TABLE 1.
Phenotypes of strains producing plasmid-borne YfgL variants
| Proteina | Mean YfgL level ± SDb | Mean LamB level ± SDb | MIC of vancomycin (μg/ml) | Growth in ΔdegP backgroundc |
|---|---|---|---|---|
| YfgL− | 0.00 | 0.46 ± 0.12 | 40 | −/+ |
| YfgL-His6 wild type | 1.00 | 1.00 | 120 | +++ |
| YfgL-His6 scramble 1 | 0.95 ± 0.03 | 0.69 ± 0.09 | 40 | −/+ |
| YfgL-His6 scramble 2 | 1.05 ± 0.01 | 0.41 ± 0.22 | 40 | −/+ |
| YfgL(L173S,L175S,R176A)-His6 | 0.82 ± 0.12 | 0.61 ± 0.09 | 40 | +/− |
| YfgL(L175S,R176A)-His6 | 0.89 ± 0.03 | 0.79 ± 0.10 | 60 | ++ |
| YfgL(L173S,R176A)-His6 | 0.95 ± 0.02 | 0.72 ± 0.13 | 80 | ++ |
| YfgL(L173S,L175S)-His6 | 0.94 ± 0.04 | 1.02 ± 0.27 | 120 | +++ |
| YfgL(R176A)-His6 | 0.92 ± 0.10 | 0.90 ± 0.04 | 120 | +++ |
| YfgL(L175S)-His6 | 0.86 ± 0.04 | 1.08 ± 0.03 | 120 | +++ |
| YfgL(L173S)-His6 | 0.92 ± 0.05 | 0.94 ± 0.14 | 120 | +++ |
| YfgL(S172A)-His6 | 0.96 ± 0.05 | 1.14 ± 0.16 | 120 | +++ |
| YfgL(S172C)-His6 | 0.72 ± 0.16 | 0.78 ± 0.26 | 80 | ++ |
| YfgL(E221A)-His6 | 0.84 ± 0.04 | 0.77 ± 0.05 | 120 | +++ |
| YfgL(D223A)-His6 | 0.94 ± 0.08 | 0.89 ± 0.13 | 120 | +++ |
| YfgL(R224A)-His6 | 0.78 ± 0.23 | 0.97 ± 0.01 | 120 | +++ |
| YfgL(D227A)-His6 | 0.65 ± 0.07 | 0.80 ± 0.06 | 80 | ++ |
| YfgL(D229A)-His6 | 0.95 ± 0.15 | 0.90 ± 0.15 | >120 | +++ |
| YfgL(S226C)-His6 | 0.86 ± 0.17 | 0.90 ± 0.03 | 120 | +++ |
a
Plasmid pBAD24 served as the negative control (YfgL−). All experiments were done with full arabinose induction (0.2% [wt/vol], final). Residue numbers are relative to the beginning of the mature YfgL protein sequence.
b
Steady-state YfgL or LamB protein levels were analyzed by Western analysis. Three independent LB cultures were quantified and averaged relative to the wild-type level.
c
Growth of a strain producing YfgL-His6 or a variant in a DegP− background at 30°C on an LB agar plate with 0.2% arabinose. −/+ indicates the strain formed extremely tiny heterogeneous colonies. +/− indicates that the strain formed small heterogeneous colonies.