Genetic Approaches to Study the Interplay Between Transcription and Nucleoid-Associated Proteins in Escherichia coli.

Bacterial nucleoid-associated proteins are important factors in regulation of transcription, in nucleoid structuring, and in homeostasis of DNA supercoiling. Vice versa, transcription influences DNA supercoiling and can affect DNA binding of nucleoid-associated proteins (NAPs) such as H-NS in Escherichia coli. Here we describe genetic tools to study the interplay between transcription and nucleoid-associated proteins in E.

Bacteria employ lysine acetylation of transcriptional regulators to adapt gene expression to cellular metabolism.

The Escherichia coli TetR-related transcriptional regulator RutR is involved in the coordination of pyrimidine and purine metabolism. Here we report that lysine acetylation modulates RutR function. Applying the genetic code expansion concept, we produced site-specifically lysine-acetylated RutR proteins.

High Abundance of Transcription Regulators Compacts the Nucleoid in Escherichia coli.

In enteric bacteria organization of the circular chromosomal DNA into a highly dynamic and toroidal-shaped nucleoid involves various factors, such as DNA supercoiling, nucleoid-associated proteins (NAPs), the structural maintenance of chromatin (SMC) complex, and macrodomain organizing proteins. Here, we show that ectopic expression of transcription regulators at high levels leads to nucleoid compaction. This serendipitous result was obtained by fluorescence microscopy upon ectopic expression of the transcription regulator and phosphodiesterase PdeL of Escherichia coli.

Deletion of FRT-sites by no-SCAR recombineering in .

Lambda-Red recombineering is the most commonly used method to create point mutations, insertions or deletions in and other bacteria, but usually an Flp recognition target (FRT) scar-site is retained in the genome. Alternative scarless recombineering methods, including CRISPR/Cas9-assisted methods, generally require cloning steps and/or complex PCR schemes for specific targeting of the genome. Here we describe the deletion of FRT scar-sites by the scarless Cas9-assisted recombineering method no-SCAR using an FRT-specific guide RNA, sgRNA, and locus-specific ssDNA oligonucleotides.

The transcription regulator and c-di-GMP phosphodiesterase PdeL represses motility in .

PdeL is a transcription regulator and catalytically active c-di-GMP phosphodiesterases (PDE) in PdeL has been shown to be a transcription autoregulator, while no other target genes have been identified so far. Here, we show that PdeL represses transcription of the flagella class II operon, , and activates encoding an extracellular anchored metalloprotease, among additional loci. DNA-binding studies and expression analyses using plasmidic reporters suggest that regulation of the and promoters by PdeL is direct.

Characterization of the pleiotropic LysR-type transcription regulator LeuO of Escherichia coli.

LeuO is a pleiotropic LysR-type transcriptional regulator (LTTR) and co-regulator of the abundant nucleoid-associated repressor protein H-NS in Gammaproteobacteria. As other LTTRs, LeuO is a tetramer that is formed by dimerization of the N-terminal DNA-binding domain (DBD) and C-terminal effector-binding domain (EBD). To characterize the Escherichia coli LeuO protein, we screened for LeuO mutants that activate the cas (CRISPR-associated/Cascade) promoter more effectively than wild-type LeuO.

Genetic Approaches to Study the Interplay Between Transcription and Nucleoid-Associated Proteins in Escherichia coli.

Bacterial nucleoid-associated proteins are important in nucleoid-structuring, homeostasis of DNA supercoiling, and in regulation of transcription. Vice versa, transcription influences DNA supercoiling and possibly DNA-binding of nucleoid-associated proteins. Here, I describe genetic tools to study the interplay between transcription and nucleoid-associated proteins such as H-NS in Escherichia coli.

Interference of transcription across H-NS binding sites and repression by H-NS.

Nucleoid-associated protein H-NS represses transcription by forming extended DNA-H-NS complexes. Repression by H-NS operates mostly at the level of transcription initiation. Less is known about how DNA-H-NS complexes interfere with transcription elongation.

