A novel family of Escherichia coli toxin-antitoxin gene pairs - PubMed (original) (raw)
A novel family of Escherichia coli toxin-antitoxin gene pairs
Jason M Brown et al. J Bacteriol. 2003 Nov.
Abstract
Bacterial toxin-antitoxin protein pairs (TA pairs) encode a toxin protein, which poisons cells by binding and inhibiting an essential enzyme, and an antitoxin protein, which binds the toxin and restores viability. We took an approach that did not rely on sequence homology to search for unidentified TA pairs in the genome of Escherichia coli K-12. Of 32 candidate genes tested, ectopic expression of 6 caused growth inhibition. In this report, we focus on the initial characterization of yeeV, ykfI, and ypjF, a novel family of toxin proteins. Coexpression of the gene upstream of each toxin restored the growth rate to that of the uninduced strain. Unexpectedly, we could not detect in vivo protein-protein interactions between the new toxin and antitoxin pairs. Instead, the antitoxins appeared to function by causing a large reduction in the level of cellular toxin protein.
Figures
FIG. 1.
Characteristics and genetic organization of bacterial TA pairs.
FIG. 2.
Ectopic expression of six candidate toxin genes causes growth inhibition. Log-phase cultures (OD600 = 0.5) were diluted in LB plus ampicillin (100 μg/ml) liquid medium to OD600 = 0.01 and grown in the presence or absence of 0.2% arabinose. The known toxin genes relE and mazF were included as positive controls.
FIG. 3.
YeeV, YkfI, and YpjF define a novel family of proteins. (A) Amino acid sequence alignment of the toxins YeeV, YkfI, and YpjF. Identical residues are highlighted in black, and chemically conserved residues are highlighted in gray. (B) Amino acid sequence alignment of the gene upstream of each toxin. (C) Chromosomal organization of yeeV, ykfI, ypjF, and the two proximal genes for each. The percent amino acid sequence identity for each homolog is indicated.
FIG. 4.
(A) Viability of strains expressing yeeV, ykfI, and ypjF. Growth in the presence (solid lines) or absence (dashed lines) of 0.2% arabinose was performed as described in the legend for Fig. 2. To quantitate CFU per milliliter, cells were diluted and plated on LB agar plus 100 μg of ampicillin/ml at the times indicated. (B) Analysis of mRNA and protein concentration for strains expressing _yeeV-_His, _ykfI-_His, _ypjF-His, or FLAG_-ypjF. Equivalent masses of total RNA and cellular protein isolated from log-phase cultures (OD600 = 0.5) shaken at 37°C for 30 min in the presence or absence of 0.2% arabinose were resolved by gel electrophoresis. mRNA was detected by hybridization with 32P-labeled DNA for each gene, and protein was detected using the appropriate antibody to each epitope tag.
FIG. 5.
Quantitation of His-tagged toxin protein. Cellular protein isolated from log-phase cultures (OD600 = 0.5) shaken at 37°C for 30 min in the presence or absence of 0.2% arabinose was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mass of total protein loaded was normalized using the optical density of each culture. Detection was performed using an anti-His6 antibody.
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