TABLE 3.

The rhuR-bhuR intergenic region exhibits RhuI-dependent promoter activity^a

Plasmids	RhuI expression	Growth conditionb	β-Galactosidase activity (Miller units)a
			−IPTG	+IPTG
pDJM31 + pRK415Δ	−	FeSO4	46.5 (1.7)	49.1 (0.5)
		EDDHA	119.0 (4.2)†	124.0 (5.5)†
pDJM31 + pERM26	+	FeSO4	1,706.8 (52.3)	3,069.8 (112.6)*
		EDDHA	2,260.0 (142.4)*†	3,958.1 (186.2)*†

^aReporter and expression plasmids were cotransformed into E. coli MGIQ1, an engineered K-12 strain that encodes a chromosomal lacI^q1 allele (V.J. Hernandez). Cells were induced at mid-log stage for 2 h by adding IPTG to a final concentration of 1 mM to the culture broth. 

^bPrimary cultures were incubated overnight in LB supplemented with 100 μM EDDHA. The Fe-stressed overnight cultures were used to inoculate secondary cultures under the indicated conditions. FeSO₄, LB supplemented with 100 μM EDDHA and 144 μM FeSO₄; EDDHA, LB supplemented with 100 μM EDDHA. 

^cReporter activity was determined by a modified version of the Miller assay (2, 27). The mean and standard deviation (in parentheses) are shown for each value. Values that are significantly different (P < 0.05) from the value for the vector control, as determined by the Student t test, are indicated by asterisks. Values that are significantly different (P < 0.05) from the value for the Fe-replete strain, as determined by the Student t test, are indicated by daggers.