TABLE 1.
Expression of growth rate-independent rrn promoters in strains with altered rrn gene dosage
| Lysogena | rrnB P1 type | Fragment end points | β-Gal activityb
|
ΔBAGH/W1485d activity ratio | β-Gal activityb
|
pNO1301/pBR322d activity ratio | ||
|---|---|---|---|---|---|---|---|---|
| ΔBAGHc | W1485 | W1485(pNO1301)e | W1485(pBR322) | |||||
| JV1100 | wt | −88, +1 | 3,960 | 2,690 | 1.47 | 1,430 | 1,930 | 0.741 |
| JV783 | wt | −50, +2 | 557 | 364 | 1.53 | 243 | 371 | 0.655 |
| JV2978 | wt | −46, +1 | 3,480 | 2,490 | 1.40 | 725 | 901 | 0.805 |
| JV901 | C-1T,C-15G | −88, +1 | 5,630 | 4,480 | 1.26 | 2,970 | 4,230 | 0.702 |
| JV1063 | Ains-22 | −50, +2 | 2,160 | 2,150 | 1.00 | 1,480 | 2,140 | 0.692 |
| JV2935 | T-33A | −48, +1 | 5,100 | 3,740 | 1.39 | 3,050 | 4,260 | 0.716 |
| JV2979 | C-1T | −46, +1 | 2,670 | 2,110 | 1.27 | 926 | 939 | 0.986 |
Lysogenic strains containing various rrnB P1-lacZ fusions on lambda prophages were the gift of Richard L. Gourse. We have maintained his numbering for identifying the prophages but we have changed the preceding initials to indicate that these prophages are in a different host background (e.g., JV1063 contains the prophage obtained from strain RLG1063; see references 2 and 6 for descriptions of the RLG strains). Lambda phages containing the rrnB P1-lacZ fusions were purified and used to infect both W1485 and ΔBAGH, and the presence of a single prophage was confirmed by PCR (16).
β-Galactosidase (β-Gal) assays were performed as described by Miller (15) except that extracts of sonicated cells were used instead of permeabilized cells. Ampicillin (final concentration, 200 μg/ml) was added to the media for assays of the plasmid-containing strains. At an OD600 of 0.6 to 0.8, each culture was divided to create triplicates that were then sonicated and measured separately for β-galactosidase. The measurements are reported as specific activity, in units per milligram of protein. Because of changes in the protein standard, values for JV2978 and JV2979 differ from the values in the plasmid-containing strains and in Table 2. The triplicates were averaged to obtain the day's value. Experiments on each strain were performed on at least two different days.
In strain W1485ΔBAGH (referred to as ΔBAGH) rrnB, rrnA, rrnG, and rrnH are inactivated by antibiotic cassette insertion and deletion) as described in reference 4.
Standard errors of ratios were less than 10%.
Plasmid pNO1301, a pBR322 derivative that contains the entire rrnB operon, was the gift of Richard L. Gourse (12).