Functional genomics of a food-related thermotolerant Acetobacter oryzifermentans strain AAB5: genetic determinants of stress response, CAZyme repertoire, and CRISPR-Cas system.

The paper presents a summary of results considering cholinergic hypothesis on the central processes of human stress response and depression caused by chronic social stress. These studies include both the work of clinicians, who are examining pathogenesis of depression, and experiments with rodents. The authors also consider the history of stress response discovery in humans and vertebrates. The paper analyses the results of early studies (1980s-1990s), which led to the hypothesis on the key role of brain cholinergic system in nonspecific stress response of the whole multicellular organism of humans and rodents. Special attention is paid to the latest ideas on acetylcholine as the most powerful neuromodulator in the brain. Acetylcholine is said to transmit large volumes of information. The authors conducted the summary of experimental and clinical studies showing that the primary reaction of humans and rodents under stress is manifested in the activation of brain cholinergic transmission, but not in subsequent changes in serotonergic system functioning. The paper analyses numerous studies, the result of which confirmed the cholinergic hypothesis on stress and depression. These facts are as follows: firstly, the increase in the acetylcholine level in the brain or in the hippocampus (physostigmine injection) causes similar changes in the behavior and physiological state of rodents under stress; secondly, single-time stress increases the acetylcholine level in several areas of the brain, thus, correlating to adaptive changes in behavior; thirdly, chronic stress that causes disturbances in rodent behavior, which are similar to depression, is accompanied by an increase of the acetylcholine level in the brain. In general, the authors come to the conclusion that ideas on the key role of cholinergic system in stress response can serve as a scientific basis for further research on the depression mechanisms both clinically and experimentally Keywords: stress, depression, acetylcholine, neuromodulator, behavior.

Sulfolobus acidocaldarius is a thermoacidophilic Crenarchaeon (78°C and pH 2-3) with established genetic systems. With these properties, S. acidocaldarius offers high potential for biotechnological applications as alternative host for the production of bulk and fine chemicals, solvents and second generation biofuels, which are, however, often toxic to cells. Biofilms, the dominant lifestyle of microorganisms, were shown to confer higher resistance towards solvent exposure and hence turn more and more into research focus for biotechnological applications. Although S. acidocaldarius is known to form biofilms, the development of suitable incubation systems is still in its beginning and also the influence of organic solvents on S. acidocaldarius has only rarely been studied. In this work, incubation systems for the cultivation of submerse biofilms of S. acidocaldarius cells on glass and polystyrene surfaces in microtiter plates, μ- and Petri-dishes as well as a flow-through system were successfully developed and optimized. These newly developed methods enabled detailed microscopic analyses and also yielded sufficient amounts of biofilms for isolation and analyses of extracellular polymeric substances (EPS) as well as for application of various -omics methods. The analyses were performed in presence and absence of organic solvents (with special respect to 1-butanol) and the results were compared to shaking cultures. S. acidocaldarius formed biofilms even in shaking cultures in Erlenmeyer flasks at the liquid/glass/air interface as a (stress)response to sublethal concentration of different solvents. In accordance with shaking cultures, in a 96-well plate static incubation system, the tendency of enhanced biofilm formation as a response to sublethal concentrations of 1-butanol (<1.5% (v/v)) was visible. By the use of an adapted resazurin assay, indicating respiratory activity, the viability of biofilm cells was confirmed colorimetrically. Furthermore, in static incubation systems e.g. on glass slides, S. acidocaldarius formed monolayered biofilms of homogenously distributed cells even in the absence of solvents as shown by crystal violet staining and subsequent light microscopy. In the presence of solvents these biofilms appeared thicker and showed an altered morphology with a heterogenous distribution and cell aggregate formation, indicated by enhanced crystal violet binding. Additionally, visualised by scanning electron microscopy, under 1.5% (v/v) 1-butanol exposure the aggregated cells were surrounded by extracellular material, probable EPS. Furthermore, the cell morphology was altered compared to the control experiments showing a diversified cell envelope with holes. In the presence of 1% (v/v) 1-butanol, less changes in biofilm morphology and no changes in cell morphology were observed by the application of atomic force microscopy. Lectin binding and subsequent confocal laser-scanning microscopy of submersed biofilms showed an increased amount of carbohydrate structures, especially α-mannopyranosyl- and α-glucopyranosyl residues. The enhanced EPS formation was also confirmed by EPS isolation and quantitative determination using colorimetric assays, which showed a 5-fold increase of carbohydrates within the extracted EPS. The amount of proteins was even 19-fold higher compared to the control without butanol, indicating that EPS formation in biofilms is significantly increased in the presence of toxic solvent concentration. Beside the influence of 1-butanol on changes in biofilm morphology and EPS formation, the response of S. acidocaldarius towards changes in lifestyle and 1-butanol was investigated by transcriptomic and proteomic studies. For this, S. acidocaldarius was cultivated as shaking culture in Erlenmeyer flasks without 1-butanol exposure and statically in Petri-dishes for biofilm and planktonic growth in the absence and presence of 1-butanol. Statically grown biofilm and planktonic cells in Petri-dishes showed only less differentially regulated genes and proteins. Lots of differentially regulated genes and proteins were found in cells of shaking cultures and statically incubated planktonic cells. In the presence of 1% (v/v) 1-butanol, genes and proteins are predominantly down-regulated, especially proteins containing transmembrane helices, confirmed by transcriptomic analyses. In planktonic as well as biofilm cells in the presence of 1-butanol, the archaellum operon, responsible for cell motility, was down-regulated. Generally, in response to 1-butanol and different lifestyles, changes in cell division and vesicle formation, stress response systems like the CRISPR-Cas and toxin-antitoxin system, transcriptional regulators and protein kinases and phosphatases (signal transduction) were observed. In conclusion, this study gives an overview of the stress response of S. acidocaldarius towards organic solvents and 1-butanol in detail. For the first time the influence of organic solvents on biofilm formation was described and investigated by various microscopic methods and EPS isolation and quantification. In addition, transcriptomic and proteomic studies were performed to investigate genome-wide and cellular responses towards different lifestyles and 1-butanol.

