Analysis of the Antimicrobial Susceptibility of the Ionizing

Ann Microbiol (2012) 62:493–500 DOI 10.1007/s13213-011-0281-y

ORIGINAL ARTICLE

Analysis of the antimicrobial susceptibility of the ionizing radiation-resistant bacterium Deinococcus radiodurans: implications for bioremediation of radioactive waste

Haïtham Sghaier & Ons Bouchami & Claus Desler & Hadeer Lazim & Mouldi Saidi & Lene Juel Rasmussen & Assia Ben Hassen

Received: 8 March 2011 /Accepted: 6 May 2011 /Published online: 24 May 2011 # Springer-Verlag and the University of Milan 2011

Abstract Deinococcus radiodurans R1 is one of the most tions using Subsystems Technology (RAST), and the resistant bacteria to chronic ionizing radiation ever identi- automatic hierarchical classification of proteins (ProtoNet) fied. Therefore, Deinococcus radiodurans shows promise servers. Among 19 retrieved sequences of D. radiodurans, in the bioremediation of mixed radioactive wastes. Nuclear six genes were functionally re-annotated as antimicrobial wastes have been demonstrated to host bacteria containing resistance genes, and only two genes were found to possess genes for antimicrobial production (Bagwell et al. PloS the radiation/desiccation response motif (RDRM). In vitro, ONE 3:e3878, 2008; Bai et al. Chem Biol 13:387–397, we found that D. radiodurans is sensitive to 40 tested 2006; Phillips et al. Int J Syst Evol Microbiol 52:933–938, antimicrobials, and it was resistant to one macrolide 2002), which may represent a constraint on the bioremedi- (spiramycin) and to three quinolones (ofloxacin, ciproflox- ation potential of Deinococcus radiodurans. In this context, acin, and nalidixic acid). The resistance of D. radiodurans the aim of this work is to investigate the antimicrobial to these quinolones was discussed based on different susceptibility of D. radiodurans through in silico and in possibilities. Moreover, in silico analyses indicated that the vitro approaches. In silico, we initiated our analyses by investigated 19 genes of D. radiodurans from ARDB, RAST, retrieving genes that are predicted to confer to D. radio- and ProtoNet encode translatable messenger ribonucleic durans resistance to antimicrobials based on the Antibiotic acids (mRNAs) related to resistance to antimicrobials. These Resistance Genes Database (ARDB), the Rapid Annota- data together with the antibiograms of D. radiodurans represent a suggestive evidence that the majority of these genes encode non-functional proteins or belong to inefficient H. Sghaier (*) : H. Lazim : M. Saidi biochemical pathways. The susceptibility of D. radiodurans Research Unit UR04CNSTN01 “Medical and Agricultural to these antimicrobials limits its potential for bioremediation Applications of Nuclear Techniques”, of nuclear waste and further genetic engineering is needed National Center for Nuclear Sciences and Technology (CNSTN), depending on the site to be depolluted. Sidi Thabet Technopark, 2020 Sidi Thabet, Tunisia e-mail: [email protected] Keywords Antimicrobial susceptibility. Bioremediation . Deinococcus radiodurans . Radioactive waste H. Sghaier e-mail: [email protected] : O. Bouchami A. Ben Hassen Introduction Laboratory of the National Bone Marrow Transplantation Centre, Street Djebel-Lakdhar, Bab-Saadoun, 1006 Tunis, Tunisia Presently, there is significant interest in microbiologically : mediated remediation because it promises to be simpler, C. Desler L. J. Rasmussen cheaper, and more environmentally friendly than the Center for Healthy Aging, Faculty of Health Sciences, Copenhagen University, more commonly used non-biological options (Lovley DK-2200 Copenhagen N, Denmark 2003). In 2002, Kineococcus radiotolerans SRS30216 494 Ann Microbiol (2012) 62:493–500 was discovered in a high-level radioactive environment at With regard to other classes of bactericidal antimicrobials, the Savannah River Site in the United States of America quinolones target DNA replication and repair by binding (USA) (Phillips et al. 2002). In 2004, Gram-positive DNA gyrase complexed with DNA, which drives double- bacteria high in G + C content, including members of strand DNA break formation and cell death as demonstrated in Arthrobacter, Bacillus, Streptomyces,andNocardioides, previous experimental work (Kohanski et al. 2007, and were among the most common genera represented among references therein). the cloned sequences from Hanford radioactive waste sites To develop bio-cleanup technologies, numerous bacteria, in the USA (Fredrickson et al. 2004). For instance, including members of the extremely radiation-resistant members of the genus Streptomyces account for 70–80% family Deinococcaceae, have been studied for their ability of secondary metabolites within the actinomycetes which to transform, detoxify, or immobilize a variety of metallic are responsible for more than two-thirds of the total and organic pollutants (Bonaventura and Johnson 1997; number of antimicrobials produced by bacteria and fungi Lovley 2003;LovleyandCoates1997;Daly2000). (more than 5,000) (Challis and Hopwood 2003). These Particularly, research aimed at developing Deinococcus discoveries, together with genomic evidence that Kine- species for bioremediation of nuclear waste began in 1997 ococcus