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FEMS Microbiology Letters 11035 (2003) 1^6 www.fems-microbiology.org

1 Retron reverse transcriptase rrtT is ubiquitous in strains of 2 Salmonella enterica serovar Typhimurium

3 Jitka Matiasovicova a, Marcela Faldynova a, Martina Pravcova a, Renata Karpiskova b, 4 Ivana Kolackova b, Jiri Damborsky c, Ivan Rychlik a;

5 a Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic 6 b National Institute of Public Health, Center for Food Chain Hygiene, Palackeho 1-3, 612 42 Brno, Czech Republic 7 c National Centre for Biomolecular Research, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic

8 Received 19 March 2003; received in revised form 5 May 2003; accepted 5 May 2003

9 First published online

10 Abstract

11 Bacterial retron reverse transcriptases are unusual enzymes which utilise the same RNA molecule as a template and also as a primer for 12 initiation of the reverse transcription. Except for their relatively frequent presence in Myxococcus spp., they are considered as quite rare 13 proteins. However, in this study we proved that retron reverse transcriptase is frequently found in certain serovars of Salmonella enterica. 14 Using polymerase chain reaction (PCR), in strains of serovar Typhimurium, the rrtT (retron reverse transcriptase Typhimurium) gene was 15 detected in 158 out of 175 tested field strains. On the other hand, in none of the 18 tested serovar Enteritidis strains the rrtT was detected 16 in their genome. Detailed computer analysis allowed us to predict the sequence of msDNA and to propose that the final msDNA is free of 17 any RNA. Furthermore, we predict that there are at least three different classes of retron reverse transcriptases. 18 ß 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. 19 20 Keywords: Retron reverse transcriptase; msDNA; Salmonella enterica serovar Typhimurium 21

22 1. Introduction inverted repeats designated as a1 and a2 play a central 38 role. Subsequently, the folded molecule is branched using 39 23 Certain bacterial species encode a speci¢c type of re- 2P-OH of guanosine residue at the 3P-end of the a2 repeat 40 24 verse transcriptases called retron reverse transcriptases and this branch is used as a primer for the initiation of 41 25 (RRTs). These have been frequently found in Myxococcus reverse transcription. As a result, cDNA complementary 42 26 sp. [6,19] while in Escherichia coli they are found in about to the loop between a1 and a2 inverted repeats is formed. 43 27 10% of ¢eld strains only [8,20]. In other , RRTs A typical end product of this process is single-stranded 44 28 have been reported very rarely. In Salmonella, RRT was cDNA (msDNA, multicopy single-stranded DNA) which 45 29 detected in four out of 70 strains of the SARB reference accumulates in bacterial cell cytoplasm [5,12]. The biolog- 46 30 collectionUNCORRECTED[30] and recently RRT (rrtE, retron reverse tran- ical role PROOF of RRT or msDNA is unknown [13]. Speculations 47 31 scriptase Enteritidis) encoded by low molecular mass plas- were presented that it may be mutagenic [26] or may con- 48 32 mid of S. Enteritidis was described [32]. Unlike retroviral trol gene expression by an antisense mechanism [27]. 49 33 reverse transcriptase, bacterial reverse transcriptases utilise RRTs are also suggested to be involved in generation of 50 34 the same RNA molecule as a template and also as a prim- promoterless cDNA of genes which can be inserted into 51 35 er for initiation of the reverse transcription. This occurs by the integron structures [29]. 52 36 a mechanism during which the template RNA molecule is Interestingly, when an integron-encoded multidrug resis- 53 37 folded into a complex secondary structure in which two tance genomic island was sequenced in Salmonella enterica 54 serovar Typhimurium (S. Typhimurium), immediately 55 downstream from it, the rrtT gene was detected on a ge- 56 1 2 * Corresponding author. Tel.: +420 (5) 41321241; netic locus resembling a phage structure [2,3]. Using stan- 57 3 Fax: +420 (5) 41211229. dard BLAST analysis with the rrtT gene sequence encoded 58 4 E-mail address: [email protected] (I. Rychlik). by the multidrug-resistant S. Typhimurium we found this 59

