The mef(A)/msr(D)-carrying streptococcal prophage Φ1207.3 encodes an SOS-like system, induced by UV-C light, responsible for increased survival and increased mutation rate

1 The mef(A)/msr(D)-carrying streptococcal prophage Φ1207.3 encodes 1 an SOS-like system, induced by UV-C light, responsible for increased 2 survival and increased mutation rate 3 4 Valeria Fox1, Francesco Santoro1,*, Carmen Apicella1, Sara Diaz-Diaz2, Josè Manuel Rodriguez-5 Martinez3, Francesco Iannelli1,*, Gianni Pozzi1 6 7 1Laboratory of Molecular Microbiology and Biotechnology, Department of Medical 8 Biotechnologies, University of Siena, 53100 Siena, Italy 9 2Unidad Clínica de Enfermedades Infecciosas, Microbiología y Medicina Preventiva, Hospital 10 Universitario Virgen Macarena, Sevilla, Spain 11 3Departamento de Microbiología, Facultad de Medicina, Universidad de Sevilla, Sevilla, Spain 12 13 *Corresponding author. 14 Mailing address: LAMMB, Biotecnologie Mediche/Università di Siena, Policlinico Le Scotte, V 15 Lotto I Piano, Viale Bracci, 53100 Siena, Italy. 16 Phone: +39-0577-233156; +39-0577-233156. Fax: +39-0577-233334. 17 E-mail: [email protected]; [email protected] 18 19 20 Keywords: SOS response, S. pneumoniae, S. pyogenes, prophage, Φ1207.3, mef(A)/msr(D), 21 mutation rate, bacterial survival, UV-C LED light, fluctuation test, DNA repair 22 23 24 2 Abstract 25 Bacterial SOS response is an inducible system of DNA repair and mutagenesis. Streptococci lack a 26 canonical SOS response, but an SOS-like response was reported in some species. The mef(A)-27 msr(D)-carrying prophage Ф1207.3 of Streptococcus pyogenes contains a region, spanning orf6 to 28 orf11, showing homology to characterized streptococcal SOS-like cassettes. Genome-wide 29 homology search showed the presence of the whole Φ1207.3 SOS-like cassette in three S. pyogenes 30 prophages, while parts of it were found in other bacterial species. To investigate whether this 31 cassette confers an SOS-mutagenesis phenotype, we constructed Streptococcus pneumoniae R6 32 isogenic derivative strains: (i) FR172, streptomycin resistant, (ii) FR173, carrying Φ1207.3, and (iii) 33 FR174, carrying a recombinant Φ1207.3, where the SOS-like cassette was deleted. These strains 34 were used in survival and mutation rate assays using a UV-C LED instrument for which we 35 designed and 3D-printed a customized equipment, constituted of an instrument support and 36 swappable-autoclavable mini-plates and lids. Upon exposure to UV fluences ranging from 0 to 6400 37 J/m2 at four different wavelengths, 255, 265, 275 and 285 nm, we found that the presence of 38 Φ1207.3 SOS-like cassette increases bacterial survival up to 34-fold. Mutation rate was determined 39 by measuring rifampicin resistance acquisition upon exposure to UV fluence of 50 J/m2 at the four 40 wavelengths by fluctuation test. The presence of Φ1207.3 SOS-like cassette resulted in a significant 41 increase in the mutation rate (up to 18-fold) at every wavelength. In conclusion, we demonstrated 42 that Φ1207.3 carries a functional SOS-like cassette responsible for an increased survival and 43 increased mutation rate in S. pneumoniae. 44 Importance 45 Bacterial mutation rate is generally low, but stress conditions and DNA damage can induce stress 46 response systems, which allow for improved survival and continuous replication at the cost of 47 elevated mutagenesis. The SOS response is a DNA repair mechanism activated by some bacteria in 48 response to stressful conditions, which leads to a temporary hypermutable phenotype and is usually 49 absent in streptococcal genomes. Here, using a reproducible and controlled UV irradiation system, 50 3 we demonstrated that the SOS-like gene cassette of prophage Φ1207.3 is functional, responsible for 51 a temporary hypermutable phenotype, and enhances bacterial survival to UV irradiation. Prophage 52 Φ1207.3 also carries erythromycin resistance genes and can lysogenize different pathogenic 53 bacteria, constituting an example of a mobile genetic element which can confer multiple phenotypes 54 to its host. 55 Introduction 56 Bacteria evolve through chromosomal rearrangements, acquisition of DNA by horizontal gene 57 transfer and mutation. Mutations arise by a process referred to as mutagenesis which is measured 58 determining the mutation frequency or the mutation rate. Mutation frequency represents the 59 proportion of mutant cells in a specific culture, while the mutation rate is the probability of a 60 mutation occurring per cell division and thus is