TABLE 1.
Analysis of substitutions isolated from the tatAF39X library by selection for chloramphenicol resistance in the presence of pSSCATa
| Codon | Substitution encoded | No. of times isolated | Growth in presence of SDS | Periplasmic TMAO reductase activity |
|---|---|---|---|---|
| Controls | ||||
| TTT | Wild type | + | 1.67 | |
| ΔtatA ΔtatE | − | 0.02 | ||
| Substitutions isolated by screening for chloramphenicol resistance | ||||
| GAT | D | 2 | + | 0.05 |
| GAA | E | 2 | +/− | 0.03 |
| GGA | G | 2 | + | 0.03 |
| CAT | H | 1 | − | 0.07 |
| ATC | I | 1 | + | 0.05 |
| ATA | I | 2 | NDb | ND |
| AAA | K | 2 | − | 0.04 |
| AAG | K | 2 | ND | ND |
| CTG | L | 2 | + | 0.04 |
| CTC | L | 2 | ND | ND |
| TTG | L | 5 | ND | ND |
| CGT | R | 2 | − | 0.12 |
| AGA | R | 2 | ND | ND |
| CGA | R | 1 | ND | ND |
| CGC | R | 1 | ND | ND |
| TCT | S | 3 | − | 0.07 |
| TCG | S | 1 | ND | ND |
| ACG | T | 1 | + | 0.09 |
| ACA | T | 2 | ND | ND |
| ACC | T | 1 | ND | ND |
| ATT | T | 3 | ND | ND |
| GTG | V | 1 | + | 0.04 |
| GTA | V | 3 | ND | ND |
| GTC | V | 1 | ND | ND |
| TAA | Stop | 1 | ND | ND |
| TGA | Stop | 2 | ND | ND |
| Remaining substitutions at the F39 codon | ||||
| GCT | A | − | 0.05 | |
| TCG | C | + | 0.08 | |
| ATG | M | + | 0.07 | |
| AAC | N | + | 0.06 | |
| CCG | P | + | 0.04 | |
| CAG | Q | − | 0.08 | |
| TGG | W | + | 0.88 | |
| TAT | Y | + | 1.05 |
Analysis of substitutions isolated from the tatAF39X library by selection for chloramphenicol resistance in the presence of pSSCAT. Chloramphenicol-resistant colonies were streaked onto fresh chloramphenicol-containing plates to confirm resistance and the entire tatA coding region analyzed by DNA sequencing. The activity of the Tat system supported by the mutated tatA genes was assessed by growth in the presence of 2% SDS (9) and by measuring TMAO reductase activity (expressed as micromoles of TMAO reduced per minute per milligram of protein) in the periplasmic fraction (29). The activities observed with wild-type tatA (strain JARV16 [ΔtatA/ΔtatE] harboring pFAT415) and the ΔtatA/ΔtatE mutant (JARV16 harboring pBluescript) are shown as controls. Chloramphenicol resistance of the ΔtatA/ΔtatE strain harboring pSSCAT producing the remaining TatA variants constructed by site-directed mutagenesis was not assessed.
ND, not determined.