Table 3. Overexpression of hypomodified or severely reduced cellular growth.
| Relevant genotype on chromosome | Wobble nucleoside in tRNA Gln, Lys, and Glu | Plasmid (Relevant genotype) | Growth on LAL- plates. (Rel. colony size (1 d)) | Growth on His-glucose–plates. (Rel. colony size (2 d)). |
|---|---|---|---|---|
| Wild type | (c)mnm5s2U34 | No plasmid | 0.9 | 1.0; |
| pUST312 (vector) | 1.0; 1.0 | 1.0 1.0 | ||
| pUST313 (glnU,Xa)) | 0.8; 0.8 | 0.6; 0.8 | ||
| pUST314 (ΔglnU,Xb)) | 1.0; | 0.9; | ||
| p815 (lysVc)) | 0.9 | 1.0 | ||
| mnmA3 | (c)mnm5U34(lacks the s2-group) | No plasmid | 1.1 | 1.1 |
| pUST312 (vector) | 1.0; 1.0 | 1.0, 1.0 | ||
| pUST313 (glnU,Xa)) | No sc; <0.1 | <0.1; <0.1 | ||
| pUST314 (ΔglnU,Xb)) | 0.6; | <0.1 | ||
| p815 (lysVc)) | 0.2 | 0.1 | ||
| tusB27 | (c)mnm5U34(lacks the s2-group) | No plasmid | 0.9 | 1,0 |
| pUST312 (vector) | 1.0; 1.0 | 1.0 | ||
| pUST313 (glnU,Xa)) | No sc; No sc | <0.1; No sc | ||
| pUST314 (ΔglnU,Xb)) | 0.5; | 0.1; | ||
| p815 (lysVc)) | 0.2 | 0.1 | ||
| tusE30 | (c)mnm5U34(lacks the s2-group) | No plasmid | 0.9 | 1.1 |
| pUST312 (vector) | 1.0 | 1.0 | ||
| pUST313 (glnU,Xa)) | No sc; No sc | 0.2; No sc | ||
| pUST314 (ΔglnU,Xb)) | ND | ND | ||
| p815 (lysVc)) | 0.2 | <0.1 | ||
| mnmE13 | s2U34(lacks the (c)mnm5-group | No plasmid | 0.9 | 1.0 |
| pUST312 (vector) | 1.0; 1.0 | 1.0; 1.0 | ||
| pUST313 (glnU,Xa)) | 0.4; 0.4 | <0.1; No sc | ||
| pUST314 (ΔglnU,Xb)) | 0.9; | 0.8 | ||
| p815 (lysVc)) | 1.2 | 1.0 | ||
| mnmG1 | s2U34(lacks the (c)mnm5-group) | No plasmid | 0.9 | 1.1 |
| pUST312 (vector) | 1.0; 1.0 | 1.0: 1.0 | ||
| pUST313 (glnU,Xa)) | 0.5; 0.4 | 0.4; 0.2 | ||
| pUST314 (ΔglnU,Xb)) | ND | ND | ||
| p815 (lysVc)) | 1.2 | 1.0 |
Indicated strains were grown at 37°C in the rich media LB overnight and then streaked out on LAL agar plates or on glucose medium containing histidine, since the host strain GT7321 requires histidine for growth. No sc, denotes that no single colonies were observed and growth only on primary and/or secondary outstreaks were observed. Numbers are the average of 10 similar sized colonies after indicated time of incubation. When two numbers are present the experiment was repeated once. The size of the colonies was determined a described in Table 2. ND = not done
a) Plasmid pUST313 carry the metT operon which contains the tRNA genes: metT-leuW-glnU-glnW-metU-glnV-glnX. The genes glnU and glnW encodes glutamine tRNA having (c)mnm5s2U34 as wobble nucleoside whereas glnV and glnX encodes glutamine tRNA having C34 as wobble nucleoside.
b) Plasmid pUST314 carry a part of the metT operon due to a spontaneous recombination between the metT and metU genes. It therefore lacks leuW-glnU-glnW genes and contains only the metT/U-glnV-glnX. The glnV and glnX genes encodes gln-tRNA having C34 as wobble nucleoside. Thus this plasmid lacks genes for the gln-tRNA havning (c)mnm5s2U as wobble nucleoside.
c) Plasmid p815 contains the valU-operon which contains the valU-valX-valY and lysV tRNA genes. The lysV gene encodes lys-tRNA having mnm5s2U34 as wobble nucleoside.