Quasi-adaptive response to alkylating agents and Ada-protein functions in <Emphasis Type="Italic">Escherichia coli</Emphasis>

In the exponentially growing E. coli cells we have described in 2005 a new fundamental genetic phenomenon, namely quasi-adaptive response to alkylating compounds, “quasi-Ada”. Phenotypic expression of “quasi-Ada” is similar to the true Ada response, however in contrast it develops in the course of pretreatment of the cells by sublethal dose of non-alkylating agent, an NO-containing dinitrosyl iron complex with glutathione (DNIC_glu). To reveal the mechanisms of quasi-adaptation and its association with the function of the regulatory protein Ada here we used a unique property of dual gene expression regulation of aidB1 gene, a part of Ada-regulon, namely its relative independence from Ada protein in anaerobic conditions. Based on the results of aidB1 gene expression analysis an EPR spectra of E.coli MV2176 cells (aidB1::lacZ) in aerobic and anaerobic conditions after the corresponding treatments we concluded that the function and the spatial structure of ^meAda and [(Cys^?)₂Fe⁺(NO⁺)₂]Ada are identical and thus the nitrosylated protein represents an Ada regulon genes expression regulator during quasi-adaptation development.     

Quasi-adaptive response to alkylating agents in Escherichia coli: a new phenomenon

An original hypothesis of a quasi-adaptive response to nitrosomethylurea (NMU) in Escherichia coli cells was verified experimentally. In contrast to the true Ada response, which is induced in cells pretreated with a sublethal dose of NMU, a quasi-adaptive response was induced using NO-containing dinitrosyl iron complex with glutathione (DNICglu). Quasi-adaptation increased expression of the Ada regulon and cell resistance to the cytotoxic and mutagenic effects of NMU. The levels of alkA, alkB, and aidB gene expression in quasi-adaptation were higher than in the true Ada response. Thus, experimental evidence was obtained for the alternative mechanism regulating the function of the Ada sensory protein in controlling expression of the Ada regulon during the adaptive response. The free iron--chelating agent o-phenanthroline (OP) facilitated degradation of DNICglu (by electron paramagnetic resonance (EPR) spectra) and considerably or completely inhibited gene expression in the quasi-adaptive response. The new phenomenon extends the functional range of NO compounds to include a role in genetic signal transduction within the Ada response system in addition to similar roles in the SoxRS, SOS, and OxyR systems in E. coli.     

Anaerobic induction of the alkylation-inducible Escherichia coli aidB gene involves genes of the cysteine biosynthetic pathway.

The Escherichia coli aidB gene is a component of the adaptive response to alkylation damage. This gene is subject to two different forms of induction: an ada-dependent alkylation induction and an ada-independent induction that occurs when cells are grown anaerobically (M. R. Volkert, L. I. Hajec, and D. C. Nguyen, J. Bacteriol. 171:1196-1198, 1989; M. R. Volkert, and D. C. Nguyen, Proc. Natl. Acad. Sci. USA 81:4110-4114, 1984). In this study, we isolated and characterized strains bearing mutations that specifically affect the anaerobic induction pathway. This pathway requires a functional cysA operon, which encodes sulfate permease. Mutations in cysA block this pathway of aidB induction. In contrast, mutations in either cysH, cysD, cysN, or cysC result in elevated levels of aidB expression during aerobic growth. These results indicate that the sulfate transport genes perform a role in anaerobic induction of the aidB gene and suggest that growth under anaerobic conditions may modify either the function or the expression of gene products encoded by the cysA operon.     

Expression and functions of adaptive response genes in Escherichia coli treated with mono- and bifunctional alkylating agents. Interference with SOS response]

The expression of genes belonging to the Ada regulon of Escherichia coli under the action of mono- and bifunctional alkylating agents--high-efficiency antitumor HMM, ACNU, and BCNU preparations--was studied. The functional specificity of the alkA, alkB, and aidB1 genes concerning both the structure and volume of DNA alkylation and the specificity of cell preadaptation was revealed. Additional experimental evidence for the role of the aidB1 gene as a unique "hazard gene", a component of the E. coli ada operon, was obtained. A phenomenon of positive interference between alternative SOS and Ada responses was observed for the first time upon gene expression.     