radiotolerans, for example, has open reading with the demonstration that, unlike most organisms, frames (ORFs) with significant homology to genes implicated their representative, D. radiodurans, can grow in the in validamycin biosynthesis (e.g., Krad_0836) (Bai et al. presence of chronic ionizing radiation at 60 grays (Gy) 2006), antimicrobial biosynthesis (e.g., Krad_2675), and per hour, comparable to the most radioactive waste sites streptomycin biosynthesis (e.g., Krad_3239, Krad_1553, of the Department of Energy (DOE) in the USA Krad_3509, Krad_4156) (Bagwell et al. 2008), strengthens (Fredrickson et al. 2000;Daly2000; Lange et al. 1998; our proposal in this work that the resistance to antimicrobials Brim et al. 2000, 2003, 2006). must be evoked when discussing bioremediation issues of The bioremediation potential of radioactive waste sites radioactive sites. Ideally, bioremediation strategies would be by Deinococcus radiodurans due to its extreme resistance designed based on knowledge of the contaminated environ- to chronic ionizing radiation (Brim et al. 2000, 2006; Daly ments (Lovley 2003). For example, knowledge of predom- 2000; Lange et al. 1998; Fredrickson et al. 2000) and the inant antimicrobial-producing organisms is important to presence in nuclear wastes of bacteria (Phillips et al. 2002) define the biological constraints for bioremediation of a with antimicrobial biosynthesis pathways (Bagwell et al. contaminated radioactive site. Consequently, an efficient 2008; Bai et al. 2006) led to this study. Here, our strategy bioremediation strategy will require the construction of for elucidating the antimicrobial susceptibility profile of D. genetic constructs related to antimicrobial resistance to be radiodurans was to retrieve and analyze, in silico, genes introduced to bioremediation bacteria. that could confer resistance to antimicrobials and to collect Previously, it was proposed that there is a common information about the functional status of these genes mechanism of cellular death underlying all classes of from the antibiograms of D. radiodurans. Based on this bactericidal antimicrobials (Kohanski et al. 2007; Liochev work, it should be possible in future to engineer D. and Fridovich 1999; Touati 2000). Briefly, highly reactive radiodurans using appropriate antimicrobial resistance hydroxyl radicals (HO•) are formed as a function of gene determinants for bioremediation within contaminated metabolism related to three reactions (1) transfer of radioactive environments. electrons from reducing substrates such as nicotinamide- adenine dinucleotide (NADH) to oxygen (O2), (2) leaching of ferrous iron(II) (Fe2+) from iron-sulfur (Fe-S) clusters, Materials and methods and (3) stimulation of the Fenton reaction—the reaction of 2+ Fe with hydrogen peroxide (H2O2) to yield ferric iron(III) Bioinformatics (Fe3+), hydroxyl anion (OH−), and HO•—which will readily damage membrane lipids, proteins, and DNA Genes of D. radiodurans related to resistance to anti- (Kohanskietal.2007; Liochev and Fridovich 1999; microbials were retrieved from the ARDB database Touati 2000;Daly2009; Chen and Schopfer 1999). Some accessible at http://ardb.cbcb.umd.edu/ (Liu and Pop antimicrobials inhibit ribosome function, targeting both 2009). Re-annotation work of the genome of D. radiodurans the 30S (tetracycline family) and 50S (macrolide family was done using the RAST server available at http://rast. and chloramphenicol) ribosome subunits. Cell-wall syn- nmpdr.org/ (Aziz et al. 2008). Proteins were retrieved from thesis inhibitors (such as beta-lactams), which interact the curated protein sequence database SWISS-PROT with penicillin-binding proteins and glycopeptides that (Bairoch and Apweiler 2000). Proteic clusters were deter- interact with peptidoglycan building blocks, interfere with mined using ProtoNet accessible at http://www.protonet.cs. normal cell-wall synthesis and induce lysis and cell death. huji.ac.il (Kaplan et al. 2005). Also, when indicated, Ann Microbiol (2012) 62:493–500 495 homology search was done using default settings and (Battista 1997). Antibiotic susceptibility testing was per- published recommendations of BLAST at NCBI (Altschul formed by using the Kirby-Bauer disc diffusion method et al. 1990; Pertsemlidis et al. 2001; Sayers et al. 2009). (Sanofi Dignostics Pasteur, France) after inoculation into Protein alignment was done using CLUSTAL W (Thompson Mueller-Hinton agar for 48 h at 30°C as recommended by et al. 1994). Prediction of the radiation/desiccation response the Comité de l’Antibiogramme de la Société Française de motif (RDRM) was made using the consensus sequence Microbiolgie (CA-SFM). Commercially available disks (WTHYGBNNNNNVCDDAN) of previously published 25 loaded with the following antibiotics were used: Penicillin motifs for D. radiodurans (Makarova et al. 2007)atthe G(6μg), oxacillin (5 μg), amoxicillin (25 μg), amoxicillin prokaryotic database of gene regulation PRODORIC (21 μg), amoxicillin + clavulanic acid (20/10 μg), cefalotin (Munch et al. 2003). (30 μg), cefotaxim (30 μg), ticarcillin (75 μg), ticarcillin + To predict whether the investigated 19 genes have a clavulanic-acid (75/10 μg), piperacillin (75 μg), probability of being expressed, we