The SOS regulon of Enterococcus faecalis is not involved in virulence traits Carrilero, L. 1, Matrat, S. 1, Thomas-Lopez, D. 1, Gutiérrez, B. 1, Santos-López, A. 1, L Ovejero, C. M1, Hidalgo, L1, Montero-Serra, N.1, Nes, I. F. 2, Gonzalez-Zorn, B. 1

1Departamento, Universidad Complutense de Madrid, Spain 2Laboratory of Microbial Gene Technology and Food Microbiology, The Norwegian University of Life Sciences, Ås, Norway. [email protected] Table 1 Differential

Locus Gene Predicted function gene SOS box expression Introduction EF0064 Hypothetical protein x 1.12 Operon EF0065 EF0065 Luciferase family protein x 1.15 CGTACATATGTTCC Enterococcus faecalis is one of the most clinically relevant nosocomial pathogens in the developed EF0066 ruvA Holliday junction DNA helicase /1.17 GGAACATATGTACG Genes related with DNA processing EF0067 ruvB Holliday junction DNA helicase x 1.27 Operon EF0067 EF0682 DNA repair exonuclease family x 1.58 CGAACAAATTTTCG EF0683 Hypothetical protein x 1.56 Operon EF0682 world. Its genetic plasticity facilitates its rapid evolution which is concerning given its role as a carrier EF0684 Cmp-binding protein x 1.81 Operon EF0682 EF0760 amino acid ABC transporter , ATP binding protein x1.22 Operon EF0761 Genes related with metabolism EF0761 amino acid ABC transporter permease protein x 1.14 CGAACATATTTTCG 1 EF0762 uvrB Excinuclease ABC (subunit B) x 1.97 CGAAAATATGTTCG and disseminator of pathogenicity determinants. Some antibiotics are inducers of the SOS response , EF0763 uvrA Excinuclease ABC (subunit A) x 2.00 Operon EF0762 EF1067 Hypothetical protein x 3.25 CAAGCTTATCTTCG 2, 3 EF1080 Structural gene for UV resistance x 7.84 CGAACGTTTGTTCG Genes related with phages which promotes the recombination and horizontal transfer of virulence factors . For the first time, we EF1081 Hypothetical protein x 6.82 Operon EF1080 EF1082 Hypothetical protein x 4.23 Operon EF1080 EF1083 Hypothetical protein x 7.68 Operon EF1080 EF1084 Universal stress protein family x 7.76 Operon EF1080 identified the genes involved in the SOS response of Enterococcus faecalis to elucidate the relationship EF1085 Hypothetical protein x 12.00 Operon EF1080 EF1112 rexB Exonuclease x 1.34 CAAACACATGTTTC EF1113 rexA Exonuclease x 1.45 Operon EF1112 Genes related with aromatics aminoacids EF1114 Hypothetical protein x 1.46 Operon EF1112 between the SOS response and virulence. EF1162 Putative helicase x 1.58 TGAACGCATGATTG EF1163 L-asparaginase x 1.21 Operon EF1162 EF1164 HD domain-containing protein x 1.49 Operon EF1162 EF1343 Sugar ABC transporter permease / 3.43 Operon EF1345 Genes that rule SOS response EF1344 Sugar ABC transporter permease / 2.27 Operon EF1345 EF1345 Sugar ABC transporter sugar-binding protein / 1.74 CGAACACATTTTAG EF1347 glycosyl hydrolase family 13 / 2.39 CTAAAATGTGTTCG EF1348 glucan 1 6-alpha-glucosidase putative / 2.78 Operon EF1347 Methods EF1349 glycosyl hydrolase family 13 / 2.22 Operon EF1347 EF1417 Integrase phage 3 x 4.30 Operon EF1418 Site directed mutagenesis of recA and lexA, the genes responsible for the regulation of the SOS EF1418 Hypothetical protein x 3.83 AGAACATACGTTCT Figure 2 EF1537 XerD Integrase/recombinase /1.24 TGAACAAGTCTTTT EF1538 ScpA Segregation and condensation protein A /1.19 Operon EF1537 EF1561 aroE shikimate 5-dehydrogenase / 2.00 ACAACAAACGTTTG response, provided us with E. faecalis strains with the SOS response constitutively activated or EF1562 phospho-2-dehydro-3-deoxyheptonate aldolase putative / 2.36 Operon EF1561 LexA-box in EF1563 aroB 3-dehydroquinate synthase / 2.69 Operon EF1561 EF1564 aroC chorismate synthase / 3.46 Operon EF1561 Firmicutes inhibited. Furthermore a transcriptomic study with a bacteria-specific microarray was performed allowing EF1565 prephenate dehydrogenase putative / 2.39 Operon EF1561 EF1566 aroA 3-phosphoshikimate 1-carboxyvinyltransferase putative / 2.91 Operon EF1561 EF1567 aroK shikimate kinase / 2.53 Operon EF1561 EF1568 chorismate mutase/prephenate dehydratase putattive / 1.84 Operon EF1561 the LexA-boxes to be identified in silico. Additionally, the resistance to ultraviolet (UV) radiation and EF1579 lexA Transcription repressor of SOS response x 1.31 CGAACGCTTGTTTG EF1686 Hypothetical protein x 9.00 CGAACACTCGTTCG EF1906 Hypothetical protein x 1.21 Operon EF1907 EF1907 maoC family protein x 1.21 AAATCGCGTGTTAG virulence of the different mutants were determined. EF2084 Hypothetical protein x 4.00 CGAACGAAAGTTCG LexA-box in EF2145 Integrase phage 5 x 5.16 CGAACATATATTCG E. faecalis V583 EF2415 Hypothetical protein x 2.73 Operon EF2416 EF2416 rpsU ribosomal protein S21 x 2.33 AAAACAGTCGTTCG EF2704 mutY A/G specific adenine glycosylase x 1.36 Operon EF2705 EF2705 recX Regulatory protein x 1.18 AGAATATATGTTCT EF2756 dinP DNA-damage-inducible protein x 2.14 AGAACGGACGTTCG Results EF3171 recA recombinase x 4.00 CGAATGTTTGTTCG

