TABLE 2.
Effects of mutations on the ability of the A. aeolicus amtB gene to complement for growth and [14C]methylammonium uptake and on the toxicity of its producta
| Plasmid | Genotype | Substitution or deletionb | Complementation
|
Toxicityc | |
|---|---|---|---|---|---|
| MA uptaked (pmol/[ml × OD600 × min]) | Doubling time (min)e | ||||
| pJES1335 | Wild type (423 aa) | 55 | 105 | Toxic | |
| pJES1377 | G314 → A | Gly105 → Asp | 40 | 120 | Toxic |
| pJES1376 | G388 → A | Ala130 → Thr | 45 | 145 | Toxic |
| pJES1378 | C758 → T | Thr253 → Ile | 55 | 100 | Toxic |
| C776 → T | Thr259 → Met | ||||
| C1211 → T | Ser404 → Phe | ||||
| pJES1379 | G1129 → A | Val377 → Ile | 55 | 120 | Toxic |
| pJES1389 | G96 → A | Trp32 → stopf | ≤3 | No growth | Not toxic |
| pJES1393 | G236 → A | Trp79 → stop | ≤3 | No growth | Not toxic |
| pJES1391 | C301 → T | Gln101 → stop | ≤3 | No growth | Not toxic |
| pJES1392 | G503 → A | Trp168 → stop | ≤3 | No growth | Weakly toxic |
| pJES1395 | G509 → A | Trp170 → stop | ≤3 | No growth | Toxic |
| pJES1394 | G791 → A | Trp264 → stop | ≤3 | No growth | Toxic |
| pJES1390 | G806 → A | Trp269 → stop | ≤3 | No growth | Toxic |
| pJES1388 | C1105 → T | Gln369 → stop | ≤3 | No growth | Toxic |
| pJES1426 | Δ2-51 (ΔTMf1) | ≤3 | No growth | Toxic | |
| pJES1427 | Δ2-87 (ΔTM1, 2) | ≤3 | No growth | Toxic | |
| pJES1428 | Δ2-143 (ΔTM1-3) | ≤3 | No growth | Toxic | |
| pJES1429 | Δ2-170 (ΔTM1-4) | ≤3 | No growth | Toxic | |
| pJES1430 | Δ2-209 (ΔTM1-5) | ≤3 | No growth | Toxic | |
| pJES1431 | Δ398-423 (ΔC terminus) | 9 | Slow growth | Toxic | |
The host strain was NCM2019 (amtB) and all plasmids were derived from pJES1335, which codes for C-terminally His-tagged A. aeolicus AmtB. All proteins carry two extra residues at the N terminus and eight at the C terminus. Positions of nucleotide bases and amino acid residues are for the native protein.
aa, amino acids. Transmembrane spanning regions predicted by the PHDhtm topology program (http://maple.bioc.columbia.edu/predictprotein/): TM1 (32-50), TM2 (64-85), TM3 (120-137), TM4 (142-165), TM5 (186-203), TM6 (219-237), TM7 (248-266), TM8 (291-315), TM9 (334-351), and TM10 (376-396).
Toxicity was assessed as described in the text.
Transport of [14C]methylammonium. Cells were grown as described in footnote c of Table 1.
Growth was assessed in liquid culture or on plates and was compared to that of NCM2019 (amtB) and NCM3404 (amtB/pJES1335). The medium was as described in footnote b of Table 1 with 10 μM IPTG.
The mutation Trp32 → stop results in reinitiation of translation at Met33 (see text).