The Bacteriophage Lambda CII Phenotypes for Complementation, Cellular Toxicity and Replication Inhibition Are Suppressed in cII-oop Constructs Expressing the Small RNA OOP
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strain Construction, Bacterial and Phage Strains Employed
2.2. Fluorescence Assays for Expressed D-GFPuv and GFPuv*
2.3. Complementation Assay for CII
2.4. Determination of Toxicity and Plasmid Loss
2.5. Oligonucleotides Used for Plasmid Construction and Sequencing
2.6. Gene Expression Plasmid
3. Results
3.1. Thermally Inducible Gene Expression from pcIpR[]Timm Plasmid
3.2. Untangling CII Activities: Cellular Toxicity and Promoting Plasmid Loss
3.3. CII Complementation
3.4. Host Modulation of CII-Dependent Cellular Toxicity and Plasmid Retention
3.5. Influence of oop RNA Expression on cII-Dependent Cellular Toxicity and Plasmid Loss
3.6. Influence of Terminally Overlapping Divergent cII and oop Transcription on CII Complementation
3.7. Plasmid Construction Variations and Influence on cII Expression
4. Discussion
5. Conclusions
- The results confirm that OOP RNA expression from the genetic element pO-oop-to can suppress high CII activity and that OOP RNA likely serves as a powerful regulatory pivot in temperate lambdoid phage development.
- Plasmids with a pO94, comprising 94 bases rightward from oop, prevented CII complementation, CII-dependent plasmid loss and suppressed CII toxicity, suggesting that the active pO promoter to produce OOP requires an extended DNA sequence, beyond that required to encode the −10 and −35 regions.
- All three CII activities were eliminated by the deletion of its COOH-terminal 20 amino acids.
- E. coli mutations were shown to influence CII activities. (a) Inactivating the hflA locus encoding HflK-HflC proteins that modulate the FtsH ATP-dependent membrane protease significantly reduced CII trans-complementation and toxicity; (b) A null allele of pcnB, encoding poly (A) polymerase I, eliminated CII complementation and increased CII toxicity; (c) Five of six rpoB point mutations significantly reduced CII trans-complementation; (d) The CII1–87 mutant, deleted for the terminal 10 amino acids, lost its ability to complement in five rpoB mutant cells.
- The results suggest that the terminal end of CII likely interacts with the β-subunit of RNA polymerase.
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Induction Time (min) | Culture up-Shift from 30 °C ± SE a | ||
---|---|---|---|
37 °C | 39 °C | 42 °C | |
0 | 0 (0.2) | 0 (4.0) | 3.2 (0.05) |
20 | 0 (0.2) | 1.4 (2.6) | 153.8 (0.5) |
40 | 0 (0.3) | 6.7 (2.0) | 415.4 (0.4) |
60 | 0.8 (0.3) | 23.2 (3.2) | 518.5 (0.5) |
90 | 0 (0.3) | 41.3 (0.3) | 624.6 (0.4) |
120 | 7.3 (14.9) | 30.0 (2.9) | 721.2 (0.4) |
150 | 10.8 (1.3) | 84.3 (3.4) | 808.4 (0.4) |
180 | 23.9 (2.6) | 136.9 (10.7) | 1039.5 (0.7) |
Host [Plasmid] a | Intensity of CII-Activated CI434 Repression at 37–39 °C b | λimm434 cII− Plaque Formation at 37–39 °C c | Cell Viability, (±SE) and [% Plasmid Loss] Per Growth Temp. of Transformants e | |
---|---|---|---|---|
39 °C | 42 °C | |||
594 [cII] = [cII1–97] | H (high) | − | 0.03 (0.001) [0] | <0.001 (0.0001) [100] |
594 [cII-oop-pO94] | 0 (none) | + d | 0.59 (0.03) [0] | 0.08 (0.03) [0] |
594 [cII1–92] | H | − | 0.73 (0.01) [0] | 0.005 (0.001) [100] |
594 [cII1–87] | H | − | 0.76 (0.02) [0] | 0.08 (0.03) [0] |
594 [cII1–77] | 0 | + d | 0.74 (0.10) [0] | 0.32 (0.10) [0] |
594 [cII-3638](38,642: A-G, M-V) | H | − | 0.62 (0.10) [0] | 0.002 (0.0004) [89] |
594 [cII-3639](38,634: A-G, Q-R) | H | − | 0.76 (0.02) [0] | <0.001 (<0.0001) [19] |
