Ann Microbiol (2013) 63:1483–1491 DOI 10.1007/s13213-013-0612-2

ORIGINAL ARTICLE

There are more small amino acids and fewer aromatic rings in proteins of ionizing radiation-resistant bacteria

Haïtham Sghaier & Steinar Thorvaldsen & Nadia Malek Saied

Received: 5 September 2012 /Accepted: 25 January 2013 /Published online: 17 February 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013

Abstract The identification of specific amino acids (AAs) Introduction or groups of functionally important AA residues in ionizing radiation-resistant bacteria (IRRB) is an important challenge Ionizing radiation (IR) resistance has been observed in a broad in understanding the biological basis of resistance to ioniz- range of eubacteria, including the Deinococcus-Thermus phy- ing radiation (IR; X-rays and gamma-rays). To address this lum (many Deinococcus sp. and Truepera radiovictrix) problem, we compared homologous sites in multiple align- (Albuquerque et al. 2005), Actinobacteria (Rubrobacter radio- ments of proteins of IRRB and IR-sensitive bacteria (IRSB) tolerans, Rubrobacter xylanophilus and Kineococcus radio- using the DeltaProt Toolbox. Substitution patterns were tolerans) (Chen et al. 2004; Ferreira et al. 1999; Phillips used as evidence for selection of certain AAs over others. et al. 2002; Yoshinaka et al. 1973), Proteobacteria Our results show that, in contrast to aromatic AAs, small/- (Methylobacterium radiotolerans and Acinetobacter radiore- tiny AAs tend to be preferred in IRRB compared to IRSB. In sistens) (Ito and Iizuka 1971; Nishimura et al. 1994), agreement with previous experimental data showing that Cyanobacteria (Chroococcidiopsis sp.) (Billi et al. 2000)and oxidation of AA residues is causative in the killing of Sphingobacteria (Hymenobacter actinosclerus) (Collins et al. irradiated cells and that IR resistance is correlated with the 2000). Presently, IR-resistant bacteria (IRRB) with completely accumulation of divalent manganese ions (Mn2+)–peptide– sequenced genomes (Liolios et al. 2010) and with published orthophosphate (Pi) complexes, we proposed a chemical information regarding their radioresistance are limited in num- interpretation based on the Hard and Soft (Lewis) Acids ber and restricted in genera: Deinococcus deserti VCD115 and Bases (HSAB) concept. These findings should assist (Baudet et al. 2010; de Groot et al. 2005; de Groot et al. future efforts in selecting mutations for rational design of 2009), Deinococcus geothermalis DSM 11300 (Makarova et proteins with enhanced IR tolerance properties. al. 2007), Deinococcus maricopensis DSM21211(Pukalletal. 2011), Deinococcus proteolyticus MRP (Copeland et al. 2012),

Keywords Amino acids . Comparative genomics . Ionizing Deinococcus radiodurans R1 (White et al. 1999), Kineococcus radiation . Resistant bacteria radiotolerans SRS30216 (Bagwell et al. 2008), Rubrobacter xylanophilus DSM 9941 (Ferreira et al. 1999; Liolios et al. Haïtham Sghaier and Steinar Thorvaldsen contributed equally to this 2010)andTruepera radiovictrix DSM 17093 (Ivanova et al. work. 2011). In particular, IR resistance of bacteria belonging to the : H. Sghaier (*) N. M. Saied three genera Deinococcus, Kineococcus and Rubrobacter has Research Unit UR04CNSTN01 “Medical and Agricultural been well studied (Bagwell et al. 2008; Daly 2009; Ferreira et ” Applications of Nuclear Techniques , National Center for Nuclear al. 1999). Presently, strong lines of evidence from different Sciences and Technology (CNSTN), Sidi Thabet Technopark, 2020 Sidi Thabet, Tunisia laboratories have converged to the conclusion that the accu- e-mail: [email protected] mulation of Mn2+–peptide (7–22 amino acids in length in D. – N. M. Saied radiodurans) Pi complexes represents a widespread strategy e-mail: [email protected] for protecting irradiated cytosolic enzymes from reactive oxy- gen species (ROS) (Daly 2012;Dalyetal.2010, and references S. Thorvaldsen therein). Thus, extreme IR resistance in bacteria consistently University of Tromsø, Breivika, 9037 Tromsø, Norway coincides with greatly diminished susceptibilities to oxidation e-mail: [email protected] of amino acid (AA) residues compared to IR-sensitive species 1484 Ann Microbiol (2013) 63:1483–1491

