The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally-acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization site-1 and site-2, which are both required for H-NS oligomerization, but the exact role of dimerization site-2 in gene silencing is unclear. To this end, we constructed an whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site-2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA, but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all these four variants formed condensed complexes on DNA, whereas wild-type H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that the dimerization site-2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing.

Excessive accumulation of polyamines causes cytotoxicity, including inhibition of cell growth and a decrease in viability. We investigated the mechanism of cytotoxicity caused by spermidine accumulation under various conditions using an Escherichia coli strain deficient in spermidine acetyltransferase (SAT), a key catabolic enzyme in controlling polyamine levels. Due to the excessive accumulation of polyamines by the addition of exogenous spermidine to the growth medium, cell growth and viability were markedly decreased through translational repression of specific proteins [RMF (ribosome modulation factor) and Fis (rRNA transcription factor) etc.] encoded by members of polyamine modulon, which are essential for cell growth and viability. In particular, synthesis of proteins that have unusual locations of the Shine–Dalgarno (SD) sequence in their mRNAs was inhibited. In order to elucidate the molecular mechanism of cytotoxicity by the excessive accumulation of spermidine, the spermidine-dependent structural change of the bulged-out region in the mRNA at the initiation site of the rmf mRNA was examined using NMR analysis. It was suggested that the structure of the mRNA bulged-out region is affected by excess spermidine, so the SD sequence of the rmf mRNA cannot approach initiation codon AUG.

Enterobacteria are able to survive under stressful conditions within animals, such as acidic conditions in the stomach, bile salts during transfer to the intestine and anaerobic conditions within the intestine. The glutamate-dependent (GAD) system plays a major role in acid resistance in Escherichia coli, and expression of the GAD system is controlled by the regulatory cascade consisting of EvgAS > YdeO > GadE. To understand the YdeO regulon in vivo, we used ChIP-chip to interrogate the E. coli genome for candidate YdeO binding sites. All of the seven operons identified by ChIP-chip as being potentially regulated by YdeO were confirmed as being under the direct control of YdeO using RT-qPCR, EMSA, DNaseI-footprinting and reporter assays. Within this YdeO regulon, we identified four stress-response transcription factors, DctR, NhaR, GadE, and GadW and enzymes for anaerobic respiration. Both GadE and GadW are involved in regulation of the GAD system and NhaR is an activator for the sodium/proton antiporter gene. In conjunction with co-transcribed Slp, DctR is involved in protection against metabolic endoproducts under acidic conditions. Taken all together, we suggest that YdeO is a key regulator of E. coli survival in both acidic and anaerobic conditions.

Cysteine synthase A encoded by cysK catalyzes the synthesis of cysteine from O-acetylserine. Expression of cysK in E scherichia coli is under the cont

Prokaryotic DNA-binding transcription factors (TFs) bind in close vicinity of the promoter and regulate transcription through interplay with the DNA-dependent RNA polymerase. Promoters associated with the genes involved in stress response have recently been found to be under the control of multiple regulators, each monitoring one specific environmental condition or factor. In order to identify TFs involved in regulation of one specific promoter, we have developed a PS-TF (promoter-specific TF) screening system, in which the binding of purified TFs to a test promoter was analysed by gel-shift assay. This PS-TF screening system was applied for detection of TFs involved in regulation of the promoter for the Escherichia coli sdiA gene encoding the master regulator of cell division and quorum sensing. After screening of a total of 191 purified TFs (two-thirds of the predicted E. coli TFs), at least 15 TFs have been identified to bind to the sdiA promoter, including five two-component system (TCS) regulators, ArcA, CpxR, OmpR, RcsB and TorR. In this study, we focus on these five TFs for detailed analysis of their regulatory roles in vivo. Under normal growth conditions in LB medium, all these TFs repressed the sdiA promoter and the repression levels correlated with their intracellular levels. Taken together, we propose that these TCS regulators repress transcription in vivo of the sdiA gene, ultimately leading to suppression of cell division.

E. coli cells encounter various environments from the mouth down to the intestines within the host animals. The pH of gastric juice is lower than 2.0, and the bacterial must quickly respond and adapt to the following environmental changes before reaching the intestines.