utilized a selection piperacillin-tazobactam (75/10 μg), cefoxitin (30 μg), strategy that we previously have demonstrated to have a cefotetan (30 μg), cefamandole (30 μg), ceftazidime high success rate in eukaryotic systems (Desler et al. 2009). (30 μg), cefepime (30 μg), cefsulodin (30 μg), latamoxef The selection strategy is based on the presence of protein (30 μg), aztreonam (30 μg), imipenem (10 μg), streptomycin domains and targeting signals in the hypothetical proteins (10 IU), gentamicin (15 μg), gentamicin (500 μg), kanamycin encoded by the 19 genes. To complement the selection (30 μg), kanamycin (10 μg), tobramycin (10 μg), netilmicin strategy, it was investigated whether each of the 19 genes (30 μg), amikacin (30 μg), erythromycin (15 IU), were part of predicted operons. The principle of the clindamycin(2IU),spiramycin(100μg), pristinamycin selection strategy is that occurrence of protein domains (15 μg), lincomycin (15 μg), tetracycline (30 IU), and targeting signals can be predicted with high probability. chloramphenicol (30 μg), rifampin (30 μg), cotimoxazole The presence of targeting signals or identifiable protein (1.25+23.75 μg), ofloxacin (5 μg), ciprofloxacin (5 μg), domains can also be present in pseudogenes as a result of pefloxacine (5 μg), nalidixic acid (30 μg), colistin gene duplication. However, if both targeting signals and (50 μg), vancomycin (30 μg), teicoplanin (30 μg), identifiable protein domains are occurring in the hypothetical fosfomycin (50 μg), fusidic acid (10 μg), novobiocin protein encoded by the gene and the gene is a part of a predicted (5 μg) (Sanofi Diagnostics Pasteur). The interpretation of operon, the risk of the gene being a pseudogene is greatly the inhibition diameters was made according to the reduced. To evaluate the fidelity of our selection strategy, the recommendations of the CA-SFM making it possible to three characterized proteins, PolA (DR_1707), RecA classify strains in three categories: sensitive (S), inter- (DR_2340), and PprA (DR_A0346), were also submitted to mediate (I) and resistant (R). Category (I) was included the selection strategy as positive controls. in category (R). The SMART program (http://smart.embl-heidelberg.de/) was used to screen the hypothetical proteins encoded by the 19 genes and the proteins encoded by the three controls. Results and discussion The SMART program identifies protein domains from a database of manually annotated known protein domains Compared to previous literature (Agha et al. 1975; Hawiger (Letunic et al. 2006; de Castro et al. 2006). The occurrence and Jeljaszewicz 1967), this is the first report that of targeting signals in the investigated proteins was investigates the antimicrobial susceptibility of an ionizing predicted using PSORTb (http://www.psort.org/psortb/). radiation-resistant bacterium based on an integrated PSORTb is a tool for prediction of subcellular localization approach. In silico, genes of D. radiodurans related to of bacterial proteins (Gardy et al. 2005). For Gram-positive resistance to antimicrobials were retrieved from the ARDB bacteria, the program generates prediction results for four database (Liu and Pop 2009). Ten genes of D. radiodurans localizations (cytoplasmic, cytoplasmic membrane, cell were found to be related to resistance to antimicrobials. In wall and extracellular). To examine if the 19 genes are part addition, the genome of D. radiodurans was accurately of operons, the DOOR database (http://csbl1.bmb.uga.edu/ re-annotated at the RAST server (Aziz et al. 2008), and OperonDB/DOOR.php) was utilized. The DOOR database eight genes related to resistance to antimicrobials were contains all predicted operons for D. radiodurans and other retrieved. Following proteic clustering, an additional gene prokaryotes (Mao et al. 2009). was retrieved from ProtoNet (Kaplan et al. 2005). Compared to previously published annotation (White et al. 1999), six Antibiograms genes [DR_0473 (ABC transporter), DR_1619 (hypothetical protein), DR_1648 (amino acid ABC transporter), DR_2145 The optimal growth temperature of D. radiodurans (strain (ABC transporter), DR_2192 (ABC transporter), and

DSM 20539 / R1 / NCIB 9279 / ATCC 13939) is 30°C DR_A0137 (amino acid ABC transporter)] were 496 Ann Microbiol (2012) 62:493–500 re-annotated as antimicrobial resistance genes (Table 1). beta-lactam antibiotic-triggered release of autolytic peptido- Information about the functional status of the 19 glycan cross-linking enzymes (Goo and Sim 2011). analyzed genes was collected from two antibiograms of Moreover, D. radiodurans is sensitive to six amino- D. radiodurans. Although technically Gram-positive, D. glycosides, five macrolides and lincosamides, one tetracy- radiodurans has a complex cell envelope similar to that of cline, four bacteriostatic antimicrobials, four quinolones, Gram-negative organisms (Battista 1997). It is considered two glycopeptides, one broad-spectrum antimicrobial, one as a negibacterium that evolved its murein walls predom- aminocoumarin antimicrobial, and one polymyxin. inantly much thicker than usual (Cavalier-Smith 2006). However, D. radiodurans is resistant to one macrolide For this reason, we did two antibiograms for D. radiodurans (spiramycin), although it is sensitive to other tested using antimicrobials that target both Gram-positive and macrolides (erythromycin, pristinamycin). The differential Gram-negative bacteria. We obtained similar antibiograms effects of various macrolides on D. radiodurans may be indicating that D. radiodurans is sensitive to all tested 19 explained by a previous work (Starosta et al. 2010) beta-lactam antimicrobials. The sensitivity of D. radiodurans indicating that macrolide antimicrobials do not inhibit to tested beta-lactams was expected since it has a predicted translation of all nascent chains similarly, but rather penicillin-binding protein (DR_0479) most probably exhibit polypeptide-specific inhibitory effects. involved in the process of synthesizing cross linked Also, D. radiodurans is resistant to three quinolones peptidoglycan. Indeed, bacterial cell death is initiated by (ofloxacin, ciprofloxacin, and nalidixic acid). Based on

Table 1 Antimicrobial resistance-related genes in Deinococcus radiodurans R1

Locus_tag / Protein_ID Protein existencea Probability of encoding a Protein description (cluster in ProtoNet)c (retrieval source) translatable proteinb

DR_0009 / Q9RYE0 (RAST) Predicted 1 Vancomycin resistance protein VanW (Cluster 3229259). DR_0025 / Q9RYC5 (RAST) Predicted 1 Vancomycin resistance protein VanW (Cluster 3229259). DR_0433 / Q9RX82 (RAST) Predicted 3 Beta-lactamase with a transpeptidase fold (Cluster 3418513). DR_0454 / Q9RX61 (ARDB) Inferred from 3 Bacitracin resistance protein BacA (Cluster 3377737). homology DR_0455 / Q9RX60 (ARDB) Predicted 2 Streptomycin 3-kinase (Cluster 3326048). DR_0472 / Q9RX43 (ARDB) Predicted 3 Tetracycline-efflux transporter (Cluster 3352459). DR_0473 / Q9RX42 (ARDB) Predicted 3 Macrolide-efflux transporter, ABC-type antimicrobial peptide transport system (Cluster 3415277). DR_0760 / Q9RWA9 (RAST) Predicted 2 Beta-lactamase with a transpeptidase fold (Cluster 3358673). DR_1004 / Q9RVM3 (ARDB) Inferred from 2 Small multidrug resistance (SugE) protein (Cluster 3389269). homology DR_1005 / Q9RVM2 (ProtoNet) Inferred from 3 Small multidrug resistance (SugE) protein (Cluster 3389269). homology DR_1619 / Q9RTY1 (RAST) Predicted 1 Vancomycin resistance protein VanW (Cluster 3229259). DR_1648 / Q9RTV6 (ARDB Predicted 2 Macrolide-efflux transporter, ABC-type polar amino acid transport system (Cluster 3415277). DR_1913 / Q9RT53 (RAST) Inferred from 2 DNA gyrase, subunit A (Cluster 3366089). homology DR_2145 / Q9RSH9 (ARDB) Predicted 2 Macrolide-Lincosamide-Streptogramin B efflux transporter, ABC-type molybdenum transport system, ATPase component/photorepair protein PhrA (Cluster 3338085). DR_2192 / Q9RSD3 (ARDB) Predicted 2 Macrolide-efflux transporter, ABC-type antimicrobial peptide transport system (Cluster 3415277). DR_2303 / Q9RS24 (ARDB) Predicted 1 Chloramphenicol acetyltransferase (Cluster 3201287). DR_A0137 / Q9RZ16 (ARDB) Predicted 3 Macrolide-efflux transporter, ABC-type proline/glycine betaine transport systems (Cluster 3415277). DR_A0218 / Q9RYU0 (RAST) Predicted 1 Beta-lactamase with a transpeptidase fold (Cluster 3370472). DR_A0241 / Q9RYR7 (RAST) Predicted 3 Beta-lactamase with a transpeptidase fold (Cluster 3271812). a Information from Swiss-Prot (Bairoch and Apweiler 2000) b Score ranges from 0 to 3 c Information from RAST (Aziz et al. 2008), ARDB (Liu and Pop 2009), or ProtoNet (Kaplan et al. 2005) Ann Microbiol (2012) 62:493–500 497 previous literature (Bearden and Danziger 2001; Jacoby Gram-negative and Gram-positive microorganisms. For 2005;Ruiz2003), three mechanisms of resistance to Gram-negative bacteria, it is the DNA gyrase, whereas in quinolones in D. radiodurans were expected: plasmids that the Gram-positives, it is the topoisomerase IV. However, protect cells from the lethal effects of quinolones, mutations some studies indicated that the DNA gyrase may act as the that alter the drug targets, and impermeability of the primary target in Gram-positive microorganisms for some membrane and/or an overexpression of efflux pump quinolones, and that some quinolones have similar affinity systems that cause decreased accumulation inside the for both targets (Ruiz 2003, and references therein). The bacteria. quinolone resistance-determining region (QRDR), between First, we analyzed the possibility that the resistance of D. positions 51 and 106 with “hot spots” for mutation at amino radiodurans to ofloxacin, ciprofloxacin, and nalidixic acid acid positions 83 and 87 (Escherichia coli numbering) is a plasmid-mediated quinolone resistance through a Qnr (Jacoby 2005, and references therein), of the A subunit of protein homologue, which could have evolved from an DNA gyrase (GyrA) of D. radiodurans was analyzed for immunity protein designed to protect DNA gyrase from a mutations. Based on E. coli numbering, two amino acid naturally occurring inhibitor (Tran and Jacoby 2002). This substitutions in the “hot spot” region of GyrA (an alanine possibility was rejected because qnr determinants were residue replaced by a serine residue in position 84 and a undetectable in the genome of D. radiodurans using the valine residue replaced by an isoleucine residue in position Basic Local Alignment Search Tool (BLAST) at the US 85) and an amino acid substitution just after the “hot spot” National Center for Biotechnology Information (NCBI) region of GyrA (a threonine residue replaced by an alanine (Altschul et al. 1990; Pertsemlidis and Fondon 2001; residue in position 88; Izumi and Aranishi 2004) were Sayers et al. 2009). observed in D. radiodurans (Fig. 1). Observed mutations in Second, some studies indicated that the DNA gyrase, the QRDR, which is near the putative active site supposed which is important for ionizing radiation resistance (Zhang to be the interaction site between the A subunit of DNA et al. 2005; Liu et al. 2003; Sghaier et al. 2008; Lipton et al. gyrase and quinolones (Izumi and Aranishi 2004; Jacoby 2002), may act as the primary target for some quinolones 2005), may contribute to the resistance of D. radiodurans (Ruiz 2003, and references therein). Interestingly, unlike to quinolones. SOS response in other bacteria, both DNA gyrase subunits Third, the five prokaryotic families of efflux transporters (DR_0906 and DR_1913) were induced with the recA-like (Li and Nikaido 2009) are present in D. radiodurans in expression pattern, suggesting that regulation of DNA which ABC (ATP binding cassette) family members supercoiling is important for repair of DNA double-strand predominate: ABC (DR_0062, DR_0095, DR_0096, breaks (Liu et al. 2003). Relevant to this, researchers DR_0163, DR_0205, DR_0283, DR_0284, DR_0406, observed that the interaction between gyrase inhibitors and DR_0473, DR_0475, DR_0511, DR_0927, DR_0957, the GyrA subunit converts DNA gyrase into a lesion- DR_1012, DR_1034, DR_1035, DR_1103, DR_1302, inducing agent, which results in widespread generation of DR_1356, DR_1550, DR_1567, DR_1568, DR_1581, double-stranded DNA breaks (Kohanski et al. 2007, and DR_1635, DR_1648, DR_2051, DR_2052, DR_2107, references therein). Thus, it is pertinent to ask whether the DR_2118, DR_2119, DR_2134, DR_2145, DR_2153, resistance of D. radiodurans to quinolones is related, in DR_2192, DR_2198, DR_2284, DR_2316, DR_2379, part, to its ability to survive the accumulation of chromo- DR_2404, DR_2469, DR_2590, DR_A0007, DR_A0137, somal DNA double strand breaks (Blasius et al. 2007). This DR_A0160, DR_A0260, DR_A0261, DR_A0280, resistance may also be related to the fact that, in the case of DR_A0324, DR_A0349, DR_B0016, DR_B0049, D. radiodurans, quinolones, to reach their targets, must DR_B0121), MF (major facilitator) (DR_0258, DR_1327, traverse an additional outer membrane barrier (Jacoby DR_A0061, DR_A0247), SMR (small multidrug resis- 2005; Battista 1997). Also, in this context, it is important tance) (DR_1004, DR_1005), MATE (multidrug and toxic to note that the quinolone targets are basically different in efflux) (DR_0107), and RND (resistance–nodulation–

Fig. 1 Alignment of amino acid sequences of the quinolone R1 (accession no. Q9RT53). Identities are denoted by asterisks and resistance-determining region (QRDR) of the gyrase A from Escher- similarities are indicated by colons. The “hot spot” region for mutation ichia coli K-12 (accession no. Q47245) and Deinococcus radiodurans is indicated by a red over-line 498 Ann Microbiol (2012) 62:493–500 division) (DR_0740). There are 60 genes in families of and at 99 bp before ATG (TTATGTTAATCACGTAA), efflux transporters of D. radiodurans. As a consequence, respectively, and may belong to the radiation–desiccation their possible reduction of quinolones accumulation in D. response regulon of D. radiodurans. Altogether, these results radiodurans, particularly that target alterations and efflux represent connotative evidence that antimicrobial resistance activation are often found together in resistant isolates genes of D. radiodurans encode non-functional proteins or (Jacoby 2005), cannot be ruled out until definitive belong to inefficient biochemical pathways. evidence. In a previous work (Kim et al. 2009), it was shown that Except its resistance to quinolones, the sensitivity of D. gamma irradiation produces radiolysis of water, resulting in radiodurans to investigated antimicrobials, although its the production of radicals such as HO•, hydrogen radical • − possession of relevant resistance genes (e.g., beta- (H ), and solvated electron (eaq ), which play a role in lactamase, chloramphenicol acetyltransferase, vancomycin degrading antimicrobials, although some scavengers of HO• − B-type resistance protein), was preliminary rationalized in and eaq , like nitrite and nitrate, significantly inhibit the terms of the presence/absence of translatable messenger radiolytic decomposition of some antimicrobials (Choi et ribonucleic acids (mRNAs). To predict whether the inves- al. 2008). Accordingly, the real effect of antimicrobials on tigated 19 genes retrieved from ARDB and RAST data- D. radiodurans in a radioactive waste with characterized bases (Table 1) have a probability of being expressed, the chemical constraints remains unclear until definitive proof. SMART and PSORTb programs, together with the entries Reciprocally, as exposure to bactericidal antimicrobials is of the DOOR database, were used to assign a score to each related to leaching of Fe2+ from Fe–S clusters (Kohanski et of the 19 genes (Table 1) and to three