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. 2002 Jun;184(12):3396–3400. doi: 10.1128/JB.184.12.3396-3400.2002

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
a

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.

b

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).

c

Toxicity was assessed as described in the text.

d

Transport of [14C]methylammonium. Cells were grown as described in footnote c of Table 1.

e

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.

f

The mutation Trp32 → stop results in reinitiation of translation at Met33 (see text).