1 0378-1097 / 03 / $22.00 ß 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies. 2 doi:10.1016/S0378-1097(03)00398-7

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60 gene also in the genome of the non-resistant completely ware was used also for primer design. The ¢rst primer pair 88 61 sequenced S. Typhimurium LT2 [28]. This suggested that (RTFor: 5P-GAA CTA TTG CTC ATC CTT CG-3P/ 89 62 the rrtT may not be a rare gene in Salmonella. We have RTRev: 5P-GTA ACG TGA CGG TTA TGT CC-3P) al- 90 63 therefore collected ¢eld strains of S. Typhimurium and lowed the detection of rrtT gene in di¡erent Salmonella 91 64 other serovars and detected the presence of rrtT by poly- serovars. The second primer pair (msDNAFor: 5P-GGA 92 65 merase chain reaction (PCR). In selected strains, produc- AGG AAG GTT ATC ATT GG-3P/msDNARev: 5P-GTC 93 66 tion of msDNA was tested. Obtained results together with TAT CCC TAA AAC TGG GG-3P) allowed for the am- 94 67 detailed sequence analysis allowed us to classify the rrtT, pli¢cation of the predicted msDNA locus and was used for 95 68 together with retrons of Vibrio cholerae, Vibrio parahae- generation of the probe used in DNA hybridisation. Sec- 96 69 molyticus, and E. coli Ec78 and Ec83, among a speci¢c ondary structure of the predicted msDNA was calculated 97 70 subclass of retron reverse transcriptases. using an algorithm available at http://www.bioinfo.rpi.e- 98 du/applications/mfold/. 99 The translated sequence of the rrtT gene from S. Typh- 100 71 2. Materials and methods imurium was searched against SWISS-PROT+TrEMBL 101 databases using BLAST P v2.0 algorithm [1] and BLO- 102 72 2.1. Bacterial strains and growth conditions SUM62 matrix. E-value threshold 0.001 was used for the 103 search. Protein sequences were aligned using Clustal X 104 73 Altogether 236 strains of S. enterica belonging to 12 v1.8 [14]. Duplicated, incomplete and non-conserved se- 105 74 serovars were included in this study. These strains be- quences were removed from the alignment (Table 1) and 106 75 longed to serovar Typhimurium (n = 175 strains), Enter- phylogenetic tree was constructed by the neighbour-join- 107 76 itidis (n = 18), Hadar (n = 13), Saintpaul (n = 10), Agona ing method [33]. Secondary elements of S. Typhimurium 108 77 (n = 5), Heidelberg (n = 6), Derby (n = 2), Stanley (n = 2), RrtT were predicted using PSI-PRED v2.4 [15]. 109 78 Schwarzengrund (n = 2), and individual strains of serovars 79 Indiana, Dublin and Gallinarum. Strains of S. Typhimuri- 2.3. Detection and initial characterisation of multicopy 110 80 um JD42 (rrtTþ, msDNAþ) and JD29 (rrtT3, msDNA3), single-stranded DNA 111 81 and serovar Enteritidis SE2159 (rrtEþ, sdsDNAþ) [32] 82 were used as controls in PCR. All the strains were grown The msDNA was isolated as described previously [19]. 112 83 in Luria Bertani (LB) broth or LB agar (Difco) at 37‡C. After electrophoresis in 13% polyacrylamide gel, the DNA 113 was visualised by staining with Sybr Gold £uorescent dye 114 84 2.2. Computer analysis and primer design (Molecular Probes, USA). To characterise the msDNA in 115 detail, it was treated with 10 U of DNase Iml 31 for 30 116 85 GeneCompar software (Applied Maths, Belgium) was min at 37‡C, 20 U of S1 nuclease for 30 min at 23‡C, 10 117 86 used for basic sequence analysis including cluster analysis Wg of RNase A ml31 for 30 min at 37‡C, 10 U of RNase 118 87 of nucleotide sequences of msDNA locus. The same soft- H for 30 min at 23‡C, and restriction endonuclease Sau3A 119

