The Pleiotropic Phenotypes Caused by an hfq Null Mutation in Vibrio harveyi
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Media
2.2. Gene Disruption and Complementation
2.3. Bacterial Growth
2.4. Swimming Ability
2.5. Extracellular Protease (ECP) Activity Assay
2.6. Measurement of Biofilm Formation
2.7. Stress Response Assays
2.8. Antibiotic Resistance
2.9. Fish Infection Assay
2.10. Comparative Transcriptome Analysis
2.11. RT-qPCR Assay
2.12. Statistical Analysis
3. Results
3.1. Growth Repression of ∆hfq
3.2. Repressed Motility of ∆hfq
3.3. Increased Extracellular Protease Activity (ECP) of ∆hfq
3.4. Reduced Biofilm Formation of ∆hfq
3.5. The hfq Mutant Increased Susceptibility to ROS
3.6. Loss of hfq Increases Sensitivity to Amphenicol Antibiotics (Chloramphenicol and Florfenicol)
3.7. Virulence Attenuation of ∆hfq
3.8. Analysis of Differentially Expressed Genes
3.9. Prediction of Hfq-Dependent sRNAs
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Strains or Plasmids | Relevant Characteristics | Sources |
---|---|---|
V. harveyi | ||
345 | The wild-type strain V. haveyi 345 | [14] |
345∆hfq | hfq null mutant strain | This study |
345:pMMB207 (WT) | Chloramphenicol resistance (Cmr); the wild-type strain V. haveyi 345 with the control plasmid pMMB207 | This study |
345∆hfq:pMMB207 (∆hfq) | Cmr; the hfq null mutant strain V. haveyi 345∆hfq with the control plasmid pMMB207 | This study |
345∆hfq:pMMB207_hfq (Chfq) | Cmr; hfq null mutant strain V. haveyi 345hfq with the complemented plasmid pMMB207_hfq | This study |
E. coli | ||
Π3813 | Emrr, Tcr, lacIQ, thi1, supE44, endA1, recA1, hsdR17, gyrA462, zei298::tn10[Tc], ΔthyA:: (erm-pir116); the intermediate host of suicide vector pSW7848 | [15] |
GEB883 | Eryr, Tetr, WT E.coli K12 ΔdapA::erm pir RP4-2 ΔrecA gyrA462, zei298::Tn10; donor strain for conjugation | [16] |
Plasmids | ||
pSW7848 | Cmr; suicide vector with an R6K origin, requiring the Pir protein for its replication, and the ccdB toxin gene | [17] |
pSW7848_∆hfq | Cmr; pSW848 containing the UP-DWON fragment of ∆hfq | This study |
pMMB207 | Cmr; expression vector | [18] |
pMMB207_hfq | Cmr; pMMB207 containing intact hfq gene | This study |
Name | Sequence (5′-3′) | Purpose | Sources |
---|---|---|---|
pSW7848-F | GTCTGATTCGTTACCAATTATGACAAC | Linearization of pSW7848 | [18] |
pSW7848-R | GAATTCGATATCAAGCTTATCGATAC | ||
hfq-UP-F | aagcttgatatcgaattcCGGCGTTGATCTACAAAG | Amplification of hfq-UP | This study |
hfq-UP-R | tcaataggaTTTATTTTCCTTATTTAATTTGTAGTTG | ||
hfq-DOWN-F | ggaaaataaaTCCTATTGAAGAACACTGTTAACC | Amplification of hfq-DOWN | This study |
hfq-DOWN-R | ttggtaacgaatcagacGCATCAACAACATGTAACAAAATG | ||
Del-check-pSW7848-F | TCACTGTCCCTTATTCGCACC | Check the assembly of recombinant plasmid pSW7848_∆hfq | This study |
Del-check-pSW7848-R | CTGCTTTTGAGCACTACCCG | ||
△hfq-check-F | CAGGCACTTGAAACCATGTCAG | Check the detection of hfq | This study |
△hfq-check-R | CTCGCTCACCGGATTCATAAC | ||
pMMB207-F | AGAAGCGGTCTGATAAAACAGAATTTGC | Linearization of pMMB207 | [18] |
pMMB207-R | GCGCAACGCAATTAATGTAAGTTAG | ||
com-hfq-F | GCGATAACATTGAACAGGCAC | Amplification of hfq | This study |
com-hfq-R | GCAATTTCTTGTGCCTTACCC | ||
com-PMMB207-check-F | CTACTGAGCGCTGCCGCACA | Check the complementation of hfq | This study |
com-PMMB207-check-R | TCGTTTTATTTGATGCCTGGCAG |
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Deng, Y.; Zang, S.; Lin, Z.; Xu, L.; Cheng, C.; Feng, J. The Pleiotropic Phenotypes Caused by an hfq Null Mutation in Vibrio harveyi. Microorganisms 2023, 11, 2741. https://doi.org/10.3390/microorganisms11112741
Deng Y, Zang S, Lin Z, Xu L, Cheng C, Feng J. The Pleiotropic Phenotypes Caused by an hfq Null Mutation in Vibrio harveyi. Microorganisms. 2023; 11(11):2741. https://doi.org/10.3390/microorganisms11112741
Chicago/Turabian StyleDeng, Yiqin, Shujun Zang, Ziyang Lin, Liwen Xu, Changhong Cheng, and Juan Feng. 2023. "The Pleiotropic Phenotypes Caused by an hfq Null Mutation in Vibrio harveyi" Microorganisms 11, no. 11: 2741. https://doi.org/10.3390/microorganisms11112741