The Transcription Factor SpoVG Is of Major Importance for Biofilm Formation of Staphylococcus epidermidis under In Vitro Conditions, but Dispensable for In Vivo Biofilm Formation
Abstract
:1. Introduction
2. Results
2.1. Genetic Organization of the yabJ-spoVG Locus in S. epidermidis 1457
2.2. Impact of the spoVG Deletion on Growth of S. epidermidis 1457
2.3. Impact of the spoVG Deletion on Biofilm Formation of S. epidermidis 1457
2.4. Impact of the spoVG Deletion on ica Transcription of S. epidermidis 1457
2.5. Interaction of SpoVG with the icaA-icaR Intergenic Region in S. epidermidis 1457
2.6. Impact of the spoVG Deletion on Infectivity of S. epidermidis 1457 in a Murine Foreign Body Infection Model
2.7. Impact of the spoVG Deletion on Chemokine Expression in S. epidermidis 1457 Infected Tissue
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains and Plasmids
4.2. Bacterial Growth Conditions
4.3. Mutant Construction
4.4. RNA Isolation and Purification, cDNA Synthesis and qRT-PCR
4.5. Biofilm Assays
4.6. Cloning, Expression and Purification of Recombinant SpoVG
4.7. Electrophoresis Mobility Shift Assay
4.8. Murine Foreign Body Infection Model
4.9. Cytokine Determinations
4.10. Statistical Analyses
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Vanepps, J.S.; Younger, J.G. Implantable device-related infection. Shock 2016, 46, 597–608. [Google Scholar] [CrossRef] [Green Version]
- de Oliveira, W.F.; Silva, P.M.S.; Silva, R.C.S.; Silva, G.M.M.; Machado, G.; Coelho, L.; Correia, M.T.S. Staphylococcus aureus and Staphylococcus epidermidis infections on implants. J. Hosp. Infect. 2018, 98, 111–117. [Google Scholar] [CrossRef] [PubMed]
- Brown, M.M.; Horswill, A.R. Staphylococcus epidermidis—Skin friend or foe? PLoS Pathog. 2020, 16, e1009026. [Google Scholar] [CrossRef] [PubMed]
- Kleinschmidt, S.; Huygens, F.; Faoagali, J.; Rathnayake, I.U.; Hafner, L.M. Staphylococcus epidermidis as a cause of bacteremia. Futur. Microbiol. 2015, 10, 1859–1879. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Joubert, I.A.; Otto, M.; Strunk, T.; Currie, A.J. Look who’s talking: Host and pathogen drivers of Staphylococcus epidermidis virulence in neonatal sepsis. Int. J. Mol. Sci. 2022, 23, 860. [Google Scholar] [CrossRef] [PubMed]
- Matarrese, A.N.; Ivulich, D.I.; Cesar, G.; Alaniz, F.; Ruiz, J.J.; Osatnik, J. Epidemiological analysis of catheter-related bloodstream infections in medical-surgical intensive care units. Medicina (B Aires) 2021, 81, 159–165. [Google Scholar]
- German Reference Center for Surveillance of Nosocomial Infections. STATIONS-KISS Module Reference Data 2017–2019. 2020. Available online: https://www.nrz-hygiene.de/fileadmin/nrz/module/station/infektionen/201701_201912_deviceref.pdf (accessed on 10 March 2022).
