Conserved and Variable Functions of the σ(E) Stress Response in Related Genomes

Bacteria often cope with environmental stress by inducing alternative sigma (σ) factors, which direct RNA polymerase to specific promoters, thereby inducing a set of genes called a regulon to combat the stress. To understand the conserved and organism-specific functions of each σ, it is necessary to be able to predict their promoters, so that their regulons can be followed across species. However, the variability of promoter sequences and motif spacing makes their prediction difficult. We developed and validated an accurate promoter prediction model for Escherichia coli σ(E), which enabled us to predict a total of 89 unique σ(E)-controlled transcription units in E. coli K-12 and eight related genomes. σ(E) controls the envelope stress response in E. coli K-12. The portion of the regulon conserved across genomes is functionally coherent, ensuring the synthesis, assembly, and homeostasis of lipopolysaccharide and outer membrane porins, the key constituents of the outer membrane of Gram-negative bacteria. The larger variable portion is predicted to perform pathogenesis-associated functions, suggesting that σ(E) provides organism-specific functions necessary for optimal host interaction. The success of our promoter prediction model for σ(E) suggests that it will be applicable for the prediction of promoter elements for many alternative σ factors

Dual Molecular Signals Mediate the Bacterial Response to Outer-Membrane Stress

In Gram-negative bacteria, outer-membrane integrity is essential for survival and is monitored by the σ[superscript E] stress-response system, which initiates damage-repair pathways. One activating signal is unassembled outer-membrane proteins. Using biochemical and genetic experiments in Escherichia coli, we found that off-pathway intermediates in lipopolysaccharide transport and assembly provided an additional required signal. These distinct signals, arising from disruptions in the transport and assembly of the major outer-membrane components, jointly determined the rate of proteolytic destruction of a negative regulator of the σ[superscript E] transcription factor, thereby modulating the expression of stress-response genes. This dual-signal system permits a rapid response to dysfunction in outer-membrane biogenesis, while buffering responses to transient fluctuations in individual components, and may represent a broad strategy for bacteria to monitor their interface with the environment.National Institutes of Health (U.S.) (Grant AI-16892)National Institutes of Health (U.S.) (Grant GM-36278

Covalent intermediate in the catalytic mechanism of the radical S-adenosyl-L-methionine methyl synthase RlmN trapped by mutagenesis.

The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical S-adenosyl-L-methionine (SAM) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity. To accomplish this reaction, RlmN and Cfr use a shared radical-mediated mechanism. In addition to the radical SAM CX(3)CX(2)C motif, both RlmN and Cfr contain two conserved cysteine residues required for in vivo function, putatively to form (cysteine 355 in RlmN) and resolve (cysteine 118 in RlmN) a covalent intermediate needed to achieve this challenging transformation. Currently, there is no direct evidence for this proposed covalent intermediate. We have further investigated the roles of these conserved cysteines in the mechanism of RlmN. Cysteine 118 mutants of RlmN are unable to resolve the covalent intermediate, either in vivo or in vitro, enabling us to isolate and characterize this intermediate. Additionally, tandem mass spectrometric analyses of mutant RlmN reveal a methylene-linked adenosine modification at cysteine 355. Employing deuterium-labeled SAM and RNA substrates in vitro has allowed us to further clarify the mechanism of formation of this intermediate. Together, these experiments provide compelling evidence for the formation of a covalent intermediate species between RlmN and its rRNA substrate and well as the roles of the conserved cysteine residues in catalysis

Lack of a robust unfoldase activity confers a unique level of substrate specificity to the universal AAA protease FtsH

