Identification of critical residues of the serotype modifying O-acetyltransferase of Shigella flexneri

Thanweer, Farzaana; Verma, Naresh K

doi:10.1186/1471-2091-13-13

BMC Biochemistry

Table 2 Effect of mutations on Oac assembly and function

From: Identification of critical residues of the serotype modifying O-acetyltransferase of Shigella flexneri

*E. coli/Shigella* strain	Mutation	Effect of mutation on Oac-PhoA-LacZα assembly in membrane (compared to wild type protein assembly)		Effect of mutation on Oac function (compared to wild type protein function)		Overall effect on Oac
*E. coli/Shigella* strain	Mutation	Western blot analysis	β-galactosidase activity in Miller units with standard deviations shown in parentheses	Slide agglutination reaction to MASF6	LPS reaction to MASF6	Overall effect on Oac
B1790	_	Not assembled (negative control)	3.8 (0.73)	N/A	N/A	N/A
SFL124	Serotype Y strain	N/A	N/A	_	_	N/A
B2012/SFL1899	Wild type Oac	Assembled (Positive control)	120.19 (5.27)	+	+	Positive control
B2266/SFL2047	SG 52-53	Appears decreased	34.85 (3.11)	+	_	Critical to assembly
B2254/SFL1909	RR 75-76	Appears decreased	79.43 (5.25)	_	_	Critical to assembly
B2260/SFL1922	GS 138-139	Appears decreased	73.87 (9.85)	+	_	Critical to assembly
B2264/SFL1936	SYG 274-276	Appears decreased	40.38 (3.40)	_	_	Critical to assembly
B2256/SFL1916	D 333	Appears decreased	46.25 (11.17)	+	+	Critical to assembly
B2263/SFL1935	GR 269-270	Appears decreased	77.17 (14.53)	+	+	Critical to assembly
B2255/SFL1911	RK 110-111	Appears increased	207.23 (16.77)	+	+	Critical to assembly
B2253/SFL1908	R 73	Appears increased	176.08 (11.77)	_	_	Critical to assembly/function
B2258/SFL1920	FP 78-79	Appears increased	156.45 (4.45)	+	+/_	Critical to assembly/function
B2261/SFL1923	WT 141-142	Appears increased	174.76 (20.79)	+	_	Critical to assembly/function
B2265/SFL1937	FPV 282-284	Appears increased	163.78 (9.49)	_	_	Critical to assembly/function
B2257/SFL1919	R 62	Appears increased	122.05 (13.34)	+	+	Non-critical
B2021/SFL1910	C 84	No effect	124.54 (7.39)	+	+	Non-critical
B2262/SFL1934	S 114	Appears increased	132.32 (10.99)	+	+	Non-critical

The table compares the overall effects of the various mutations on Oac assembly and function. Both qualitative and quantitative assays were considered to represent the effect on the protein assembly. Firstly, Western immunoblotting was used as a qualitative assay to assess the assembly of wild type and mutant Oac-PhoA-LacZα proteins. Levels of Oac- PhoA-LacZα protein assembled by the mutant constructs were then quantified and compared with the wild type protein using the BG (β-galactosidase) assay. Results are an average of two independent experimental repeats with two internal replicates. The table also shows the results of the qualitative functional assays conducted for the Oac mutants. Preliminary slide agglutination tests were confirmed by LPS Western immunoblotting using MASF6 antibodies. N/A refers to not applicable.

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ISSN: 1471-2091

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