TABLE 1.
Complementation analysis of MurJ depletion by S. pyogenes ytgP alleles
| Strain | Relevant genotypeb | Efficiency of platinga |
||
|---|---|---|---|---|
| MurJ+ YtgP− | MurJ− YtgP+ (40 μM IPTG) | MurJ− YtgP+ (60 μM IPTG) | ||
| NR1305 | NR1152(pCA24Not) | 1.0 ± 0.2 | <1.2 × 10−4 | <1.2 × 10−4 |
| NR3064 | NR1152(pHIS-YtgP) | 1.0 | 0.4 ± 0.1 | 2.1 ± 1.6 |
| NR3067 | NR1152(pHIS-YtgP/R29A) | 0.9 ± 0.3 | <10−4 | <10−4 |
| NR3068 | NR1152(pHIS-YtgP/R29K) | 0.4 ± 0. 3 | 2.0 (± 1.8) × 10−3 | 0.1 ± 0.0 |
| NR3073 | NR1152(pHIS-YtgP/R152A) | 5.7 ± 4.3 | <10−4 | <10−4 |
| NR3074 | NR1152(pHIS-YtgP/E171A) | 0.9 ± 0.4 | <10−4 | <10−4 |
| NR3069 | NR1152(pHIS-YtgP/R175A) | 2.3 ± 1.5 | <10−4 | <10−4 |
| NR3070 | NR1152(pHIS-YtgP/R175K) | 1.1 ± 0.1 | 2.2 ± 1.8 | 2.0 ± 1.8 |
| NR3071 | NR1152(pHIS-YtgP/K301A) | 4.2 ± 2.9 | <10−4 | <10−4 |
| NR3072 | NR1152(pHIS-YtgP/K301R) | 2.0 ± 1.8 | 0.4 ± 0.3 | 2.0 ± 1.6 |
Efficiency of plating values for growth of the arabinose-dependent MurJ depletion strain (NR1152) expressing His-ytgP alleles carrying the indicated substitutions were calculated as described in Materials and Methods. A value of 1 (set by the wild-type allele) indicates full complementation by His-ytgP alleles; a value of <1.2 × 10−4 indicates no complementation, as this is the highest value obtained from the strain carrying the control plasmid pCA24Not; a value of <1 to >1.2 × 10−4 indicates partial complementation. For MurJ+ YtgP− conditions, cells were grown on LB plates with arabinose to induce the expression of wild-type E. coli murJ. For MurJ− YtgP+ conditions, cells were grown on LB plates with either 40 or 60 μM IPTG to induce the expression of the S. pyogenes His-ytgP alleles.
NR1152 is the arabinose-dependent MurJ depletion strain. Plasmid pHIS-YtgP expresses His-ytgP upon induction with IPTG; pCA24Not is the vector control lacking His-ytgP.