A simple and unified protocol to purify all seven Escherichia coli RNA polymerase sigma factors.

RNA polymerase sigma factors are indispensable in the process of bacterial transcription. They are responsible for a given gene's promoter region recognition on template DNA and hence determine specificity of RNA polymerase and play a significant role in gene expression regulation. Here, we present a simple and unified protocol for purification of all seven Escherichia coli RNA polymerase sigma factors.

LIC_12757 from the pathogenic spirochaete Leptospira interrogans encodes an autoregulated ECF σ-type factor.

ECF (extracytoplasmic function) σ factors, members of the σ-family, are the largest class of alternative σ factors which are stimulated in the presence of specific signals and direct RNA polymerase to transcribe a defined subset of genes. Thanks to them, bacterial pathogens can effectively reprogram their gene expression and, consequently, survive in the host and establish infection in a relatively short time. The number of ECF σ factors encoded within bacterial genomes is different depending on a given species and it reflects the likelihood that these bacteria will encounter harsh environmental conditions.

Increased Levels of (p)ppGpp Correlate with Virulence and Biofilm Formation, but Not with Growth, in Strains of Uropathogenic .

Urinary tract infections are one of the most frequent bacterial diseases worldwide. UPECs are the most prominent group of bacterial strains among pathogens responsible for prompting such infections. As a group, these extra-intestinal infection-causing bacteria have developed specific features that allow them to sustain and develop in their inhabited niche of the urinary tract.

Molecular basis for lethal cross-talk between two unrelated bacterial transcription factors - the regulatory protein of a restriction-modification system and the repressor of a defective prophage.

Bacterial gene expression depends on the efficient functioning of global transcriptional networks, however their interconnectivity and orchestration rely mainly on the action of individual DNA binding proteins called transcription factors (TFs). TFs interact not only with their specific target sites, but also with secondary (off-target) sites, and vary in their promiscuity. It is not clear yet what mechanisms govern the interactions with secondary sites, and how such rewiring affects the overall regulatory network, but this could clearly constrain horizontal gene transfer.

Three Microbial Musketeers of the Seas: , and , and Their Adaptation to Different Salinity Probed by a Proteomic Approach.

Osmotic changes are common challenges for marine microorganisms. Bacteria have developed numerous ways of dealing with this stress, including reprogramming of global cellular processes. However, specific molecular adaptation mechanisms to osmotic stress have mainly been investigated in terrestrial model bacteria.

Minigene as a Novel Regulatory Element in Toxin-Antitoxin Systems.

The type II toxin-antitoxin (TA) system is characterized by a complex and multilayered mode of gene expression regulation. Precise and tight control of this process is crucial to keep the toxin in an appropriate balance with the cognate antitoxin until its activation is needed for the cell. In this report, we provide evidence that a minigene encoded within the operon influences translation of the Txe toxin.

Virus-Host Interaction Gets . PART II: Functional Transcriptomics of the DksA-Deficient Cell upon Phage P1 Infection.

The virus-host interaction requires a complex interplay between the phage strategy of reprogramming the host machinery to produce and release progeny virions, and the host defense against infection. Using RNA sequencing, we investigated the phage-host interaction to resolve the phenomenon of improved lytic development of P1 phage in a DksA-deficient host. Expression of the and P1 genes in the wild-type host was the highest among all and most probably leads to phage virulence.

Virus-Host Interaction Gets . PART I: Phage P1 Enhanced Development in an DksA-Deficient Cell.

Bacteriophage P1 is among the best described bacterial viruses used in molecular biology. Here, we report that deficiency in the host cell DksA protein, an global transcription regulator, improves P1 lytic development. Using genetic and microbiological approaches, we investigated several aspects of P1 biology in an attempt to understand the basis of this phenomenon.

Therapeutic Implications of Targeting Heat Shock Protein 70 by Immunization or Antibodies in Experimental Skin Inflammation.

Heat shock proteins (Hsp) are constitutive and stress-induced molecules which have been reported to impact innate and adaptive immune responses. Here, we evaluated the role of Hsp70 as a treatment target in the imiquimod-induced, psoriasis-like skin inflammation mouse model and related assays. We found that immunization of mice with Hsp70 resulted in decreased clinical and histological disease severity associated with expansion of T cells in favor of regulatory subtypes (CD4FoxP3/CD4CD25 cells).

Insights into Transcriptional Repression of the Homologous Toxin-Antitoxin Cassettes and .

Transcriptional repression is a mechanism which enables effective gene expression switch off. The activity of most of type II toxin-antitoxin (TA) cassettes is controlled in this way. These cassettes undergo negative autoregulation by the TA protein complex which binds to the promoter/operator sequence and blocks transcription initiation of the TA operon.

Estimates of Rel, Mesh1, and SAH Hydrolysis of (p)ppGpp and (p)ppApp by Thin Layer Chromatography and NADP/NADH Coupled Assays.

