Lrp Acts as Both a Positive and Negative Regulator for Type 1 Fimbriae Production in Salmonella enterica Serovar Typhimurium

Abstract

Leucine-responsive regulatory protein (Lrp) is known to be an indirect activator of type 1 fimbriae synthesis in Salmonella enterica serovar Typhimurium via direct regulation of FimZ, a direct positive regulator for type 1 fimbriae production. Using RT-PCR, we have shown previously that fimA transcription is dramatically impaired in both lrp-deletion (Δlrp) and constitutive-lrp expression (lrpC) mutant strains. In this work, we used chromosomal PfimA-lacZ fusions and yeast agglutination assays to confirm and extend our previous results. Direct binding of Lrp to PfimA was shown by an electrophoretic mobility shift assay (EMSA) and DNA footprinting assay. Site-directed mutagenesis revealed that the Lrp-binding motifs in PfimA play a role in both activation and repression of type 1 fimbriae production. Overproduction of Lrp also abrogates fimZ expression. EMSA data showed that Lrp and FimZ proteins independently bind to PfimA without competitive exclusion. In addition, both Lrp and FimZ binding to PfimA caused a hyper retardation (supershift) of the DNA-protein complex compared to the shift when each protein was present alone. Nutrition-dependent cellular Lrp levels closely correlated with the amount of type 1 fimbriae production. These observations suggest that Lrp plays important roles in type 1 fimbriation by acting as both a positive and negative regulator and its effect depends, at least in part, on the cellular concentration of Lrp in response to the nutritional environment.

The metabolic regulator CodY links Listeria monocytogenes metabolism to virulence by directly activating the virulence regulatory gene prfA

“…Other regulatory proteins bind to different targets in the effector-bound (holo) (Wunsche et al, 2012) and effector-unbound (apo) states (Balderas-Martinez et al, 2013). For example, Lrp (a regulatory protein in Gram-negative bacteria that controls both metabolic and virulence genes) and the Fur regulators have been shown to be active in both their holo and apo forms (Newman and Lin, 1995;van der Woude et al, 1995;Cho et al, 2008;Carpenter et al, 2009;Baek et al, 2011;Butcher et al, 2012). In fact, Lrp was shown to repress genes in its holo form, while activating others in the apo form (Chen et al, 2005).…”

Overproduction of the multidrug efflux pump MexEF‐OprN does not impair Pseudomonas aeruginosa fitness in competition tests, but produces specific changes in bacterial regulatory networks

“…While this approach is highly valuable to delimit the potential MexT targets, expression of MexT from a plasmid does not reflect the mechanism by which MexT activation leads to overexpression of MexEF‐OprN and hence to antibiotic resistance in clinical settings; therefore, it may not be suitable for assessing fitness costs. Furthermore, the fact that some transcriptional regulators can be both positive and negative regulators of the same gene depending on their concentration highlights the need of performing these experiments using models as close as possible to natural isolates (Baek et al ., 2011).…”

The Bacterial Two-Hybrid System Uncovers the Involvement of Acetylation in Regulating of Lrp Activity in Salmonella Typhimurium

“…The fim operon of S . Typhimurium encodes type 1 fimbriae and can be directly and positively regulated by Lrp (McFarland et al, 2008; Baek et al, 2011). Therefore, fimZ and fimA were selected as representative genes to determine the effect of acetylation of K36 in vivo .…”

“…The expression of the type 1 fimbriae can be evaluated by the yeast agglutination assay (Kukkonen et al, 1993; Baumler et al, 1997). Since Lrp can positively regulate the expression of the type 1 fimbriae (Blomfield et al, 1993; Baek et al, 2011), we used the yeast agglutination assay to confirm the role of acetylation in regulating activity of Lrp. The chromosome K36Q, K36R, K36A mutants along with the wild type strain were subcultured twice for overnight static growth, respectively.…”