Expression of different ParE toxins results in conserved phenotypes with distinguishable classes of toxicity

Ames, Jessica R.; Muthuramalingam, Meenakumari; Murphy, Teri J.; Najar, Fares Z.

doi:10.1002/mbo3.902

Cited by 12 publications

(14 citation statements)

References 76 publications

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“…Antitoxin contacts deduced from superposition of the AtYoeB structure (PDB ID 6N90) onto the E. coli YoeB-YefM complex (PDB ID 2A6Q) (Kamada and Hanaoka, 2005). more than 10 h of overexpression (Ames et al, 2019). We are able to produce recombinant AtYoeB in the absence of antitoxin with yields of 3-5 milligrams per liter of culture, and with greater than 7 milligrams per liter of culture when the antitoxin is co-expressed (see Materials and Methods section for induction details).…”

Section: Atyoeb Is Toxic To Agrobacterium But Not Escherichia Culturesmentioning

confidence: 99%

Identifying a Molecular Mechanism That Imparts Species-Specific Toxicity to YoeB Toxins

et al. 2020

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The ribosome-dependent E. coli (Ec) mRNase toxin YoeB has been demonstrated to protect cells during thermal stress. Agrobacterium tumefaciens (At), a plant pathogen, also encodes a YoeB toxin. Initial studies indicated that AtYoeB does not impact the growth of Ec, but its expression is toxic to the native host At. The current work examines this species-specific effect. We establish the highly similar structure and function of Ec and AtYoeB toxins, including the ability of the AtYoeB toxin to inhibit Ec ribosomes in vitro. Comparison of YoeB sequences and structures highlights a four-residue helix between β-strands 2 and 3 that interacts with mRNA bases within the ribosome. This helix sequence is varied among YoeB toxins, and this variation correlates with bacterial classes of proteobacteria. When the four amino acid sequence of this helix is transplanted from EcYoeB onto AtYoeB, the resulting chimera gains toxicity to Ec cells and lessens toxicity to At cells. The reverse is also true, such that EcYoeB with the AtYoeB helix sequence is less toxic to Ec and gains toxicity to At cultures. We suggest this helix sequence directs mRNA sequence-specific degradation, which varies among proteobacterial classes, and thus controls growth inhibition and YoeB toxicity.

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Section: Atyoeb Is Toxic To Agrobacterium But Not Escherichia Culturesmentioning

confidence: 99%

Identifying a Molecular Mechanism That Imparts Species-Specific Toxicity to YoeB Toxins

et al. 2020

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“…We have previously measured the toxicity of AtYoeB to E. coli and noted a delayed impact on colony forming units, with minimal impact on existing cells until more than ten hours of overexpression. [63] AtYoeB in the absence of antitoxin could reproducibly yield 3-5 milligrams per liter of LB culture, and with greater than 7 milligrams per liter of culture when the antitoxin is co-expressed (see Methods section for induction details).…”