Quasi-Adaptive Response to Alkylating Agents in <Emphasis Type="Italic">Escherichia coli</Emphasis>: A New Phenomenon

An original hypothesis of a quasi-adaptive response to nitrosomethylurea (NMU) in Escherichia coli cells was verified experimentally. In contrast to the true Ada response, which is induced in cells pretreated with a sublethal dose of NMU, a quasi-adaptive response was induced using NO-containing dinitrosyl iron complex with glutathione (DNIC_glu). Quasi-adaptation increased expression of the Ada regulon and cell resistance to the cytotoxic and mutagenic effects of NMU. The levels of alkA, alkB, and aidB gene expression in quasi-adaptation were higher than in the true Ada response. Thus, experimental evidence was obtained for the alternative mechanism regulating the function of the Ada sensory protein in controlling expression of the Ada regulon during the adaptive response. The free iron—chelating agent o-phenanthroline (OP) facilitated degradation of DNIC_glu (by electron paramagnetic resonance (EPR) spectra) and considerably or completely inhibited gene expression in the quasi-adaptive response. The new phenomenon extends the functional range of NO compounds to include a role in genetic signal transduction within the Ada response system in addition to similar roles in the SoxRS, SOS, and OxyR systems in E. coli.__________Translated from Genetika, Vol. 41, No. 5, 2005, pp. 607–613.Original Russian Text Copyright © 2005 by Vasilieva, Moschkovskaya.     

The sources of inorganic sulfur in the process of cluster protein Fnr[4Fe-4S]2+ reconstruction in Escherichia coli cells cultivated with NO-donating agents

Dinitrosyl iron complexes (DNICs) with thiol ligands--binuclear and mononuclear--inhibited aidB gene expression in E. coli cells. This process is due to the nitrosylation of the active center in iron-sulfur protein Fnr [4Fe-4S]2+ by low-molecular DNICs. The next step is transformation of the above DNICs into the DNICs with the thiol groups in the apo-form of Fnr protein. These nitrosylated proteins are characterized by the EPR signal with g perpendicular = 2.04 and g parallel 1 = 2,014. An addition of sulfur containing L-Cys or N-A-L-Cys as well as Na2S to the cells lead to the increasing in the aidB gene expression simultaneously with an appearance of the EPR signal with g perpendicular = 2.04 and g parallel = 2.02 as the characteristics of the DNICs with persulfide (R-S-S-) ligands. We suppose that the recovery of the aidB gene activity was due to the accumulation of inorganic sulfur in the cells and reconstruction of the active center in Fnr[4Fe-4S]2+. It appears that the above process is the function of L-cysteine-desulfurase protein which repaired the active center of Fnr[4Fe-4S]2+ protein using the sulfur from L-Cys or N-A-L-Cys after its deacetylation. On the other side the ions of inorganic sulfur being reacted with SH-groups led to the transformation of DNIC with thiol ligands into the persulfides. Na2S was the most potent activator of the aidB gene expression in our experiments.     

Induction of the Escherichia coli aidB gene under oxygen-limiting conditions requires a functional rpoS (katF) gene.

The Escherichia coli aidB gene is regulated by two different mechanisms, an ada-dependent pathway triggered by methyl damage to DNA and an ada-independent pathway triggered when cells are grown without aeration. In this report we describe our search for mutations affecting the ada-independent aidB induction pathway. The mutant strain identified carries two mutations affecting aidB expression. These mutations are named abrB (aidB regulator) and abrD. The abrB mutation is presently poorly characterized because of instability of the phenotype it imparts. The second mutation, abrD1, reduces the expression of aidB observed when aeration is ceased and oxygen becomes limiting. Genetic and phenotypic analysis of the abrD1 mutation demonstrates that it is an allele of rpoS. Thus, aidB is a member of the family of genes that are transcribed by a sigma S-directed RNA polymerase holoenzyme. Examination of aidB expression in an rpoS insertion mutant strain indicates that both rpoS13::Tn10 and abrD1 mutations reduce aidB expression under oxygen-limiting conditions that prevail in unaerated cultures, reduce aidB induction by acetate at a low pH, but have little or no effect on the ada-dependent alkylation induction of aidB.     

