Quasi-Adaptive Response to Alkylating Agents in Escherichia coli: A New Phenomenon

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<ul><li><p> 1022-7954/05/4105- 2005 Pleiades Publishing, Inc.0484</p><p>Russian Journal of Genetics, Vol. 41, No. 5, 2005, pp. 484489. Translated from Genetika, Vol. 41, No. 5, 2005, pp. 607613.Original Russian Text Copyright 2005 by Vasilieva, Moschkovskaya.</p><p>INTRODUCTIONPotentially mutagenic alkylating agents occur both</p><p>in the environment and in the living cell, as metabolicproducts. An important source of endogenous alkyla-tion is bacterial nitrosation of amino acids and peptideswith endogenous nitric oxide [1, 2]. All organismseubacteria, archaebacteria, and eukaryoteshavemechanisms protecting cells from toxic and genotoxiceffects of alkylation [3]. In </p><p>Escherichia coli</p><p>, the protec-tive mechanisms involve the </p><p>tag</p><p> and </p><p>ogt</p><p> genes, whichare expressed constitutively, and the </p><p>ada, alkA</p><p>, </p><p>alkB</p><p>,</p><p>and </p><p>aid</p><p> genes, expression of which is associated withinduction of an adaptive response (Ada) [4]. The prod-ucts of the </p><p>tag</p><p> and </p><p>alkA</p><p> genes act as glycosylases and,although slightly differing in specificity, both exciseN</p><p>3</p><p>-meA from DNA to yield apurinic sites [5].The </p><p>alkB</p><p> gene belongs to the same operon as the</p><p>ada</p><p> gene, and its expression is similarly controlled bythe </p><p>ada</p><p> promoter [6]. The AlkB protein is involved inrepairing lesions induced by SN2-methylating agents insingle-stranded DNA, mostly in the replication forkand in transcribed regions [7]. The AlkB protein is iron-dependent and utilizes a unique mechanism of oxidativedemethylation to directly repair N</p><p>1</p><p>-meA and N</p><p>3</p><p>-meC,eliminating the methyl group in the form of formalde-hyde [8].</p><p>The multifunctional protein AidB is regulated viatwo pathways: one is Ada-dependent, while the other isAda-independent and realized in response to acidifica-tion of the medium [9, 10]. The </p><p>aidB</p><p> gene is an alarmgene of the </p><p>ada</p><p> operon: it is expressed to a lower levelin response to nitrosomethylurea (NMU) as compared</p><p>with the other genes and is the only gene providing forrepair of interstrand crosslinks induced in DNA bychlorine-substituted nitrosoalkylurea (ACNU) [11].</p><p>Among all alkylation products of DNA bases, O</p><p>6</p><p>-</p><p>meG occupies a special place because of its preferentialpairing with T rather than C during DNA replication[12, 13]. Though a minor adduct, O</p><p>6</p><p>-meG plays a cru-cial role in mutagenesis, carcinogenesis, apoptosis, andthe clustogenic effect of methylating and chlorethylat-ing agents. Moreover, O</p><p>6</p><p>-meG is a lethal lesion, and itselimination depends absolutely on the postreplicativemismatch repair system. Although O</p><p>6</p><p>-meG is the mostdangerous cyto- and genotoxic adduct, only one or twoproteins repair it without mistakes via a unique irrevers-ible stochiometric reaction [3]. In mammals, this func-tion is performed by O</p><p>6</p><p>-meG DNA-methyltransferase(MGMT), whose amount is strongly controlled andmaintained at 10</p><p>5</p><p> molecules per cell [14].A nonadapted </p><p>E. coli</p><p> cell contains approximately60 molecules of the Ada protein. However, completeinduction of this protein takes place within 1 h of incu-bation in the presence of an alkylating agent used in anontoxic dose [15]. In addition, </p><p>E. coli</p><p> cells constitu-tively express the product of the </p><p>ogt</p><p> gene to approxi-mately 30 molecules per cell. This protein is an alterna-tive of MGMT, is similar to the Ada protein in biochem-ical characteristics, but is not induced by alkylatingagents [16]. The Ada sensory protein, which regulatestranscription of the Ada regulon, is not an enzyme: Adadirectly repairs O</p><p>6</p><p>-meG and O</p><p>4</p><p>-meT via an irreversiblestochiometric reaction [3]. Since direct structural stud-ies of the proteinDNA complex are still unfeasible,</p><p>Quasi-Adaptive Response to Alkylating Agents in </p><p>Escherichia