TABLE 3.
Protease secretion assays
| Strain or genotype | ΔFU/min per OD600a | Complementation | ΔFU/min per OD600d |
|---|---|---|---|
| TRH7000 | 77.7 ± 2.3 | ||
| ΔepsC | 0 ± 0.3 | ΔepsC + pEpsC | 78.0 ± 4.4 |
| ΔepsD | 1.3 ± 0.7 | ΔepsD + pEpsD | 53.9 ± 5.0 |
| ΔepsD + pEpsD + IPTGb | 71.7 ± 3.8 | ||
| ΔepsG | 2.6 ± 0.6 | ΔepsG + pEpsG | 90.8 ± 10.0 |
| ΔepsL | 0 ± 0.7 | ΔepsL + pEpsL | 65.1 ± 3.6 |
| PU3 (epsM mutant) | 6.0 ± 1.1 | PU3 (epsM mutant) + pEpsM | 71.8 ± 1.5 |
| gfp-epsC | 76.4 ± 3.5 | ||
| gfp-epsC ΔepsD | 0.7 ± 1.1 | gfp-epsC ΔepsD + pEpsD | 46.6 ± 2.5 |
| gfp-epsC ΔepsD + pEpsD + IPTGb | 76.5 ± 4.8 | ||
| gfp-epsC ΔepsL | 0 ± 0.5 | gfp-epsC ΔepsL + pEpsL | 71.9 ± 2.4 |
| gfp-epsC epsM mutant | 5.4 ± 2.1 | gfp-epsC epsM mutant + pEpsM | 73.8 ± 1.7 |
| gfp-epsM | 76.3 ± 5.7 | ||
| gfp-epsM ΔepsC | 1.3 ± 1.2 | gfp-epsM ΔepsC + pEpsC | 80.0 ± 2.0 |
| gfp-epsM ΔepsD | 0 ± 0.9 | gfp-epsM ΔepsD + pEpsD | 42.5 ± 2.3 |
| gfp-epsM ΔepsD + pEpsD + IPTGb | 65.9 ± 4.5 | ||
| gfp-epsM ΔepsL | 0.8 ± 1.2 | gfp-epsM ΔepsL + pEpsL | 93.6 ± 2.8 |
| PBAD::epsc | 70.3 ± 0.7 | ||
| PBAD::eps gfp-epsCc | 65.9 ± 2.3 | ||
| PBAD:: eps gfp-epsC ΔepsDc | 0 ± 3.9 | PBAD::eps gfp-epsC ΔepsD + pEpsDc | 26.8 ± 0.5 |
| PBAD::eps gfp-epsC ΔepsD + pEpsD + IPTGb,c | 63 ± 2.0 | ||
| PBAD:: eps gfp-epsMc | 62.9 ± 2.6 | ||
| PBAD:: eps mcherry-epsC gfp-epsMc | 63.4 ± 4.5 |
a
ΔFU indicates the protease activity detected in the supernatants of overnight cultures for TRH7000 and deletion mutant strains. Protease activity in overnight culture supernatants was assayed by measuring methylcoumarin fluorescence generated from Boc-Gln-Ala-Arg-7-amido-4-methylcoumarin hydrolysis, and rates were normalized to an OD600. The average of at least three experiments is presented ± the standard error of the mean.
b
10 μM IPTG.
c
0.01% arabinose.
d
ΔFU indicates the protease activity for the TRH7000 and gfp fusion mutant strains upon introduction of plasmids.