control genes using a al. 2007) and as the concentration of intracellular Fe ions is previously described selection strategy (Desler et al. 2009). negatively related to the degree of ionizing radiation polA, recA, and pprA were chosen as control genes because resistance (Daly et al. 2004; Daly 2009), one can speculate they have been shown to be functional and they are that the exposure of D. radiodurans to antimicrobials will crucially involved in resistance mechanisms of D. sensitize it to ionizing radiation. Clearly, Fe homeostasis is radiodurans (Heinz and Marx 2007; Kim and Cox 2002; crucial for bacterial cell survival under antibiotics or Narumi et al. 2004; Lipton et al. 2002). The score ranges ionizing radiation (Daly et al. 2004; Kohanski et al. 2007; from 0 being the lowest score and 3 the highest score Daly 2009; Yeom et al. 2010). possible, indicating that the investigated gene has, respectively, a low or a high probability of encoding a translatable mRNA. One point was added to the score of an investigated Conclusion gene if a protein domain could be identified in the encoded proteins. Another point was added if the encoded proteins For the treatment of mixed radioactive wastes, the proposed were predicted to localize in the cytosolic compartment, use of D. radiodurans represents a realistic approach (Daly cytoplasmic membrane, cell wall, or in the extracellular 2000), especially that D. radiodurans-like strains have been space. A final point was added if the gene was part of a isolated from a radionuclide-contaminated environment in predicted operon. For the three control genes known to be the USA (Fredrickson et al. 2004). To optimize this expressed in D. radiodurans, polA and recA have a perfect treatment, our results highlight the need to improve the score of 3 while pprA has a score of 2. The 19 investigated resistance of D. radiodurans to antimicrobials. Fortunately, genes all have a score of at least 1, indicating an increased D. radiodurans is transformable with a remarkable genome probability of the gene encoding a translatable mRNA. Of plasticity (Daly 2000). For example, D. radiodurans has the 19 investigated genes, 7 yielded a score of 3 while 7 been engineered to express metal-remediating and organic yielded a score of 2. A total of 14 genes therefore have compound-degrading genes in highly irradiating environ- scores comparable to the scores obtained for polA, recA, ments (Brim et al. 2000; Lange et al. 1998). Also, previous and pprA. Hence, our bioinformatics analysis, based on a results showed that D. radiodurans expressing Pseudomo- previously used selection strategy (Desler et al. 2009), nas putida tod and xyl operons is capable of mineralizing supports the possibility of the 19 ORFs all encoding toluene and other fuel hydrocarbons, and that energy translatable mRNAs whereof 14 of these have an increased derived from toluene catabolism is coupled to its native probability of encoding translatable mRNAs (Table 1). More- hexavalent chromium Cr(VI)-reducing capabilities. More- over, among the 19 investigated genes, only a beta-lactamase over, D. radiodurans, in combination with humic acids or (DR_0433) and the subunit A of DNA gyrase (DR_1913) synthetic electron shuttle agents, can reduce uranium (U) were selected by the screen using a previously discovered and technetium (Tc) (Brim et al. 2006; Fredrickson et al. consensus sequence of the RDRM in D. radiodurans 2000). In the future, we believe that a more efficient use of (Makarova et al. 2007). DR_0433 and DR_1913 have D. radiodurans for bioremediation will necessitate anterior RDRMs in the coding region (ATTCGCGGCCTGCAAAA) microbiological and chemical analyses of the contaminated Ann Microbiol (2012) 62:493–500 499 site to determine the potential antimicrobial drugs that may Brim H, Osborne JP, Kostandarithes HM, Fredrickson JK, Wackett LP, be present, their radiolytic decomposition rate, and their Daly MJ (2006) Deinococcus radiodurans engineered for complete toluene degradation facilitates Cr(VI) reduction. Microbiol toxic effects on D. radiodurans. Consequently, researchers 152(8):2469–2477 interested in implementing an efficacious bioremediation Cavalier-Smith T (2006) Rooting the tree of life by transition strategy will need to design the most potent genetic analyses. Biol Direct 1:19 constructs to be introduced to D. radiodurans. Challis GL, Hopwood DA (2003) Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proc Nat Acad Sci USA Acknowledgements We would like to thank the reviewers for their 100(2):14555–14561 comments that helped to improve the manuscript. This work was Chen SX, Schopfer P (1999) Hydroxyl-radical production in physiological performed under the auspices of the Tunisian National Center for reactions. A novel function of peroxidase. Eur J Biochem 260 Nuclear Sciences and Technology (CNSTN) in collaboration with the (3):726–735 Laboratory of the National Bone Marrow Transplantation Center Choi D, Yu S, Lee M (2008) The effects of inorganic ions on (Tunisia) and the Center for Healthy Aging in Copenhagen University radiolytic decomposition of oxytetracycline. Appl Chem 12 (Denmark). A part of this work was supported by the NORDEA (2):397–400 foundation (Denmark). Special