Table 1 Sequences of the retron reverse transcriptases retrieved from SWISS-PROT+TrEMBL and analysed in this study 1 SWISS-PROT Code Protein name Organism Ref. 2 Q9FDH6 RT_ST reverse transcriptase, RrtT S. Typhimurium [2] 3 Q9S1F2 RET_VC reverse transcriptase V. cholerae [34] 4 NAa RET_VP reverse transcriptase V. parahaemolyticus NPb 5 Q47526 RET_EC reverse transcriptase, retron Ec83 E. coli [23] 6 Q46666 RET_EC2 reverse transcriptase, retron Ec78 E. coli [25] 7 P23070 RT86_EC RNA-directed DNA polymerase, retron EC86 E. coli [24] 8 Q99S60UNCORRECTED SAV2209_SA hypothetical protein SAV2209 PROOFStaphylococcus aureus [17] 9 Q8TP23 MA2102_MA hypothetical protein MA2102 Methanosarcina acetivorans [7] 10 P23071 RT65_MX RNA-directed DNA polymerase, retron MX65 [10] 11 P23072 RT16_MX RNA-directed DNA polymerase, retron MX162 M. xanthus [11] 12 Q53751 RT163_SA reverse transcriptase aurantiaca [9] 13 Q50210 RT_ML reverse transcriptase Melittangium lichenicola [31] 14 Q8RGW6 FN0161_FN RNA-directed DNA polymerase Fusobacterium nucleatum [16] 15 Q05804 RT107_EC RNA-directed DNA polymerase, retron EC107 E. coli [21] 16 Q8P4T0 XCC3626_XC RNA-directed DNA polymerase Xanthomonas campestris [4] 17 P21325 RT67_EC RNA-directed DNA polymerase, retron EC67 E. coli [21] 18 Q8VRM1 RET_NE reverse transcriptase RT-Ne144 Nannocystis exedens NP 19 Q9L7Z6 RRT_SE retron reverse transcriptase, RrtE S. Enteritidis [32] 20 aSWISS-PROT code not available (BAC06578). 21 bNP, not published.

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120 for 30 min at 37‡C. Samples were electroblotted without 121 denaturation onto a nylon membrane (Zeta-Probe; Bio- 122 Rad, USA). Using a PCR Master Mix kit (Qiagen, Ger- 123 many), a PCR product (520 bp in size, for msDNA prim- 124 ers see above) spanning the expected low molecular mass 125 DNA locus was ampli¢ed, labelled by the ECL Direct 126 Nucleic Acid Labelling kit (Amersham, UK), and used 127 as a probe in hybridisation according to the instructions 128 of the manufacturer.

