Topological characterisation and identification of critical domains within glucosyltransferase IV (GtrIV) of Shigella flexneri

Table 1 Analysis of gtrIV-phoA/lacZ fusions and gtrIV-phoA/lacZ-gtrIV sandwich fusions for GtrIV topology determination

Sample ID	AA¹	Colour²	Average AP³	Average BG⁴	%AP⁵	%BG⁶	NAR⁷ (AP:BG)	Location on Model⁸
Random C-terminal Fusions
B1925	A15	Purple	311 ± 9	25 ± 2	32.6%	16.1%	2:1	t.1
B1910	N29	Blue	356 ± 47	1^#	37.3%	0.6%	52:1	p.2
B1924	P38	Blue	357 ± 44	2^#	37.5%	1.3%	24:1	p.2
B1907	N41	Blue	296 ± 47	1^#	31.1%	0.6%	38:1	p.2
B1935	G42	Blue	195 ± 28	3 ± 1	20.5%	1.3%	20:1	p.2
B1906	L56	Blue	133 ± 29	4 ± 1	14.0%	2.6%	5:1	p.2
B1931	F60	Blue	383 ± 25	1^#	40.2%	0.6%	57:1	p.2
B1912	Q186	Purple	9 ± 91	15 ± 2	0.9%	9.7%	1:11	t.4
B1919	I200	Red	22 ± 4	132 ± 6	2.3%	85.1%	1:34	t.4
B1913	N235	Blue	292 ± 51	0^#	30.6%	0.0%	> 100:1	p.6
B1926	L240	Blue	394 ± 20	1^#	41.4%	0.7%	58:1	p.6
B1904	L246	Blue	660 ± 39	1^#	69.3%	0.7%	97:1	p.6
B1921	N251	Blue	962* ± 59	1^#	100.9%	0.9%	> 100:1	p.6
B1922	N255	Blue	553 ± 30	0^#	58.1%	0.1%	> 100:1	p.6
B1908	D261	Blue	385 ± 55	2^#	40.4%	1.2%	33:1	p.6
B1905	S300	Blue	426 ± 21	4 ± 1	43.6%	2.8%	16:1	p.6
B1923	M310	Blue	589 ± 15	1 ± 1	61.8%	0.1%	> 100:1	p.6
B1920	V359	Purple	269 ± 34	9 ± 1	28.2%	5.5%	5:1	t.6
B1911	Q393	Purple	12 ± 3	8 ± 2	1.2%	4.9%	1:4	t.7
PCR-Constructed Fusions
B2216	V102	Red	2^#	121 ± 17	0.2%	78.2%	1: > 100	c.3
B1937	D146	Blue	953 ± 35	7 ± 1	100.0%	4.3%	23:1	re (p.4)
B2217	V155	Red	6 ± 2	71 ± 9	0.6%	45.8%	1:76	re (c.4)
B2405	K162	Red	5^#	11^#	0.5%	7.1%	1:14	re (c.4)
B2225	D169	Red	3^#	79 ± 17	0.3%	51.1%	1: > 100	re (c.4)
B1936	D406	Blue	177 ± 40	1^#	18.6%	0.9%	20:1	p.8
B1914	K437	Red	5 ± 1	155** ± 8	0.5%	100.0%	1: > 100	c.9
Sandwich Fusions
B2187	R93	Red	0^#	10 ± 2	0.1%	21.3%	1: > 100	c.3
B2190	K117	Red	0^#	14 ± 2	0.3%	29.4%	1: > 100	c.3
B2143	A181	Blue	135⁺ ± 25	0^#	100.0%	0.3%	> 100:1	re (p.4)
B2192	R375	Red	1 ± 1	47⁺⁺ ± 7	0.4%	100.0%	1: > 100	c.7

¹ Position of the last residue of GtrIV followed by phoA/lacZ, ² Colour of colony as seen on dual indicator plates, ^3,4 Activities of the fusions, average of four independent experiments with standard deviations of each activity, ^5,6 Percentages of AP and BG activities measured relative to the maximum activity in the set, ⁷Normalised AP:BG activity ratio (NAR) rounded to the nearest integer. ⁸Location of the fusion on the adjusted topological model of GtrIV(Figure 1); c, cytoplasm; p, periplasm; t, transmembrane helix; re, re-entrant loop. *Highest truncation AP, **Highest truncation BG, ⁺Highest Sandwich fusion AP, ⁺⁺Highest sandwich fusion BG. Since the random C-terminal fusions and PCR mediated fusions brought about truncated protein fused to the dual reporter, they were taken as one data set and the highest respective AP and BG values between them was used to calculate the NAR values. The NAR values for the sandwich fusions were calculated separately based on the highest AP and BG values obtained from all the sandwich fusions alone. ^#Standard deviations were less than 0.5 and not included.

ISSN: 1471-2091