SlyA can complement other regulators for SPI-2 expression 
The number of inputs at each regulator shown in Figure 6B corresponds to the number of regulators that directly or indirectly control its expression.
The number of regulators that act on slyA was surprising.
Quantitative RT-PCR confirmed that slyA transcription was reduced in 11 of 14 mutant backgrounds (Figure 6A).
slyA transcriptional activation by PhoP has been reported [63],[64].
The complementation result suggests that many of the regulators may function on SPI-2 through SlyA activation of ssrB or alternatively via both regulators (slyA and ssrB) acting together.
To determine epistatic relationships, we introduced a slyA-expressing plasmid, pBAD30SlyA, into each regulatory mutant and examined complementation in each mutant background compared to an empty vector control (Figure 7C).
The construct strongly complemented ompR/envZ, phoP/phoQ, csrA and himD, suggesting that the effect of these regulators on SPI-2 expression may be indirect via regulation of slyA.
Surprisingly, the expression of SPI-2 genes depended upon slyA even in an ssrA/ssrB deleted strain.
This suggests that slyA can regulate expression of SPI-2 in a way that is independent of ssrB.
To test this possibility we constructed double deletions of ssrAB/ompRenvZ, ssrAB/phoPQ, ssrAB/csrA and ssrAB/himD.
As shown in Figure 7D, slyA is capable of activating expression of SPI-2 genes independently of ssrB, ompR/envZ, phoP/phoQ, csrA and himD (ihf), although the effects are not as strong as ssrB.
Furthermore there was a clear dichotomy in expression between the first two SPI-2 operons encoding ssaB-ssaE and sseA-sseG and the operons further downstream (ssaG-ssaQ) suggesting that slyA may act at different sites within SPI-2.
However, these experiments are based on over-expression of slyA that may complicate the analysis because of binding to sites that would normally not be occupied.
