Table 3.
Functional analysis of mutations detected in S. pneumoniae R6M1TC-5 and R6M2TC-4
| Straina | Mutated alleles |
MIC (mg/L)b |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| rpsJ |
patA |
patB | TET | TET + R | CIP | CIP + R | Etbr | Etbr + R | ||
| promotor | CDS | |||||||||
| R6 | WT | WT | WT | WT | 0.125 | 0.125 | 0.5 | 0.5 | 2 | 0.25 |
| R6rpsL+ | WT | WT | WT | WT | 0.125 | 0.125 | 0.5 | 0.5 | 2 | 0.25 |
| R6M1TC-5 | R6M1TC-5 | R6M1TC-5 | R6M1TC-5 | WT | 8 | 2 | 4 | 0.5 | 16 | 0.25 |
| R6M2TC-4 | R6M2TC-4 | R6M2TC-4 | R6M2TC-4 | R6M2TC-4 | 8 | 1 | 8 | 0.5 | 16 | 0.25 |
| R6rpsJ(R6M1TC-1) | R6M1TC-1 | WT | WT | WT | 0.5 | 0.5 | 0.5 | 0.5 | 2 | 0.25 |
| R6rpsJ(R6M1TC-1)-rpsL+ | R6M1TC-1 | WT | WT | WT | 0.5 | ND | 0.5 | ND | ND | ND |
| R6rpsJ(R6M1TC-1)-rev(R6WT)-rpsL+ | WT | WT | WT | WT | 0.125 | ND | 0.5 | ND | ND | ND |
| R6rpsJ(R6M1TC-5) | R6MITC-5 | WT | WT | WT | 1 | 1 | 0.5 | 0.5 | 2 | 0.25 |
| R6rpsJ(R6M1TC-5)-rpsL+ | R6MITC-5 | WT | WT | WT | 1 | ND | 0.5 | ND | ND | ND |
| R6rpsJ(R6M1TC-5)-rev(R6WT)-rpsL+ | WT | WT | WT | WT | 0.125 | ND | 0.5 | ND | ND | ND |
| R6patA(R6M1TC) | WT | R6M1TC-5 | R6M1TC-5 | WT | 0.5 | 0.125 | 2 | 0.5 | 16 | 0.25 |
| R6p_patA(R6M1TC)-rpsL+ | WT | R6M1TC-5 | WT | WT | 0.25 | 0.125 | 1 | 0.5 | 8 | 0.25 |
| R6CDSpatA(R6M1TC)-rpsL+ | WT | WT | R6M1TC-5 | WT | 0.25 | 0.125 | 1 | 0.5 | 8 | 0.25 |
| R6CDSpatA(G510A)(R6M1TC)-rpsL+ | WT | WT | R6M1TC(G510A) | WT | 0.25 | 0.125 | 1 | 0.5 | 4 | 0.25 |
| R6CDSpatA(C883A)(R6M1TC)-rpsL+ | WT | WT | R6M1TC(C883A) | WT | 0.125 | 0.125 | 1 | 0.5 | 8 | 0.25 |
| R6rpsJ(R6M1TC)-patA(R6M1TC) | R6M1TC-5 | R6M1TC | R6M1TC-5 | WT | 2 | 0.5 | 2 | 0.5 | 16 | 0.25 |
| R6rpsJ(R6M2TC) | R6M2TC-4 | WT | WT | WT | 0.5 | 0.5 | 0.5 | 0.5 | 2 | 0.25 |
| R6patA-patB(R6M2TC) | WT | R6M2TC-4 | R6M2TC-4 | R6M2TC-4 | 0.25 | 0.125 | 1 | 0.5 | 16 | 0.25 |
| R6patA-patB(R6M2TC)-rpsL+ | WT | R6M2TC-4 | R6M2TC-4 | R6M2TC-4 | 0.25 | ND | 1 | ND | ND | ND |
| R6patA-patB(R6M2TC)-rev(patA)-rpsL+ | WT | WT | WT | R6M2TC-4 | 0.125 | ND | 0.5 | ND | ND | ND |
| R6p_patA(R6M2TC)-rpsL+ | WT | R6M2TC-4 | WT | WT | 0.25 | 0.125 | 1 | 0.5 | 8 | 0.25 |
| R6CDSpatA(R6M2TC)-rpsL+ | WT | WT | R6M2TC-4 | WT | 0.25 | 0.125 | 1 | 0.5 | 4 | 0.25 |
| R6CDSpatB(R6M2TC)-rpsL+ | WT | WT | WT | R6M2TC-4 | 0.125 | 0.125 | 0.5 | 0.5 | 2 | 0.25 |
| R6CDSpatA-patB(R6M2TC)-dhfr+ | WT | WT | R6M2TC-4 | R6M2TC-4 | 0.25 | 0.125 | 1 | 0.5 | 8 | 0.25 |
| R6rpsJ(R6M2TC)-patA(R6M2TC) | R6M2TC-4 | R6M2TC-4 | R6M2TC-4 | WT | 0.5 | 0.25 | 2 | 0.5 | 16 | 0.25 |
| R6rpsJ(R6M2TC)-patA(R6M2TC)-patB(R6M2TC) | R6M2TC-4 | R6M2TC-4 | R6M2TC-4 | R6M2TC-4 | 0.5 | 0.25 | 2 | 0.5 | 16 | 0.25 |
TET, tetracycline; R, reserpine (20 mg/L); CIP, ciprofloxacin; Etbr, ethidium bromide; ND, not done; CDS, coding sequence.
arpsL+ and dhfr+ indicate an allele conferring resistance to streptomycin and trimethoprim, respectively, that was co-transformed along with the PCR fragment of interest for selection purposes.
bMICs are the results of three independent biological replicates. MICs in bold are significantly different from the parent strain without the mutation.