In the present study, we have investigated the mechaases, a class that includes the eukaryotic 26S proteanism of degradation by FtsH. We previously showed that some and its structurally related prokaryotic counterFtsH recognized and degraded proteins with nonpolar parts. In the bacterium E. coli, five such ATP-dependent carboxy-terminal tails in vivo (Herman et al., 1998). Two proteases have thus far been identified: ClpAP, ClpXP, classes of nonpolar tails were identified: those recogHslUV, Lon, and FtsH (HflB). Among these, FtsH is the nized solely by FtsH in vivo (e.g., 108; to their overall thermodynamic stability. This was also FtsH is a membrane-anchored metallo-protease with true for the degradation of a membrane protein and its its active site facing the cytoplasm. It contains a wellvariants studied in vivo. Thus, unlike other well-studied conserved 200 amino acid motif called the "AAA" motif ATP-dependent proteases, FtsH appears to lack robust (so named because the diverse functions of its member unfoldase activity. Instead, our experiments are consisproteins are ATPase associated with activity [reviewed tent with the idea that ATP hydrolysis by FtsH is mainly in Ogura and Wilkinson, 2001]). FtsH degrades both inteused to translocate unfolded substrates sequentially gral membrane and cytoplasmic proteins. FtsH performs from the recognition signal to the active site. We propose that lack of a robust unfoldase enables FtsH to discriminate among proteins based on their thermodynamic sta

Sequential Vinorelbine and Docetaxel in Advanced Non-small Cell Lung Cancer Patients Age 70 and Older and/or with a Performance Status of 2: A Phase II Trial of the Southwest Oncology Group (S0027)

BackgroundThis phase II study (S0027) evaluated the efficacy and tolerability of planned sequential single-agent chemotherapy with vinorelbine followed by docetaxel in patients with advanced non-small cell lung cancer (NSCLC) age 70 and older and/or a performance status (PS) of 2.MethodsPatients with stage IIIB (pleural effusion) or stage IV NSCLC, age 70 and older with a PS of 0-1 or 2, any age, received three cycles of vinorelbine 25 mg/m2 days 1 and 8 every 21 days followed by three cycles of docetaxel 35 mg/m2 days 1, 8, and 15 every 28 days.ResultsA total of 125 patients entered the study; 117 patients were assessable for response, survival, and toxicity. Seventy-five patients were in stratum1 (age 70 and older, PS 0-1) and 42 patients in stratum 2 (PS 2, any age). Objective response was 19% (95% confidence interval [CI]: 11%–30%) and 11% (95% CI: 3%–25%) in strata 1 and 2, respectively. Median survival was 9.1 months (95% CI: 7.1–12.7) and 5.5 months (95% CI: 3.1–6.5) in strata 1 and 2, respectively. Survival at 12 months was 41% and 13% in strata 1 and 2, respectively. Grade 3/4 neutropenia was seen in 32% and 31% of patients in strata 1 and 2, respectively. Three deaths probably related to treatment were noted: one in stratum 1 and two in stratum 2.ConclusionSequential vinorelbine and docetaxel is a well-tolerated and effective regimen in comparison with reports of other treatments tested in patients with advanced NSCLC age 70 and older and/or with a PS of 2

Module-Based Analysis of Robustness Tradeoffs in the Heat Shock Response System

Biological systems have evolved complex regulatory mechanisms, even in situations where much simpler designs seem to be sufficient for generating nominal functionality. Using module-based analysis coupled with rigorous mathematical comparisons, we propose that in analogy to control engineering architectures, the complexity of cellular systems and the presence of hierarchical modular structures can be attributed to the necessity of achieving robustness. We employ the Escherichia coli heat shock response system, a strongly conserved cellular mechanism, as an example to explore the design principles of such modular architectures. In the heat shock response system, the sigma-factor σ(32) is a central regulator that integrates multiple feedforward and feedback modules. Each of these modules provides a different type of robustness with its inherent tradeoffs in terms of transient response and efficiency. We demonstrate how the overall architecture of the system balances such tradeoffs. An extensive mathematical exploration nevertheless points to the existence of an array of alternative strategies for the existing heat shock response that could exhibit similar behavior. We therefore deduce that the evolutionary constraints facing the system might have steered its architecture toward one of many robustly functional solutions

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.National Human Genome Research Institute (U.S.) (Grant P50HG006193

Коло Марусі Чурай

In this article Marusya Churay*s (a character famous in story and song) life history is researched. On the basis of real events and historical facts the author tells about people who were related to the life of this personality

High-throughput, quantitative analyses of genetic interactions in E. coli.

Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli