Table 3

Spontaneous suppressor mutations of YopE4S–A-Npt were selected by plating Y. enterocolitica harboring plasmid pDA54 (YopE4S–A-Npt) on tryptic soy agar plates with neomycin (50 μg/ml). Plasmid was purified from individual colonies and transformed into W22703, and plasmid transformants were selected on chloramphenicol plates. Individual isolates were tested for resistance to neomycin [minimal inhibitory concentration (MIC) for 105 cells], relative concentration of mRNA ([x]/[yopH]), synthesis ([X]/[YopH]), and secretion of hybrid Npt proteins. Mutational changes of the suppressors were determined by DNA sequencing. ND, not determined.

PlasmidCodons 2 to 7Codon 12MIC (μg/ml)Secretion (%)[X]/ [YopH][x]/ [yopH]
pDA46 wild typeAAA AUA UCA UCA UUU AAUCCC250 501.21.7
pDA 54 (4S-A)AAA AUA GCA UCA UUU AAUCCC25ND00.7
1AAA AUA GCA UCA UUU AAUCCA250421.02.3
2AAA AUA UCA UCA UUU AAUCCA250460.80.8
3, 4AAA AUA UCA GAA UUU AAUCCA500460.91.6
5–8AAA AUA GCA UAU  AAUCCA500431.0 1.9
9AAG AUA GCA UAU  AAUCCA500491.0 1.5