TABLE 2.
Chloramphenicol selection pressure assaysa
| Integron | Cm concn (mg/liter) on day: |
Level of gene cassette rearrangementsd | ||||
|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | ||
| Integrons 1S (initial Cm MIC: 32 mg/liter) | ||||||
| p1S (PcS, IntI1R32_N39) | 50 | 75 | 100 | 300 | 400 | + |
| p1S + pBad-intI1*P32_H39 | 100 | 250 | 400 | + | ||
| p1S + pBad-intI1*R32_H39 | 100 | 300 | 400 | +++ | ||
| p1S + pBad-intI1*R32_N39b | 100 | 300 | 400 | + | ||
| p1SLc | 100 | 150 | 400 | + | ||
| p1SintI1Y312F | 50 | 75 | 100 | 150 | − | |
| Integrons 1W (initial Cm MIC: 8 mg/liter) | ||||||
| p1W (PcW, IntI1R32_H39) | 5 | 5 | 15 | 75 | + | |
| p1W + pBad-intI1*P32_H39 | ND | ND | ND | ND | ND | ND |
| p1W + pBad-intI1*R32_H39b | 5 | 10 | 75 | +++ | ||
| p1W + pBad-intI1*R32_N39 | ND | ND | ND | ND | ND | ND |
| p1WLc | 5 | 15 | 60 | + | ||
| Integrons 1WTGN-10 (initial Cm MIC: 16 mg/liter) | ||||||
| p1WTGN-10 (PcWTGN-10, IntI1P32_H39) | 25 | 25 | 100 | 250 | 350 | + |
| p1WTGN-10 + pBad-intI1*P32_H39b | 25 | 100 | 250 | 350 | + | |
| p1WTGN-10 + pBad-intI1*R32_H39 | 50 | 150 | 350 | +++ | ||
| p1WTGN-10 + pBad-intI1*R32_N39 | ND | ND | ND | ND | ND | ND |
| p1WTGN-10Lc | 25 | 50 | 300 | + | ||
a
Values indicate Cm concentrations reached in BHI broth each day of the protocol using DH5α strains. The native integron organization, without intI1 overexpression, and its corresponding values are presented in bold. Each experiment was performed at least 3 times. ND, not determined.
b
intI1 overexpression was obtained with the same integrase as those present in the synthetic integron.
c
“L” indicates a mutated LexA-binding site.
d
To locate the position of the catB9 gene cassette within the integron, 100 colonies were analyzed each day by PCR and sequencing. Symbols: +, single rearrangement with integron intI1-aac(6′)-Ib-catB9; +++, multiple rearrangements; −, no rearrangement.