- Pinto, M.; Borges, V.; Nascimento, M.; Martins, F.; Pessanha, M.A.; Faria, I.; Rodrigues, J.; Matias, R.; Gomes, J.P.; Jordao, L. Insights on catheter-related bloodstream infections: A prospective observational study on the catheter colonization and multi-drug resistance. J. Hosp. Infect. 2022, 123, 43–51. [Google Scholar] [CrossRef]
- Strasheim, W.; Kock, M.; Ueckermann, V.; Hoosien, E.; Dreyer, A.W.; Ehlers, M.M. Surveillance of catheter-related infections: The supplementary role of the microbiology laboratory. BMC Infect. Dis. 2015, 15, 5. [Google Scholar] [CrossRef] [Green Version]
- Buetti, N.; Priore, E.L.; Atkinson, A.; Widmer, A.F.; Kronenberg, A.; Marschall, J. Catheter-related infections: Does the spectrum of microbial causes change over time? A nationwide surveillance study. BMJ Open 2018, 8, e023824. [Google Scholar] [CrossRef] [Green Version]
- Weiner-Lastinger, L.M.; Abner, S.; Edwards, J.R.; Kallen, A.J.; Karlsson, M.; Magill, S.S.; Pollock, D.; See, I.; Soe, M.M.; Walters, M.S.; et al. Antimicrobial-resistant pathogens associated with adult healthcare-associated infections: Summary of data reported to the National Healthcare Safety Network, 2015–2017. Infect. Control Hosp. Epidemiol. 2020, 41, 1–18. [Google Scholar] [CrossRef] [Green Version]
- Kranjec, C.; Angeles, D.M.; Mårli, M.T.; Fernández, L.; García, P.; Kjos, M.; Diep, D. Staphylococcal biofilms: Challenges and novel therapeutic perspectives. Antibiotics 2021, 10, 131. [Google Scholar] [CrossRef] [PubMed]
- Schilcher, K.; Horswill, A.R. Staphylococcal biofilm development: Structure, regulation, and treatment strategies. Microbiol. Mol. Biol. Rev. 2020, 84, e00026-19. [Google Scholar] [CrossRef] [PubMed]
- França, A.; Pérez-Cabezas, B.; Correia, A.; Pier, G.B.; Cerca, N.; Vilanova, M. Staphylococcus epidermidis biofilm-released cells induce a prompt and more marked in vivo inflammatory-type response than planktonic or biofilm cells. Front. Microbiol. 2016, 7, 1530. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Arciola, C.R.; Campoccia, D.; Ravaioli, S.; Montanaro, L. Polysaccharide intercellular adhesin in biofilm: Structural and regulatory aspects. Front. Cell. Infect. Microbiol. 2015, 5, 7. [Google Scholar] [CrossRef] [Green Version]
- Deighton, M.; Borland, R. Regulation of slime production in Staphylococcus epidermidis by iron limitation. Infect. Immun. 1993, 61, 4473–4479. [Google Scholar] [CrossRef] [Green Version]
- Rachid, S.; Cho, S.; Ohlsen, K.; Hacker, J.; Ziebuhr, W. Induction of Staphylococcus epidermidis biofilm formation by environmental factors: The possible involvement of the alternative transcription factor SigB. Adv. Exp. Med. Biol. 2000, 485, 159–166. [Google Scholar] [CrossRef]
- Knobloch, J.K.-M.; Jäger, S.; Horstkotte, M.A.; Rohde, H.; Mack, D. RsbU-Dependent regulation of Staphylococcus epidermidis biofilm formation is mediated via the alternative sigma factor SigB by repression of the negative regulator gene icaR. Infect. Immun. 2004, 72, 3838–3848. [Google Scholar] [CrossRef] [Green Version]
- Tormo, M.A.; Martí, M.; Valle, J.; Manna, A.C.; Cheung, A.L.; Lasa, I.; Penadés, J.R.; Sar, A. Is an essential positive regulator of Staphylococcus epidermidis biofilm development. J. Bacteriol. 2005, 187, 2348–2356. [Google Scholar] [CrossRef] [Green Version]