coli</p><p>: A New Phenomenon</p><p>S. V. Vasilieva and E. Ju. Moschkovskaya</p><p>Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, 117334 Russia;fax: (095)137-41-01; e-mail: svasilieva@polymer.chph.ras.ru</p><p>Received April 30, 2004</p><p>Abstract</p><p>An original hypothesis of a quasi-adaptive response to nitrosomethylurea (NMU) in </p><p>Escherichiacoli</p><p> cells was verified experimentally. In contrast to the true Ada response, which is induced in cells pretreatedwith a sublethal dose of NMU, a quasi-adaptive response was induced using NO-containing dinitrosyl ironcomplex with glutathione (DNIC</p><p>glu</p><p>). Quasi-adaptation increased expression of the Ada regulon and cell resis-tance to the cytotoxic and mutagenic effects of NMU. The levels of </p><p>alkA, alkB,</p><p> and</p><p> aidB</p><p> gene expression inquasi-adaptation were higher than in the true Ada response. Thus, experimental evidence was obtained for thealternative mechanism regulating the function of the Ada sensory protein in controlling expression of the Adaregulon during the adaptive response. The free ironchelating agent </p><p>o</p><p>-phenanthroline (OP) facilitated degra-dation of DNIC</p><p>glu</p><p> (by electron paramagnetic resonance (EPR) spectra) and considerably or completely inhib-ited gene expression in the quasi-adaptive response. The new phenomenon extends the functional range of NOcompounds to include a role in genetic signal transduction within the Ada response system in addition to similarroles in the SoxRS, SOS, and OxyR systems in</p><p> E. coli.</p><p>GENETICS OF MICROORGANISMS</p></li><li><p> RUSSIAN JOURNAL OF GENETICS</p><p>Vol. 41</p><p>No. 5</p><p>2005</p><p>QUASI-ADAPTIVE RESPONSE TO ALKYLATING AGENTS 485</p><p>several models have been advanced to explain the con-version of alkylated Ada into a factor regulating tran-scription of the Ada regulon genes [3] and repair ofO</p><p>6</p><p>-meG [17].Today, a crystal structure is available and known in</p><p>detail only for one Ada protein, </p><p>E. coli</p><p> Ada [18]; struc-tural analysis of its mammalian counterpart is close tocompletion [3]. Since adaptive response proteins ofmammals and </p><p>E. coli</p><p> are similar in general structure,have homologous C-terminal domains, and are regu-lated similarly [19], bacterial cells can be used as amodel to study the common features of the molecularmechanisms underlying the interaction of adaptiveresponse proteins with potential inductors and to iden-tify new promising regulators of adaptive processes.Since cysteine methylation at SH groups is the crucialfactor of Ada activation, we assumed that the Ada activ-ity can be alternatively regulated using NO compounds,with S-nitrosylcysteine functioning in place of S-meth-ylcysteine in key position Cys-69. In this work, weexperimentally verified our assumption.</p><p>MATERIALS AND METHODS</p><p>Bacterial strains.</p><p> We used </p><p>E. coli</p><p> strains MV1571</p><p>(</p><p>alkA</p><p>51</p><p>::</p><p>Mu</p><p>-</p><p>dl</p><p>) (</p><p>bla</p><p>lac</p><p>), </p><p>MV</p><p>1601 (</p><p>alkB</p><p>52</p><p>::</p><p>Mu</p><p>-</p><p>dl</p><p>)(</p><p>bla</p><p>lac</p><p>)</p><p>, and MV2176 </p><p>(</p><p>aidB</p><p>1</p><p>::</p><p>Mu</p><p>-</p><p>dl</p><p>) (</p><p>bla</p><p>lac</p><p>)</p><p>, whichwere constructed and kindly provided by M. Volkert(University of Massachusetts, United States). Thesestrains contain the </p><p>lacZ</p><p> structural gene for </p><p>-galactosi-dase under the control of the </p><p>ada</p><p> promoter, while thechromosomal </p><p>lac </p><p>operon is deleted. Thus, expression ofthe genes under study was assayed indirectly by the </p><p>-</p><p>galactosidase activity, which was measured colorimet-rically. Genetic characteristics of the strains weredescribed in [20].</p><p>Reagents.</p><p> We used </p><p>o-</p><p>nitrophenyl </p><p>-D-galactopyra-noside (ONPG), cysteine, glutathione, and HEPES(Sigma, United States). A dinitrosyl iron complex withglutathione (DNIC</p><p>glu</p><p>) was used as a dimer and wasobtained by treating 5.4 mM FeSO</p><p>4</p><p> and 10.8 mM glu-tathione (ironthiol ratio 1 : 2 (w/w)) with gaseousnitric oxide in solution (15 mM HEPES, pH 7.6) in aTunberg flask at 200300 mm Hg. NMU was synthe-sized at the Institute of Chemical Physics and dissolvedin a phosphate buffer (pH 6.0). In addition, we used</p><p>E. coli</p><p>-galactosidase (5000 units, Sigma).