also an estimation of the probability of a mutation 61 occurring during the life of a bacterial cell (1–3). Mutation rates differ among bacterial species and 62 among strains of the same species, and are influenced by the fidelity of DNA replication, DNA 63 damage and repair pathways, and exposure to chemical or physical agents. In Escherichia coli DNA 64 replication is carried out by high fidelity polymerases which misinsert nucleotides at a rate of 10-4-65 10-6 per base pair (bp), but possess a 3’-exo proofreading activity that reduces mutation load by 66 about 100-fold (4). E. coli has also evolved the SOS response, which is inducible by DNA damage 67 and allows for improved survival and continuous replication at the cost of elevated mutagenesis (5, 68 6). The main proteins involved in SOS response are LexA, a homodimeric transcriptional repressor 69 of the SOS regulon, and RecA, which, upon DNA damage, binds to ssDNA, becomes activated, and 70 promotes the autocleavage of LexA and subsequent transcription of the SOS genes (7). The SOS 71 response induces the expression of genes involved in DNA-damage tolerance and repair, including 72 the Translesion Synthesis (TLS) system (8). TLS is involved in the tolerance to DNA damage, and 73 can be both error-free or error-prone, when the polymerase V is recruited. Polymerase V is encoded 74 by the umuDC operon, where umuC encodes a translesion DNA polymerase, of the Y family, while 75 4 umuD encodes a regulatory protein. The low fidelity polymerase V has a high rate of nucleotide 76 misincorporation (10-3-10-4 per bp) (9) and also lacks proofreading activity (10). 77 Streptococci lack a canonical SOS response (11), but SOS-like gene cassettes are present in the 78 genomes, either on the chromosome or on Mobile Genetic Elements, of Streptococcus uberis, 79 Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus sanguinis, Streptococcus mitis, 80 and Streptococcus thermophilus (12). In S. uberis, the SOS-like cassette contains 4 genes arranged 81 in an operon including (i) hdiR, coding for a protein homologous to the HdiR regulator of 82 Lactococcus lactis, induced by heat shock and DNA-damage (13), (ii) umuC encoding an UmuC-83 like protein, (iii) 2 genes of unknown function. The HdiR regulator of S. uberis undergoes 84 spontaneous self-cleavage at alkaline pH, similar to the RecA-mediated self-cleavage of LexA-like 85 proteins. HdiR specifically binds to an inverted repeat sequence downstream the hdiR putative 86 promoter, suggesting a role as transcriptional repressor of the SOS-like cassette. The pneumococcal 87 Integrative and Conjugative Element (ICE) Tn5253 confers UV resistance to the cell, inducing a 88 mutagenic SOS response (14). Tn5253 contains an SOS-like cassette similar to that described in S. 89 uberis, comprising: (i) orf69, which codes for an LexA-like repressor, (ii) orf70, which codes for an 90 UmuC-like protein, (iii) orf71 and orf72 of unknown function (15–17). In S. pyogenes, a gene 91 cassette homologous to the already known streptococcal SOS-like cassettes is associated with the 92 mef(A)-msr(D)-carrying prophages Ф1207.3 (18–21), Φ10394.4, Φm46.1 and its variant 93 VP_00501.1 (22–25). Ф1207.3 is a functional bacteriophage capable to lysogenize different strains 94 of the human pathogens S. pyogenes and S. pneumoniae, which colonize the upper respiratory tract 95 (26). Since both of these species can cause invasive infections, the transmission of antibiotic 96 resistance genes by lysogenic transfer of Ф1207.3 assumes clinical relevance. In this work, we 97 transferred Ф1207.3 prophage from the original S. pyogenes host to the S. pneumoniae standard 98 laboratory strain R6 to investigate if the SOS-like cassette carried by the Ф1207.3 prophage is able 99 to activate an SOS-like response, which could increase the acquisition of mutations associated to 100 antimicrobial resistance. 101 5 Materials and methods 102 Bioinformatic analysis 103 Homology searches were conducted in public nucleotide and protein databases, namely Microbial 104 Genomes Database (https://www.ncbi.nlm.nih.gov/genome/microbes/), Conserved Domain 105 Database (CDD, https://www.ncbi.nlm.nih.gov/cdd/) and Pfam protein family database 106 (https://pfam.xfam.org/), using default parameters, and only alignment with E-values <0.001 were 107 considered. Protein sequence analysis was conducted with the tools available at Softberry website 108 (http://www.softberry.com/berry.phtml) and with the software Phyre2 109 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) (27). Nucleotide sequence of the orf6-110 orf11 region was used as a probe for Microbial Genomes Database searches, while ORF predicted 111 amino acid sequences were used for protein databases searches. 