Project description:The Hha and YdgT proteins are suggested to modulate the expression of horizontally acquired genes by interacting with H-NS and StpA, which play central roles in the transcriptional silencing of such genes. However, it is also possible that Hha/YdgT repress gene expression independently of H-NS/StpA, as we have not fully understood the molecular mechanism through which Hha/YdgT modulate H-NS/StpA activity. To gain further insight into the basic functions of Hha/YdgT, we analysed the impact of hha/ydgT double inactivation on the transcriptome profile of Escherichia coli K-12, and compared the effects with that of hns/stpA double inactivation. In addition, we examined the effects of hha/ydgT inactivation on the chromosomal binding of H-NS, and conversely the effects of hns/stpA inactivation on the chromosomal binding of Hha. Our results demonstrated that the chromosomal binding of Hha requires H-NS/StpA, and is necessary for the repression of a subset of genes in the H-NS/StpA regulon. Furthermore, the distribution of H-NS binding around Hha/YdgT-dependent and -independent genes suggests that Hha/YdgT proteins modulate formation of the H-NS/StpA-DNA complex.
Project description:In enterobacteria, AT-rich horizontally acquired genes, including virulence genes, are silenced through the actions of at least three nucleoid-associated proteins (NAPs): H-NS, StpA and Hha. These proteins form gene-silencing nucleoprotein filaments through direct DNA binding by H-NS and StpA homodimers or heterodimers. Both linear and bridged filaments, in which NAPs bind one or two DNA segments, respectively, have been observed. Hha can interact with H-NS or StpA filaments, but itself lacks a DNA-binding domain. Filaments composed of H-NS alone can inhibit transcription initiation and, in the bridged conformation, slow elongating RNA polymerase (RNAP) by promoting backtracking at pause sites. How the other NAPs modulate these effects of H-NS is unknown, despite evidence that they help regulate subsets of silenced genes in vivo (e.g. in pathogenicity islands). Here we report that Hha and StpA greatly enhance H-NS-stimulated pausing by RNAP at 20°C. StpA:H-NS or StpA-only filaments also stimulate pausing at 37°C, a temperature at which Hha:H-NS or H-NS-only filaments have much less effect. In addition, we report that both Hha and StpA greatly stimulate DNA-DNA bridging by H-NS filaments. Together, these observations indicate that Hha and StpA can affect H-NS-mediated gene regulation by stimulating bridging of H-NS/DNA filaments.
Project description:The Hha family of proteins is involved in the regulation of gene expression in enterobacteria by forming complexes with H-NS-like proteins. Whereas several amino acid residues of both proteins participate in the interaction, some of them play a key role. Residue D48 of Hha protein is essential for the interaction with H-NS, thus the D48N substitution in Hha protein abrogates H-NS/Hha interaction. Despite being a paralog of H-NS protein, StpA interacts with HhaD48N with higher affinity than with the wild type Hha protein. To analyze whether Hha is capable of acting independently of H-NS and StpA, we conducted transcriptomic analysis on the hha and stpA deletion strains and the hhaD48N substitution strain of Salmonella Typhimurium using a custom microarray. The results obtained allowed the identification of 120 genes regulated by Hha in an H-NS/StpA-independent manner, 38% of which are horizontally acquired genes. A significant number of the identified genes are involved in functions related to cell motility, iron uptake, and pathogenicity. Thus, motility assays, siderophore detection and intra-macrophage replication assays were performed to confirm the transcriptomic data. Our findings point out the importance of Hha protein as an independent regulator in S. Typhimurium, highlighting a regulatory role on virulence.
Project description:Transcriptomic analysis in a Salmonella enterica Serovar Typhimurium SL 1344 that constitutively expresses stdE and stdF compared with a strain carrying an stdEF deletion A four chip study using total RNA recovered from two separate cultures of Salmonella enterica Serovar Typhimurium SL 1344 constitutively expressing stdE and stdF and two separate cultures of Salmonella enterica Serovar Typhimurium SL 1344 lacking stdE and stdF.
Project description:The H-NS family of proteins has been shown to participate in the regulation of a large number of genes in Gram-negative bacteria in response to environmental factors. In recent years, it has become apparent that proteins of the Hha family are essential elements for H-NS-regulated gene expression. Hha has been shown to bind H-NS, although the details for this interaction are still unknown. In the present paper, we report fluorescence anisotropy and NMR studies of the interaction between Hha and H-NS64, a truncated form of H-NS containing only its N-terminal dimerization domain. We demonstrate the initial formation of a complex between one Hha and two H-NS64 monomers in 150 mM NaCl. This complex seems to act as a nucleation unit for higher-molecular-mass complexes. NMR studies suggest that Hha is in equilibrium between two different conformations, one of which is stabilized by binding to H-NS64. A similar exchange is also observed for Hha in the absence of H-NS when temperature is increased to 37 degrees C, suggesting a key role for intrinsic conformational changes of Hha in modulating its interaction with H-NS.