(Daly 2012). Here, our aim is to investigate functionally im- Materials and methods portant AAs in IRRB compared to IR-sensitive bacteria (IRSB). In the present study, we determined whether the AA compo- In the context of understanding the biological basis of sition inferred for a set of proteins in three IRRB different resistance phenotypes, recent research in computa- [Deinococcus radiodurans R1 (White et al. 1999); tional biology has allowed the identification of the pheno- Kineococcus radiotolerans SRS30216 (Phillips et al. 2002) types of organisms based on the propensity of AAs to enter and Rubrobacter xylanophilus DSM 9941 (BioProject acces- more frequently into the proteins of thermophiles/hyperther- sion PRJNA58057)] was similar to the composition of an mophiles (Di Giulio 2000), barophiles/non-barophiles (Di identical set of proteins in three IRSB [Escherichia coli K- Giulio 2005a), acidophiles/alkaliphiles (Di Giulio 2005b), 12 (Blattner et al. 1997); Thermus thermophilus HB27 (Henne aerobic/anaerobic cells (Archetti and Di Giulio 2007), mes- et al. 2004)andPseudomonas putida F1 (BioProject acces- ophiles/psychrophiles (Thorvaldsen et al. 2007) and meth- sion PRJNA58355)]. In brief, a total of 31 basal DNA repair- anophiles/non-methanophiles (Di Giulio 2009). In addition, related proteins of D. radiodurans were selected (Sghaier et the current literature provides substitution preferences of al. 2008). Orthologs were obtained using the default settings AAs that lead to the adaptation of proteins to temperature of the Basic Local Alignment Search Tool (BLAST) at the (McDonald 2001; Metpally and Reddy 2009; McDonald National Center for Biotechnology Information (NCBI) web- 2010; Saunders et al. 2003; Pasamontes and Garcia-Vallve site (Altschul et al. 1990). Multiple alignments were con- 2006; Nakashima et al. 2003). Similarly, identification of structed using the ClustalW multiple sequence alignment AAs, which if damaged by radiolysis leads to loss of enzy- program (Thompson et al. 1994). Each of the six types of matic activity, and the determination of whether particular organisms was represented with only one sequence in each AA are favored by selection at high IR doses over others, alignment by assuming no horizontal gene transfer and, hence, have long been an objective in radiobiology and protein obtained a reasonably independent dataset in the sense that all evolution research (Garrison 1987; Rea et al. 2011). sequences belonged to different phylogenetic orders. All Radiation-induced decomposition of water generates several alignments were inspected and verified manually for a mini- reactive species [e.g., hydrogen peroxide (H2O2), hydroxyl mum cut-off score of 20 % identity with all other sequences. • • radical (HO ), hydroperoxyl radical (HO 2), hydrated elec- Five proteins (DR_0856, DR_1244, DR_0099, DR_2285, − + tron (e aq) and proton (H )], which, in the presence of and DR_2584) with low identity had to be eliminated because • •− oxygen, react further so that HO and superoxide (O2 ) of uncertain alignments. The final dataset consisted of multi- are essentially the only species present in oxygenated aque- ple alignments of 26 proteins; each of 3 IRRB plus 3 IRSB ous solutions (Draganić 2005). HO• is known to be far more AA sequences. We also grouped the AAs into 12 property •− • reactive than O2 ; therefore, the major fraction of HO is groups (Taylor 1986) as follows: the acidic AA group includ- removed by the most reactive sites. In proteins, aromatic, ed aspartic acid (D) and glutamic acid (E); aliphatic: isoleu- sulfur-containing and aliphatic AA side chains are known to cine (I), leucine (L) and valine (V); aromatic: histidine (H), be the main targets of HO•-mediated