112 Bacterial strains and growth 113 Streptococcal strains used in this work and their relevant properties are reported in Table 1. Strains 114 were grown in Tryptic Soy Broth (TSB, BD) at 37°C. Starter cultures were taken at an optical 115 density at 590 nm (OD590) ranging from 0.2 to 0.3 and were frozen in 10% glycerol at -70°C. Solid 116 media were obtained by supplementing TSB with 1.5% agar (BD Difco) and 3% defibrinated horse 117 blood (Liofilchem). When required, both liquid and solid media were supplemented with antibiotics 118 at the following concentrations: 500 µg/ml kanamycin, 500 µg/ml streptomycin, 0.5 µg/ml 119 erythromycin, 5 µg/ml chloramphenicol and 20 µg/ml rifampicin. 120 Lysogenization assays 121 Transfer of bacteriophage Φ1207.3 was obtained through a plate mating experiment, as previously 122 described (28). Briefly, donor cells, carrying the phage, and recipient cells were grown separately in 123 TSB in the presence of the appropriate antibiotics. Upon reaching the end of the exponential phase, 124 cells were mixed at a donor-recipient 1:10 ratio, centrifuged, and the pellet was plated on TSA 125 plates supplemented with 5% horse blood. Plates were incubated at 37°C in the presence of 5% CO2 126 for 4h, cells were then recovered with a cotton swab and resuspended in TSB/10% glycerol. To 127 6 select for lysogens, cell suspension was plated following a multilayer plating procedure in presence 128 of erythromycin and streptomycin which select for lysogens and counterselect the Φ1207.3 donor 129 cells. To confirm the lysogens phenotype, a genetic analysis was performed as reported (16,28), 130 while PCR and direct Sanger sequencing of PCR template were used for genotype analysis 131 according to the protocol already described (32). 132 PCR Gene SOEing mutagenesis 133 To generate a knock-out mutant of orf6-orf11 region, PCR Gene Splicing by Overlap Extension 134 (SOEing) was used to produce a mutagenic construct containing the kanamycin resistance cassette 135 (876 bp) flanked by the DNA fragments located upstream of orf6 (555 bp) and downstream of orf11 136 (829 bp) as previously described (29). The primer pair IF149/IF210 was used to amplify the 876-bp 137 ami/aphIII kanamycin resistance cassette (30), while IF242/IF239 and IF240/IF241 amplified the 138 flanking fragments. Assembly of the final construct (2,260 bp) was obtained with the IF242/IF241 139 primer pair. PCR was carried out as previously described (31). The PCR products were separated in 140 a 0.8% agarose gel at 200 V for 30 minutes, stained for 15 minutes in a 0.1% Ethidium Bromide 141 solution and visualized with UV light. PCR products after gel purification (NucleoSpin Gel and 142 PCR Clean-up kit, Macherey-Nagel) were quantified using the Qubit 2.0 Fluorometer (Thermo 143 Fisher Scientific). Oligonucleotide primers are listed in Table S1. The PCR mutagenic construct 144 was directly used as donor DNA in transformation experiments. , Since integration of Φ1207.3 into 145 S. pneumoniae chromosome leads the disruption of the celB competence gene with a consequent 146 impairment of genetic transformation, we constructed transformable S. pneumoniae Rx1 derivative 147 strains carrying Φ1207.3 integrated elsewhere in pneumococcal chromosome to be used as 148 tranformation recipients (Table 1) (28). Pneumococcal competent cells were prepared as previously 149 described (29). Competent cells were thawed, supplemented with 25 ng/ml Competence Stimulating 150 Peptide (CSP) and 1 μg/ml of the purified mutagenic construct, and the transformation mixture was 151 incubated at 37°C for 45 minutes. The resulting recombinant strains were selected for acquisition of 152 kanamycin resistance by multilayer plating (28). The deletion of the orf6-11 region (nucleotides 153 7 6,189 to 9,297, GenBank accession no. AY657002) was confirmed by PCR and direct sequencing 154 of the PCR products (32). 