not function as an importer of known compatible solutes under physiological conditions and does not contribute to the virulence defect of a σ^E1-null strain. The sole in vitro phenotype associated with genetic disruption of this putative transport system is reduced growth in the presence of high Li⁺ ion concentrations. A crystal structure of B. abortus YehZ revealed a class II periplasmic binding protein fold with significant structural homology to Archaeoglobus fulgidus ProX, which binds glycine betaine. However, the structure of the YehZ ligand-binding pocket is incompatible with high-affinity binding to glycine betaine. This is consistent with weak measured binding of YehZ to glycine betaine and related compatible solutes. We conclude that YehZYXW is a conserved, stress-regulated transport system that is phylogenetically and functionally distinct from quaternary ammonium-compatible solute importers.IMPORTANCEBrucella abortus σ^E1 regulates transcription in response to stressors encountered in its mammalian host and is necessary for maintenance of chronic infection in a mouse model. The functions of the majority of genes regulated by σ^E1 remain undefined. We present a functional/structural analysis of a conserved putative membrane transport system (YehZYXW) whose expression is strongly activated by σ^E1 Though annotated as a quaternary ammonium osmolyte uptake system, experimental physiological studies and measured ligand-binding properties of the periplasmic binding protein (PBP), YehZ, are inconsistent with this function. A crystal structure of B. abortus YehZ provides molecular insight into differences between bona fide quaternary ammonium osmolyte importers and YehZ-related proteins, which form a distinct phylogenetic and functional group of PBPs.

Keywords: ABC transporter; Alphaproteobacteria; EcfG; RpoS; YehZ; YehZYXW; general stress response; lithium; sigma factor.

FIG 1

Regulated expression of the B. abortus yehZYXW operon (bab1_0223-bab1_0226). (A) (Top) Differential expression of the yehZYXW operon under oxidative stress in B. abortus ΔrpoE1 relative to the wild type (WT) measured by RNA-seq. A log₂ negative fold change indicates genes that had decreased transcription levels in a ΔrpoE1-null mutant relative to the wild type (24, 25). The error bars indicate standard deviations (SD). (Bottom) Cartoon representation of the yehZYXW promoter region, with the predicted σ^E1 binding site. (B) Cartoon representation of the YehZYXW ABC transport system. When present in the periplasmic space, an unknown ligand specifically interacts with the periplasmic binding protein YehZ (Bab1_0226) (blue). Ligand-bound YehZ then contacts the YehYW transmembrane permease (Bab1_0225, Bab1_0223) and delivers the molecule into the cytoplasmic space. Ligand translocation from the periplasm to the cytoplasm is energized by ATP hydrolysis in the nucleotide-binding domain protein YehX (Bab1_0224). OM, outer membrane; IM, inner membrane.

FIG 2

Growth of B. abortus wild-type (WT), ΔrpoE1, and ΔyehZ strains under different osmotic conditions (Biolog PM9 plate). The concentration of NaCl was increased stepwise from 1 to 10%, and 23 possible osmoprotective molecules were tested in the presence of 6% (1 M) NaCl. The area under each growth curve was calculated to quantify growth. The percentage of growth was calculated for each strain under each condition; 1% NaCl was considered 100% (maximum growth) and 10% NaCl was considered 0% (no growth; subtracted from each reading). The error bars represent SD.

FIG 3

Brucella growth on solid agar in the presence of glycine betaine and NaCl. After 5 days of incubation at 37°C on TSA plates, growth of B. abortus wild-type and ΔyehZ strains in the presence of 200 or 300 mM NaCl either with (+) or without (−) 20 mM glycine betaine was determined. Growth of B. abortus wild-type and ΔyehZ strains on plain TSA plates (0 mM NaCl) with (+) or without (−) 20 mM glycine betaine is also shown.

FIG 4

In vitro and in vivo infection assays. (A) Infection of differentiated THP-1 cells with wild-type B. abortus strain 2308 (WT) and the ΔyehZ strain. Inactivated macrophages (IM), activated macrophages (AM), or immature dendritic cells (IDC) were infected. The numbers of B. abortus CFU recovered from the different THP-1-derived cell types at 1, 24, 48, and 72 h postinfection are plotted. Each infection was performed in triplicate; the error bars represent standard errors of the mean (SEM). (B) Mice (n = 5 per strain per time point) were injected intraperitoneally with 5 × 10⁴ brucellae (wild type, ΔyehZ, and ΔyehZ ectopically complemented). Spleens were harvested, weighed (top), and plated for CFU enumeration (bottom) at 1, 2, 4, and 8 weeks postinfection. The error bars represent SEM.