</p><p>Gene expression.</p><p> Expression of the </p><p>alkA</p><p>, </p><p>alkB</p><p>, and</p><p>aidB</p><p> genes was assayed according to a published pro-tocol [21]. Bacterial cells were treated with inductorsfor 30 min. The cell suspension was diluted 50-foldwith LB, supplemented with the chromogen ONPG,and incubated at 37</p><p>C for 2 h. The buffer for </p><p>-galac-tosidase activity assays was as described in [8]. The </p><p>-</p><p>galactosidase activity was measured colorimetrically at420 nm, using a Specord UVVIS spectrophotometer(Germany), and computed as </p><p>E</p><p> = 1000</p><p>D</p><p>420</p><p>/</p><p>t</p><p>, where</p><p>D</p><p> is the absorbance at 420 nm and </p><p>t</p><p> is the time of incu-bation in the presence of the chromogen.</p><p>Adaptive response</p><p> to NMU was induced by treatingcells with </p><p>10</p><p>5</p><p> M NMU (a sublethal concentration) inthe liquid L medium with aeration at 37</p><p>C for 1 h. Toinduce a quasi-adaptive response, cells were pretreatedwith DNIC</p><p>glu</p><p> used at a sublethal concentration in thedark. In some experiments, cells were exposed at thesublethal NMU concentration before treatment withDNIC</p><p>glu</p><p>.</p><p>UV absorption spectra</p><p> of NMU and DNIC</p><p>glu</p><p> solu-tions and their mixtures were recorded with theSpecord UVVIS spectrophotometer.</p><p>Electron paramagnetic resonance (EPR) study.</p><p>Cells were grown aerobically in the L medium toOD</p><p>6</p><p>00</p><p> 0.4</p><p>. To prepare a sample, a culture was centri-fuged at 7000 </p><p>g</p><p> and concentrated to 5 ml after 30-min pre-treatment with DNIC</p><p>glu</p><p> and, when indicated, </p><p>o</p><p>-phenan-throline (OP). Cells were collected by centrifugation,resuspended in 0.3 ml of the L medium, and quicklyfrozen in calibrated ampules for EPR examination. EPRspectra were recorded in the X range, using a Radiopanspetrometer (Poland) at modulation amplitude 5 mT,temperature 77 K, and microwave power 5 mW.</p><p>-Galactosidase activity assays with a cell-free sys-tem</p><p> were performed with the chromogenic substrateONPG in the presence of NMU and DNIC</p><p>glu</p><p>. A sample(2 ml) was incubated at 37</p><p>C for 1 h, combined with0.4 ml of buffer B containing ONPG [21, 22], and incu-bated for 20 min until it yellowed. The reaction was ter-minated by adding 1 M Na2CO3 [21]. All samples freefrom DNICglu were tested for D420 spectrophotometri-cally against the buffer. Samples containing DNICglu,which is yellowish in solution, were tested against0.2 mM DNICglu.</p><p>RESULTSAt the first step to verification of our hypothesis of</p><p>quasi-adaptation, we compared the level of inducedexpression of the adaptive response genes for the trueadaptive and quasi-adaptive responses to NMU. Cellswere exposed at sublethal concentrations of NMU orDNICglu. Preliminary experiments showed that DNICgluhad a relatively low cytotoxic effect on E. coli: its sub-lethal concentrations upon a 30-min exposure wereestimated at 0.1 mM for the alkA mutant, 0.2 mM forthe aidB mutant, and 0.5 mM for the alkB mutant. Anal-ysis of the NMU dose dependences showed that thelevel of expression of the Ada regulon genes increasedconsiderably both in the classical and in the quasi-adap-tive response. In the presence of 1.0 mM NMU, the lev-els of gene expression increased twofold in the trueadaptive response and 3.5-fold in the quasi-adaptiveresponse in alkA::lacZ and alkB::lacZ E. coli cells(Figs. 1, 2). The highest quasi-adaptive response wasobserved in cells with a high content of the Ada proteinafter preliminary adaptation at a sublethal concentra-</p></li><li><p>486</p><p>RUSSIAN JOURNAL OF GENETICS Vol. 41 No. 5 2005</p><p>VASILIEVA, MOSCHKOVSKAYA</p><p>tion of NMU. The level of alkB gene expression in suchcells was more than fivefold higher than in control cellsand more than twofold higher than in true adaptation toNMU (Fig. 2).