155 UV fluence measurement 156 UV-C light experiments were carried out using a PearlLab Micro UV-C LED instrument 157 (AquiSense Technologies, Erlanger, Kentucky, USA). The instrument is a compact device equipped 158 with 4 interchangeable USB UV-C LEDs emitting at different wavelengths (255, 265, 275 and 285 159 nm) and a dimmer switch that allows irradiation with 10 different intensities. For our purposes, the 160 lower portion of the instrument was un-screwed, and the lamp was used as a general light source. A 161 customized equipment, constituted of an instrument support and swappable and autoclavable mini-162 plates and lids, was 3D-designed and printed in Nylon-12 (Figure S1, 163 https://doi.org/10.5281/zenodo.8208699). The irradiance was measured 3 times with a ILT1400 164 radiometer and SED240/W detector (International Light Technologies, Peabody, MA). Mean and 165 standard deviation for each intensity of the different wavelengths used are reported in Table S2. The 166 UV fluence (J/m2) is calculated as “irradiance (W/m2) x exposure time (sec)”, thus the same value 167 of the UV fluence can be obtained varying the values of the 2 parameters. Since the PearlLab Micro 168 UV-C LED instrument emits light at 10 different intensities, allowing for 10 different values of 169 irradiance for each of the 4 wavelengths, different combinations of intensity and time were possible. 170 The choice of the optimal parameters to be used in UV-C light survival and fluctuation assays was 171 based on a preliminary survival experiment where an UV fluence of 50 J/m2 was obtained varying 172 the irradiance and the time. Since the combination of high intensities and low exposure times, or 173 low intensities and high exposure times, resulted in a wide variability of survival among the 174 technical replicates, we chose, when possible, to maintain similar exposure times across all 175 wavelengths and to use intermediate intensities for the 50 J/m2 UV fluence for fluctuation assays 176 (Table S3). 177 Survival assay 178 8 Bacterial survival upon UV-C light irradiation at different wavelengths (255 to 285 nm) was 179 assessed by plating the cells after exposure to different UV fluences from 0 to 6400 J/m2 (0, 25, 50, 180 100, 200, 400, 800, 1600, 3200, 6400) and by calculating the cell viability for each strain. Cells 181 were grown at 37°C in 10 ml of TSB until reaching an OD590 of 0.6 (about 1 x 108 CFU/ml), when 182 they were centrifuged at 5,000 x g for 15 minutes, washed with sterile phosphate buffered saline 183 (PBS, Sigma Aldrich) and resuspended in 10 ml of PBS. UV-C survival experiments were carried 184 out by placing 1 ml of cells in the sterile Nylon-12 mini-plate and exposing them to different 185 fluences, under continuous stirring. UV-C irradiated and untreated control bacterial cells were 186 plated by multilayer plating. Survival was calculated as the ratio between UV-C irradiated and not 187 irradiated control cells. 188 Luria-Delbrück fluctuation test 189 Mutation rate was determined by fluctuation analysis in absence or presence of UV-C light at 190 different wavelengths ranging from 255 to 285 nm. Strains were grown in TSB at 37°C until an 191 OD590 of 0.6 and the number of total and rifampicin-resistant CFUs was determined prior to 192 freezing at -70°C in 1 ml-aliquots containing 10% glycerol. Rifampicin was used at a final 193 concentration of 20 µg/ml in the third layer of TSA, when appropriate (28). For mutation rate 194 determination, a set of 10 independent cultures was obtained by inoculating 5 ml of TSB with 1 x 195 105 CFU of the same 1 ml frozen aliquot. For mutation rate determination following UV-C light 196 treatment, the 1 ml frozen aliquot was irradiated at an UV fluence of 50 J/m2 before inoculum. 197 Then, to prepare 10 independent cultures, an inoculum corresponding to 1 x 105 CFU was calculated 198 by inferring bacterial viability after irradiation from the data obtained with the UV-C light survival 199 assay. Cultures were grown at 37°C until they reached the mid-log phase, corresponding to about 12 200 generations, the number of total and rifampicin-resistant CFUs was determined by plating 0.1 ml of 201 culture and incubating the plates at 37°C for 48 h. For each strain and condition, 4 replicates of the 202 fluctuation test were carried out and the rifampicin-resistant CFUs numbers of the 40 cultures were 203 pooled into a single data set (33). The mutation rate determination and the statistical analysis were 204 9 carried out using the R package rSalvador (34). The expected number of mutations per culture (m) 205 was first obtained with the use of the function newton.LD.plating, which computes the maximum 206 likelihood estimates (MLE) of m, adjusting to the plating efficiency, i.e., when only a portion of the 207 culture is plated. Once the estimate of m was obtained, the mutation rate (µ) was determined 208 dividing m by the number of cell divisions that had taken place. Since the final number of cells in a 209 culture, Nt, arises from Nt-1 divisions, the mutation rate is: 210 µ= 𝑚 𝑁𝑡 − 1 ≈ 𝑚 𝑁𝑡 where Nt -1 is equal to the number of generations and can be approximated to Nt (3, 34). 