TABLE 5.
Mutations that affect proximal regulation of the ttr operon
| Strain | β-Galactosidase level in cells grown as indicatedb
|
|||||
|---|---|---|---|---|---|---|
| Relevant genotypea
|
Aerobic
|
Anaerobic
|
||||
| ttr mutation testedd | lac fusion | Medium alone
|
Medium plus tetrathionate
|
Medium alone
|
Medium plus tetrathionate
|
|
| − Tc (+Tc) | − Tc (+Tc) | − Tc (+Tc) | − Tc (+Tc) | |||
| TT18694 | ttrB123::MudJ | A | 1 | 23 | 3 | 1,775 |
| TT22471 | ttrR132(del)::Cm(swap) | A | 3 | 3 | 3 | 4 |
| TT20440 | ttrS117::Tn10dTc | A | 1 | 2 | 6 | 12 |
| TT20433 | ttrS108::Tn10dTc | A | 1 | 2 | 7 | 7 |
| TT22348 | ttrS130::Tn10dCm | A | 2 | 2 | 815 | 297 |
| TT22350 | ttrS130::Tn10dCm ttrS108::Tn10dTc | A | 2 | 2 | 325 | 106 |
| TT22352 | ttrS130::Tn10dCm ttrS117::Tn10dTc | A | 2 | 2 | 5 | 5 |
| TT22467 | ttrS130::Tn10dCm oxrA2::Tn10 | A | 3 | 3 | 19 | 11 |
| TT22373 | ttrS133(del)::Cm(swap)c | A | 4 | 4 | 1,545 | 141 |
| TT18683 | ttrA121::MudJ | B | 3 | 25 | 13 | 1,734 |
| TT22356 | ttrP128::Tn10dTPOP2 | B | 34 (1,438) | 10 (1,455) | 17 (724) | 12 (1,676) |
| TT22354 | ttrP127::Tn10dTPOP2 | B | 9 (216) | 6 (139) | 10 (51) | 8 (163) |
All strains carried a ttr::MudJ insertion that fuses lac transcription to the ttrBCA promoter. Strains designated as A carry ttrB123::MudJ; those designated B carry ttrA121::MudJ.
The level of β-galactosidase activity is presented in units defined by Miller (37). Enzyme levels in parentheses were observed in cells grown in the presence of the antibiotic tetracycline (Tc), which induces promoters within the TPOP transposon.
All of the coding sequence unique to ttrS was removed and replaced by the chloramphenicol resistance gene and distal Tn10dCm transposon, followed by the 10 bases adjacent to the distal end of this Tn10dCm element in strain ttrS130::Tn10dCm.
Each of the ttr control mutations was tested for its effect on anaerobic acid production and growth in strains carrying a wild-type ttrBCA operon. The wild-type strain (LT2), as well as TT20369 (ttrS130::Tn10dCm), TT22349 (ttrS108::Tn10dTc ttrS130::Tn10dCm), and TT22372 [ttrS133(del)::Cm(swap)], formed red patches (produced acid) when grown on MacConkey-tetrathionate (10 mM) medium. All other strains formed white patches. Strains with an insertion in the promoter region (ttrP128::Tn10dTPOP2 or ttrP127::Tn10dTPOP2) formed red patches when grown on this medium in the presence of the antibiotic tetracycline (Tc), which induces promoters within the TPOP transposon. Growth was tested anaerobically on solid medium with propanediol or ethanolamine as the sole carbon source and 10 mM tetrathionate; LT2, as well as TT20369 (ttrS130::Tn10dCm), TT22349 (ttrS108::Tn10dTc, ttrS130::Tn10dCm), and TT22372 [ttrS133(del)::Cm(swap)], produced visible patches on this media. All other strains failed to grow under these conditions. One of the strains with an insertion in the promoter region (ttrP128::Tn10dTPOP) produced a visible patch when grown on this medium in the presence of tetracycline.