Assessment of Bacillus subtilis Plasmid pLS20 Conjugation in the Absence of Quorum Sensing Repression
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains, Plasmids, and Primers
2.2. Plasmid and Strain Construction
2.3. Conjugation
2.4. Flow Cytometric Analysis
2.5. Microscopy
3. Results
3.1. Conventional Colony Formation Assay
- (i)
- The CFU/OD600 values of both donors and recipients tended to decrease when the donors had been grown in the presence of IPTG compared to its absence.
- (ii)
- In the presence of IPTG, the CFU/OD600 of transconjugants was higher only when the concentration of recipients exceeded that of donors, whereas, in the absence of IPTG, the CFU/OD600 of transconjugants gave an apparent peak at recipients: donors = 25:25, and the value similar to that were found in the presence of IPTG.
- (iii)
- As the number of donors exceeded that of recipients, the CFU/OD600 of transconjugants showed a tendency to be lower in the presence of IPTG than in its absence.
3.2. Microscopic Observation
3.3. Assessment by Fluorescence-Activated Flow Cytometry
3.4. Accurate Assessment of Conjugation Efficiency
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Appendix A
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Strain | Description | Source or Reference |
---|---|---|
E. coli | ||
C600 | Plasmid cloning host | Laboratory stock |
B. subtilis | ||
168 | trpC2 | Laboratory stock |
YNB102 | trpC2, amyE::(Pspank-rappLS20 spc), pLS20catΔoriT | [31] |
KV7 | trpC2, ΔcomK::tet | This study |
KV9 | trpC2, amyE::(Pspank-rappLS20 spc), pGR16B, pLS20catΔoriT | This study |
KV10 | trpC2, ΔcomK::tet, aprE::(PrpsO-mKATE2 kan) | This study |
KV11 | trpC2, amyE::(Pspank-rappLS20 spec), pGR16B_sfGFP, pLS20catΔoriT | This study |
Plasmid | ||
pLS20catΔoriT | pLS20cat lacking the oriT region | [31] |
pGR16B | erm, amp | [32] |
pGR16B_sfGFP | pGR16B carrying sfGFP | This study |
pDG-SG51 | mKATE2 | [33] |
pANPCK | kan | [34] |
pOGW | tet | [35] |
Primer | Sequence (5′-3′) |
---|---|
aprE_D_F | cggaaggatactacatcctggttaatcaacgtacaagcagctgcac |
aprE_D_R | ggccgagcagtattcgaatgtcaag |
aprEU_F3_nested | caccgagctcatagcttgtcgcgatcacctcatcc |
aprE_D_R2_nested | tgctttcgctgattacaacattggtgacgctgcct |
aprE_U_F | ccggtacttgccaccacatcataac |
aprE_U_PrpsO_R | aatttgcgtgcgttgcaagttatttccgcactctcgctatttccgtagagactcg |
comK_up_F | tgaaggattggcttattcgctctgc |
comK_up_R | cagtatttcatcacttatacaacaactaataatctatcatctgtttttg |
comK_Down_F | gcctggcagttccctactctcgcatgcgtgagctcggggaacggtattag |
comK_Down_R | atcgaagatctgcctactgaacaaatc |
comK_nested_F | gcttgagcgctgcatattctttagagagcg |
comK_nested_R | gttgtaaaagcggcgcttccgtatttgccg |
Kan_F | ggatagactccaccagaagagccgcaagcttacgataaacccagc |
Kan_terminator_R | ccaggatgtagtatccttccgaaaaaatcccgccgctggcgggattttaactaggtactaaaacaattcatcc |
mKate2_F | gggctaaatatgatttggaggtgaaacaggatgtcagaactaatcaaagagaatatg |
mKate2_Kan_R | gctcttctggtggagtctatcctataaacgcagaaaggcccacccgaag |
tetR_F | ttgttgtataagtgatgaaatactg |
tetR_R | atgcgagagtagggaactgccaggc |
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Mori, K.; Verrone, V.; Amatsu, R.; Fukui, K.; Meijer, W.J.J.; Ishikawa, S.; Wipat, A.; Yoshida, K.-i. Assessment of Bacillus subtilis Plasmid pLS20 Conjugation in the Absence of Quorum Sensing Repression. Microorganisms 2021, 9, 1931. https://doi.org/10.3390/microorganisms9091931
Mori K, Verrone V, Amatsu R, Fukui K, Meijer WJJ, Ishikawa S, Wipat A, Yoshida K-i. Assessment of Bacillus subtilis Plasmid pLS20 Conjugation in the Absence of Quorum Sensing Repression. Microorganisms. 2021; 9(9):1931. https://doi.org/10.3390/microorganisms9091931
Chicago/Turabian StyleMori, Kotaro, Valeria Verrone, Ryotaro Amatsu, Kaho Fukui, Wilfried J. J. Meijer, Shu Ishikawa, Anil Wipat, and Ken-ichi Yoshida. 2021. "Assessment of Bacillus subtilis Plasmid pLS20 Conjugation in the Absence of Quorum Sensing Repression" Microorganisms 9, no. 9: 1931. https://doi.org/10.3390/microorganisms9091931