211 Differences in mutation rates were compared using the rSalvador likelihood ratio test (LRT) (34–212 36), which takes into consideration both the plating efficiency and the final cell number Nt, which 213 differs among different strains or experimental conditions, by applying the compare.LD.plating 214 function pairwise among all conditions of each strain and among the different strains. The p.adjust 215 R function was then used to correct the overall false positive rate of the obtained p values for 216 multiple comparisons. Differences in the mutation rate were considered significant only when the 217 adjusted p-values were less than 0.05 (33). The comparison was also observed graphically by 218 checking the overlapping of the 84% confidence intervals (C.I.s) (36). 219 Results 220 Φ1207.3 carries an SOS-like cassette 221 The Φ1207.3 DNA region located between the mef(A)-msr(D) erythromycin resistance cassette (20) 222 and the type II restriction-modification cassette (37), contains 6 open reading frames (namely orf6 223 to orf11) likely arranged in an operon (Figure 1). Homology search revealed that orf11 predicted 224 gene product is homologous to the HdiR LexA-like transcriptional repressor of Lactococcus lactis, 225 orf8 is homologous to the Streptococcus pneumoniae Tn5253 umuC/orf70, while orf7 gene product 226 is homologous to the Bacillus subtilis YolD-like protein, which is predicted to be a functional 227 equivalent of UmuD (38) (Table 2). The deduced amino acid sequences of orf6 to orf11 share 228 16 Effect of the Φ1207.3 SOS-like cassette on the mutation rate in S. pneumoniae upon UV-C light 361 irradiation was investigated in absence of Φ1207.3 prophage, in presence of Φ1207.3 and in the 362 presence of the recombinant Φ1207.3ΔSOS-like cassette. Irradiation was carried out with UV-C 363 light at wavelengths 255 nm, 265 nm, 275 nm, and 285 nm at a fluence of 50 J/m2. Strain FR173, 364 carrying Φ1207.3, showed a significant increase in the mutation rate (up to 18.38-fold at 285 nm) 365 upon UV-C light treatment at every wavelength, whereas no significant increase was observed for 366 strain FR172, not carrying Φ1207.3, and strain FR174, carrying the recombinant Φ1207.3∆SOS-367 like cassette. The mutation rate was assessed by measuring the acquisition of rifampicin resistance. 368 Results are reported as means of four independent experiments with 84% confidence intervals. *** 369 adjusted p-value <0.001. 370 Figure S1. UV-C LED instrument and 3D-designed and printed customized equipment. A) 371 The PearlLab Micro UV-C LED instrument is equipped with 4 interchangeable USB UV-C LEDs 372 emitting at different wavelengths (255, 265, 275 and 285 nm) and a dimmer switch that allows 373 irradiation with 10 different intensities. The instrument was used as a UV-C LED light source to 374 irradiate bacteria contained in a customized equipment. B) The customized equipment was 3D-375 designed and printed in Nylon-12 and is constituted by an instrument support and swappable and 376 autoclavable mini-plates and lids. Length (X), width (Y), height (Z) of the items, reported in 377 millimeters (mm), are as follows: support: 76 x 76 x 27.5 mm, mini-plate: 24 x 24 x 10 mm, lid: 20 378 x 20 x 3.4 mm. The files for 3D printing of the equipment are available at 379 https://doi.org/10.5281/zenodo.8208699. 380 Figure S2. Schematic comparison of the Φ1207.3 SOS-like cassette with sequences found in 381 complete bacterial genomes. Blast homology search analysis revealed the presence of a cassette 382 100% homologous to Φ1207.3 cassette in 3 S. pyogenes genomes associated with mef(A)-msr(D)-383 carrying prophages. A copy of the cassette containing a short deletion at the 5’ end of orf11 was 384 found in S. gallolyticus, S. dysgalactiae, Streptococcus spp., and S. suis genomes. Different 385 fragments of the cassette are present in the other genomes. A fragment of the cassette spanning orf6 386 17 to the partial 3’ end of orf8 is present into the genomes of S. pneumoniae, different streptococcal 387 species, and Gemella sanguinis. The fragment belongs to the mef(A)-msr(D)-carrying element 388 Tn1207.1 in the S. pneumoniae INV200 genome and to the mef(E)-msr(D)-carrying elements in the 389 remaining genomes. ORFs and their direction of transcription are represented by arrows, The scale 390 is in kilobases. Homologous regions are indicated by lines, the name of the bacterial species is 391 reported on the right and the number of genomes on the left. Regions shorter than 500 nts are not 392 shown. 