damage. Aliphatic AAs phenylalanine (F), W and tyrosine (Y); basic: arginine (R), H, become more reactive with an increasing number of methyl and lysine (K); charged: R, D, E, H and K; hydrophilic: D, E, groups, but the reaction with aromatic AA residues is faster K, asparagine (N), glutamine (Q) and R; hydrophobic: alanine and easier for addition of HO• to double bonds, with tryp- (A), cysteine (C), F, I, L, methionine (M), valine (V), Wand Y; tophan (W) residues being the most prone to oxidative neutral: glycine (G), Q, serine (S) and threonine (T); non- damage by ROS (Stadtman and Levine 2003). Thus, resis- polar: A, C, G, I, L, M, F, P, V, W and Y; polar: R, N, D, E, Q, tance to radical-induced damage might be related to the H, K, S and T; small: A, C, D, G, N, P, S and V; and tiny: A, G substitution of residues highly prone to oxidative damage and S. We applied the new methods of comparative statistical with residues less prone. In order to support this hypothesis, bioinformatics as described in a recent paper by Thorvaldsen we examined different AA compositions of proteins of et al. (2010), and AA composition and substitution were IRRB and IRSB. calculated and analyzed using the DeltaProt Toolbox. Using orthologous sequences clustering along with the To analyze changes in AA composition, we applied the DeltaProt, a software toolbox for comparative genomics, we nonparametric Wilcoxon paired test, which was not restrict- conducted an analysis of multiple alignments of DNA repair ed to the assumption of statistical independence within each proteins of IRRB and IRSB to examine a possible correla- group. We also computed the cumulated substitution matrix tion of AA substitution patterns with adaptation to their between the two groups of sequences, and analyzed it sta- respective optimal growth conditions under IR. In this pa- tistically with the chi-square test by assuming independence per, we discuss the results from comparative analysis of 26 of the data within each group. However, the Pseudomonas proteins from three members of IRRB and three members of and Escherichia sp. in the IRSB group were somewhat IRSB. related types of proteobacteria (same phylogenetic class, Ann Microbiol (2013) 63:1483–1491 1485 but different order). Therefore, we also calculated a substi- IRRB and IRSB proteins were compared using multiple align- tution matrix consisting of mean values between the groups ments (Fig. 1). Indeed, based on previous similar research and analyzed this dataset by allowing statistical dependence (Archetti and Di Giulio 2007; Di Giulio 2000, 2005a, b, within each group to determine whether the two approaches 2009; Thorvaldsen et al. 2007), we expected that differences corresponded. Similar results will add weight to the conclu- in AA composition between IRRB and IRSB may be symp- sions. Multiple testing can potentially increase the number tomatic for IR resistance. We made a comparison of the mean of false positives (Type I error) and, hence, the significance AA compositional changes calculated on the basis of the levels were set low (at 0.01), and individual P values were orthologous proteins observed in the direction from IRSB to reported. IRRB. A summary of overall sequence identities is shown in Table 1.Figure2 represents a plot of compositional changes observed in our dataset of 26 protein families. Using the Results paired Wilcoxon test, the AAs (A, R, N, D, Q, E, G, I, L, K, M, F, S, T, Y and V) sustained significant changes (P values< To examine if there were detectable trends in the AA compo- 0.01). The P values demonstrated significant preferences in sition related to IR resistance, AA compositional changes of frequencies of AA occurrences and property groups in IRRB

