Different csrA Expression Levels in C versus K-12 E. coli Strains Affect Biofilm Formation and Impact the Regulatory Mechanism Presided by the CsrB and CsrC Small RNAs
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacteria, Plasmids, and Growth Media
2.2. Gene Expression Determination
2.3. Luciferase Activity Assay
2.4. PNAG Detection
2.5. Western Blotting
2.6. Statistical Analysis
3. Results
3.1. E. coli C Produces More PNAG than E. coli K-12
3.2. CsrA-Dependent Regulation of pgaABCD Operon Is More Stringent in E. coli K-12 than in E. coli C
3.3. PNPase-Dependent Regulation of pga Operon Is More Stringent in E. coli C than in E. coli K-12
3.4. Expression Profile of the csrA Gene and of sRNAs Regulating CsrA Activity in E. coli C and K-12
3.5. Compensatory Regulation of CsrB and CsrC Does Not Occur in E. coli C
3.6. Ectopically Expressed RNase II Restores CsrB and CsrC Production in C-1a Δpnp
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Name | Relevant Characters a | Source/Reference |
---|---|---|
Bacterial Strains | ||
C-1a | E. coli C, prototrophic | [29] |
C-5691 | C-1a ∆pnp751 | [30] |
C-5736 | ∆pgaC | This work |
C-5737 | C-5691 ∆pgaC | This work |
C-5738 | C-5736 ∆csrA::kan | This work |
C-5739 | C-5737 ∆csrA::kan | This work |
C-5741 | C-1a csrA::kan | This work |
C-5936 | ∆pgaC::kan | [25] |
C-5937 | C-5691 ∆pgaC::kan | [25] |
C-5938 | C-1a ∆csrA::kan | This work |
C-5939 | C-1a ∆csrA | This work |
C-5940 | ∆csrB::kan | [25] |
C-5942 | C-5691 ∆csrB::kan | [25] |
C-5944 | ∆csrC::cat | [25] |
C-5946 | C-5691 ∆csrC::cat | [25] |
JW1007 | BW25113 ∆pgaC::kan | [31] |
KG-206 | MG1655 ∆pnp751::kan | This work |
KG-211 | MG1655 ∆pnp751 | This work |
KG-292 | MG1655 ∆pgaC::kan | This work |
KG-293 | KG-211 ∆pgaC::kan | This work |
KG-294 | MG1655∆pgaC ∆csrA::kan | This work |
KG-295 | KG-211 ∆pgaC ∆csrA::kan | This work |
KG-299 | MG1655∆csrC::cat | This work |
KG-302 | MG1655∆csrB::kan | This work |
KG-305 | KG-211 ∆csrB | This work |
KG-307 | MG1655 ∆pgaC | This work |
KG-308 | KG-211 ∆pgaC | This work |
KG-309 | KG-211 ∆csrC::cat | This work |
MG1655 | rph- | [32] |
MG1655 csrA::kan | csrA::kan | [15] |
Plasmids | ||
pBAD21Δ1 | Cloning vector | [25] |
pBADpnp | pBAD24 derivative; carries the pnp gene | [25] |
pBADrnb | pBAD24 derivative; carries the rnb gene | [25] |
pBADrnr | pBAD24 derivative; carries the rnr gene | [25] |
pCP20 | FLP encoding plasmid | [27] |
pCSRA | pGZ119HE derivative; carries 28191692818929 region of MG1655 genome under pTAC promoter | This work |
pΔLpga | pJAMA8 derivative, harbors the −116 to +32 region relative to the pgaA transcription start site cloned into the SphI/XbaI sites | [25] |
pGZ119HE | Cloning vector | [33] |
pJAMA8 | Cloning vector | [34] |
pKD13 | RED mutagenesis system plasmid | [27] |
pKD3 | RED mutagenesis system plasmid | [27] |
pKD46 | RED mutagenesis system plasmid | [27] |
pLpga2 | pJAMA8 derivative; carries -116 to +249 relative to transcription start of pgaA promoter translationally fused with luxA | [25] |
Phage P1 hft | High transduction frequency phage P1 derivative | [35] |
E. coli C | E. coli K-12 | |||||||
---|---|---|---|---|---|---|---|---|
- | wt | Δpnp | ΔcsrA | Δpnp ΔcsrA | wt | Δpnp | ΔcsrA | Δpnp ΔcsrA |
LUX R.A. a | 1.0 | 14.0 ± 0.3 | 26.3 ± 3.0 | 37.9 ± 1.2 | 0.1 ± 0.0 | 0.3 ± 0.0 | 42.0 ± 13.5 | 65.1 ± 27.2 |
Pwt b | na | 9 × 10−8 | 6 × 10−5 | 4 × 10−7 | na | 1 × 10−5 | 3 × 10−3 | 7 × 10−3 |
PC-K c | 3 × 10−8 | 7 × 10−8 | ns | ns | 3 × 10−8 | 7 × 10−8 | ns | ns |
Gene | RNA Relative Amount a | |
---|---|---|
C-5691 | MG1655 | |
csrAb | 0.3 ± 0.1 | 3.5 ± 1.3 |
csrB | 0.2 ± 0.2 | 57.6 ± 9.9 |
csrC | 0.1 ± 0.1 | 10.5 ± 5.2 |
mcaS | 0.7 ± 0.1 | 0.2 ± 0.1 |
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Carzaniga, T.; Falchi, F.A.; Forti, F.; Antoniani, D.; Landini, P.; Briani, F. Different csrA Expression Levels in C versus K-12 E. coli Strains Affect Biofilm Formation and Impact the Regulatory Mechanism Presided by the CsrB and CsrC Small RNAs. Microorganisms 2021, 9, 1010. https://doi.org/10.3390/microorganisms9051010
Carzaniga T, Falchi FA, Forti F, Antoniani D, Landini P, Briani F. Different csrA Expression Levels in C versus K-12 E. coli Strains Affect Biofilm Formation and Impact the Regulatory Mechanism Presided by the CsrB and CsrC Small RNAs. Microorganisms. 2021; 9(5):1010. https://doi.org/10.3390/microorganisms9051010
Chicago/Turabian StyleCarzaniga, Thomas, Federica A. Falchi, Francesca Forti, Davide Antoniani, Paolo Landini, and Federica Briani. 2021. "Different csrA Expression Levels in C versus K-12 E. coli Strains Affect Biofilm Formation and Impact the Regulatory Mechanism Presided by the CsrB and CsrC Small RNAs" Microorganisms 9, no. 5: 1010. https://doi.org/10.3390/microorganisms9051010