- Conlon, K.M.; Humphreys, H.; O’Gara, J.P. icaR encodes a transcriptional repressor involved in environmental regulation of ica operon expression and biofilm formation in Staphylococcus epidermidis. J. Bacteriol. 2002, 184, 4400–4408. [Google Scholar] [CrossRef] [Green Version]
- Liu, Q.; Fan, J.; Niu, C.; Wang, D.; Wang, J.; Wang, X.; Villaruz, A.E.; Li, M.; Otto, M.; Gao, Q. The Eukaryotic-type serine/threonine protein kinase Stk is required for biofilm formation and virulence in Staphylococcus epidermidis. PLoS ONE 2011, 6, e25380. [Google Scholar] [CrossRef]
- Sadykov, M.R.; Hartmann, T.; Mattes, T.A.; Hiatt, M.; Jann, N.J.; Zhu, Y.; Ledala, N.; Landmann, R.; Herrmann, M.; Rohde, H.; et al. CcpA coordinates central metabolism and biofilm formation in Staphylococcus epidermidis. Microbiology 2011, 157, 3458–3468. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Rowe, S.E.; Mahon, V.; Smith, S.G.; O’Gara, J.P. A novel role for SarX in Staphylococcus epidermidis biofilm regulation. Microbiology 2011, 157, 1042–1049. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chang, Y.-M.; Jeng, W.-Y.; Ko, T.-P.; Yeh, Y.-J.; Chen, C.K.-M.; Wang, A.H.-J. Structural study of TcaR and its complexes with multiple antibiotics from Staphylococcus epidermidis. Proc. Natl. Acad. Sci. USA 2010, 107, 8617–8622. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hoang, T.-M.; Zhou, C.; Lindgren, J.K.; Galac, M.R.; Corey, B.; Endres, J.E.; Olson, M.E.; Fey, P.D. Transcriptional regulation of icaADBC by both IcaR and TcaR in Staphylococcus epidermidis. J. Bacteriol. 2019, 201, 201. [Google Scholar] [CrossRef] [Green Version]
- Wu, Y.; Meng, Y.; Qian, L.; Ding, B.; Han, H.; Chen, H.; Bai, L.; Qu, D.; Wu, Y. The vancomycin resistance-associated regulatory system VraSR modulates biofilm formation of Staphylococcus epidermidis in an ica-dependent manner. mSphere 2021, 6, e0064121. [Google Scholar] [CrossRef]
- Lerch, M.F.; Schoenfelder, S.M.; Marincola, G.; Wencker, F.; Eckart, M.; Förstner, K.U.; Sharma, C.M.; Thormann, K.M.; Kucklick, M.; Engelmann, S.; et al. A non-coding RNA from the intercellular adhesion (ica) locus ofStaphylococcus epidermidiscontrols polysaccharide intercellular adhesion (PIA)-mediated biofilm formation. Mol. Microbiol. 2019, 111, 1571–1591. [Google Scholar] [CrossRef]
- Schoenfelder, S.M.K.; Lange, C.; Prakash, S.A.; Marincola, G.; Lerch, M.F.; Wencker, F.D.R.; Förstner, K.U.; Sharma, C.M.; Ziebuhr, W. The small non-coding RNA RsaE influences extracellular matrix composition in Staphylococcus epidermidis biofilm communities. PLoS Pathog. 2019, 15, e1007618. [Google Scholar] [CrossRef] [Green Version]
- Ziebuhr, W.; Krimmer, V.; Rachid, S.; Lossner, I.; Gotz, F.; Hacker, J. A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: Evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. Mol. Microbiol. 1999, 32, 345–356. [Google Scholar] [CrossRef]
- Jutras, B.L.; Chenail, A.M.; Rowland, C.L.; Carroll, D.; Miller, M.C.; Bykowski, T.; Stevenson, B. Eubacterial SpoVG homologs constitute a new family of site-specific DNA-binding proteins. PLoS ONE 2013, 8, e66683. [Google Scholar] [CrossRef] [Green Version]
