Molecular structure and function of the novel BrnT/BrnA toxin-antitoxin system of Brucella abortus

Abstract

Type II toxin-antitoxin (TA) systems are expressed from two-gene operons that encode a cytoplasmic protein toxin and its cognate protein antitoxin. These gene cassettes are often present in multiple copies on bacterial chromosomes, where they have been reported to regulate stress adaptation and persistence during antimicrobial treatment. We have identified a novel type II TA cassette in the intracellular pathogen Brucella abortus that consists of the toxin gene, brnT, and its antitoxin, brnA. BrnT is coexpressed and forms a 2:2 tetrameric complex with BrnA, which neutralizes BrnT toxicity. The BrnT(2)-BrnA(2) tetramer binds its own promoter via BrnA, and autorepresses its expression; its transcription is strongly induced in B. abortus by various stressors encountered by the bacterial cell during infection of a mammalian host. Although highly divergent at the primary sequence level, an atomic resolution (1.1 Å) crystal structure of BrnT reveals a secondary topology related to the RelE family of type II ribonuclease toxins. However, overall tertiary structural homology to other RelE family toxins is low. A functional characterization of BrnT by site-directed mutagenesis demonstrates a correspondence between its in vitro activity as a ribonuclease and control of bacteriostasis in vivo. We further present an analysis of the conserved and variable features of structure required for RNA scission in BrnT and the RelE toxin family. This structural investigation informs a model of the RelE-fold as an evolutionarily flexible scaffold that has been selected to bind structurally disparate antitoxins, and exhibit distinct toxin activities including RNA scission and DNA gyrase inhibition.

FIGURE 1.

The brnTA operon. A, the brnT and brnA genes overlap by 1 bp. B, clustal multiple sequence alignment of B. abortus BrnT toxin and homologous toxin sequences from 10 other Gram-negative species including several human pathogens. Ba, B. abortus (GI: 82615932); Ec, E. coli (GI: 309704811); Yp, Yersinia pestis (GI: 22126312); Ypt, Yersinia pseudotuberculosis (GI: 170024520); Bpp, Bordatella parapertussis (GI: 33596571); Nm, Neisseria meningitidis (GI: 161870004); Hi, Haemophilus influenzae (GI: 270670382); Bp, Burkholderia pseudomallei (GI: 217424545); Vc, Vibrio cholerae (GI: 15601097); Cb, Coxiella burnetii (GI: 154705785); Cc, Caulobacter crescentus (GI: 16124744). Boxes shaded black are amino acids identical in 40% of sequences shown. Boxes shaded gray are similar in 40% of sequences.

FIGURE 2.

B. abortus BrnTA is a toxin-antitoxin system. A, antitoxin (brnA, blue), toxin (brnT, red), or brnTA operon (green) were expressed in E. coli and optical density at 600 nm (A₆₀₀) was measured at 30-min intervals. B, starting at the time of induction (marked with black arrows) colony forming units were quantified on LB agar. C, growth curves of B. abortus 2308 + pSRK-Km (empty vector control, black), pSRK-brnT (red), pSRK-brnA (blue), or pSRK-brnTA (green). A₆₆₀ was measured every hour; expression of genes from pSRK was induced after 4 h (black arrow). D, B. abortus viable colony forming units after induction of gene expression were enumerated by plating on Schaedler blood agar. E, E. coli Plac-brnT + Para-brnA or Plac-brnT + Para empty vector control (EVC) were grown in the presence or absence of 0.5 mm IPTG for increasing amounts of time. Cells were then plated on LB agar containing ampicillin and kanamycin ± 0.2% arabinose. Solid lines indicate the presence of arabinose in agar; dashed lines indicate agar without arabinose. + or − indicates induction of specific protein. Error bars represent S.D.

FIGURE 3.

