br Enzymes repairing alkylated DNA br Induction
Enzymes repairing alkylated DNA
Induction of the Ada regulon Methylated Ada is a strong activator for ada and alkA promoters. Although much weaker than the methylated form, unmethylated Ada acts alone as an activator for ada and alkA promoters. In contrast, the unmethylated protein present in E. coli in quantities of >200 molecules, inhibits methylated Ada activated transcription from the ada promoter. In the process of inhibition the 67 amino Raloxifene HCl chemical long C-terminal part of the Ada protein plays a pivotal role .
Ada response in different microorganisms Although the Ada response has been the subject of detailed study in E. coli, , , , it is also expressed in other distantly related taxa; nevertheless, its organization differs. Ada regulon organization characteristic of E. coli is widespread among other Enterobacteria including Shigella, Salmonella, Citrobacter, Enterobacter and Klebsiella. For instance, the alkB gene of K. pneumoniae renders the E. coli alkB− resistant to MMS (unpublished data). The Salmonella typhimurium Ada protein complements the E. coli ada− mutant and repairs O6meG with the same efficiency as E. coli Ada . However, S. typhimurium does not adapt to challenging doses of MNNG and its Ada protein only induces the β-galactosidase reporter 12 times that observed in the uninduced control, as compared with 38-fold in the case of E. coli Ada. Weak transcriptional activity of S. typhimurium Ada protein presumably results from the lack of an acidic amino acid residue in position 102 of the polypeptide chain . Using polyclonal antibodies against the E. coli Ada protein, two groups of microorganisms have been identified according to prevalence of the Ada response. Although the eukaryotic fungus A. nidulans lacked the response, it has preserved adaptability to alkylating agents. In some species, in induced and non-induced cells, the level of O6meG methyltransferase activity was as high as in others after induction. This type of response was elicited from Streptococcus aureus ATCC12598, for example. Other species, such as Pseudomonas aeruginosa ATCC27853 and Xanthomonas maltophilia ATCC13637, induced the Ada response as much as 10-fold that in E. coli. Bacillus subtilis M9CT responded similarly to E. coli but, surprisingly, the cell extract from adapted B. subtilis was no more active against 3meA lesions than from non-adapted cells , . Regarding the induction of E. coli alkA promoter, three categories of Gram negative bacteria have been identified: Considerable differences could be seen within the Pseudomonadaceae family, including species in all three categories . Fig. 6 illustrates a short description of Ada response organization in different microorganisms.
Concluding remarks and future perspectives In their natural environment, bacteria are constantly exposed to changing conditions. Nutrient shortage forces them to remain in the stationary growth phase during most of their life cycle. Under stressful conditions the Ada response is induced to exploit water/soil contaminants. Otherwise, in these bacteria, low fidelity DNA polymerases are synthesized, causing more errors in DNA synthesis, which leads to an accumulation of mutations, allowing the bacteria to overcome the growth barrier to permit survival. These mutations are termed adaptive or stationary phase , . Our investigations of substrate specificity and regulation of Ada proteins identified differences among bacterial species. In contrast to E. coli AlkA, P. putida AlkA shows the broadest substrate specificity of the Ada proteins. On the other hand, the AlkB protein, which is extremely valuable in the protection of E. coli against alkylation, remains outside the Ada operon in P. putida. Expressed constitutively, it played only a minor role in vivo in repairing the lesions tested . In vivo and in silico analysis of various microorganisms shows the widespread existence and versatile organization of Ada regulon genes, including not only ada, alkA, alkB and aidB but also COG3826, alkD and other genes, the role of which still remains to be elucidated in the repair of alkylated DNA. The broadly considered model of the Ada regulon corroborates the high flexibility of microorganisms and their adaptability to the challenge of a rapidly changing environment.