FIG 5

Comparison of B. abortus YehZ to osmolyte-binding PBPs. (A) Neighbor-joining phylogenetic tree of YehZ-related proteins and osmolyte-binding proteins. YehZ sequences from B. abortus, E. coli, and A. tumefaciens and sequences of the osmolyte-binding proteins A. fulgidus ProX, B. subtilis OpuCC and OpuBC, L. lactis OpuAC, B. burgdorferi ProX, S. meliloti ChoX and EhuB, B. abortus ChoX and Bab2_502, and E. coli ProX are included. Species names and the ligands bound by the proteins are indicated; PBPs with no known ligand are labeled with a question mark. IbpA from C. crescentus was used as an outgroup. The bootstrap values for 100 replicates are shown next to the nodes. YehZ proteins from B. abortus, E. coli, and A. tumefaciens (blue shading) and A. fulgidus ProX and B. subtilis OpuCC and OpuBC proteins (green shading) were used for the multiple-sequence alignment. (B) Multiple-sequence alignment of B. abortus (Bra), E. coli (Esc) (PDB ID 4WEP), and A. tumefaciens (Agt) (PDB ID 4NE4) YehZ proteins (blue shading) and A. fulgidus ProX (Arf) (PDB ID 1SW2) and B. subtilis OpuCC (Bs1) (PDB ID 3PPP) and OpuBC (Bs2) (PDB ID 3R6U) proteins (green shading). Residue numbering starts with the first residue present in the corresponding structure in the PDB. Identical residues are shaded in black, and homologous residues are shaded in gray. Secondary structures (β-strands and α-helices) are indicated above the alignment and numbered. Residues interacting with the ligand in the ligand-bound structures are highlighted in red and green. The asparagine residue (N127) of B. abortus YehZ that interacts with the arginine or the histidines of the His tag is marked with a purple dot.

FIG 6

Ligand interaction assays. (A) Purified B. abortus YehZ (25 μM) was progressively denatured in the presence (blue circles) or absence (gray circle) of different concentrations (0.25 to 250 mM) of potential ligands. As a positive control, C. crescentus IbpA (25 μM) was thermally denatured in the presence (green circles) or absence (gray circle) of increasing concentrations of myo-inositol. Average temperatures of denaturation are indicated on the graph for the proteins alone and the proteins with tested molecules. The SD, if significant, is also shown as light-colored circles. The melting profile of each protein was independently measured 3 times for each condition. (B) (Left) Low concentrations; representative isothermal titration calorimetry data from purified B. abortus YehZ. Ligands (glycine betaine, choline, ectoine, and carnitine) at 1 mM concentration were injected (1-μl injections) every 3 min in the calorimetric cell containing 200 μl of protein (at 100 μM). (Right) High concentrations; representative isothermal titration calorimetry curve from purified B. abortus YehZ with 40 mM glycine betaine injected (2 μl) every 2 min in the calorimetric cell containing 200 μl of 100 μM protein.

FIG 7

1.6-Å X-ray crystal structure of B. abortus YehZ. (A) (Top) Simplified representation of YehZ presenting an N-terminal signal peptide (M1 to A24) followed by the first portion of lobe 1 (Q25 to P122), the first segment of the hinge (S123 to T128), lobe 2 (W129 to D235), the second segment of the hinge (K236 to Q243), and the second portion of lobe 1 (P244 to K304). (Bottom) Open apo structure of B. abortus YehZ (Q25 to K304) showing the two lobes (1 and 2 [light blue]) articulated around a two-segment hinge that form the cavity (light red) (opening angle, ∼59°). β-Strands and α-helices are colored yellow and blue, respectively. (B) The YehZ cavity is negatively charged (red). Residues from the His tag are found in each cavity of the two molecules present in the asymmetric unit. (Top) Two histidines (green) from the His tag are present in the molecule A cavity. (Bottom) The arginine from the His tag (green) is present in the molecule B cavity. (C) Structural superposition of B. abortus YehZ (blue sticks) and A. fulgidus ProX (orange sticks) (PDB ID 1SW5) (33) cavities. Residues forming the aromatic betaine-binding box (represented as a green surface) and residues interacting with the ligand carboxylate group in the A. fulgidus ProX structure are shown. Equivalent residues in B. abortus YehZ are also displayed. For both cavities, residues not directly involved in ligand interaction but important to preserve the physicochemical characteristics of the cavities are shown as orange lines (A. fulgidus ProX) and blue lines (B. abortus YehZ) (compare the multiple-sequence alignment in Fig. 5B). (D) Model of the B. abortus YehZ cavity in a closed conformation built using the liganded A. fulgidus ProX (PDB ID 1SW2) as a template. The same color code as in panel C was used. Residues W129 and N84 in the B. abortus YehZ cavity can adopt multiple conformations, but only two rotamers for each residue are compatible with the closed-form model. Interactions between glycine betaine (GB) (yellow) and the residues present in the A. fulgidus ProX cavity (PDB ID 1SW2) are represented by dashed lines. The multiple-sequence alignment in Fig. 5B shows a sequence comparison.