- Huang, Q.; Zhang, Z.; Liu, Q.; Liu, F.; Liu, Y.; Zhang, J.; Wang, G. SpoVG is an important regulator of sporulation and affects biofilm formation by regulating Spo0A transcription in Bacillus cereus 0–9. BMC Microbiol. 2021, 21, 172. [Google Scholar] [CrossRef]
- Burke, T.P.; Portnoy, D.A. SpoVG is a conserved RNA-binding protein that regulates Listeria monocytogenes lysozyme resistance, virulence, and swarming motility. MBio 2016, 7, e00240-16. [Google Scholar] [CrossRef] [Green Version]
- Meier, S.; Goerke, C.; Wolz, C.; Seidl, K.; Homerova, D.; Schulthess, B.; Kormanec, J.; Berger-Bächi, B.; Bischoff, M. SigmaB and the SigmaB-dependent arlRS and yabJ-spoVG loci affect capsule formation in Staphylococcus aureus. Infect. Immun. 2007, 75, 4562–4571. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Schulthess, B.; Bloes, D.A.; Francois, P.; Girard, M.; Schrenzel, J.; Bischoff, M.; Berger-Bächi, B. The σB-dependent yabJ-spoVG operon is involved in the regulation of extracellular nuclease, lipase, and protease expression in Staphylococcus aureus. J. Bacteriol. 2011, 193, 4954–4962. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Zhu, Q.; Liu, B.; Sun, B. SpoVG modulates cell aggregation in Staphylococcus aureus by regulating sasC expression and extracellular DNA release. Appl. Environ. Microbiol. 2020, 86, 86. [Google Scholar] [CrossRef] [PubMed]
- Bischoff, M.; Brelle, S.; Minatelli, S.; Molle, V. Stk1-mediated phosphorylation stimulates the DNA-binding properties of the Staphylococcus aureus SpoVG transcriptional factor. Biochem. Biophys. Res. Commun. 2016, 473, 1223–1228. [Google Scholar] [CrossRef] [PubMed]
- Mack, D.; Siemssen, N.; Laufs, R. Parallel induction by glucose of adherence and a polysaccharide antigen specific for plastic-adherent Staphylococcus epidermidis: Evidence for functional relation to intercellular adhesion. Infect. Immun. 1992, 60, 2048–2057. [Google Scholar] [CrossRef] [Green Version]
- Mäder, U.; Nicolas, P.; Depke, M.; Pané-Farré, J.; Debarbouille, M.; Van Der Kooi-Pol, M.M.; Guérin, C.; Dérozier, S.; Hiron, A.; Jarmer, H.; et al. Staphylococcus aureus transcriptome architecture: From laboratory to infection-mimicking conditions. PLoS Genet. 2016, 12, e1005962. [Google Scholar] [CrossRef]
- Ungphakorn, W.; Malmberg, C.; Lagerbäck, P.; Cars, O.; Nielsen, E.I.; Tängdén, T. Evaluation of automated time-lapse microscopy for assessment of in vitro activity of antibiotics. J. Microbiol. Methods 2017, 132, 69–75. [Google Scholar] [CrossRef]
- Seidl, K.; Goerke, C.; Wolz, C.; Mack, D.; Berger-Bächi, B.; Bischoff, M. Staphylococcus aureus CcpA affects biofilm formation. Infect. Immun. 2008, 76, 2044–2050. [Google Scholar] [CrossRef] [Green Version]
- Pätzold, L.; Brausch, A.-C.; Bielefeld, E.-L.; Zimmer, L.; Somerville, G.A.; Bischoff, M.; Gaupp, R. Impact of the histidine-containing phosphocarrier protein HPr on carbon metabolism and virulence in Staphylococcus aureus. Microorganisms 2021, 9, 466. [Google Scholar] [CrossRef]
- Mandakhalikar, K.D.; Rahmat, J.N.; Chiong, E.; Neoh, K.G.; Shen, L.; Tambyah, P.A. Extraction and quantification of biofilm bacteria: Method optimized for urinary catheters. Sci. Rep. 2018, 8, 8069. [Google Scholar] [CrossRef]