129 3. Results and discussion

130 3.1. Presence of rrtT in di¡erent serovars of S. enterica

131 Using PCR, the presence of the rrtT gene in di¡erent 132 serovars of S. enterica was investigated. This gene was 133 present in the vast majority of S. Typhimurium. Quite Fig. 1. Phylogenetic analysis of protein sequences of selected retron re- 1 134 frequently it was found also among strains belonging to verse transcriptases (RRTs). Group 1, retrons probably producing 2 135 the serovar Saintpaul. Occurrence of the rrtT in other RNA-less msDNA; group 2, retrons producing msDNA bound to 3 msRNA; group 3, heterogeneous group producing both msDNA and/or 4 136 serovars was not so frequent. A single positive strain sdsDNA. Protein codes are listed in Table 1. 5 137 was found in serovar Schwarzengrund. rrtT was absent 138 in all investigated strains belonging to serovars Agona, 139 Heidelberg, Enteritidis, Hadar, Derby, Indiana, Stanley, identity, 62% similarity) [25], and retron Ec83 (44% iden- 162 140 Dublin and Gallinarum (Table 2). Although it is expected tity, 62% similarity) [23]. A considerable drop in homology 163 141 that outside the genus Myxococcus the RRTs are quite was observed in the next homologous retron Ec86 (30% 164 142 rare [18], our screening showed very frequent presence of identity and 49% similarity) [22]. The recently character- 165 143 the rrtT in S. Typhimurium. Together with results obtained ised retron rrtE encoded by plasmid of S. Enteritidis [32] 166 144 in other Salmonella serovars it seems that rrtT is present was only very weakly similar (22% identity, 45% similar- 167 145 especially in serogroup B strains, and is absent in serovars ity). The multiple alignment (not shown) of protein se- 168 146 of group D1 (Enteritidis, Dublin, Gallinarum) and C2 quences revealed high conservation of the retron sequences 169 147 (Hadar). The genetic locus, of which the rrtT gene is in the regions of predicted secondary elements and strict 170 148 part, resembles an integrated prophage [2,3]. It is therefore conservation of the catalytic residues. The catalytic triad 171 149 possible that the distribution of rrtT in di¡erent serovars essential for enzymatic activity (Asp99, Asp185 and 172 150 of S. enterica is dependent on the ability of the phage to Asp186) was strictly conserved in all proteins in the align- 173 151 infect and integrate into the genome of only certain sero- ment including the RrtT from S. Typhimurium. Phyloge- 174 152 vars. Strains of group B might be sensitive to this phage netic analysis (Fig. 1) clearly separated the retrons produc- 175 153 while strains of groups D1 and C2 may be resistant. ing msDNA bound to msRNA (retrons Mx162, Mx65 or 176 Ec86) and the retrons producing RNA-less msDNA (ret- 177 154 3.2. Sequence comparison rons Ec83, Ec78, RrtT and Vibrio retrons). Remaining 178 retron elements comprising both the retrons known to 179 155 Protein BLAST analysis of the RrtT originating from produce the single-stranded msDNA and also the retron 180 156 the multidrug-resistant S. Typhimurium DT104 con¢rmed of S. Enteritidis the ¢nal product of which is the double- 181 157 the presenceUNCORRECTED of the identical gene in the completely se- stranded PROOF sdsDNA were quite heterogeneous and may be 182 158 quenced S. Typhimurium LT2 only [28]. The closest similar reclassi¢ed in the future. 183 159 retrons were those of V. cholerae (48% identity and 65% 160 similarity at protein level) [34], V. parahaemolyticus (44% 161 identity, 62% similarity) and E. coli retron Ec78 (46%

Table 2 Presence of the rrtT gene in di¡erent serovars of S. enterica 1 Typhim Saintp Schw Agona Enter Hadar Heidelb Derby Stanley Indiana Gall Dublin 2 rrtTþ 15861000000000 3 rrtT3 174151813622111 4 Typhim, Typhimurium; Saintp, Saintpaul; Schw, Schwarzengrund; Heidelb, Heidelberg; Gall, Gallinarum.

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1 Fig. 2. Multiple alignment of the msDNA gene loci of E. coli retrons Ec83 and Ec78, retron of V. cholerae (RET_VC), retron of V. parahaemolyticus 2 (RET_VP) and rrtT retron of S. Typhimurium (RT_ST). Functional sequences are marked by boxes and described above. Branching G, and 5P- and 3P- 3 ends of predicted msDNA are marked by arrows. Asterisks were used to label sequences conserved among all ¢ve retrons.