Activation of leuO by LrhA in Escherichia coli.

LeuO is a conserved LysR-type transcription factor of pleiotropic function in Enterobacteria. Regulation of the leuO gene has been best studied in Escherichia coli and Salmonella enterica. Its expression is silenced by the nucleoid-associated proteins H-NS and StpA, autoregulated by LeuO, and in E.

Correlation of Antagonistic Regulation of Transcription with the Cellular Levels of BglJ-RcsB and LeuO in .

LeuO is a conserved and pleiotropic transcription regulator, antagonist of the nucleoid-associated silencer protein H-NS, and important for pathogenicity and multidrug resistance in . Regulation of transcription of the gene is complex. It is silenced by H-NS and its paralog StpA, and it is autoregulated.

Single-cell characterization of metabolic switching in the sugar phosphotransferase system of Escherichia coli.

The utilization of several sugars in Escherichia coli is regulated by the Phosphotransferase System (PTS), in which diverse sugar utilization modules compete for phosphoryl flux from the general PTS proteins. Existing theoretical work predicts a winner-take-all outcome when this flux limits carbon uptake. To date, no experimental work has interrogated competing PTS uptake modules with single-cell resolution.

Interaction of the RcsB Response Regulator with Auxiliary Transcription Regulators in Escherichia coli.

The Rcs phosphorelay is a two-component signal transduction system that is induced by cell envelope stress. RcsB, the response regulator of this signaling system, is a pleiotropic transcription regulator, which is involved in the control of various stress responses, cell division, motility, and biofilm formation. RcsB regulates transcription either as a homodimer or together with auxiliary regulators, such as RcsA, BglJ, and GadE in Escherichia coli.

YjjQ Represses Transcription of flhDC and Additional Loci in Escherichia coli.

Unlabelled: The presumptive transcriptional regulator YjjQ has been identified as being virulence associated in avian pathogenic Escherichia coli (APEC). In this work, we characterize YjjQ as transcriptional repressor of the flhDC operon, encoding the master regulator of flagellar synthesis, and of additional loci. The latter include gfc (capsule 4 synthesis), ompC (outer membrane porin C), yfiRNB (regulated c-di-GMP synthesis), and loci of poorly defined function (ybhL and ymiA-yciX).

Amount of colicin release in Escherichia coli is regulated by lysis gene expression of the colicin E2 operon.

The production of bacteriocins in response to worsening environmental conditions is one means of bacteria to outcompete other microorganisms. Colicins, one class of bacteriocins in Escherichia coli, are effective against closely related Enterobacteriaceae. Current research focuses on production, release and uptake of these toxins by bacteria.

Transcriptional regulation by BglJ-RcsB, a pleiotropic heteromeric activator in Escherichia coli.

The bacterial Rcs phosphorelay signals perturbations of the bacterial cell envelope to its response regulator RcsB, which regulates transcription of multiple loci related to motility, biofilm formation and various stress responses. RcsB is unique, as its set of target loci is modulated by interaction with auxiliary regulators including BglJ. The BglJ-RcsB heteromer is known to activate the HNS repressed leuO and bgl loci independent of RcsB phosphorylation.

RcsB-BglJ-mediated activation of Cascade operon does not induce the maturation of CRISPR RNAs in E. coli K12.

Prokaryotic immunity against foreign nucleic acids mediated by clustered, regularly interspaced, short palindromic repeats (CRISPR) depends on the expression of the CRISPR-associated (Cas) proteins and the formation of small CRISPR RNAs (crRNAs). The crRNA-loaded Cas ribonucleoprotein complexes convey the specific recognition and inactivation of target nucleic acids. In E.

RcsB-BglJ activates the Escherichia coli leuO gene, encoding an H-NS antagonist and pleiotropic regulator of virulence determinants.