- O’Gara, J.P. icaand beyond: Biofilm mechanisms and regulation in Staphylococcus epidermidis and Staphylococcus aureus. FEMS Microbiol. Lett. 2007, 270, 179–188. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Dobinsky, S.; Kiel, K.; Rohde, H.; Bartscht, K.; Knobloch, J.K.-M.; Horstkotte, M.A.; Mack, D. Glucose-related dissociation between icaADBC transcription and biofilm expression by Staphylococcus epidermidis: Evidence for an additional factor required for polysaccharide intercellular adhesin synthesis. J. Bacteriol. 2003, 185, 2879–2886. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Jeng, W.-Y.; Ko, T.-P.; Liu, C.-I.; Guo, R.-T.; Liu, C.-L.; Shr, H.-L.; Wang, A.H.-J. Crystal structure of IcaR, a repressor of the TetR family implicated in biofilm formation in Staphylococcus epidermidis. Nucleic Acids Res. 2008, 36, 1567–1577. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Heilmann, C.; Schweitzer, O.; Gerke, C.; Vanittanakom, N.; Mack, D.; Götz, F. Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis. Mol. Microbiol. 1996, 20, 1083–1091. [Google Scholar] [CrossRef] [PubMed]
- Mozos, I.R.D.L.; Vergara-Irigaray, M.; Segura, V.; Villanueva, M.; Bitarte, N.; Saramago, M.; Domingues, S.; Arraiano, C.; Fechter, P.; Romby, P.; et al. Base Pairing interaction between 5′- and 3′-UTRs Controls icaR mRNA translation in Staphylococcus aureus. PLoS Genet. 2013, 9, e1004001. [Google Scholar] [CrossRef]
- Jefferson, K.K.; Pier, D.B.; Goldmann, D.A.; Pier, G.B. The Teicoplanin-associated locus regulator (TcaR) and the Intercellular adhesin locus regulator (IcaR) are transcriptional inhibitors of the ica locus in Staphylococcus aureus. J. Bacteriol. 2004, 186, 2449–2456. [Google Scholar] [CrossRef] [Green Version]
- Rupp, M.E.; Ulphani, J.S.; Fey, P.D.; Bartscht, K.; Mack, D. Characterization of the importance of polysaccharide intercellular adhesin/hemagglutinin of Staphylococcus epidermidis in the pathogenesis of biomaterial-based infection in a mouse foreign body infection model. Infect. Immun. 1999, 67, 2627–2632. [Google Scholar] [CrossRef] [Green Version]
- Le, K.Y.; Park, M.D.-Y.; Otto, M. Immune evasion mechanisms of Staphylococcus epidermidis biofilm infection. Front. Microbiol. 2018, 9, 359. [Google Scholar] [CrossRef]
- Mack, D.; Riedewald, J.; Rohde, H.; Magnus, T.; Feucht, H.H.; Elsner, H.-A.; Laufs, R.; Rupp, M.E. Essential functional role of the polysaccharide intercellular adhesin of Staphylococcus epidermidis in hemagglutination. Infect. Immun. 1999, 67, 1004–1008. [Google Scholar] [CrossRef] [Green Version]
- Fredheim, E.G.A.; Granslo, H.N.; Flaegstad, T.; Figenschau, Y.; Rohde, H.; Sadovskaya, I.; Mollnes, T.E.; Klingenberg, C. Staphylococcus epidermidis polysaccharide intercellular adhesin activates complement. FEMS Immunol. Med. Microbiol. 2011, 63, 269–280. [Google Scholar] [CrossRef] [PubMed]
