1d) We therefore concluded that the newly identified genes are u

1d). We therefore concluded that the newly identified genes are under the direct control of MlrA. Note that besides the csgD promoter, the genes for two additional transcription factors, CadC and YrbA,

are also under the control of MlrA. If this is the case, Dabrafenib MlrA is located at a higher level in the hierarchy of the transcription factor network (Ishihama, 2010). To confirm the activating role of MlrA on the csgD promoter, we next measured csgD mRNA using the primer extension assay. The level of csgD P1 mRNA was low in cells at steady-state at the exponential phase of growth in LB medium (Fig. 4, lane 1). When MlrA was overexpressed using arabinose-inducible expression vector, csgD mRNA markedly increased even in the wild-type E. coli (Fig. 4, lanes 1 and 2). The enhancing effect of MlrA overexpression was also examined for a set of E. coli mutants, each lacking one of the transcription factors that are involved in the regulation of the csgD promoter (Ogasawara et al., 2010). In the presence of MlrA overexpression,

an increase of csgD mRNA was observed in the mutants lacking Crl (Fig. 4, lanes 5 and 6), H-NS (Fig. 4, lanes 7 and 8), RstA (Fig. 4, lanes 11 and 12), CpxR (Fig. 4, lanes 13 and 14) and RcsB (Fig. 4, lanes 15 and 16), indicating that MlrA functions independent of these regulators. In contrast, however, enhancement of the csgD promoter was not detected for mutants see more lacking OmpR (Fig. 4, lanes 3 and

4) and IHF (Fig. 4, lanes 17–20), both binding near the MlrA-binding site (see Fig. 2; also see Fig. 6). One explanation for the lack of csgD promoter activation in the mutants lacking OmpR and IHF is that MlrA requires these two activators for function. Promoter P1 is known to be recognized by RpoS sigma factor (Ogasawara et al., 2007a), and the activity Ureohydrolase of RpoS sigma is controlled by an accessory protein Crl (Bougdour et al., 2004). The induction level of csgD mRNA by MlrA overexpression in both the rpoS (data not shown) and the crl mutant (Fig. 4, lane 6) was as high as that in wild-type E. coli (Fig. 4, lane 2). This is not unexpected given that the csgD promoter P1 is recognized by both RpoD and RpoS (Ogasawara et al., 2007a). Escherichia coli contains an uncharacterized protein YcgE that exhibits an overall similarity of 76% to MlrA (Brown et al., 2001). We also tested possible functional replacement of MlrA by YcgE, but overexpression of YcgE did not affect the csgD mRNA level (data not shown). Together, these observations indicate that MlrA is a DNA-binding positive regulator of csgD transcription, but its homologue YcgE is not involved in csgD regulation. Both the reporter assay and primer extension analysis indicated the stimulatory role of MlrA on csgD transcription. If this is the case, the set of genes under the control of the CsgD global regulator must be activated in the presence of MlrA overexpression.

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