Part:BBa_K3512002

CcdA Antitoxin

CcdA antitoxin is part of the CcdA-CcdB toxin-antitoxin system. The target of CcdB is the GyrA subunit of DNA gyrase, an essential type II topoisomerase in Escherichia coli. Gyrase alters DNA topology by effecting a transient double-strand break in the DNA backbone, passing the double helix through the gate and resealing the gaps. The CcdB toxin acts by trapping DNA gyrase in a cleaved complex with the gyrase A subunit covalently closed to the cleaved DNA, causing DNA breakage and cell death in a way closely related to quinolones antibiotics.

In absence of the antitoxin CcdA, the CcdB toxin traps DNA-gyrase cleavable complexes, inducing breaks into DNA and cell death.

Contribution: XHD-Wuhan-Pro-China 2021

Toxicity and antitoxicity plate assays

To test the toxicity of the cloned CcdBO157 and CcdBF variants, the corresponding pBAD33-ccdB constructs were transformed in MG1655. The resulting transformants were plated on LB plates containing chloramphenicol with or without arabinose (1%). The CcdB variants were considered to be functional (toxic) when transformants were able to grow only in the absence of arabinose. To test the ability of the cloned CcdAO157 and CcdAF variantsto counteract the toxicity of CcdBO157 and CcdBF, respectively,the corresponding pKK-ccdA constructs were transformed in MG1655 expressing the reference ccdBO157 orccdBF genes from the pBAD33 vector. The resulting transformants were plated on LB plates containing chloramphenicol and ampicillin with arabinose (1%). Basal expression of ccdA from the pTac promoter of pKK223-3 in MG1655 is sufficient to test the antitoxicity phenotype. The CcdA variants were considered to be functional when the toxicity of CcdBO157 or CcdBF protein was counteracted, i.e., when strains coexpressing a ccdA variant with the ccdBO157 or ccdBF reference genes were able to grow in the presence of arabinose while strains expressing only the ccdBO157 or ccdBF reference gene were not.

Result

We identified a small sequence that presents identity with the 39 region of ccdAO157 and two putative ORFs (Figure 4, A and B). The 2704-bp IR corresponds to that of 1425 bp with the insertion of an IS621 in the sequence presenting identity with ccdAO157.

Table 3 shows the amino acid sequences of the corresponding CcdA proteins. The 47 antitoxin proteins presented very few variations, except in the case of CcdAO138. Overall, seven classes of alleles could be identified. The most prevalent one was identical to the ccdAO157 gene of the O157:H7 EDL933 reference strain (37/47 isolates). Five classes, representing 9/47 isolates, presented single point variations (S76R, A54T, D72E, T47I, or T34M). The capacity of 1 representative protein of each class to antagonize the toxic activity of the reference CcdBO157 protein was tested, using the antitoxicity plate assay. The single point variations did not affect the capacity of the CcdA variants to antagonize CcdBO157 activity (data not shown, Table 3). The last class (1 isolate) presented a frameshift mutation caused by a 1-nt deletion. This led to a major modification of the carboxy terminus of CcdAO138 that affects the antitoxic activity of this variant. Indeed, when co-expressed with CcdBO157, this protein was unable to restore viability, showing that CcdAO138 is inactive (Figure 5A). This result was expected since it has been shown that the carboxy-terminal domain of CcdAF is responsible for the antitoxin activity (Bernard and Couturier 1991).

Discussion

Interestingly, the evolution of ccdAO157-like antitoxins and of the flanking apaH and folA genes appears to be much more constrained. Inactivation of the toxin gene prior to the antitoxin gene presumably constitutes the first and safer step of TA systems degradation.

Indeed, the inactive CcdBO138 variant is coupled either to the inactive CcdAO138 as in the case of the ccdO138 variant or to an active antitoxin as in the case of the ccdO153 variant. Thus, the situation at present strongly indicates a decay of the ccdO157 system. An alternative hypothesis is that the antitoxin might play an antiaddictive role as shown for the ccdEch system (Saavedra De Bast et al. 2008) although not against CcdBF-like toxin since CcdAO157 antitoxin does not protect against ccdF addiction (Wilbaux et al. 2007).

Reference

Mine, N., Guglielmini, J., Wilbaux, M. and Van Melderen, L., 2009. The Decay of the Chromosomally Encoded ccdO157 Toxin–Antitoxin System in the Escherichia coli Species. Genetics, 181(4), pp.1557-1566.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]