Section: Resultsmentioning

confidence: 99%

A YoeB toxin from A. tumefaciens has metal-dependent DNA cleaving activity

McGillick

Ames

Murphy

et al. 2019

Preprint

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9Toxin-antitoxin (TA) systems, including YoeB-YefM, are important mediators of bacterial 10 physiological changes. Agrobacterium tumefaciens YoeB and YefM are similar to that from E. 11 coli, and interact as a tight heterotetramer with a KD of 653 pM. We have verified that AtYoeB 12 can perform both ribosome-dependent and -independent RNA cleavage. We have also 13 characterized a newly described metal-dependent and pH-sensitive DNA cleaving ability. We 14 note that this DNA cleaving ability is observed at toxin concentrations as low as 150 nM. The 15 dose-dependence of in vitro ribosome-independent RNA and metal-dependent DNA cleavage is 16 equivalent, and requires a ten-fold increase in toxin concentration as opposed to in the presence 17 of the ribosome. The toxin concentration inside bacterial cells is unknown and according to 18 current models, should increase upon activation of YoeB through degradation of the YefM 19 and chromosomes.[1] Type II TA systems are the most widely studied and contain both a protein 44 toxin and antitoxin.[4, 17] Type II toxins generally inhibit translation (HipA,[16] VapC,[18] 45 RelE,[19, 20] Doc,[21] HicA,[22] and MazF,[23, 24]), while a few toxins such as Par#,[25] 46 CcdB,[26] and Fic,[27] inhibit the DNA supercoiling gyrase enzyme. Despite the apparent 47Primary antibodies were an anti-pentaHis mouse antibody (Qiagen) and an anti-Strep mouse 106 antibody (IBA), while the secondary antibody as a fluorescently labeled goat anti-mouse 107 antibody (BioRad). Blots were imaged on using a ChemiDoc (BioRad). 108Crystallization of AtYoeB. Crystallization screening was carried out at 293 K in a 96-well plate 109 using sitting drop vapor diffusion crystallography with the aid of a Mosquito crystallization robot 110 (TPP LabTech). The JSCG+, PACT 1, and PACT 2 crystallization screens (Molecular 111 Dimensions) were first tested. Small, intergrown needle-like crystals appeared after 16-18 hours 112 from several conditions with polyethylene glycol (PEG) 3350 as a precipitant. Unfortunately, 113 further optimization by altering the pH, salt and precipitant concentration doesn't improve shape 114

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“…This superfamily contains toxins that assume a similar fold, although three different biochemical activities have been described for their members. Toxins of the RelE family (including YafQ and YoeB) inhibit translation by mRNA cleavage (Harms et al, 2018), toxins of the ParE family have been reported to interfere with DNA replication by inhibiting gyrase (Ames et al, 2019), and the Vibrio parahaemolyticus toxin Vp1843 has been shown to have a DNA nicking endonuclease activity (Zhang et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

The Cysteine Protease MaOC1, a Prokaryotic Caspase Homolog, Cleaves the Antitoxin of a Type II Toxin-Antitoxin System

2021

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The bloom-forming cyanobacterium Microcystis aeruginosa is known for its global distribution and for the production of toxic compounds. In the genome of M. aeruginosa PCC 7806, we discovered that the gene coding for MaOC1, a caspase homolog protease, is followed by a toxin-antitoxin module, flanked on each side by a direct repeat. We therefore investigated their possible interaction at the protein level. Our results suggest that this module belongs to the ParE/ParD-like superfamily of type II toxin-antitoxin systems. In solution, the antitoxin is predominantly alpha-helical and dimeric. When coexpressed with its cognate toxin and isolated from Escherichia coli, it forms a complex, as revealed by light scattering and affinity purification. The active site of the toxin is restricted to the C-terminus of the molecule. Its truncation led to normal cell growth, while the wild-type form prevented bacterial growth in liquid medium. The orthocaspase MaOC1 was able to cleave the antitoxin so that it could no longer block the toxin activity. The most likely target of the protease was the C-terminus of the antitoxin with two sections of basic amino acid residues. E. coli cells in which MaOC1 was expressed simultaneously with the toxin-antitoxin pair were unable to grow. In contrast, no effect on cell growth was found when using a proteolytically inactive MaOC1 mutant. We thus present the first case of a cysteine protease that regulates the activity of a toxin-antitoxin module, since all currently known activating proteases are of the serine type.

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Expression of different ParE toxins results in conserved phenotypes with distinguishable classes of toxicity

Cited by 12 publications

References 76 publications

Identifying a Molecular Mechanism That Imparts Species-Specific Toxicity to YoeB Toxins

Identifying a Molecular Mechanism That Imparts Species-Specific Toxicity to YoeB Toxins

A YoeB toxin from A. tumefaciens has metal-dependent DNA cleaving activity

The Cysteine Protease MaOC1, a Prokaryotic Caspase Homolog, Cleaves the Antitoxin of a Type II Toxin-Antitoxin System

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