BrnTA forms a tetramer consisting of two antitoxins and two toxins. A, c(S) distribution calculated from sedimentation velocity measurements of the purified BrnTA complex. BrnT-BrnA sediments as a single 2.9 ± 0.3 S species (root mean square deviation = 0.006). Resolution of this purified complex by 14% SDS-PAGE reveals multiple Coomassie-stained bands; mass spectrometry of tryptic peptides from these excised bands demonstrates that they contain either BrnT or BrnA, labeled as A or T to the right of each band. B, top, nESI-MS of purified BrnA-BrnT identifies a major complex with a molecular weight that matches a tetramer with 2:2 stoichiometry. B, bottom, 70-V CID spectrum of the 15+ tetramer ion species reveals monomeric and trimeric product ions, supporting a model in which the precursor is a 2:2 tetramer. C, size exclusion chromatography of BrnT (red) and BrnA (blue) and SDS-PAGE of major peaks. D, c(S) distribution from AUC of BrnT (red) and BrnA (blue), BrnT is 13.2 kDa, root mean square deviation = 0.022; frictional ratio = 1.2. BrnA is 29.3 kDa, root mean square deviation = 0.006, frictional ratio = 1.8.

FIGURE 4.

Structure of BrnT. A, ribbon structure of BrnT. β-Strands, pink; α-helices, light blue. B, electrostatic surface map of BrnT; basic, blue; acidic, red. C, conservation surface map of BrnT reveals the structural position of residues that are conserved among BrnT homologs (DUF497 in the Conserved Domain Database). Coordinates of B. abortus BrnT have been deposited in the Protein Data Bank (PDB ID 3U97).

FIGURE 5.

BrnT is a ribonuclease toxin. A, quantification of in vivo protein synthesis in E. coli 30 min after induction of brnA, brnT, or the brnTA operon; expression shown in counts per minute (cpm). Error bars represent S.D; * = p < 0.01 (one-way analysis of variance, Tukey post test). B, degradation of lacZ RNA incubated with water or buffer controls or purified BrnT, BrnA, or BrnT + BrnA (see “Experimental Procedures”). Stars indicate equal concentration of BrnT.

FIGURE 6.

Structure/function analysis of BrnT. A, amino acid sequence of B. abortus BrnT. Mutated residues are colored in red and green depending on their effect on BrnT toxicity (no effect or strong effect, respectively). Sequence position of secondary structure elements are labeled above the alignment. Conserved residues are highlighted with black (identical residues) and gray (similar residues) squares; shading is thresholded at 50% (based on alignment with 97 homologous sequences). Residues predicted to be involved in RNA binding as assessed by three different algorithms are highlighted with purple (RNABindR), pink (BindN), and orange (PiRaNhA) squares. B, ribbon structure of BrnT. β-Strands, pink; α-helices, light blue. Side chains of mutated residues are shown as sticks and colored in red and green depending on their effect on BrnT toxicity (no effect or strong effect, respectively). C, site-directed mutagenesis of conserved charged amino acids in BrnT reveals residues required for full toxicity when compared with wild-type toxin. Red bars indicate toxicity similar to wild-type, green bars indicate diminished toxicity, and white bars indicate uninduced controls. D, RNase activity of purified wild-type BrnT or BrnT point mutants was assessed by monitoring uncleaved RNA on an agarose gel. Error bars represent S.D.

FIGURE 7.

Structural comparison between BrnT and members of the RelE toxin family. For each toxin (BrnT, RelE, YoeB, and MqsR) a secondary structure diagram and a surface rendered model (white) are shown. Residues that are known to be required for RNA cleavage in each of these structures are highlighted in red. PDB codes of each structure are in parentheses.

FIGURE 8.

BrnA negatively autoregulates the brnTA operon; brnTA transcription is activated by multiple stressors. Purified antitoxin (A) and TA complex (B), but not toxin alone (C) bind to ³²P-labeled probe corresponding to the 50 nucleotides upstream of the brnT start codon, and cause a gel shift in EMSA. D, β-galactosidase assay of wild-type B. abortus or B. abortus ΔbrnTA carrying a PbrnTA-lacZ promoter fusion plasmid. E, TaqMan assay quantifying brnT transcript in wild-type B. abortus in various stress conditions including 5 mm H₂O₂ in Gerhardt's minimal media, heat shock (44 °C), pH 4.0, and 200 μg/ml of chloramphenicol. Each sample is normalized to 16 S RNA and then compared with a relevant non-stressed control. Error bars represent S.D.