Fig. 1 Example of a multiple alignment used to analyze AA composition and substitution by the DeltaProt Toolbox. AAs are highlighted as follows: red aromatic residues (F, H, W, Y); blue small residues (A, C, D, N, G, P, S, V) and black other residues. All sequences are listed with their GenBank identifiers followed by the species abbreviation and bacterial phenotype. Species abbreviations: Dra Deinococcus radiodurans R1, Eco Escherichia coli K-12, Kra Kineococcus radiotolerans SRS30216, Ppu Pseudomonas putida F1, Rxy Rubrobacter xylanophilus DSM 9941, Tth Thermus thermophilus HB27. Phenotype abbreviations: S ionizing radiation-sensitive bacteria, R ionizing radiation- resistant bacteria. Gaps are denoted by the ‘-’ character. Other abbreviations are as indicated in the text 1486 Ann Microbiol (2013) 63:1483–1491

Table 1 Sequence identity

Ecoli Thet2 Psepi Deira Kinpd Rubxd Mean sequence length

Ecoli 1.000 804 Thet2 0.388 1.000 640 Psepi 0.679 0.372 1.000 806 Deira 0.362 0.587 0.349 1.000 663 Kinpd 0.374 0.445 0.379 0.409 1.000 686 Rubxd 0.449 0.505 0.430 0.471 0.518 1.000 644

Overall sequence identity and mean sequence length of the genes from Escherichia coli K-12 (Ecoli), Thermus thermophilus HB27 (Thet2), Pseudomonas putida F1 (Psepi), Deinococcus radiodurans R1 (Deira), Kineococcus radiotolerans SRS30216 (Kinpd) and Rubrobacter xylano- philus DSM 9941 (Rubxd). Organisms in bold are IR resistant proteomes as compared to IRSB proteomes or vice versa (aliphatic, hydrophobic and small) (P=0.0003). The internal (Table 2). As indicated by the P values from Table 2,there substitutions between the similar residues L and I were also were many AAs, including aliphatic (I, L, V), tiny (A, G, S) significantly less frequent than expected (Table 3; Fig. 3). and small AA residues (N, D, C, P, V), which were signifi- Figure 4 presents the results of the average substitution cantly preferred in IRRB as compared to IRSB. On the other counts between the two groups and is in good agreement hand, aromatic AA residues (H, F, W, Y) were significantly with Fig. 3, although the outcomes of the statistical tests, less favored in IRRB proteomes. No significant changes (P in general, were weaker because each group was treated values<0.01) were detected for the group of hydrophobic or as one observation. charged AA residues (Table 2). Figure 3 represents AA substitutions between IRSB and IRRB. For a given pair of AAs, the “forward” substitution Discussion refers to the direction between the two groups: from IRSB to IRRB. These accumulated counts were tested against a null Our objective in this study was to analyze the compositional hypothesis obtained by the same type of counts within one variation and substitution preferences of AAs in proteomes group (IRSB) in which there was no direction. We counted of IRRB compared to the proteomes of IRSB to investigate using the Jones method (Jones et al. 1992), in which the general proteome wide characteristics for IR adaptation. As sequences with the highest identities between the groups were indicated in Table 1, the overall sequence identity between paired, and observed AA exchanges were tallied in a matrix. two species largely reflects phylogenetic congruence rather The cumulative counts were determined by forming the max- than phenotypic similarity (Sghaier et al. 2011; Pride et al. imum number of sequence pairs, where each sequence was 2003). For exemple, D. radiodurans and T. thermophilus involved only once to avoid oversampling of the data. belong to a distinct bacterial clade with a high overall Interestingly, the most frequent favored significant substitu- sequence identity equal to 0.587 (Table 1) but have remark- tion (IRSB → IRRB) was L (aliphatic and hydrophobic) to V ably different phenotypes (Omelchenko et al. 2005).

4

3

2

1

0 Percentage -1

-2

-3 A R N D C Q E G H I L K M F P S T W Y V

Fig. 2 Change in composition of AAs. Comparison of the mean AA the empirical standard deviations. By the paired Wilcoxon test the AAs compositional changes calculated on the basis of 26 orthologous pro- (A, R, N, D, Q, E, G, I, L, K, M, F, S, T, Y and V) have a significant teins observed in the direction IR-sensitive → IR-resistant group, with change (P values<0.01). Abbreviations are as indicated in the text 3 sequences from different species in each group. Error bars represent Ann Microbiol (2013) 63:1483–1491 1487

Table 2 Change in composition

Amino acid Aromatic Aliphatic Tiny Small Hydrophobic Negative Positive group (HFW Y) (I LV) (A GS) (ANDCGPSV) (A G I LMF P WV) (D E) (R K)

Direction of change ↘↘↗↗ ––– P-value .0009 <10−4 <10−4 <10−4 .02 .22 .21

Changes in composition of merged groups of amino acids as observed in the direction from proteins of IRSB to proteins of IRRB. P values from Wilcoxon paired tests. Abbreviations are as indicated in the text