thanks to Kira Makarova who Daly MJ (2000) Engineering radiation-resistant bacteria for environ- provided helpful comments that improved an initial draft. mental biotechnology. Curr Opin Biotechnol 11(3):280–285 Daly MJ (2009) A new perspective on radiation resistance based on Deinococcus radiodurans. Nat Rev 7(3):237–245 Daly MJ, Gaidamakova EK, Matrosova VY, Vasilenko A, Zhai M, References Venkateswaran A, Hess M, Omelchenko MV, Kostandarithes HM, Makarova KS, Wackett LP, Fredrickson JK, Ghosal D (2004) Accumulation of Mn(II) in Deinococcus radiodurans facili- Agha N, Salih FM, Auda H, Al-Barwari S (1975) The effect of tates gamma-radiation resistance. Science 306(5698):1025–1028 ionizing radiation on the antibiotic sensitivity of Micrococ- de Castro E, Sigrist CJ, Gattiker A, Bulliard V, Langendijk-Genevaux cus radiodurans strains RI and RII5. Int J Appl Radiat Isot 26 PS, Gasteiger E, Bairoch A, Hulo N (2006) ScanProsite: (6–7):387–391 detection of PROSITE signature matches and ProRule- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic associated functional and structural residues in proteins. Nuclec local alignment search tool. J Mol Biol 215(3):403–410 Acids Res 34:362–365 Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Desler C, Suravajhala P, Sanderhoff M, Rasmussen M, Rasmussen LJ Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, (2009) In Silico screening for functional candidates amongst Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, hypothetical proteins. BMC Bioinform 10:289 Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Fredrickson JK, Kostandarithes HM, Li SW, Plymale AE, Daly MJ (2000) Vonstein V, Wilke A, Zagnitko O (2008) The RAST Server: rapid Reduction of Fe(III), Cr(VI), U(VI), and Tc(VII) by Deinococcus annotations using subsystems technology. BMC Genomics 9:75 radiodurans R1. Appl Environ Microbiol 66(5):2006–2011 Bagwell CE, Bhat S, Hawkins GM, Smith BW, Biswas T, Hoover TR, Fredrickson JK, Zachara JM, Balkwill DL, Kennedy D, Li SM, Saunders E, Han CS, Tsodikov OV, Shimkets LJ (2008) Survival Kostandarithes HM, Daly MJ, Romine MF, Brockman FJ (2004) in nuclear waste, extreme resistance, and potential applications Geomicrobiology of high-level nuclear waste-contaminated vadose gleaned from the genome sequence of Kineococcus radiotolerans sediments at the hanford site, washington state. Appl Environ SRS30216. PLoS ONE 3(12):e3878 Microbiol 70(7):4230–4241 Bai L, Li L, Xu H, Minagawa K, Yu Y, Zhang Y, Zhou X, Floss HG, Gardy JL, Laird MR, Chen F, Rey S, Walsh CJ, Ester M, Brinkman Mahmud T, Deng Z (2006) Functional analysis of the validamycin FS (2005) PSORTb v. 2.0: expanded prediction of bacterial protein biosynthetic gene cluster and engineered production of validoxyl- subcellular localization and insights gained from comparative amine A. Chem Biol 13(4):387–397 proteome analysis. Bioinformatics 21(5):617–623 Bairoch A, Apweiler R (2000) The SWISS-PROT protein sequence Goo KS, Sim TS (2011) Designing new beta-lactams: implications database and its supplement TrEMBL in 2000. Nuclec Acids Res from their targets, resistance factors and synthesizing enzymes. 28(1):45–48 Curr Comput Aided Drug Des 7(1):53–80 Battista JR (1997) Against all odds: the survival strategies of Hawiger J, Jeljaszewicz J (1967) Antibiotic sensitivity of Micrococcus Deinococcus radiodurans. Annu Rev Microbiol 51:203–224 radiodurans. Appl Microbiol 15(2):304–306 Bearden DT, Danziger LH (2001) Mechanism of action of and Heinz K, Marx A (2007) Lesion bypass activity of DNA polymerase resistance to quinolones. Pharmacotherapy 21(10):224S–232S A from the extremely radioresistant organism Deinococcus Blasius M, Buob R, Shevelev IV, Hubscher U (2007) Enzymes radiodurans. J Biol Chem 282(15):10908–10914 involved in DNA ligation and end-healing in the radioresistant Izumi S, Aranishi F (2004) Relationship between gyrA mutations and bacterium Deinococcus radiodurans. BMC Mol Biol 8:69 quinolone resistance in Flavobacterium psychrophilum isolates. Bonaventura C, Johnson FM (1997) Healthy environments for healthy Appl Environ Microbiol 70(7):3968–3972 people: bioremediation today and tomorrow. Environ Health Jacoby GA (2005) Mechanisms of resistance to quinolones. Clin Perspect 105(1):5–20 Infect Dis 41(2):S120–S126 Brim H, McFarlan SC, Fredrickson JK, Minton KW, Zhai M, Wackett Kaplan N, Sasson O, Inbar U, Friedlich M, Fromer M, Fleischer H, LP, Daly MJ (2000) Engineering Deinococcus radiodurans for Portugaly E, Linial N, Linial M (2005) ProtoNet 4.0: a metal remediation in radioactive mixed waste environments. Nat hierarchical classification of one million protein sequences. Biotechnol 18(1):85–90 Nuclec Acids Res 33:216–218 Brim H, Venkateswaran A, Kostandarithes HM, Fredrickson JK, Daly MJ Kim JI, Cox MM (2002) The RecA proteins of Deinococcus (2003) Engineering Deinococcus geothermalis for bioremediation radiodurans and Escherichia coli promote DNA strand ex- of high-temperature radioactive waste environments. Appl Environ change via inverse pathways. Proc Natl Acad Sci USA 99 Microbiol 69(8):4575–4582 (12):7917–7921 500 Ann Microbiol (2012) 62:493–500