- Vuong, C.; Kocianova, S.; Voyich, J.M.; Yao, Y.; Fischer, E.R.; DeLeo, F.; Otto, M. A Crucial role for exopolysaccharide modification in bacterial biofilm formation, immune evasion, and virulence. J. Biol. Chem. 2004, 279, 54881–54886. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kristian, S.A.; Birkenstock, T.A.; Sauder, U.; Mack, D.; Götz, F.; Landmann, R. Biofilm Formation Induces C3a Release and protects Staphylococcus epidermidis from IgG and complement deposition and from neutrophil-dependent killing. J. Infect. Dis. 2008, 197, 1028–1035. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Skovdal, S.M.; Hansen, L.K.; Ivarsen, D.M.; Zeng, G.; Büttner, H.; Rohde, H.; Jørgensen, N.P.; Meyer, R.L. Host factors abolish the need for polysaccharides and extracellular matrix-binding protein in Staphylococcus epidermidis biofilm formation. J. Med. Microbiol. 2021, 70, 001287. [Google Scholar] [CrossRef]
- Mack, D.; Rohde, H.; Dobinsky, S.; Riedewald, J.; Nedelmann, M.; Knobloch, J.K.-M.; Elsner, H.-A.; Feucht, H.H. Identification of three essential regulatory gene loci governing expression of Staphylococcus epidermidis polysaccharide intercellular adhesin and biofilm formation. Infect. Immun. 2000, 68, 3799–3807. [Google Scholar] [CrossRef] [Green Version]
- Knobloch, J.K.-M.; Bartscht, K.; Sabottke, A.; Rohde, H.; Feucht, H.-H.; Mack, D. Biofilm formation by Staphylococcus epidermidis depends on functional RsbU, an Activator of the sigB operon: Differential activation mechanisms due to ethanol and salt stress. J. Bacteriol. 2001, 183, 2624–2633. [Google Scholar] [CrossRef] [Green Version]
- Handke, L.D.; Slater, S.R.; Conlon, K.M.; O’Donnell, S.T.; Olson, M.E.; Bryant, K.A.; Rupp, M.E.; O’Gara, J.; Fey, P.D. SigmaB and SarA independently regulate polysaccharide intercellular adhesin production in Staphylococcus epidermidis. Can. J. Microbiol. 2007, 53, 82–91. [Google Scholar] [CrossRef]
- Kreiswirth, B.N.; Löfdahl, S.; Betley, M.J.; O’Reilly, M.; Schlievert, P.; Bergdoll, M.S.; Novick, R. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 1983, 305, 709–712. [Google Scholar] [CrossRef]
- Nedelmann, M.; Sabottke, A.; Laufs, R.; Mack, D. Generalized transduction for genetic linkage analysis and transfer of transposon insertions in different Staphylococcus epidermidis strains. Zent. Bakteriol. 1998, 287, 85–92. [Google Scholar] [CrossRef]
- Monk, I.R.; Tree, J.; Howden, B.; Stinear, T.P.; Foster, T.J. Complete bypass of restriction systems for major Staphylococcus aureus lineages. MBio 2015, 6, e00308-15. [Google Scholar] [CrossRef] [Green Version]
- Geiger, T.; Francois, P.; Liebeke, M.; Fraunholz, M.; Goerke, C.; Krismer, B.; Schrenzel, J.; Lalk, M.; Wolz, C. The stringent response of Staphylococcus aureus and its impact on survival after phagocytosis through the induction of intracellular PSMs expression. PLoS Pathog. 2012, 8, e1003016. [Google Scholar] [CrossRef] [PubMed]
- Brückner, R. Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus. FEMS Microbiol. Lett. 1997, 151, 1–8. [Google Scholar] [CrossRef]
- Bae, T.; Schneewind, O. Allelic replacement in Staphylococcus aureus with inducible counter-selection. Plasmid 2006, 55, 58–63. [Google Scholar] [CrossRef] [PubMed]
- Gaupp, R.; Wirf, J.; Wonnenberg, B.; Biegel, T.; Eisenbeis, J.; Graham, J.; Herrmann, M.; Lee, C.Y.; Beisswenger, C.; Wolz, C.; et al. RpiRc is a pleiotropic effector of virulence determinant synthesis and attenuates pathogenicity in Staphylococcus aureus. Infect. Immun. 2016, 84, 2031–2041. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Pfaffl, M.W. Relative quantification. In Real-Time PCR; Tevfik Dorak, M., Ed.; Taylor & Francis: Abingdon, UK, 2006; pp. 63–82. [Google Scholar]
