TABLE 1.
The amt genes of A. aeolicus and M. jannaschii complement an E. coli amtB mutant for growth at low ammonium and [14C]methylammonium uptake
| Straina | Doubling timeb (min) at IPTG concn (μM)
|
MA uptakec (pmol/[ml × OD600 × min]) | ||
|---|---|---|---|---|
| 0 | 10 | 100d | ||
| NCM1458 (10) (wild type) | 80 | NAe | NA | 80 |
| NCM2019 (10) (amtB) | 600 | NA | NA | ≤3 |
| NCM3404 (amtB/pJES1335 [A. aeolicus AmtB-His6]) | 160 | 100 | 270 | 100 |
| NCM3421 (amtB glnA/pJES1335 [A. aeolicus AmtB-His6]) | NA | NA | NA | ≤3 |
| NCM3452 (amtB/pJES1348 [M. jannaschii Amt1-His6]) | 110 | 110 | No growth | 155 |
| NCM3453 (amtB/pJES1349 [M. jannaschii Amt2-His6]) | 320 | 250 | No growth | 13 |
| NCM3896 (amtB/pJES1452 [E. coli AmtB-His6])f | 300 | No growth | No growth | 200 |
Plasmids pJES1335, pJES1348, and pJES1349 were derived by subcloning from pJES1331, pJES1343, and pJES1344, respectively, in which the amt genes are under the control of a T7 promoter and ribosome binding site. The latter were used for overexpression. All plasmids yield C-terminally His-tagged proteins.
Cells were grown in N- and C-free medium at pH 5 with 0.2% glucose as the carbon source and 0.5 mM NH4Cl as the nitrogen source (10) and with IPTG as indicated.
Transport of [14C]methylammonium (MA). Cells were grown in N- and C-free medium at pH 7 with 0.4% glucose as the carbon source and 2 mM glutamine as the nitrogen source and with 10 μM IPTG. Growth on glutamine induces the synthesis of glutamine synthetase and of AmtB in the wild-type strain.
Slowing of growth at high IPTG concentrations is due to toxicity of the Amt proteins. It is also observed with the E. coli protein, whether or not it carries a His tag, under similar circumstances.
NA, not applicable.
pJES1452 carries the gene that codes for E. coli AmtB-His6 in the same vector used for the genes from hyperthermophiles. Plasmid pJES1452 is toxic even on enriched medium at pH 7, probably because the E. coli protein is highly expressed. The E. coli gene carries only five rare codons, whereas the genes from hyperthermophiles carry between 21 and 36 rare codons (mostly ATA for isoleucine). Vectors from which the E. coli AmtB-His6 protein is apparently less well expressed are less toxic. They complement fully for growth and less well for [14C]methylammonium uptake (data not shown).