Previous research has indicated that the amount of dam- which consist mainly of peptides bound to Mn2+ and orthophos- age of AA residues in irradiated cells was strongly influ- phate (Pi) (Daly et al. 2010). At 50,000 Gy, Mn2+–peptide– enced by antioxidant status, where yields of IR-induced Pi complexes preserved 50 % activity of the dodecameric en- protein oxidation can be more than 100-fold greater in zyme glutamine synthetase, which is normally inactivated by hypersensitive bacteria than in extremely resistant bacteria. 150 Gy (Daly et al. 2010). IR resistance in bacteria appears to be dependent on the Here, our results indicated that the merged group of small importance of the function of a targeted AA sequence, its AAs, including sulfur-containing residues, is a preferred abundance, and how susceptible it is to carbonylation, a group of residues in IRRB as compared to IRSB (Table 2), severe and permanent form of oxidation (Daly 2012, 2009, which might be explained particularly by the functional and and references therein). Interspecies comparisons of irradi- structural importance of sulfur-containing AA residues— ated bacteria showed that the levels of damage of AA proteic disulfide bonds, although Fe-S is a preferred proteic sequences are not only quantitatively related to the efficien- target of ROS (Chatgilialoglu et al. 2011). Briefly, coordi- cy of DNA repair and survival, but were mechanistically nation binding between metals and organic molecules in- linked to the accumulation of divalent manganese ions cluding AAs is based on the interaction between an electron (Mn2+)(Daly2012;Dalyetal.2007). Ensuing studies donor (Lewis base) and an electron acceptor (Lewis acid). showed that protein-free cell extracts of IRRB are armed with This principle was established by Lewis in 1923 and later, in low-molecular-weight ROS-scavenging Mn2+ complexes, 1963, modified by Pearson who categorized both acids and

591 200 ~ ~ V V Y 493 Y W W 166 T T S S P 394 P 133 F F M M K K L 296 L 100 I I H H G G 197 67

Replacement amino acid E E Replacement amino acid Q Q C C D D 99 33 N N R R A A 0 A R N D C Q E G H I L K M F P S T W Y V ~ A R N D C Q E G H I L K M F P S T W Y V ~ Replaced amino acid Replaced amino acid

Fig. 3 Substitutions. Visualization of the cumulated number of pair- Fig. 4 Substitutions. Visualization of the mean number of pair- wise substitutions in a comparison of 26 alignments of orthologous wise substitutions observed between the two groups, when com- proteins from two groups. Each group consists of 3 sequences from 3 paring 26 alignments of orthologous proteins. The size and colour organisms, and the organisms are assumed to be independent. The size of each marker indicates the magnitude of the substitution (see and colour of each marker indicates the magnitude of the substitution colour-bar). A tilde (~) indicates deletion/insertion. Favoured sub- (see colour-bar). A tilde (~) indicates deletion/insertion. Favoured stitutions with P values<0.05 in the direction non-irradiated hab- substitutions with P values<0.05 in the direction non-irradiated habitat itat → irradiated-habitat are marked with upward-pointing → irradiated-habitat are marked with upward-pointing triangles, and triangles, and the non-favoured substitutions with P values<0.05 the non-favoured substitutions with P values<0.05 are marked with are marked with downward-pointing triangles. Abbreviations are downward-pointing triangles. Abbreviations are as indicated in the text as indicated in the text 1488 Ann Microbiol (2013) 63:1483–1491