Strain | Generation Time (min) 1 | p Value 2 |
---|---|---|
1457 | 44.69 ± 6.77 | |
ΔspoVG | 42.53 ± 1.90 | 0.999 |
ΔspoVG::spoVG | 42.48 ± 5.85 | 0.610 |
Strain | Description 1 | Reference or Source |
---|---|---|
S. aureus | ||
RN4220 | NCTC8325-4 derivative, acceptor of foreign DNA | [59] |
S. epidermidis | ||
1457 | Central venous catheter isolate, high level PIA producer | [37] |
1457 M10 | Biofilm-negative 1457 icaA-Tn917 transposon mutant; ErmR | [60] |
1457 M15 | rsbU-Tn917 mutant of 1457 used as recipient by electroporation; ErmR | [56] |
1457 ΔspoVG | 1457 ΔspoVG::ermB; ErmR | This study |
1457 ΔspoVG::spoVG | cis-complemented 1457 ΔspoVG derivative | This study |
E. coli | ||
BL21 Star (DE3) | Protein expression strain | Invitrogen |
DH5α | Cloning strain | Invitrogen |
IM08B | E. coli DC10B derivative harboring hsdS of S. aureus strain NRS384, Δdcm | [61] |
TOP10 | E. coli derivative ultra-competent cells used for general cloning | Invitrogen |
Plasmids | ||
pBASE6 | E. coli–Staphylococcus temperature-sensitive suicide shuttle vector, secY counterselection; bla cat | [62] |
pBASE6_spoVG_comp | pBASE6 derivative harboring the C-terminal region of purR, yabJ-spoVG, and the N-terminal region of glmU | This study |
pBT2 | Temperature sensitive E. coli-Staphylococcus shuttle plasmid; bla, cat | [63] |
pBT2_spoVG_KO | pBT2 derivative harboring spoVG up-ermB-spoVG do; bla, cat, ermB | This study |
pDEST R4-R3 | Gateway-destination vector, contains ccdB and attR4/R3 sites; bla, cat | Invitrogen |
pDEST_spoVG_ko | pDEST R4-R3 derivative harboring spoVG up-ermB-spoVG do; bla, cat, ermB | This study |
pDONR 221 | Entry vector to clone attB1 and attB2 flanked PCR products; ccdB+, cat | Invitrogen |
pDONR P2R-P3 | Entry vector to clone attB2 and attB3 flanked PCR products | Invitrogen |
pDONR P4-P1R | Entry vector to clone attB4 and attB1 flanked PCR products | Invitrogen |
pENTRY_ermB | pDONR 221 derivative harboring attB1 and attB2 flanked ermB | This study |
pENTRY_spoVG_up | pDONR P4-P1R derivative harboring attB4 and attB1 flanked spoVG up | This study |
pENTRY_spoVG_do | pDONR P2R-P3 derivative harboring attB2 and attB3 flanked spoVG do | This study |
HAT-HA-EpiSpoVG_pDon221 | pDONR 221 derivative harboring ATTB1, Shine-Dalgarno and Kozac sequences, a HAT clontech tag, a HA tag, a linker region and spoVG; cat | This study |
pRSF-HAT-HA-SpoVG | pRSF RfA derivative harboring the spoVG ORF N-terminally fused to HAT and HA tags; kan | This study |
pRSF RfA | Gateway-destination vector; kan | Montpellier Genomic Collection (MGC) |
Primer | Direction | Sequence (5′-3′) 1 |
---|---|---|
Cloning primer | ||
spoVG up | forward | GGGGACAACTTTGTATAGAAAAGTTGTTAAAGTGGAACCAGGCAAC |
reverse | GGGGACTGCTTTTTTTGACAAACTTGCTAGCGTAATGGAAACGAGTG | |
spoVG do | forward | GGGGACAGCTTTCTTGTACAAAGTGGTCAGATAACGAAGAATCAGAC |
reverse | GGGGACAACTTTGTATAATAAAGTTGGAAAACGAGGTCATCAAACC | |
ermB | forward | GGGGACAAGTTTGTACAAAAAAGCAGGCTGACGGTGACATCTCTCTATTG |
reverse | GGGGACCACTTTGTACAAGAAAGCTGGGTGAAAAGGTACCATAAACGGTCG | |
spoVG_KO | forward | atggtactgcagTTAAAGTGGAACCAGGCAAC |
