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Gene Review

ccdB  -  CcdB

Escherichia coli B171

 
 
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Disease relevance of ccdB

 

High impact information on ccdB

  • The sequence was fused at the C-terminal end of the CcdB and CcdA proteins encoded by plasmid F [3].
  • Every residue of the 101-aa Escherichia coli toxin CcdB was substituted with Ala, Asp, Glu, Lys, and Arg by using site-directed mutagenesis [4].
  • At all buried positions, introduction of Asp results in an inactive phenotype at all CcdB transcriptional levels [4].
  • CcdA has two binding sites for CcdB and vice versa, permitting soluble hexamer formation but also causing precipitation, especially at CcdA:CcdB ratios close to one [5].
  • Modulation of DNA supercoiling activity of Escherichia coli DNA gyrase by F plasmid proteins. Antagonistic actions of LetA (CcdA) and LetD (CcdB) proteins [6].
 

Biological context of ccdB

  • The system is based on a plasmid construct containing an inducible marker gene ccdB ("killer" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements [7].
  • Based on these results, we propose a model in which the ratio between CcdA and CcdB regulates the repression state of the ccd operon [2].
  • All missense mutations which inactivate CcdB killer activity are located in the region coding for the last three C-terminal residues [8].
  • We show that the gyrA462 mutation suppresses this SOS activation, indicating that SOS induction is a consequence of DNA damages promoted by the CcdB protein on gyrase-DNA complexes [9].
  • The crystal structure of CcdB and the dimerization domain of the A subunit of gyrase (GyrA14) dictates an open conformation for the catalytic domain of gyrase when CcdB is bound [10].
 

Anatomical context of ccdB

 

Associations of ccdB with chemical compounds

  • However, in the presence of ATP, or the non-hydrolysable analogue 5'-adenylyl beta,gamma-imidodiphosphate, microcin B17 stabilises a gyrase-dependent DNA cleavage complex in a manner reminiscent of quinolones, Ca(2+), or the bacterial toxin CcdB [11].
  • Quinolones, coumarins, cyclothialidines, CcdB and microcin B17 inhibit DNA gyrase [12].
  • Two substitutions lead to CcdB-promoted killing: glutamine 33-->cysteine and glutamine 33-->phenylalanine [13].
  • The mutants can be directly selected on LB plates containing IPTG, through which the toxic CcdB protein is induced, thereby eliminating cells carrying wild-type parental plasmids [14].
 

Analytical, diagnostic and therapeutic context of ccdB

  • Crystallization of CcdB in complex with a GyrA fragment [1].
  • By using the Escherichia coli cytotoxin CcdB as a model system, simple procedures for generating Ts mutants at high frequency through site-directed mutagenesis were developed [15].
  • Surface plasmon resonance shows that CcdB binds to the N-terminal domain of GyrA with high affinity [16].
  • An initial complex (alpha) is formed by direct interaction between GyrA and CcdB; this complex can be detected by affinity column and gel-shift analysis, and has a proteolytic signature which is characterised by a 49 kDa fragment of GyrA [16].
  • These vectors are based on the control of cell death CcdB direct selection technology and are well adapted to the cloning of blunt-ended PCR products that were generated by using thermostable polymerases that provide proofreading activity [17].

References

  1. Crystallization of CcdB in complex with a GyrA fragment. Dao-Thi, M.H., Van Melderen, L., De Genst, E., Buts, L., Ranquin, A., Wyns, L., Loris, R. Acta Crystallogr. D Biol. Crystallogr. (2004) [Pubmed]
  2. The ratio between CcdA and CcdB modulates the transcriptional repression of the ccd poison-antidote system. Afif, H., Allali, N., Couturier, M., Van Melderen, L. Mol. Microbiol. (2001) [Pubmed]
  3. Bacteriophage Mu repressor as a target for the Escherichia coli ATP-dependent Clp Protease. Laachouch, J.E., Desmet, L., Geuskens, V., Grimaud, R., Toussaint, A. EMBO J. (1996) [Pubmed]
  4. Mutagenesis-based definitions and probes of residue burial in proteins. Bajaj, K., Chakrabarti, P., Varadarajan, R. Proc. Natl. Acad. Sci. U.S.A. (2005) [Pubmed]
  5. Intricate interactions within the ccd plasmid addiction system. Dao-Thi, M.H., Charlier, D., Loris, R., Maes, D., Messens, J., Wyns, L., Backmann, J. J. Biol. Chem. (2002) [Pubmed]
  6. Modulation of DNA supercoiling activity of Escherichia coli DNA gyrase by F plasmid proteins. Antagonistic actions of LetA (CcdA) and LetD (CcdB) proteins. Maki, S., Takiguchi, S., Miki, T., Horiuchi, T. J. Biol. Chem. (1992) [Pubmed]
  7. Inhibition of DNA topoisomerase II may trigger illegitimate recombination in living cells: Experiments with a model system. Umanskaya, O.N., Lebedeva, S.S., Gavrilov, A.A., Bystritskiy, A.A., Razin, S.V. J. Cell. Biochem. (2006) [Pubmed]
  8. F plasmid CcdB killer protein: ccdB gene mutants coding for non-cytotoxic proteins which retain their regulatory functions. Bahassi, E.M., Salmon, M.A., Van Melderen, L., Bernard, P., Couturier, M. Mol. Microbiol. (1995) [Pubmed]
  9. Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. Bernard, P., Couturier, M. J. Mol. Biol. (1992) [Pubmed]
  10. Molecular basis of gyrase poisoning by the addiction toxin CcdB. Dao-Thi, M.H., Van Melderen, L., De Genst, E., Afif, H., Buts, L., Wyns, L., Loris, R. J. Mol. Biol. (2005) [Pubmed]
  11. The antibiotic microcin B17 is a DNA gyrase poison: characterisation of the mode of inhibition. Heddle, J.G., Blance, S.J., Zamble, D.B., Hollfelder, F., Miller, D.A., Wentzell, L.M., Walsh, C.T., Maxwell, A. J. Mol. Biol. (2001) [Pubmed]
  12. Effect of different classes of inhibitors on DNA gyrase from Mycobacterium smegmatis. Chatterji, M., Unniraman, S., Mahadevan, S., Nagaraja, V. J. Antimicrob. Chemother. (2001) [Pubmed]
  13. The antidote and autoregulatory functions of the F plasmid CcdA protein: a genetic and biochemical survey. Salmon, M.A., Van Melderen, L., Bernard, P., Couturier, M. Mol. Gen. Genet. (1994) [Pubmed]
  14. One-step, highly efficient site-directed mutagenesis by toxic protein selection. Xu, W., Zhang, Y., Yeh, L.Y., Ruprecht, C.R., Wong-Staal, F., McFadden, B.A., Reddy, T.R., Ruprecht, R.M. BioTechniques (2002) [Pubmed]
  15. Design of temperature-sensitive mutants solely from amino acid sequence. Chakshusmathi, G., Mondal, K., Lakshmi, G.S., Singh, G., Roy, A., Ch, R.B., Madhusudhanan, S., Varadarajan, R. Proc. Natl. Acad. Sci. U.S.A. (2004) [Pubmed]
  16. The interaction of DNA gyrase with the bacterial toxin CcdB: evidence for the existence of two gyrase-CcdB complexes. Kampranis, S.C., Howells, A.J., Maxwell, A. J. Mol. Biol. (1999) [Pubmed]
  17. Positive selection vectors to generate fused genes for the expression of his-tagged proteins. Van Reeth, T., Drèze, P.L., Szpirer, J., Szpirer, C., Gabant, P. BioTechniques (1998) [Pubmed]
 
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