393 18 References 394 1. Pope CF, O’Sullivan DM, McHugh TD, Gillespie SH. 2008. A Practical Guide to Measuring Mutation 395 Rates in Antibiotic Resistance. AAC 52:1209–1214. 396 2. Rosche WA, Foster PL. 2000. Determining Mutation Rates in Bacterial Populations. Methods 20:4–17. 397 3. Foster PL. 2006. Methods for Determining Spontaneous Mutation Rates, p. 195–213. In Methods in 398 Enzymology. Elsevier. 399 4. Goodman MF. 2016. Better living with hyper-mutation: Hypermutation. Environ Mol Mutagen 400 57:421–434. 401 5. Radman M. 1975. SOS Repair Hypothesis: Phenomenology of an Inducible DNA Repair Which is 402 Accompanied by Mutagenesis, p. 355–367. In Hanawalt, PC, Setlow, RB (eds.), Molecular 403 Mechanisms for Repair of DNA. Springer US, Boston, MA. 404 6. Maslowska KH, Makiela‐Dzbenska K, Fijalkowska IJ. 2019. The SOS system: A complex and tightly 405 regulated response to DNA damage. Environ Mol Mutagen 60:368–384. 406 7. Baharoglu Z, Mazel D. 2014. SOS, the formidable strategy of bacteria against aggressions. FEMS 407 Microbiol Rev 38:1126–1145. 408 8. Fijalkowska IJ, Schaaper RM, Jonczyk P. 2012. DNA replication fidelity in Escherichia coli: a multi-409 DNA polymerase affair. FEMS Microbiology Reviews 36:1105–1121. 410 9. Tang M, Pham P, Shen X, Taylor J-S, O’Donnell M, Woodgate R, Goodman MF. 2000. Roles of E. 411 coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis. Nature 412 404:1014–1018. 413 10. Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA, Ellenberger T. 2006. DNA Repair and 414 Mutagenesis. ASM press. 415 19 11. Gasc AM, Sicard N, Claverys JP, Sicard AM. 1980. Lack of SOS repair in Streptococcus pneumoniae. 416 Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 70:157–165. 417 12. Varhimo E, Savijoki K, Jalava J, Kuipers OP, Varmanen P. 2007. Identification of a Novel 418 Streptococcal Gene Cassette Mediating SOS Mutagenesis in Streptococcus uberis. JB 189:5210–5222. 419 13. Savijoki K, Ingmer H, Frees D, Vogensen FK, Palva A, Varmanen P. 2003. Heat and DNA damage 420 induction of the LexA-like regulator HdiR from Lactococcus lactis is mediated by RecA and ClpP: 421 RecA and CIpP modulate the expression of HdiR. Molecular Microbiology 50:609–621. 422 14. Munoz-Najar U, Vijayakumar MN. 1999. An Operon That Confers UV Resistance by Evoking the 423 SOS Mutagenic Response in Streptococcal Conjugative Transposon Tn5252. J Bacteriol 181:2782–424 2788. 425 15. Iannelli F, Santoro F, Oggioni MR, Pozzi G. 2014. Nucleotide sequence analysis of integrative 426 conjugative element Tn5253 of Streptococcus pneumoniae. Antimicrob Agents Chemother, 58:1235–427 1239. 428 16. Santoro F, Romeo A, Pozzi G, Iannelli F. 2018. Excision and Circularization of Integrative 429 Conjugative Element Tn5253 of Streptococcus pneumoniae. Front Microbiol 9:1779. 430 17. Santoro F, Fox V, Romeo A, Lazzeri E, Pozzi G, Iannelli F. 2021. Chromosomal integration of Tn5253 431 occurs downstream of a conserved 11-bp sequence of the rbgA gene in Streptococcus pneumoniae and 432 in all the other known hosts of this integrative conjugative element (ICE). Mobile DNA 12:25. 433 18. Santagati M, Iannelli F, Cascone C, Campanile F, Oggioni MR, Stefani S, Pozzi G. 2003. The Novel 434 Conjugative Transposon Tn1207.3 Carries the Macrolide Efflux Gene mef(A) in Streptococcus 435 pyogenes. Microbial Drug Resistance 9:243–247. 436 19. Iannelli F, Santagati M, Santoro F, Oggioni MR, Stefani S, Pozzi G. 2014. Nucleotide sequence of 437 conjugative prophage Φ1207.3 (formerly Tn1207.3) carrying the mef(A)/msr(D) genes for efflux 438 resistance to macrolides in Streptococcus pyogenes. Front Microbiol 5:687–687. 439 20 20. Iannelli F, Santoro F, Santagati M, Docquier J-D, Lazzeri E, Pastore G, Cassone M, Oggioni MR, 440 Rossolini GM, Stefani S, Pozzi G. 2018. Type M Resistance to Macrolides Is Due to a Two-Gene 441 Efflux Transport System of the ATP-Binding Cassette (ABC) Superfamily. Front Microbiol 9:1670. 442 21. Fox V, Santoro F, Pozzi G, Iannelli F. 2021. Predicted transmembrane proteins with homology to 443 Mef(A) are not responsible for complementing mef(A) deletion in the mef(A)–msr(D) macrolide efflux 444 system in Streptococcus pneumoniae. BMC Res Notes 14:432. 