Kim HY, Yu SH, Lee MJ, Kim TH, Kim SD (2009) Radiolysis of Pertsemlidis A, Fondon JW, 3rd (2001) Having a BLAST with selected antibiotics and their toxic effects on various aquatic bioinformatics (and avoiding BLASTphemy). Genome Biol 2 organisms. Radiat Phys Chem 78:267–272 (10):reviews2002.1–reviews2002.10 Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ (2007) Phillips RW, Wiegel J, Berry CJ, Fliermans C, Peacock AD, White A common mechanism of cellular death induced by bactericidal DC, Shimkets LJ (2002) Kineococcus radiotolerans sp. nov., a antibiotics. Cell 130(5):797–810 radiation-resistant, gram-positive bacterium. Int J Syst Evol Lange CC, Wackett LP, Minton KW, Daly MJ (1998) Engineering a Microbiol 52(3):933–938 recombinant Deinococcus radiodurans for organopollutant degra- Ruiz J (2003) Mechanisms of resistance to quinolones: target dation in radioactive mixed waste environments. Nat Biotechnol alterations, decreased accumulation and DNA gyrase protection. 16(10):929–933 J Antimicrob Chemother 51(5):1109–1117 Letunic I, Copley RR, Pils B, Pinkert S, Schultz J, Bork P (2006) Sayers EW, Barrett T, Benson DA, Bryant SH, Canese K, Chetvernin SMART 5: domains in the context of genomes and networks. V, Church DM, DiCuccio M, Edgar R, Federhen S, Feolo M, Nucleic Acids Res 34:D257–D260 Geer LY, Helmberg W, Kapustin Y, Landsman D, Lipman DJ, Li XZ, Nikaido H (2009) Efflux-mediated drug resistance in bacteria: Madden TL, Maglott DR, Miller V, Mizrachi I, Ostell J, Pruitt an update. Drugs 69(12):1555–1623 KD, Schuler GD, Sequeira E, Sherry ST, Shumway M, Liochev SI, Fridovich I (1999) Superoxide and iron: partners in crime. Sirotkin K, Souvorov A, Starchenko G, Tatusova TA, Wagner IUBMB Life 48(2):157–161 L, Yaschenko E, Ye J (2009) Database resources of the Lipton MS, Pasa-Tolic L, Anderson GA, Anderson DJ, Auberry DL, National Center for Biotechnology Information. Nuclec Acids Battista JR, Daly MJ, Fredrickson J, Hixson KK, Kostandarithes Res 37:D5–D15 H, Masselon C, Markillie LM, Moore RJ, Romine MF, Shen Y, Sghaier H, Ghedira K, Benkahla A, Barkallah I (2008) Basal DNA Stritmatter E, Tolic N, Udseth HR, Venkateswaran A, Wong KK, repair machinery is subject to positive selection in ionizing- Zhao R, Smith RD (2002) Global analysis of the Deinococcus radiation-resistant bacteria. BMC Genomics 9:297 radiodurans proteome by using accurate mass tags. Proc Natl Starosta AL, Karpenko VV, Shishkina AV, Mikolajka A, Sumbatyan NV, Acad Sci USA 99(17):11049–11054 Schluenzen F, Korshunova GA, Bogdanov AA, Wilson DN (2010) Liu B, Pop M (2009) ARDB–Antibiotic Resistance Genes Database. Interplay between the ribosomal tunnel, nascent chain, and macro- Nuclec Acids Res 37:443–447 lides influences drug inhibition. Chem Biol 17(5):504–514 Liu Y, Zhou J, Omelchenko MV, Beliaev AS, Venkateswaran A, Stair J, Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Wu L, Thompson DK, Xu D, Rogozin IB, Gaidamakova EK, Zhai improving the sensitivity of progressive multiple sequence M, Makarova KS, Koonin EV, Daly MJ (2003) Transcriptome alignment through sequence weighting, position-specific gap dynamics of Deinococcus radiodurans recovering from ionizing penalties and weight matrix choice. Nuclec Acids Res 22 radiation. Proc Natl Acad Sci USA 100(7):4191–4196 (22):4673–4680 Lovley DR (2003) Cleaning up with genomics: applying molecular Touati D (2000) Iron and oxidative stress in bacteria. Arch Biochem biology to bioremediation. Nat Rev 1(1):35–44 Biophys 373(1):1–6 Lovley DR, Coates JD (1997) Bioremediation of metal contamination. Tran JH, Jacoby GA (2002) Mechanism of plasmid-mediated Curr Opin Biotechnol 8(3):285–289 quinolone resistance. Proc Natl Acad Sci USA 99(8):5638–5642 Makarova KS, Omelchenko MV, Gaidamakova EK, Matrosova VY, White O, Eisen JA, Heidelberg JF, Hickey EK, Peterson JD, Dodson Vasilenko A, Zhai M, Lapidus A, Copeland A, Kim E, Land M, RJ, Haft DH, Gwinn ML, Nelson WC, Richardson DL, Moffat Mavrommatis K, Pitluck S, Richardson PM, Detter C, Brettin T, KS,QinH,JiangL,PamphileW,CrosbyM,ShenM, Saunders E, Lai B, Ravel B, Kemner KM, Wolf YI, Sorokin A, Vamathevan JJ, Lam P, McDonald L, Utterback T, Zalewski C, Gerasimova AV, Gelfand MS, Fredrickson JK, Koonin EV, Daly Makarova KS, Aravind L, Daly MJ, Minton KW, Fleischmann MJ (2007) Deinococcus geothermalis: the pool of extreme RD, Ketchum KA, Nelson KE, Salzberg S, Smith HO, Venter JC, radiation resistance genes shrinks. PLoS ONE 2(9):e955 Fraser CM (1999) Genome sequence of the radioresistant Mao F, Dam P, Chou J, Olman V, Xu Y (2009) DOOR: a database for bacterium Deinococcus radiodurans R1. Science 286 prokaryotic operons. Nuclec Acids Res 37:D459–D463 (5444):1571–1577 Munch R, Hiller K, Barg H, Heldt D, Linz S, Wingender E, Jahn D Yeom J, Imlay JA, Park W (2010) Iron homeostasis affects antibiotic- (2003) PRODORIC: prokaryotic database of gene regulation. mediated cell death in Pseudomonas species. J Biol Chem 285 Nuclec Acids Res 31(1):266–269 (29):22689–22695 Narumi I, Satoh K, Cui S, Funayama T, Kitayama S, Watanabe H Zhang C, Wei J, Zheng Z, Ying N, Sheng D, Hua Y (2005) Proteomic (2004) PprA: a novel protein from Deinococcus radiodurans that analysis of Deinococcus radiodurans recovering from gamma- stimulates DNA ligation. Mol Microbiol 54(1):278–285 irradiation. Proteomics 5(1):138–143