TABLE 2.

In vivo transcription evidence that EutT synthesizes AdoCbl

Strain	Genotypec	β-Galactosidase (active tetramers/CFU) activitya in cells grown in minimal mediumb with the indicated supplement(s)
		None	EA	EA, (CN)2Cbi	EA, HOCbl
JE7145	cobA+	1,720 ± 100	1,960 ± 60	4,960 ± 400	6,330 ± 640
JE7179	cobA	400 ± 30	440 ± 30	980 ± 60	810 ± 70
JE7202	cobA/pVOCd	NDe	ND	770 ± 30	2,120 ± 80
JE7203	cobA/peutT+f	ND	ND	4,750 ± 20	12,210 ± 450

^aThe number of active tetramers of β-galactosidase enzyme was determined as described in Materials and Methods.

^bNCE medium was supplemented with MgCl₂, NH₄Cl, glycerol (as a carbon and energy source), methionine, and trace minerals. Also added were ethanolamine (EA; 50 mM), CN₂Cbi (0.2 μM), 200 nM HOCbl (0.2 μM), AdoCbl (0.2 μM) and l-(+)-arabinose (200 μM). After 2 h of growth at 37°C, 1 ml of culture was centrifuged and cells were resuspended in 0.3 ml of sterile saline. CFU counts were determined by dilution plating on nutrient agar plates.

^cComplete genotypes of the strains are listed in Table 1. All strains carried a chromosomal eutE18::MudJ(lacZ⁺) element and mutations metE205 and ara-9.

^dpVOC, pBAD24 (17).

^eND, not determined.

^fpeutT⁺, pEUT7.