Specific DNA binding and regulation of its own expression by the AidB protein in Escherichia coli

Upon exposure to alkylating agents, Escherichia coli increases expression of aidB along with three genes (ada, alkA, and alkB) that encode DNA repair proteins. While the biological roles of the Ada, AlkA, and AlkB proteins have been defined, despite many efforts, the molecular functions of AidB remain largely unknown. In this study, we focused on the biological role of the AidB protein, and we demonstrated that AidB shows preferential binding to a DNA region that includes the upstream element of its own promoter, PaidB. The physiological significance of this specific interaction was investigated by in vivo gene expression assays, demonstrating that AidB can repress its own synthesis during normal cell growth. We also showed that the domain architecture of AidB is related to the different functions of the protein: the N-terminal region, comprising the first 439 amino acids (AidB "I-III"), possesses FAD-dependent dehydrogenase activity, while its C-terminal domain, corresponding to residues 440 to 541 (AidB "IV"), displays DNA binding activity and can negatively regulate the expression of its own gene in vivo. Our results define a novel role in gene regulation for the AidB protein and underline its multifunctional nature.     

Characterization of a novel tetracycline resistance that functions only in aerobically grown Escherichia coli.

A tetracycline resistance (Tcr) gene that was found originally on two Bacteroides plasmids (pBF4 and pCP1) confers tetracycline resistance on Escherichia coli, but only when it is grown aerobically. Using maxicells, we have identified a 44-kilodalton protein which is encoded by the region that carries the Tcr gene and which may be the Tcr gene product. Localization experiments indicate that this 44-kilodalton protein is cytoplasmic. To determine whether the tetracycline resistance gene is expressed under anaerobic conditions, we have constructed a protein fusion between the Tcr gene and lacZ. In strains of E. coli carrying the fusion, beta-galactosidase activity was the same when the cells were grown under anaerobic conditions as when the cells were grown under aerobic conditions. This indicates that the tetracycline resistance gene product is made under anaerobic conditions but does not work. The failure of the Tcr protein to function under anaerobic conditions was not due to a requirement for function of the anaerobic electron transport system, because neither nitrate nor fumarate added to anaerobic media restored tetracycline resistance. Inhibition of the aerobic electron transport system with potassium cyanide did not prevent growth on tetracycline of cells containing the Tcr gene. A heme-deficient mutant, E. coli SHSP19, which carries the Tcr gene, was still resistant to tetracycline even when grown in heme-free medium. These results indicate that functioning of the Tcr gene product is not dependent on the aerobic electron transport system. Thus the requirement for aerobic conditions appears to reflect a requirement for oxygen. Spent medium from an E. coli strain carrying the Tcr gene, which was grown in medium containing tetracycline (50 micrograms/ml), did not inhibit growth of a tetracycline-susceptible strain of E. coli. Thus, the Tcr gene product may be detoxifying tetracycline.     

Cloning and characterization of the Salmonella typhimurium ada gene, which encodes O6-methylguanine-DNA methyltransferase.

The ada gene of Escherichia coli encodes O6-methylguanine-DNA methyltransferase, which serves as a positive regulator of the adaptive response to alkylating agents and as a DNA repair enzyme. The gene which can make an ada-deficient strain of E. coli resistant to the cell-killing and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the gene potentially encoded a protein with a calculated molecular weight of 39,217. Since the nucleotide sequence of the cloned gene shows 70% similarity to the ada gene of E. coli and there is an ada box-like sequence (5'-GAATTAAAACGCA-3') in the promoter region, we tentatively refer to this cloned DNA as the adaST gene. The gene encodes Cys-68 and Cys-320, which are potential acceptor sites for the methyl group from the damaged DNA. The multicopy plasmid carrying the adaST gene significantly reduced the frequency of mutation induced by MNNG both in E. coli and in S. typhimurium. The AdaST protein encoded by the plasmid increased expression of the ada'-lacZ chromosome fusion about 5-fold when an E. coli strain carrying both the fusion operon and the plasmid was exposed to a low concentration of MNNG, whereas the E. coli Ada protein encoded by a low-copy-number plasmid increased it about 40-fold under the same conditions. The low ability of AdaST to function as a positive regulator could account for the apparent lack of an adaptive response to alkylation damage in S. typhimurium.     

Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase

The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned and placed under the control of the lac regulatory region on a multicopy runaway plasmid, thereby yielding a hybrid plasmid pYN3059. Ada protein with a molecular weight of about 38,000 was overproduced when cells harboring pYN3059 were incubated at 42 degrees C in the presence of a lac inducer, isopropyl-beta-D-thiogalactoside. Taking advantage of overproduction of Ada protein, we purified the protein to apparent physical homogeneity. The purified 38,000-dalton Ada protein transferred the methyl group from the O6-methylguanine residue of alkylated DNA to the Ada protein, per se. Although the Ada protein was degraded to smaller polypeptides when crude extracts or partially purified preparations were incubated in a high ionic-strength buffer at neutral pH, the purified Ada protein remained stable under the same conditions, indicating that the Ada protein may not undergo autodegradation. An amino-terminal sequence and total amino acid composition of the purified Ada protein were in accord with nucleotide sequence of the ada gene, determined by the dideoxy method using M13 phage. It was deduced that Ada protein comprises 354 amino acids and its molecular weight is 39,385. The promoter for the ada gene was determined by S1 nuclease mapping.     

Anaerobic regulation by an atypical Arc system in Shewanella oneidensis

Shewanella oneidensis strain MR-1 is well known for its respiratory versatility, yet little is understood about how it regulates genes involved in anaerobic respiration. The Arc two-component system plays an important role in this process in Escherichia coli; therefore, we determined its function in S. oneidensis. arcA from S. oneidensis complements an E. coli arcA mutant, but the Arc regulon in S. oneidensis constitutes a different suite of genes. For example, one of the strongest ArcA-regulated gene clusters in E. coli, sdh, is not regulated by the Arc system in S. oneidensis, and the cyd locus, which is induced by ArcA in E. coli under microaerobic conditions, is repressed by ArcA in S. oneidensis under anaerobic conditions. One locus that we identified as being potentially regulated by ArcA in S. oneidensis contains genes predicted to encode subunits of a dimethyl sulphoxide (DMSO) reductase. We demonstrate that these genes encode a functional DMSO reductase, and that an arcA mutant cannot fully induce their expression and is defective in growing on DMSO under anaerobic conditions. While S. oneidensis lacks a highly conserved full-length ArcB homologue, ArcA is partially activated by a small protein homologous to the histidine phosphotransfer domain of ArcB from E. coli, HptA. This protein alone is unable to compensate for the lack of arcB in E. coli, indicating that another protein is required in addition to HptA to activate ArcA in S. oneidensis.     

Complementation of sensitivity to alkylating agents in Escherichia coli and Chinese hamster ovary cells by expression of a cloned bacterial DNA repair gene.

Dual expression vectors derived from pSV2gpt and encoding all or part of the Escherichia coli ada+ gene have been constructed. Following transformation into an E. coli ada strain or transfection and stable integration into the genome of Chinese hamster ovary (CHO) cells, plasmid vectors containing the whole ada+ gene conferred resistance to both killing and mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Thus, the bacterial DNA repair gene was functionally expressed in the mammalian cells. Plasmids containing an N-terminal fragment of the ada+ gene which encoded only one of the two methyltransferase activities of the Ada protein did not significantly protect E. coli or CHO cells against MNNG. These results are consistent with the central role of the intact ada+ gene in controlling the adaptive response to alkylating agents in E. coli. However, the data further suggest that some alkylation lesions in DNA, such as O6-methylguanine, may exert partly different biological effects in E. coli and mammalian cells.