445 22. Banks DJ, Porcella SF, Barbian KD, Martin JM, Musser JM. 2003. Structure and Distribution of an 446 Unusual Chimeric Genetic Element Encoding Macrolide Resistance in Phylogenetically Diverse 447 Clones of Group A Streptococcus. J Infect Dis 188:1898–1908. 448 23. Brenciani A, Bacciaglia A, Vignaroli C, Pugnaloni A, Varaldo PE, Giovanetti E. 2010. Φm46.1, the 449 Main Streptococcus pyogenes Element Carrying mef (A) and tet (O) Genes. Antimicrob Agents 450 Chemother 54:221–229. 451 24. Di Luca MC, D’Ercole S, Petrelli D, Prenna M, Ripa S, Vitali LA. 2010. Lysogenic Transfer of mef(A) 452 and tet(O) Genes Carried by Φm46.1 among Group A Streptococci. Antimicrob Agents Chemother 453 54:4464–4466. 454 25. Vitali LA, Di Luca MC, Prenna M, Petrelli D. 2016. Correlation between genetic features of the 455 mef(A)- msr(D) locus and erythromycin resistance in Streptococcus pyogenes. Diagnostic 456 Microbiology and Infectious Disease 84:57–62. 457 26. Santoro F, Pastore G, Fox V, Petit M-A, Iannelli F, Pozzi G. 2023. Streptococcus pyogenes Φ1207.3 Is 458 a Temperate Bacteriophage Carrying the Macrolide Resistance Gene Pair mef(A)-msr(D) and Capable 459 of Lysogenizing Different Streptococci. Microbiol Spectr e04211-22. 460 27. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. 2015. The Phyre2 web portal for protein 461 modeling, prediction and analysis. Nat Protoc 10:845–858. 462 21 28. Iannelli F, Santoro F, Fox V, Pozzi G. 2021. A Mating Procedure for Genetic Transfer of Integrative 463 and Conjugative Elements (ICEs) of Streptococci and Enterococci. MPs 4:59. 464 29. Iannelli F, Pozzi G. 2004. Method for Introducing Specific and Unmarked Mutations Into the 465 Chromosome of Streptococcus pneumoniae. MB 26:81–86. 466 30. Pearce BJ, Iannelli F, Pozzi G. 2002. Construction of new unencapsulated (rough) strains of 467 Streptococcus pneumoniae. Research in Microbiology 5. 468 31. Santoro F, Oggioni MR, Pozzi G, Iannelli F. 2010. Nucleotide sequence and functional analysis of the 469 tet(M)-carrying conjugative transposon Tn5251 of Streptococcus pneumoniae. FEMS Microbiol Lett. 470 32. Iannelli F, Giunti L, Pozzi G. 1998. Direct sequencing of long polymerase chain reaction fragments. 471 Mol Biotechnol 10:183–185. 472 33. Silayeva O, Engelstädter J, Barnes AC. 2020. Evolutionary epidemiology of Streptococcus iniae: 473 Linking mutation rate dynamics with adaptation to novel immunological landscapes. Infect Genet Evol 474 85:104435. 475 34. Zheng Q. 2017. rSalvador: An R Package for the Fluctuation Experiment. G3 (Bethesda) 7:3849–3856. 476 35. Zheng Q. 2015. Methods for comparing mutation rates using fluctuation assay data. Mutation 477 Research/Fundamental and Molecular Mechanisms of Mutagenesis 777:20–22. 478 36. Zheng Q. 2016. Comparing mutation rates under the Luria–Delbrück protocol. Genetica 144:351–359. 479 37. Euler CW, Ryan PA, Martin JM, Fischetti VA. 2007. M.SpyI, a DNA methyltransferase encoded on a 480 mefA chimeric element, modifies the genome of Streptococcus pyogenes. J Bacteriol, 2006/11/03 ed. 481 189:1044–1054. 482 38. Permina EA, Mironov AA, Gelfand MS. 2002. Damage-repair error-prone polymerases of eubacteria: 483 association with mobile genome elements. Gene 293:133–140. 484 22 39. Santagati M, Iannelli F, Oggioni MR, Stefani S, Pozzi G. 2000. Characterization of a Genetic Element 485 Carrying the Macrolide Efflux Gene mef(A) in Streptococcus pneumoniae. 9. Antimicrob Agents 486 Chemother 44:2585–2587. 487 40. Pozzi G, Iannelli F, Oggioni M, Santagati M, Stefani S. 2004. Genetic Elements Carrying Macrolide 488 Efflux Genes in Streptococci. 3. CDTID 4:203–206. 489 41. Del Grosso M, Scotto d’Abusco A, Iannelli F, Pozzi G, Pantosti A. 2004. Tn2009, a Tn916-Like 490 Element Containing mef(E) in Streptococcus pneumoniae. Antimicrob Agents Chemother 48:2037–491 2042. 492 42. Del Grosso M, Camilli R, Iannelli F, Pozzi G, Pantosti A. 2006. The mef(E)-Carrying Genetic Element 493 (mega) of Streptococcus pneumoniae: Insertion Sites and Association with Other Genetic Elements. 494 Antimicrob Agents Chemother 50:3361–3366. 495 43. Shoemaker NB, Guild WR. 1974. Destruction of low efficiency markers is a slow process occurring at 496 a heteroduplex stage of transformation. Molec Gen Genet 128:283–290. 497 44. Guild WR, Shoemaker NB. 1976. Mismatch correction in pneumococcal transformation: donor length 498 and hex-dependent marker efficiency. J Bacteriol 125:125–135. 499 45. Claverys J-P, Prats H, Vasseghi H, Gherardi M. 1984. Identification of Streptococcus pneumoniae 500 mismatch repair genes by an additive transformation approach. Mol Gen Genet 196:91–96. 