184 3.3. Operon analysis species allowed us to further extend previous observations 215 and de¢ne a speci¢c class of retron reverse transcriptases 216 185 Grouping of retrons of V. cholerae, V. parahaemolyti- which all synthesise msDNA which is probably cut out of 217 186 cus, rrtT, Ec78 and Ec83 stimulated us to a detailed se- msRNA in the terminal step of their biosynthesis by an 218 187 quence comparison. Immediately upstream from the rrtT unknown mechanism. The fact that retrons Ec78 and Ec83 219 188 we were unable to identify a1 and a2 inverted repeats. 189 Instead, 3 bp upstream the rrtT start codon, the stop 190 codon of another open reading frame (ORF) was located. 191 A similar ORF was also recorded in the Ec86 retron (30% 192 identity and 44% similarity) and in the phiR73 retron 193 phage (25% identity, 43% similarity). Upstream from this 194 extra ORF, a typical msDNA locus was identi¢ed. Multi- 195 ple alignment of this msDNA locus and msDNA loci of 196 related retrons is presented in Fig. 2. Out of the alignment 197 and together with previous ¢ndings for retrons Ec78 and 198 Ec83, we were able to predict a1 and a2 repeats upstream 199 the additional ORF present in rrtT operon, branching G, 200 3P-end of msDNA and cleavage site close to 5P-end of 201 msDNA (Figs. 2 and 3). The same sequence analysis 202 alsoUNCORRECTED allowed us to postulate the characteristics of this class PROOF 203 of retrons. These retrons (i) have absolutely conserved, 7 204 bp long, a1 and a2 repeats of CTC TTT A/T AAA AGA 205 sequence, (ii) contain a long (over 20 bp) inverted repeat 206 in the centre of the msDNA, and (iii) contain a conserved 207 sequence TTA TTG A specifying termination of msDNA 208 synthesis (3P-end of msDNA). Although the sequence of 209 long inverted repeat is not conserved, its 5P-end always 210 starts with GCG and its 3P-end consists of complementary 211 trinucleotide CGC. Although some of the conserved se- 212 quences between retrons Ec78 and Ec83 have been sug- 213 gested earlier [25], having the possibility to compare se- Fig. 3. Predicted secondary structure of msDNA produced by S. Typhi- 1 214 quence from three di¡erent bacterial genera and four murium. Sau3A restriction site is indicated. 2

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1 Fig. 4. Detection of msDNA and its initial characterisation. msDNA was treated with restriction endonuclease Sau3A (lane 1), RNase A (lane 2), S1 2 nuclease (lane 3), RNase H (lane 4), DNase I(lane 5). Lane 6, non-treated control msDNA. Lane M, 20 bp ladder. A: Polyacrylamide gel electropho- 3 resis. B: The result after hybridisation.

