Table 2

Effect of mutations on Oac assembly and function
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
Western blot analysis β-galactosidase activity in Miller units with standard deviations shown in parentheses Slide agglutination reaction to MASF6 LPS reaction to MASF6
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.

Thanweer and Verma

Thanweer and Verma BMC Biochemistry 2012 13:13   doi:10.1186/1471-2091-13-13

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