Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate

Abstract

Microbial niches contain toxic chemicals capable of forcing organisms into periods of intense natural selection to afford survival. Elucidating the mechanisms by which microbes evade environmental threats has direct relevance for understanding and combating the rise of antibiotic resistance. In this study we used a toxic small-molecule, bromoacetate, to model the selective pressures imposed by antibiotics and anthropogenic toxins. We report the results of genetic selection experiments that identify nine genes from Escherichia coli whose overexpression affords survival in the presence of a normally lethal concentration of bromoacetate. Eight of these genes encode putative transporters or transmembrane proteins, while one encodes the essential peptidoglycan biosynthetic enzyme, UDP-N-acetylglucosamine enolpyruvoyl transferase (MurA). Biochemical studies demonstrate that the primary physiological target of bromoacetate is MurA, which becomes irreversibly inactivated via alkylation of a critical active-site cysteine. We also screened a comprehensive library of E. coli single-gene deletion mutants and identified 63 strains displaying increased susceptibility to bromoacetate. One hypersensitive bacterium lacks yliJ, a gene encoding a predicted glutathione transferase. Herein, YliJ is shown to catalyze the glutathione-dependent dehalogenation of bromoacetate with a k(cat)/K(m) value of 5.4 × 10(3) M(-1) s(-1). YliJ displays exceptional substrate specificity and produces a rate enhancement exceeding 5 orders of magnitude, remarkable characteristics for reactivity with a nonnatural molecule. This study illustrates the wealth of intrinsic survival mechanisms that can be exploited by bacteria when they are challenged with toxins.

Fig. 1.

MurA inactivation by alkylation with bromoacetate. (A) MALDI mass spectrometry of MurA tryptic digest. The mass peak is from the peptide containing C115 of control (foreground, expected mass 3421.9 Da) and bromoacetate inactivated enzyme (background, mass increase of 57.7 Da). (B) Time-dependent loss of MurA activity in the presence of bromoacetate and 1 mM UDP-GlcNAc. Bromoacetate concentrations were 0.1 mM (•), 0.2 mM (▾), 0.3 mM (▪), and 0.4 mM (♦). Inset is a replot of the first-order rate constants (k_obs) vs. bromoacetate concentrations. Data were analyzed as described in SI Material and Methods and yielded a second-order inactivation rate constant (k_inact) of 11.4 ± 0.2 M^-1 s^-1. (C) Growth of E. coli BW25113 when overproducing wild-type, C115D or C115A MurA after 48 h incubation at 37 °C on LB-agar supplemented with bromoacetate (0.9 mM), chloramphenicol and IPTG.

Scheme 1.

(A) MurA catalyzed reaction. (B) GstB catalyzed reaction.

Fig. 2.

The E. coli genes gstB and gshA provide resistance to bromoacetate. (A) Michaelis–Menten plot of GstB catalyzed conjugation of glutathione to bromoacetate under saturating glutathione. This plot yields a k_cat value of 27 s^-1 a K_m value for bromoacetate of 5 mM and a second-order rate constant of 5.4 × 10³ M^-1 s^-1. (B) Growth of gstB- and gshA- strains on LB-agar supplemented with bromoacetate (0.45 mM), iodoacetate (0.125 mM) or bromoacetamide (0.175 mM) compared to the parental strain (BW25113).