Table 3 Substitutions Favored substitutions Non-favored substitutions

Pair Forward Reverse P value Pair Forward Reverse P value ~G 409 260 0.0002 ~Q 78 180 0.00007 LV 591 352 0.0003 ~L 225 366 0.0003 EG 213 112 0.004 DN 46 94 0.002 The most biased AA replace- ments in the IRSB → IRRB di- AG 336 253 0.004 IL 325 342 0.003 rection (P values<0.01). The ER 303 196 0.005 AK 82 161 0.003 dataset consists of 26 align- AD 186 137 0.008 AQ 123 225 0.003 ments, each of 6 ortholog pro- QK 55 70 0.003 teins, and the P values are obtained from the chi-square test EQ 192 220 0.005 in DeltaProt (Thorvaldsen et al. AL 184 355 0.005 2010). Abbreviations are as in- RL 102 184 0.009 dicated in the text bases in hard, soft and borderline. The interaction between which can remove ROS generated by the Fenton reaction and Lewis acids and Lewis bases is especially strong when other physicochemical redox processes and provide global pro- strong acids interact with strong bases and week acids tection of irradiated proteins. Indeed, a previous study interact with weak bases (Pearson 1963, and references (McNaughton et al. 2010) revealed a striking correlation be- therein). Our hypothesis, based on published in vitro data tween the in vivo concentration of Pi complexes of Mn2+ with (Daly 2012, 2009, and references therein), our in silico oxidative stress resistance, thereby supporting previous in vitro results and the Pearson acid base concept (Hard and Soft studies that demonstrated the superoxide dismutase (SOD) ac- Lewis Acids and Bases (HSAB) concept; Pearson 1963), is tivity of the Mn2+–Pi complex (Barnese et al. 2008). However, 2+ 2+ •− that the action of Mn ,aPearson’s Hard Lewis Acid Mn –Pi removes O2 rapidly and catalytically from aqueous (PHLA), and Pi, a Pearson’s Hard Lewis Base (PHLB), solution via a disproportionation mechanism that is entirely occur at two levels (Fig. 5): (1) Mn2+ replaces Fe2+,a different from those of the SOD enzymes (Barnese et al. 2008). Pearson’s Borderline Lewis Acid (PBLA) cofactor, and other Furthermore, our results indicated that aromatic AAs tend divalent cations to preserve active sites—mainly aliphatic (I, L, to be avoided in IRRB (Table 2). Indeed, all AA residues in V)andsmall(N,D,C,P,V)/tiny(A,G,S)AAsthroughtheir proteins are subject to ROS attacks; principally, short-lived − • PHLB (R-NH2,HO)—of irradiated enzymes from oxidative HO generated by IR; however, the aromatic AAs are most damage; and (2) surplus Mn2+ binds to Pi, a PHLB, to form sensitive to oxidation (Stadtman 1993). In accordance with a ROS-scavenging complexes with various other metabolites, previous classification of AAs that explained mutation data

Fig. 5 Model of ionizing-radiation-driven Mn action for global pro- indirectly, to damage all biomolecules (2). To protect proteins, Mn2+, tection of proteins. Compared to a non-irradiated cell (left) containing aPearson’s Hard Lewis Acid (PHLA), replaces Fe2+, a Pearson’s ferrous iron proteins (Protein-Fe), manganese (Mn) and orthophos- Borderline Lewis Acid (PBLA), as a cofactor of proteins to preserve phate (Pi), in an irradiated cell (right), γ irradiation induces the active sites (3). Then, surplus Mn2+ binds to Pi, a Pearson’s Hard Lewis production of ROS (1) which have the ability, either directly or Base (PHLB), to form ROS-scavenging complexes (4) Ann Microbiol (2013) 63:1483–1491 1489 through correlation mainly with their size (Taylor 1986), Competing interests The authors declare that they have no compet- together with the classification of Pearson (1963), we hy- ing interests. pothesize that aromatic rings of AAs are not efficiently 2+ shielded by Mn mainly because they have relatively large Authors’ contributions H.S. and S.T. conceived the study and com- dimensions and they are not PHLB. piled the data. S.T. developed algorithms and performed calculations. The substitution pattern in the orthologous proteins of H.S. and S.T. analyzed the results and wrote the paper. N.M.S. performed chemical analyses. All authors have approved the final version. IRRB and IRSB confirmed previous observations and showed several interesting features (Table 3; Figs. 3, 4) that are not seen in the change in composition of AAs (Fig. 2). Our findings indicated that the larger aliphatic AAs (L, I) References were decreased, but the smaller aliphatic AA (V) was in- creased in the IRRB proteomes as compared to IRSB pro- Albuquerque L, Simoes C, Nobre MF, Pino NM, Battista JR, Silva MT, teomes (Table 3;Figs.3, 4). The most frequent favored Rainey FA, da Costa MS (2005) Truepera radiovictrix gen. nov., substitution from IRSB to IRRB was L (big) to V (small). sp. nov., a new radiation resistant species and the proposal of Trueperaceae fam. nov. FEMS Microbiol Lett 247(2):161–169 Among the most frequent non-favored substitutions from Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic IRSB to IRRB were A (tiny) to L (big) together with the local alignment search tool. 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