. 1997 Apr 29;94(9):4560–4565. doi: 10.1073/pnas.94.9.4560

Table 1.

Phenotypic properties of FepA charge substitution mutants

FepA mutant	Name	Ferric enterobactin				Colicin B		Colicin D
		Binding^* and transport^†				Killing,^‡ ++	Binding^§K_d	Killing,^‡ ++	Binding^§K_d
		K_d	K_m	V_max	Nutrition	Killing,^‡ ++	Binding^§K_d	Killing,^‡ ++	Binding^§K_d
++	FepA	22	155	85	20	100	145	100	169
R283A	A	23	253	97	20	100	210	100	184
R286A	B	19	571	98	22	100	140	20	188
R313A	C	26	522	109	20	100	190	100	187
R316A	D	27	309	97	21	20	180	4	173
R274A	E	21	152	79	20	100	108	100	177
R283/286A	AB	28	524	70	21	100	220	80	133
R283/313A	AC	25	186	73	20	100	290	100	174
R283/316A	AD	27	161	64	21	10	250	2	312
R274/283A	AE	20	287	96	20	100	142	100	227
R286/313A	BC	21	335	87	21	100	226	20	192
R274/286A	BE	29	108	74	20	100	148	100	152
R313/316A	CD	23	268	101	21	20	170	80	303
R274/313A	EC	32	132	105	20	100	510	100	215
R274/316A	DE	26	145	76	20	20	142	2	232
R/283/286/313A	ABC	12	1676	77	21	25	120	10	215
R283/313/316A	ACD	44	316	93	20	100	200	12.5	408
R274/R313/R316A	ECD	22	354	150	20	10	462	2.5	275
R286/316A	BD	701	2,745	24	17	2	220	0.2	517
R283/286/316A	ABD	1,586	2,834	18	15	5	210	2	865
R286/313/316A	BCD	1,708	>10,000	37	14	2	280	1	1,065
R233/286/313/316A	ABCD	>5,000	>10,000	0	9	0.1	145	0.2	1,699

K_d values (nM) were determined from the concentration-dependence of ⁵⁹Fe-enterobactin binding; the mean values from four independent experiments were plotted with grafit (Elsevier), using the “Bound versus Total” equation. The mean SE of the fitted curves that produced the K_d values was 32%, with a SD of 8.6%.

^†

The K_m (nM) and V_max (pmol/min per 10⁹ cells) of transport were determined from ⁵⁹Fe-enterobactin uptake assays (26); mean values from two independent experiments were plotted with grafit to yield the kinetic parameters. The mean SE of the fitted curves that produced the K_m values was 46%, with a SD of 17%. The diameters of growth halos in siderophore nutrition assays are reported in millimeters. The tabulated values represent a single experiment, but little variation was observed among several trials.

^‡

Bacteria were plated in top agar, and serial dilutions of colicin B or D were spotted onto the plates to determine the colicin titer on the various mutants, relative to wild-type FepA. The tabulated values represent a single experiment, but little variation was observed among several independent trials.

^§

Combined data from two independent binding experiments were fit with grafit, using the Bound versus Total equation. For colicin B, the mean SE of the fitted curves that produced the K_d values (nM) was 30%, with a SD of 6%, whereas for colicin D, the mean SE was 21%, with a SD of 7.5%.