The LysR-type transcription factor LeuO is involved in regulation of pathogenicity determinants and stress responses in Enterobacteriaceae, and acts as antagonist of the global repressor H-NS. Expression of the leuO gene is repressed by H-NS, and it is upregulated in stationary phase and under amino acid starvation conditions. Here, we show that the heterodimer of the FixJ/NarL-type transcription regulators RcsB and BglJ strongly activates expression of leuO and that RcsB-BglJ regulates additional loci.

RNase III initiates rapid degradation of proU mRNA upon hypo-osmotic stress in Escherichia coli.

Hyper-osmotic stress strongly induces expression of the Escherichia coli proU operon encoding a high affinity uptake system for the osmoprotectants glycine betaine and proline betaine. Osmoregulation of proU takes place at the transcriptional level by upregulation of the promoter at high osmolarity and repression of transcription by the nucleoid-associated protein H-NS at low osmolarity. In the present study, we describe an additional level of proU osmoregulation that is independent of transcriptional regulation.

Nonlinear fitness landscape of a molecular pathway.

Genes are regulated because their expression involves a fitness cost to the organism. The production of proteins by transcription and translation is a well-known cost factor, but the enzymatic activity of the proteins produced can also reduce fitness, depending on the internal state and the environment of the cell. Here, we map the fitness costs of a key metabolic network, the lactose utilization pathway in Escherichia coli.

BglJ-RcsB heterodimers relieve repression of the Escherichia coli bgl operon by H-NS.

RcsB is the response regulator of the complex Rcs two-component system, which senses perturbations in the outer membrane and peptidoglycan layer. BglJ is a transcriptional regulator whose constitutive expression causes activation of the H-NS- and StpA-repressed bgl (aryl-β,D-glucoside) operon in Escherichia coli. RcsB and BglJ both belong to the LuxR-type family of transcriptional regulators with a characteristic C-terminal DNA-binding domain.

H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO.

The recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E.

Fate of the H-NS-repressed bgl operon in evolution of Escherichia coli.

In the enterobacterial species Escherichia coli and Salmonella enterica, expression of horizontally acquired genes with a higher than average AT content is repressed by the nucleoid-associated protein H-NS. A classical example of an H-NS-repressed locus is the bgl (aryl-beta,D-glucoside) operon of E. coli.

Characterization of a beta-glucoside operon (bgc) prevalent in septicemic and uropathogenic Escherichia coli strains.

Escherichia coli strains, in general, do not ferment cellobiose and aryl-beta-D-glucosidic sugars, although "cryptic" beta-d-glucoside systems have been characterized. Here we describe an additional cryptic operon (bgc) for the utilization of cellobiose and the aryl-beta-d-glucosides arbutin and salicin at low temperature. The bgc operon was identified by the characterization of beta-glucoside-positive mutants of an E.

Fine-tuned growth phase control of dps, encoding a DNA protection protein, by FIS and H-NS.

Dps, a ferritin-like DNA-binding protein, protects bacterial DNA against oxidative stress and hyper-compacts the nucleoid into a crystalline-like structure. In Escherichia coli, transcription of dps from a single promoter is directed by sigma S-RNA polymerase in stationary phase and by sigma 70-RNA polymerase in fast-dividing cells exposed to oxidative stress. In this issue of Molecular Microbiology, Grainger and co-workers demonstrate that the nucleoid-associated proteins, FIS and H-NS, control sigma factor selection at the dps promoter by a novel mechanism.

Characterization of a yjjQ mutant of avian pathogenic Escherichia coli (APEC).

Infections with extraintestinal avian pathogenic Escherichia coli (APEC) cause significant economic losses in the poultry industry worldwide. In a previous study we applied signature-tagged transposon mutagenesis and identified 28 virulence-associated genes in APEC strain IMT5155 (O2 : H5 : K1). One of them, yjjQ, encodes a putative transcriptional regulator whose function and role in pathogenesis are still unknown.