594 [cII-3638–3639] | H | − | 0.65 (0.04) [0] | <0.001 (<0.0001) [6] |
594 hflA::kan [cII] | S (slight) | + | 0.89 (0.01) [0] | 0.008 (<0.0001) [100] |
594 hflA::kan [cII1–92] | S | + | 0.69 (0.01) [0] | <0.001 (<0.0001) [100] |
594 hflA::kan [cII1–87] | S | + | 0.85 (0.02) [0] | 0.24 (<0.0001) [0] |
594 hflA::kan [cII1–77] | 0 | + d | 0.86 (0.10) [0] | 0.58 (0.01) [0] |
594 hflA::kan [cII-oop-pO94] | 0 | + d | 0.63 (<0.0001) [0] | 0.001 (0.0002) [100] |
594 pcnB::kan [cII] | 0 | + d | <0.001 (0.01) [67] | <0.001 (0.0001) [100] |
594 pcnB::kan [cII-oop-pO94] | 0 | + d | 0.46 (0.01) [64] | <0.001 (0.0003) [89] |
Host(s) Strains a | CII-Construct Plasmid(s) with Thermally Inducible Expression of cII Allele | Intensity of CII-Activated CI434 Repression at 37–39 °C b | λimm434 cII− Plaque Formation at 37–39 °C b |
---|---|---|---|
594 rpoB B1 | cII; cII1–92 | H | − |
594 rpoB B8, C1, C4, C10, D2, D6 | cII; cII1–92 | S | + |
594 rpoB B1, C1 | cII1–87 | S | + |
594 rpoB B8, C4, C10, D2, D6 | cII1–87 | 0 | + c |
Host Strains with rpoB Alleles | Cell Viability (±SE) [% Plasmid Loss] Per Growth of Transformant at 42 °C | |
---|---|---|
[cII] a | [cII-oop-pO94] b | |
594 c | <0.001 (0.001) [100] | 0.08 (0.03) [0] |
594 rpoB B1 | <0.001 (<0.0001) [100] | 0.07 (0.01) [0] |
594 rpoB B8 | 0.01 (<0.0001) [78] | 0.13 (0.03) [0] |
594 rpoB C1 | 0.007 (0.10) [100] | 0.36 (<0.0001) [0] |
594 rpoB C4 | 0.003 (0.010) [0] | 0.07 (<0.0001) [0] |
594 rpoB C10 | 0.004 (0.001) [100] | 1.00 (0.01) [0] |
594 rpoB D2 | 0.38 (<0.0001) [0] | 0.10 (<0.0001) [81] |
594 rpoB D6 | 0.02 (0.01) [6] | 0.03 (0.01) [86] |
CII-Construct Plasmids with Thermally Inducible Expression of cII Allele | Intensity of CII-Activated CI434 Expression at 37–39 °C a | λimm434 cII− Plaque Formation at 37–39 °C a | Cell Viability (±SE) [% Plasmid Loss] Per Growth Temp. of Transformants | |
---|---|---|---|---|
39 °C | 42 °C | |||
sR-38339-pE-cII (p747) | H | − | 0.62 (0.10) [0] | <0.001 (<0.004) [94] |
sR-38339-pE-cII-oop-pO94 (p748) | 0 | + d | 0.82 (0.02) [0] | 0.001 (<0.0001) [94] |
sR-38339-pE-cII-cy3048 in −10 pE (p767) | 0-S b | + | 0.89 (0.01) [0] | 0.08 (<0.0001) [0] |
sR-38339-pE-cII-cy2001 in −10 pE (p765) | 0-S b | + | 0.69 (<0.0001) [0] | 0.01 (<0.0001) [0] |
sR-38339-pE-cII-cy42 in −35 pE (p764) | 0-S b | + | 1.00 (0.020) [0] | 1.00 (<0.0001) [42] |
sR-38339-pE-cII-cy3001 in −35 pE (p766) | H | − | 0.72 (<0.0001) [0] | <0.001 (0.001) [8] |
cII-oop-pO45WT (p763) | H | − | 0.72 (0.10) [0] | 0.07 (<0.0001) [0] |
cII-oop-pO45-38,683-87MH2 in −10 pO (p759) | H | − | 0.67 (0.10) [0] | <0.001 (0.002) [100] |
cII-oop-pO45-38683LK in −10 pO (p762) | H | − | 0.78 (0.20) [0] | <0.001 (<0.0001) [50] |
cII-oop-pO94-38683-87MH (p681) c | H | − | 0.71 (<0.0001) [0] | <0.001 (<0.0001) [86] |
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Rajamanickam, K.; Hayes, S. The Bacteriophage Lambda CII Phenotypes for Complementation, Cellular Toxicity and Replication Inhibition Are Suppressed in cII-oop Constructs Expressing the Small RNA OOP. Viruses 2018, 10, 115. https://doi.org/10.3390/v10030115
Rajamanickam K, Hayes S. The Bacteriophage Lambda CII Phenotypes for Complementation, Cellular Toxicity and Replication Inhibition Are Suppressed in cII-oop Constructs Expressing the Small RNA OOP. Viruses. 2018; 10(3):115. https://doi.org/10.3390/v10030115
Chicago/Turabian StyleRajamanickam, Karthic, and Sidney Hayes. 2018. "The Bacteriophage Lambda CII Phenotypes for Complementation, Cellular Toxicity and Replication Inhibition Are Suppressed in cII-oop Constructs Expressing the Small RNA OOP" Viruses 10, no. 3: 115. https://doi.org/10.3390/v10030115