reverse | atggtaggatccGAAAACGAGGTCATCAAACC | |
MBH603 | forward | gtcgagctCATGCTACAATGTGGTGCTG |
MBH607 | reverse | gacgagaTCTTTAATCGCACGTTCTGAGTC |
Verification primer | ||
erm_v | forward | AATTGGAACAGGTAAAGGGC |
spoVG_do_v | reverse | GGTTTGATAATTTTAGAAATTC |
MBH608 | forward | GGTACCCTAAGCACTAGGCCCATATTCGC |
qRT-PCR primer | ||
gyrB | forward | CTAATGCTGATTTACGACGCGTAA |
reverse | TCTGTAGGACGCATTATTGTTGAAA | |
icaD | forward | GTATTGTATCGTTGTGATGAT |
reverse | ACTTTCCATTTGAGAATTGAT | |
icaR | forward | ATGGTACTACACTTGATGATA |
reverse | GTAATGATAATATAGACTAGCCTTT | |
yabJ | forward | AAAGCGACAATCTATATTTCTGA |
reverse | ACCTATCAATTCAATTTCTACCTT | |
spoVG | forward | GCAGTGATGAAAGTATATGATGA |
reverse | CTTCGTCTGATTCTTCGTTATC | |
EMSA primer | ||
icaA p | forward | TAACAACATTCTATTGCAAATTGAAATACTTTCGATTAGCATAT-GCTTTACAACCTAACTAACGAAAGGTAGGTGAAAAA |
reverse | TTTTTCACCTACCTTTCGTTAGTTAGGTTGTAAAGCATATGCTAA-TCGAAAGTATTTCAATTTGCAATAGAATGTTGTTA | |
icaR p | forward | GAAACAGTAATATTTGTAATTTTAACTTAATTTTTCTGTAATAT-ATTTTTAGAAAATTGAATAAGGGGGAGATTTTAGAA |
reverse | TTCTAAAATCTCCCCCTTATTCAATTTTCTAAAAATATATTACA-GAAAAATAAGTTAAAATTACAAATATTACTGTTTC | |
secA p | forward | AGTATAATTTTCTAACTATAAATGATAAGATATATTGTTGTAG-GCCAAACAGTTTTTTAGCTAAAGGAGCGAACGAAATG |
reverse | CATTTCGTTCGCTCCTTTAGCTAAAAAACTGTTTGGCCTACAA- CAATATATCTTATCATTTATAGTTAGAAAATTATACT |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Benthien, H.; Fresenborg, B.; Pätzold, L.; Elhawy, M.I.; Huc-Brandt, S.; Beisswenger, C.; Krasteva-Christ, G.; Becker, S.L.; Molle, V.; Knobloch, J.K.; et al. The Transcription Factor SpoVG Is of Major Importance for Biofilm Formation of Staphylococcus epidermidis under In Vitro Conditions, but Dispensable for In Vivo Biofilm Formation. Int. J. Mol. Sci. 2022, 23, 3255. https://doi.org/10.3390/ijms23063255
Benthien H, Fresenborg B, Pätzold L, Elhawy MI, Huc-Brandt S, Beisswenger C, Krasteva-Christ G, Becker SL, Molle V, Knobloch JK, et al. The Transcription Factor SpoVG Is of Major Importance for Biofilm Formation of Staphylococcus epidermidis under In Vitro Conditions, but Dispensable for In Vivo Biofilm Formation. International Journal of Molecular Sciences. 2022; 23(6):3255. https://doi.org/10.3390/ijms23063255
Chicago/Turabian StyleBenthien, Hannah, Beate Fresenborg, Linda Pätzold, Mohamed Ibrahem Elhawy, Sylvaine Huc-Brandt, Christoph Beisswenger, Gabriela Krasteva-Christ, Sören L. Becker, Virginie Molle, Johannes K. Knobloch, and et al. 2022. "The Transcription Factor SpoVG Is of Major Importance for Biofilm Formation of Staphylococcus epidermidis under In Vitro Conditions, but Dispensable for In Vivo Biofilm Formation" International Journal of Molecular Sciences 23, no. 6: 3255. https://doi.org/10.3390/ijms23063255
APA StyleBenthien, H., Fresenborg, B., Pätzold, L., Elhawy, M. I., Huc-Brandt, S., Beisswenger, C., Krasteva-Christ, G., Becker, S. L., Molle, V., Knobloch, J. K., & Bischoff, M. (2022). The Transcription Factor SpoVG Is of Major Importance for Biofilm Formation of Staphylococcus epidermidis under In Vitro Conditions, but Dispensable for In Vivo Biofilm Formation. International Journal of Molecular Sciences, 23(6), 3255. https://doi.org/10.3390/ijms23063255