</p><p>Regardless of adaptation, the aidB gene is usuallyexpressed to a far lower level than the alkA and alkBgenes in experiments with NMU [11]. As shown in Fig. 3,the highest level of aidB expression was observed at0.2 mM NMU in intact cells and at 1 mM, a fivefoldhigher concentration, in adapted cells. Still expressionof the aidB gene increased in all variants with preadap-tation.</p><p>By definition, the phenotypic expression of the Adaresponse to alkylating agents in E. coli involves anincrease in the resistance of adapted cells to toxic andmutagenic affects of such agents [4]. Experiments onquasi-adaptation were carried out with the E. colialkB::lacZ strain and showed that cell resistanceincreased more than in true adaptation, at least in termsof the parameters under study (Table 1).</p><p>The cytotoxic effects of NO and its compounds aremostly associated with inactivation of enzymes in thecell. Since we used the inducible -galactosidase activ-ity to indirectly characterize the level of gene expres-sion, control experiments were performed to estimatethe effects of MNU and DNICglu on the -galactosidaseactivity in a cell-free system. The inductors were usedat concentrations allowing the maximum levels of geneexpression (Table 2). Neither NMU nor DNICgluaffected the -galactosidase activity in these experi-ments.</p><p>To achieve our aim, cells were exposed to a com-bined effect of the genotoxic agents MNU and DNICgluin most experiments. In view of this, we studied theinteraction of the two agents by UV absorption spectraof their solutions and mixtures (Fig. 4). Present in a</p><p>solution, DNICglu had no effect on the UV absorptionspectrum of NMU. It is of interest that NMU andDNICglu had similar absorption peaks in the region of200250 nm. EPR spectroscopy was used to check thepermeability of E. coli cells to DNICglu and to study thechanges in DNICglu concentration and structure in thecell. DNICglu is converted from the dimeric into themonomeric form and displays paramagnetic propertiesin the presence of free thiols in solution or upon bindingwith SH groups of proteins. Cells incubated withDNICglu showed an EPR spectrum characteristic of thisagent, with anisotropic g-factors g| = 2.03 and g|| =</p><p>30</p><p>25</p><p>20</p><p>15</p><p>10</p><p>5</p><p>0.2 0.4 0.6 0.8 1.0</p><p> -Galactosidase activity, MU</p><p>NMU, mM0</p><p>1</p><p>2</p><p>3</p><p>4</p><p>5</p><p>Fig. 1. NMU-dependent induction of alkA gene expressionin E. coli MV 1571. Cells were used (1) before and afterpretreatment with (2) 0.01 mM NMU; (3) 0.1 mM DNICglu;(4) 0.01 mM NMU 0.1 mM DNICglu; or (5) OP variants 1, 3, and 4.</p><p>30</p><p>25</p><p>20</p><p>15</p><p>10</p><p>5</p><p>0.2 0.4 0.6 0.8 1.0</p><p> -Galactosidase activity, MU</p><p>NMU, mM0</p><p>1</p><p>2</p><p>3</p><p>4</p><p>5</p><p>40</p><p>35</p><p>Fig. 2. NMU-dependent induction of alkB gene expressionin E. coli MV 1601. Cells were used (1) before and afterpretreatment with (2) 0.01 mM NMU; (3) 0.5 mM DNICglu;(4) 0.01 mM NMU 0.5 mM DNICglu; or (5) OP variants 1, 3, and 4.</p><p>30</p><p>25</p><p>20</p><p>15</p><p>10</p><p>5</p><p>0.2 0.4 0.6 0.8 1.0</p><p>-Galactosidase activity, MU</p><p>NMU, mM0</p><p>1</p><p>2</p><p>34</p><p>35</p><p>Fig. 3. NMU-dependent induction of aidB gene expressionin E. coli MV 2176. Cells were used (1) before and afterpretreatment with (2) 0.01 mM NMU; (3) 0.2 mM DNICglu;(4) 0.01 mM NMU 0.2 mM DNICglu.</p></li><li><p>RUSSIAN JOURNAL OF GENETICS Vol. 41 No. 5 2005</p><p>QUASI-ADAPTIVE RESPONSE TO ALKYLATING AGENTS 487</p><p>2.014 (Fig. 5). A single washing of the cells with water(variant 2) halved the signal intensity on average (Fig. 5),testifying that a considerable portion of the donor wasabsorbed on the cell surface and lost as a result of cellwashing. Preincubation of cells with 0.1 mM OP for30 min reduced tenfold the intensity of the EPR signalat g| = 2.03 in cells exposed to DNICglu as comparedwith the initial level. The shape of the spectrum alsochanged, suggesting decomposition of DNICglu. Whencells pretreated with OP were washed once with water(variant 4), the intensity of the EPR signal at g| = 2.03increased twofold, to the level c...</p></li></ul>