Roles of the Crp/Fnr Family Regulator ArcR in the Hemolysis and Biofilm of Staphylococcus aureus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains, Plasmids, Primers, and Growth Conditions
2.2. Construction of the S. aureus Mutant and Complementary Strains
2.3. Detection of Biofilm Production
2.4. Confocal Laser Scanning Microscopy (CLSM) for the Observation of Biofilm Production
2.5. Determination of Hemolytic Activity
2.6. Total RNA Extraction, cDNA Generation, qRT-PCR, and RNA-seq
2.7. Construction of LacZ Reporter Vectors
2.8. β-Galactosidase Activity Assay
2.9. PIA Detection
2.10. ArcR Protein Expression and Purification
2.11. Electrophoretic Mobility Shift Assay (EMSA)
2.12. Statistical Analysis
3. Results
3.1. Deletion of arcR Increased the Hemolytic Activity in S. aureus
3.2. Deletion of arcR Increased Biofilm Formation in S. aureus
3.3. Gene Expression Profiling of the ΔarcR Mutant
3.4. ArcR Regulates the Transcription of HEMOLYTIC Genes
3.5. ArcR Influences Biofilm Formation through ica Operon
3.6. ArcR Could Bind to the Promoter Regions of the ica Operon and hla
4. Discussion
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Strain or Plasmid | Relevant Characteristic | Source or Reference |
---|---|---|
S. aureus | ||
RN4220 | 8325-4, r−, restriction-deficient mutagenized RN450 | [34] |
NCTC8325 WT | NCTC8325 wild-type strain | NASA a |
NCTC8325 ΔarcR | NCTC8325 arcR deletion mutant | [33] |
NCTC8325 ΔarcR ΔicaA | NCTC8325 arcR icaA double mutant strain | This study |
USA300 | CA-MRSA, wild-type | NARSA |
USA300 ΔarcR | USA300 arcR deletion mutant | This study |
E. coli | ||
DH5α | E. coli host for cloning | Vazyme |
BL21 (DE3) | Expression strain; F− ompT hsdSB (rB− mB−) gal dcm (DE3) | Vazyme |
Plasmid | ||
pBTs | temp-sensitive plasmid, E. coli and S. aureus shutle vector, Apr, Cmr, for the construction of allelic-exchange mutants | [35] |
pBTs-arcR | pBTs derivative, for arcR deletion | This study |
pBTs-icaA | pBTs derivative, for icaA deletion | This study |
pCN51 | E. coli/S. aureus expression vector, Apr, Emr | [36] |
pCN51-arcR | Complementary arcR in S. aureus | This study |
pET28a | Expression vector with His tag in E. coli, Kmr | Addgene |
pET28a-arcR | His6-ArcR expression vector | This study |
pOS1-lacZ | E. coli and S. aureus shuttle vector, with lacZ ORF lacking first 6 amino acids, Apr, Cmr. | [37] |
pOS1hla | pOS1-lacZ plasimd derivative, including 546-bp sequence of hla | This study |
pOS1hlb | pOS1-lacZ plasimd derivative, including 412-bp sequence of hlb | This study |
pOS1sbi | pOS1-lacZ plasimd derivative, including 311-bp sequence of sbi | This study |
pOS1vraX | pOS1-lacZ plasimd derivative, including 287-bp sequence of vraX | This study |
pOS1splA | pOS1-lacZ plasimd derivative, including sequence of splA | This study |
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Fu, T.; Fan, Z.; Li, Y.; Li, Z.; Zhao, H.; Feng, Y.; Xue, G.; Cui, J.; Yan, C.; Gan, L.; et al. Roles of the Crp/Fnr Family Regulator ArcR in the Hemolysis and Biofilm of Staphylococcus aureus. Microorganisms 2023, 11, 1656. https://doi.org/10.3390/microorganisms11071656
Fu T, Fan Z, Li Y, Li Z, Zhao H, Feng Y, Xue G, Cui J, Yan C, Gan L, et al. Roles of the Crp/Fnr Family Regulator ArcR in the Hemolysis and Biofilm of Staphylococcus aureus. Microorganisms. 2023; 11(7):1656. https://doi.org/10.3390/microorganisms11071656
Chicago/Turabian StyleFu, Tongtong, Zheng Fan, Yujie Li, Zhoufei Li, Hanqing Zhao, Yanling Feng, Guanhua Xue, Jinghua Cui, Chao Yan, Lin Gan, and et al. 2023. "Roles of the Crp/Fnr Family Regulator ArcR in the Hemolysis and Biofilm of Staphylococcus aureus" Microorganisms 11, no. 7: 1656. https://doi.org/10.3390/microorganisms11071656
APA StyleFu, T., Fan, Z., Li, Y., Li, Z., Zhao, H., Feng, Y., Xue, G., Cui, J., Yan, C., Gan, L., Feng, J., Yuan, J., & You, F. (2023). Roles of the Crp/Fnr Family Regulator ArcR in the Hemolysis and Biofilm of Staphylococcus aureus. Microorganisms, 11(7), 1656. https://doi.org/10.3390/microorganisms11071656