Microarray experiments lead to the characterization of the SOS regulon. 54 genes have been identified, Table 1: Genes constituting the E. faecalis SOS regulon identified by microarray experiments and in silico analysis. Figure 2: LexA-box in Firmicutes and in Enterococcus faecalis. 37 being overexpressed and 17 repressed in the bacteria with the SOS response constitutively expressed. Some of these genes, for example those involved in DNA repair (recA, lexA, uvrBA), had Conclusions been previously identified as part of the SOS regulon of other bacteria. In contrast, some typical SOS This study identifies the SOS regulon of E. faecalis for the first time to date. The SOS response regulon genes, such as low fidelity polymerases, are not present in the Enterococcus faecalis SOS follows a typical system involving the proteins RecA and LexA, however a number of unique genes regulon. Genes involved in the metabolism of aromatic aminoacids, which are involved in the virulence of were identified as part of the regulon. This study also allowed for the precise identification of the E. Listeria spp., are suppressed in the SOS regulon of E. faecalis. The resistance to UV radiation was faecalis LexA-box consensus sequence. Interstit6 ngly, we did not observe any involvement of the increased when the SOS was expressed and there were no virulence differences between the WT strain SOS response in the virulence of E. faecalis. and the lexA- mutant in vivo. References Figure 1 Figure 1 A: Growth after exposure to different intensities of UV light. 1Miller C. et al. SOS response induction by b-lactams and bacterial defense against antibiotic lethality. Science. 2004. 305. - ind- A B WT, JH2-2 wild type; lexA , insertional mutant of lexA; lexA , mutant 1629-1631. with a LexA protein that can´t be cleaved; lexAcom, complemented 2Beaber J.W. et al. SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature. 2004 Jan 1;427(6969):72-4. WT insertional lexA mutant; recA-, insertional mutant for recA. B: Virulence Epub 2003 Dec 21. LexA- assays performed in female BALB/c mice 3-6 weeks old. Mice were 3Aranda, J. et al. Acinetobacter baumannii RecA protein in repair of DNA damage, antimicrobial resistance, general stress response, Ind LexA - inoculated with 5x108 CFU of E. faecalis JH2-2 and individual CFU and virulence. 2011, Journal of Bacteriology, 193(15):3740-7. Com 4 LexA (black dots) in log10 and median (black bars) in kidneys at day 7 of Janky R. et al. Evaluation of phylogenetic footprint discovery for predicting bacterial cis-regulatory elements and revealing their RecA- mice are shown. WT, wild type; lexA-, insertional mutant for lexA. No evolution. BMC Bioinformatics. 2008. 9 (37). significant differences are observed in CFU (Man-Whitney test, α=0,05) between WT and lexA-, thus no differences in the virulence of FEMS Meeting Attendance Grant WT and lexA- strains were observed. FPU Grant