Abstract
Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada) response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS) or N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i) the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii) the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB), characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB), PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems against alkylating agents of exo- and endogenous origin.
Highlights
Alkylating agents of endogenous and exogenous origin introduce a variety of damages to DNA
The genomes of P. putida species code more putative proteins dealing with alkyl-DNA damages than E. coli In E. coli, the adaptive response results in increased expression of four genes, ada, alkB, alkA and aidB [5,6]
In order to establish whether a similar response occurs in pseudomonads, we searched for the presence of E. coli Ada regulon orthologs in P. putida genome and examined the ability of alkylating agents to induce transcription of these genes
Summary
Alkylating agents of endogenous (by-products of cellular metabolism) and exogenous (environmental chemicals) origin introduce a variety of damages to DNA. A crucial role in this process is played by adaencoded Ada methyltransferase, functioning as a transcriptional activator inducing expression of the ada-alkB operon and the alkA and aidB genes. Self-methylation at Cys-38 results in transcriptional activation of Ada protein and in an increase in specific DNA binding affinity to genes containing the sequence of ada operator in their promoters. High concentration of unmethylated Ada protein (>200 molecules per cell) inhibits transcriptional activation [9], whereas MPTs act as molecular sensors for changing the levels of DNA alkylation in bacteria [5]. Induced within the Ada response AlkA and expressed constitutively Tag proteins are, respectively, 3meA DNA glycosylase II and I. It has been proven that AlkAs from Archaeoglobus fulgidus [13] and Deinococcus radiodurans [14] show activity towards the main substrates of AlkB dioxygenase, 3meC and 1meA
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
