Rli27 is necessary for upregulation of Lmo0514 protein levels in intracellular bacteria by a mechanism that involves interaction with the <i>lmo0514</i> long 5′-UTR isoform.

<p>(A) Real-time qPCR assays showing no changes in relative levels of the <i>lmo0514</i> ORF (fragment amplified with Lmo0514-F and Lmo0514-R primers) or the isoform bearing the long 5′-UTR (fragment amplified with UTR-B and UTR-1R primers) in intracellular bacteria that lack Rli27 or are defective for the <i>rli27</i>-flanking <i>lmo0411</i> gene. (B) Decrease in levels of Lmo0514 protein produced by intracellular bacteria, caused by absence of Rli27. No such effect was seen in <i>lmo0411</i>- or <i>lmo0412</i>-defective strains. Levels of InlA, another cell wall-bound LPXTG protein, were monitored as loading control. (C) RT-PCR assays showing expression of Rli27 and mutated versions Rli27-mut1 and Rli27-mut3 produced in trans from the pP1 plasmid. (D) Western blot assays showing the effect of the mut3 mutation in Rli27, which impedes upregulation of the Lmo0514 protein in intracellular bacteria. InlA levels were monitored as loading control. (E) Real-time qPCR showing that lack of Rli27 does not affect <i>lmo0514</i> transcript stability in extracellular bacteria grown in BHI medium to stationary phase. Primers correspond to those that map in the coding region (Lmo0514-F and Lmo0514-R). Data are derived from a minimum of three independent experiments. (F) Western blot showing that Rli27 is not necessary for Lmo0514 protein production by extracellular bacteria. Due to the small amount of Lmo0514 protein produced by extracellular bacteria (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004765#pgen-1004765-g001" target="_blank">Fig. 1B</a>), the anti-Lmo0514 antibody-treated membrane was overexposed. Levels of InlH, another cell wall-bound LPXTG protein, were monitored as loading control. Densitometry values are indicated in Western blots beneath panels B, D, and F.