501 46. Hoskins J, Alborn WE, Arnold J, Blaszczak LC, Burgett S, DeHoff BS, Estrem ST, Fritz L, Fu D-J, 502 Fuller W, Geringer C, Gilmour R, Glass JS, Khoja H, Kraft AR, Lagace RE, LeBlanc DJ, Lee LN, 503 Lefkowitz EJ, Lu J, Matsushima P, McAhren SM, McHenney M, McLeaster K, Mundy CW, Nicas TI, 504 Norris FH, O’Gara M, Peery RB, Robertson GT, Rockey P, Sun P-M, Winkler ME, Yang Y, Young-505 Bellido M, Zhao G, Zook CA, Baltz RH, Jaskunas SR, Rosteck PR, Skatrud PL, Glass JI. 2001. 506 Genome of the Bacterium Streptococcus pneumoniae Strain R6. J Bacteriol 183:5709–5717. 507 23 47. Wehrli W. 1983. Rifampin: Mechanisms of Action and Resistance. Clinical Infectious Diseases 508 5:S407–S411. 509 48. Goldstein BP. 2014. Resistance to rifampicin: a review. J Antibiot 67:625–630. 510 49. Sundin GW, Weigand MR. 2007. The microbiology of mutability. FEMS Microbiology Letters 511 277:11–20. 512 50. Gressel J, Levy AA. 2010. Stress, Mutators, Mutations and Stress Resistance, p. 471–483. In Pareek, 513 A, Sopory, SK, Bohnert, HJ (eds.), Abiotic Stress Adaptation in Plants: Physiological, Molecular and 514 Genomic Foundation. Springer Netherlands, Dordrecht. 515 51. Jolivet-Gougeon A, Kovacs B, Le Gall-David S, Le Bars H, Bousarghin L, Bonnaure-Mallet M, Lobel 516 B, Guillé F, Soussy C-J, Tenke P. 2011. Bacterial hypermutation: clinical implications. Journal of 517 Medical Microbiology 60:563–573. 518 52. Tanaka MM, Bergstrom CT, Levin BR. 2003. The Evolution of Mutator Genes in Bacterial 519 Populations: The Roles of Environmental Change and Timing. Genetics 164:843–854. 520 53. Marles-Wright J, Lewis RJ. 2007. Stress responses of bacteria. Current Opinion in Structural Biology 521 17:755–760. 522 54. Negri M-C, Morosini M-I, Baquero M-R, Campo R del, Blázquez J, Baquero F. 2002. Very Low 523 Cefotaxime Concentrations Select for Hypermutable Streptococcus pneumoniae Populations. AAC 524 46:528–530. 525 55. Gould CV, Sniegowski PD, Shchepetov M, Metlay JP, Weiser JN. 2007. Identifying Mutator 526 Phenotypes among Fluoroquinolone-Resistant Strains of Streptococcus pneumoniae Using Fluctuation 527 Analysis. Antimicrob Agents Chemother 51:3225–3229. 528 56. Cortes PR, Piñas GE, Albarracin Orio AG, Echenique JR. 2008. Subinhibitory concentrations of 529 penicillin increase the mutation rate to optochin resistance in Streptococcus pneumoniae. Journal of 530 Antimicrobial Chemotherapy 62:973–977. 531 24 57. Henriques-Normark B, Blomberg C, Dagerhamn J, Bättig P, Normark S. 2008. The rise and fall of 532 bacterial clones: Streptococcus pneumoniae. Nat Rev Microbiol 6:827–837. 533 58. Giraud A, Matic I, Radman M, Fons M, Taddei F. 2002. Mutator Bacteria as a Risk Factor in 534 Treatment of Infectious Diseases. Antimicrob Agents Chemother 46:863–865. 535 59. Labat F, Pradillon O, Garry L, Peuchmaur M, Fantin B, Denamur E. 2005. Mutator phenotype confers 536 advantage in Escherichia coli chronic urinary tract infection pathogenesis. FEMS Immunology & 537 Medical Microbiology 44:317–321. 538 60. Denamur E, Matic I. 2006. Evolution of mutation rates in bacteria. Mol Microbiol 60:820–827. 539 61. Oliver A, Mena A. 2010. Bacterial hypermutation in cystic fibrosis, not only for antibiotic resistance. 540 Clinical Microbiology and Infection 16:798–808. 541 62. Gifford DR, Berríos-Caro E, Joerres C, Galla T, Knight CG. 2019. Mutators drive evolution of multi-542 resistance to antibiotics. bioRxiv 643585. 543 63. Nguyen SV, McShan WM. 2014. Chromosomal islands of Streptococcus pyogenes and related 544 streptococci: molecular switches for survival and virulence. Frontiers in Cellular and Infection 545 Microbiology 4. 546 64. Huang J, Dai X, Wu Z, Hu X, Sun J, Tang Y, Zhang W, Han P, Zhao J, Liu G, Wang X, Mao S, Wang 547 Y, Call DR, Liu J, Wang L. 2023. Conjugative transfer of streptococcal prophages harboring antibiotic 548 resistance and virulence genes. ISME J 1–15. 549 65. Claverys JP, Lacks SA. 1986. Heteroduplex Deoxyribonucleic Acid Base Mismatch Repair in Bacteria. 550 Microbiol Rev 50:33. 551 66. Salles C, Créancier L, Claverys JP, Méjean V. 1992. The high level streptomycin resistance gene from 552 Streptococcus pneumoniae is a homologue of the ribosomal protein S12 gene from Escherichia coli. 553 22. Nucleic Acids Res 20:6103–6103. 554 25 67. Muñoz R, Bustamante M, de la Campa AG. 1995. Ser-127-to-Leu substitution in the DNA gyrase B 555 subunit of Streptococcus pneumoniae is implicated in novobiocin resistance. Journal of Bacteriology 556 177:4166–4170. 557 558 559