Novel roles of LeuO in transcription regulation of E. coli genome: antagonistic interplay with the universal silencer H-NS

LeuO, the regulator of leucine biosynthesis operon of Escherichia coli, is involved in the regulation of as yet unspecified genes affecting the stress response and pathogenesis expression. To get insights into the regulatory role(s) of LeuO, Genomic SELEX screening has been performed to identify the whole set of its regulation targets. A total of 140 LeuO‐binding sites were identified on the E. coli genome, of which as many as 133 (95%) were found to contain the binding sites of H‐NS, the universal silencer of stress‐response genes, supporting the concept that LeuO plays an antagonistic role with anti‐silencing activity. Western blot analysis indicated that H‐NS predominates in growing phase; however, after prolonged culture for 1 week, H‐NS decreased instead LeuO increased, supporting the anti‐silencing role of LeuO. In concert with this model, a set of stress‐response genes including cryptic chaperone/usher‐type fimbriae operons are under the control of antagonistic interplay between LeuO and H‐NS. Confocal laser scanning microscopic observation in flow‐chambers showed that the mutants lacking leuO and some fimbriae genes are defective in biofilm formation or form altered biofilm architecture. Taken together we propose that LeuO is a major player in antagonistic interplay against the universal silencer H‐NS.     

Mlc regulation of Salmonella pathogenicity island I gene expression via hilE repression

The global regulator Mlc is a repressor of several genes and operons that are involved in sugar uptake and metabolism. A Salmonella enterica serovar Typhimurium mlc mutant showed reduced levels of invasion and cytotoxicity compared to the wild-type, and exhibited reduced expression levels of hilD, hilA and invF, which are regulatory genes in the Salmonella pathogenicity island 1 (SPI1). However, the effects of Mlc on hilD expression and bacterial invasiveness were not seen in the hilE mutant, and hilE expression was increased in the mlc mutant, which suggests that Mlc exerts positive effects on the expression of SPI1 genes by reducing the expression of HilE, which is known to down-regulate the expression of SPI1 genes through direct interaction with HilD. We found that the two known promoters of hilE were not modulated by Mlc, and we identified a third promoter, designated P3, which was repressed by Mlc. The gel mobility shift assay and footprinting analysis revealed that Mlc repressed hilE in a direct manner by binding to two distinct sites in the hilE P3 promoter region. The specific down-regulation of hilD observed in the presence of Mlc regulon-inducible sugars, such as glucose and mannose, could not be detected in the mlc mutant. Based on these results, we propose that Mlc functions to sense the availability of sugars and is linked to virulence gene regulation by its ability to control hilE expression in Salmonella.     

FliZ regulates expression of the Salmonella pathogenicity island 1 invasion locus by controlling HilD protein activity in Salmonella enterica serovar typhimurium

A prerequisite for Salmonella enterica to cause both intestinal and systemic disease is the direct injection of effector proteins into host intestinal epithelial cells via a type three secretion system (T3SS); the T3SS genes are carried on Salmonella pathogenicity island 1 (SPI1). These effector proteins induce inflammatory diarrhea and bacterial invasion. Expression of the SPI1 T3SS is tightly regulated in response to environmental signals through a variety of global regulatory systems. We have previously shown that three AraC-like regulators, HilD, HilC, and RtsA, act in a complex feed-forward regulatory loop to control the expression of the hilA gene, which encodes the direct regulator of the SPI1 structural genes. In this work, we characterize a major positive regulator of this system, the flagellar protein FliZ. Through genetic and biochemical analyses, we show that FliZ posttranslationally controls HilD to positively regulate hilA expression. This mechanism is independent of other flagellar components and is not mediated through the negative regulator HilE or through FliZ-mediated RpoS regulation. We demonstrate that FliZ controls HilD protein activity and not stability. FliZ regulates HilD in the absence of Lon protease, previously shown to degrade HilD. Indeed, it appears that FliZ, rather than HilD, is the most relevant target of Lon as it relates to SPI1 expression. Mutants lacking FliZ are significantly attenuated in their ability to colonize the intestine but are unaffected during systemic infection. The intestinal attenuation is partially dependent on SPI1, but FliZ has additional pleiotropic effects.