220 are capable of complementation [25] further supports our References 249 221 hypothesis on de¢nition of this speci¢c class of retron 222 reverse transcriptases. [1] Altschul, S.F., Madden, T.L., Scha¡er, A.A., Zhang, J., Zhang, Z., 250 Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI- 251 BLAST: a new generation of protein database search programs. Nu- 252 223 3.4. Detection and characterisation of predicted msDNA cleic Acids Res. 25, 3389^3402. 253 [2] Boyd, D., Peters, G.A., Cloeckaert, A., Boumedine, K.S., Chaslus- 254 224 In selected strains we have detected and initially char- Dancla, E., Imberechts, H. and Mulvey, M.R. (2001) Complete nu- 255 225 acterised the predicted msDNA. In 35 strains of S. Typh- cleotide sequence of a 43-kilobase genomic island associated with the 256 226 imurium positive in PCR for the rrtT, presence of msDNA multidrug resistance region of Salmonella enterica serovar Typhimu- 257 rium DT104 and its identi¢cation in phage type DT120 and serovar 258 227 was con¢rmed (not shown). In eight S. Typhimurium Agona. J. Bacteriol. 183, 5725^5732. 259 228 strains negative in rrtT by PCR, no production of [3] Boyd, D.A., Peters, G.A., Ng, L. and Mulvey, M.R. (2000) Partial 260 229 msDNA was observed. The presence of the gene in the characterization of a genomic island associated with the multidrug 261 230 genome of individual strains therefore correlated with resistance region of Salmonella enterica Typhimurium DT104. FEMS 262 231 the production of msDNA. The msDNA was resistant Microbiol. Lett. 189, 285^291. 263 [4] da Silva, A.C., Ferro, J.A., Reinach, F.C., Farah, C.S., Furlan, L.R., 264 232 to RNase A, RNase H, and sensitive to DNase I. S1 Quaggio, R.B., Monteiro-Vitorello, C.B., Van Sluys, M.A., Almeida, 265 233 nuclease degraded the predicted loop and single-stranded N.F., Alves, L.M., do Amaral, A.M., Bertolini, M.C., Camargo, 266 234 5P- and 3P-ends (see Fig. 3) and left intact the double- L.E., Camarotte, G., Cannavan, F., Cardozo, J., Chambergo, F., 267 235 stranded core stem sequence. This resulted in an increase Ciapina, L.P., Cicarelli, R.M., Coutinho, L.L., Cursino-Santos, 268 236 in polyacrylamide gel electrophoresis mobility (Fig. 4). J.R., El Dorry, H., Faria, J.B., Ferreira, A.J., Ferreira, R.C., Ferro, 269 M.I., Formighieri, E.F., Franco, M.C., Greggio, C.C., Gruber, A., 270 237 The stem sequence behaved also like a template for Katsuyama, A.M., Kishi, L.T., Leite, R.P., Lemos, E.G., Lemos, 271 238 Sau3A restriction endonuclease. Hybridisation with M.V., Locali, E.C., Machado, M.A., Madeira, A.M., Martinez-Ros- 272 239 msDNA speci¢c probe con¢rmed the identity of msDNA si, N.M., Martins, E.C., Meidanis, J., Menck, C.F., Miyaki, C.Y., 273 240 (Fig. 4). These characteristics were consistent with the ex- Moon, D.H., Moreira, L.M., Novo, M.T., Okura, V.K., Oliveira, 274 241 pected structure of single-stranded DNA molecule with M.C., Oliveira, V.R., Pereira, H.A., Rossi, A., Sena, J.A., Silva, C., 275 de Souza, R.F., Spinola, L.A., Takita, M.A., Tamura, R.E., Teixeira, 276 242 complex secondary structure and con¢rmed the theoretical UNCORRECTEDE.C., PROOF Tezza, R.I., Trindade dos, S.M., Tru⁄, D., Tsai, S.M., White, 277 243 predictions. F.F., Setubal, J.C. and Kitajima, J.P. (2002) Comparison of the ge- 278 nomes of two Xanthomonas pathogens with di¡ering host speci¢cities. 279 Nature 417, 459^463. 280 244 Acknowledgements [5] Dhundale, A., Lampson, B., Furuichi, T., Inouye, M. and Inouye, S. 281 (1987) Structure of msDNA from Myxococcus xanthus: evidence for 282 a long, self-annealing RNA precursor for the covalently linked, 283 245 We would like to thank Michaela Dekanova for the branched RNA. Cell 51, 1105^1112. 284 246 technical assistance. This work has been supported by [6] Dhundale, A.R., Furuichi, T., Inouye, S. and Inouye, M. (1985) 285 247 EU project QLK2-CT2000-01126 and the grant of the Distribution of multicopy single-stranded DNA among 286 248 Czech Ministry of Agriculture M03-99-01. and related species. J. Bacteriol. 164, 914^917. 287 [7] Galagan, J.E., Nusbaum, C., Roy, A., Endrizzi, M.G., Macdonald, 288 P., FitzHugh, W., Calvo, S., Engels, R., Smirnov, S., Atnoor, D., 289 Brown, A., Allen, N., Naylor, J., Stange-Thomann, N., DeArellano, 290

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