The Mesh1 class of hydrolases found in bacteria, metazoans and humans was discovered as able to cleave an intact pyrophosphate residue esterified on the 3'hydroxyl of (p)ppGpp in a Mn dependent reaction. Here, thin layer chromatography (TLC) qualitative evidence is presented indicating the substrate specificity of Mesh1 from and human MESH1 also extends to the (p)ppApp purine analogs. More importantly, we developed real time enzymatic assays, coupling ppNpp hydrolysis to NADH oxidation and pppNpp hydrolysis to NADP reduction, which facilitate estimation of kinetic constants.

Mutational analysis of Escherichia coli GreA protein reveals new functional activity independent of antipause and lethal when overexpressed.

There is a growing appreciation for the diverse regulatory consequences of the family of proteins that bind to the secondary channel of E. coli RNA polymerase (RNAP), such as GreA, GreB or DksA. Similar binding sites could suggest a competition between them.

Autologous extracellular Hsp70 exerts a dual role in rheumatoid arthritis.

Extracellular heat shock proteins (Hsp) influence the adaptive immune response and may ameliorate pathogenesis of autoimmune diseases. While some preclinical observations suggest that highly conserved bacterial and/or murine Hsp70 peptides have potential utility in treatment of rheumatoid arthritis (RA) via induction of T regulatory cells (Treg), the role of extracellular inducible human Hsp70 in adaptive immune processes requires further investigation. The present study evaluated Hsp70 influence on inflammatory cytokine-mediated modulation of T cell immunophenotype in ways that influence RA onset and severity.

Identification of σ-Dependent Promoter Upstream of from the Pathogenic Spirochaete by Applying an Two-Plasmid System.

There is limited information on gene expression in the pathogenic spirochaete and genetic mechanisms controlling its virulence. Transcription is the first step in gene expression that is often determined by environmental effects, including infection-induced stresses. Alterations in the environment result in significant changes in the transcription of many genes, allowing effective adaptation of to mammalian hosts.

Autoregulation of Expression Relies on GraL Rather than on Promoter Region.

GreA is a well-characterized transcriptional factor that acts primarily by rescuing stalled RNA polymerase complexes, but has also been shown to be the major transcriptional fidelity and proofreading factor, while it inhibits DNA break repair. Regulation of gene expression itself is still not well understood. So far, it has been shown that its expression is driven by two overlapping promoters and that leader encodes a small RNA (GraL) that is acting on mRNA.

RSH Enzyme Synthesizes (p)ppGpp and pppApp and , and Leads to Discovery of pppApp Synthesis in .

In bacteria, the so-called stringent response is responsible for adaptation to changing environmental conditions. This response is mediated by guanosine derivatives [(p)ppGpp], synthesized by either large mono-functional RelA or bi-functional SpoT (synthesis and hydrolysis) enzymes in β and γproteobacteria, such as . In Firmicutes and α, δ, and 𝜀proteobacteria, large bifunctional Rel-SpoT-homologs (RSH), often accompanied by small (p)ppGpp synthetases and/or hydrolases devoid of regulatory domains, are responsible for (p)ppGpp turnover.

[50th anniversary of discovering „magic spots” – the latest advances in (p)ppGpp research].

About 50 years ago, "magic spots" - mediators of the bacterial stringent response, were discovered and were later identified as guanosine tetra- and pentaphosphate (ppGpp and pppGpp, jointly referred to as (p)ppGpp). At first, it seemed that stringent response is associated only with bacterial response to amino acid starvation, however, it soon turned out that (p)ppGpp is synthesized in response to other stresses as well. The mentioned alarmones are found to exist in all known bacterial species, as well as in plants.

Structure-function comparisons of (p)ppApp vs (p)ppGpp for Escherichia coli RNA polymerase binding sites and for rrnB P1 promoter regulatory responses in vitro.

Precise regulation of gene expression is crucial for bacteria to respond to changing environmental conditions. In addition to protein factors affecting RNA polymerase (RNAP) activity, second messengers play an important role in transcription regulation, such as well-known effectors of the stringent response: guanosine 5'triphosphate-3'diphosphate and guanosine 3', 5'-bis(diphosphate) [(p)ppGpp]. Although much is known about importance of the 5' and 3' moieties of (p)ppGpp, the role of the guanine base remains somewhat cryptic.

A search for the in trans role of GraL, an Escherichia coli small RNA.

Small RNA are very important post-transcriptional regulators in both, bacteria and eukaryotes. One of such sRNA is GraL, encoded in the greA leader region and conserved among enteric bacteria. Here, we conducted a bioinformatics search for GraL's targets in trans and validated our findings in vivo by constructing fusions of probable targets with lacZ and measuring their activity when GraL was overexpressed.

From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis.

Unlabelled: The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E.

Differential regulation by ppGpp versus pppGpp in Escherichia coli.

Both ppGpp and pppGpp are thought to function collectively as second messengers for many complex cellular responses to nutritional stress throughout biology. There are few indications that their regulatory effects might be different; however, this question has been largely unexplored for lack of an ability to experimentally manipulate the relative abundance of ppGpp and pppGpp. Here, we achieve preferential accumulation of either ppGpp or pppGpp with Escherichia coli strains through induction of different Streptococcal (p)ppGpp synthetase fragments.