Fig. 5: The T1 cluster is regulated by a recombination-mediated phase variation mechanism. | Nature Communications

Fig. 5: The T1 cluster is regulated by a recombination-mediated phase variation mechanism.

From: Structure, biosynthesis and regulation of the T1 antigen, a phase-variable surface polysaccharide conserved in many Salmonella serovars

Fig. 5

a Genetic organization of the genomic regions encompassing genetic switches for T1 antigen biosynthesis, and the expression of E. coli type I fimbriae (shown from MG1655; U00096.3), and a putative autotransporter (HyxB) encoded on E. coli pathogenicity island PAI-X (from E. coli O157:H7; EDL933). STM0716 is an ortholog of FimB and FimX, which are tyrosine recombinases responsible for flipping a promoter region preceding the fim and hyxRAB operons. In each case, the invertible region is flanked by nine base-pair inverted repeats (IR1 and IR2). b Schematic showing the experimental strategy to identify ON and OFF states of the invertible switch using qPCR. Primer P1 (in the middle of the switch) serves as the forward primer or reverse primer, depending on the switch orientation and acts with one of the two primers (P2 or P3) located outside the switch to generate a diagnostic amplification product. Standard curves were constructed using synthesized (fixed) templates representing the OFF or ON states, and used to calculate the relative amounts of each state from genomic DNA templates. Bar graphs represent the average amounts detected, with individual data points plotted on top of the bar graph. Samples and standards were analyzed in triplicate and error bars indicating standard deviation is shown using the average DNA quantity detected in each sample as the center point. qPCR was performed on two separate genome preparations with comparable results. c T1 antigen phenotypes resulting from engineered ON and OFF states of the T1 phase switch. Whole-cell lysates were examined by SDS-PAGE and western immunoblotting. pWQ1128 (carrying STM0716-STM0716) was cloned to contain the switch region locked in either the ON or OFF state and STM0716 was deleted to create pWQ1133 (ON) and pWQ1134 (OFF). Each plasmid was also co-transformed with a plasmid carrying STM0716 in trans. The presence of the T1 antigen was then probed by silver staining and western blotting with the specific antisera. These analyses were performed in triplicate. Source data are provided as a Source Data file. d Sanger sequencing results for the switch regions from plasmids recovered from the cultures examined in c. No phase variation was detected in the absence of STM0716, evident in the uniform chromatographic species. Expression of STM0716 resulted in a mixed population of species reflecting both orientations of the switch. Most importantly, expression of STM0716 in the ON state led to a predominant OFF state, whereas expression of STM0716 in the OFF state resulted in a minor amount of ON state.

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