SUMMARYFunctional genomics is the use of systematic gene perturbation approaches to determine the contributions of genes under conditions of interest. Although functional genomic strategies have been used in bacteria for decades, recent studies have taken advantage of CRISPR (clustered regularly interspaced short palindromic repeats) technologies, such as CRISPRi (CRISPR interference), that are capable of precisely modulating expression of all genes in the genome. Here, we discuss and review the use of CRISPRi and related technologies for bacterial functional genomics. We discuss the strengths and weaknesses of CRISPRi as well as design considerations for CRISPRi genetic screens. We also review examples of how CRISPRi screens have defined relevant genetic targets for medical and industrial applications. Finally, we outline a few of the many possible directions that could be pursued using CRISPR-based functional genomics in bacteria. Our view is that the most exciting screens and discoveries are yet to come.

Exploiting SNPs for biallelic CRISPR mutations in the outcrossing woody perennial Populus reveals 4-coumarate:CoA ligase specificity and redundancy.

Serotonin, one of the first neurotransmitters to be identified, is an evolutionarily old molecule that is highly conserved across the animal kingdom, and widely used throughout the brain. Despite this, ascribing a specific set of functions to brain serotonin and its receptors has been difficult and controversial. The 2A subtype of serotonin receptors (5-HT2A receptor) is the major excitatory serotonin receptor in the brain and has been linked to the effects of drugs that produce profound sensory and cognitive changes. Numerous studies have shown that this receptor is upregulated by a broad variety of stressors, and have related 5-HT2A receptor function to associative learning. This review proposes that stress, particularly stress related to danger and existential threats, increases the expression and function of 5-HT2A receptors. It is argued that this is a neurobiological adaptation to promote learning and avoidance of danger in the future. Upregulation of 5-HT2A receptors during stressful events forms associations that tune the brain to environmental cues that signal danger. It is speculated that life-threatening situations may activate this system and contribute to the symptoms associated with post-traumatic stress disorder (PTSD). 3,4-Methylenedioxymethamphetamine, which activates 5-HT2A receptors, has been successful in the treatment of PTSD and has recently achieved status as a breakthrough therapy. An argument is presented that 3,4-methylenedioxymethamphetamine may paradoxically act through these same 5-HT2A receptors to ameliorate the symptoms of PTSD. The central thematic contention is that a key role of serotonin may be to function as a stress detection and response system.

Chapter Nineteen - Neuropeptide Regulation of the Locus Coeruleus and Opiate-Induced Plasticity of Stress Responses

Anticonceptivos hormonales orales, coagulación y trombosis

A CRISPR view on autophagy.

Since the evolution of the cellular phenotype, prokaryotic and eukaryotic cells have had to cope with adverse changes in their environment. Although cells have evolved many distinct stress responses, this chapter will focus on three major, highly conserved, response systems, i.e., the genotoxic response system, which is activated by DNA damage; the oxidative stress response system, which is activated by excess reactive oxygen species (ROS) and imbalances in the oxidant/antioxidant status within cells; and the heat shock response, which is activated by exposure to heat and other agents that adversely affect protein folding (Fig. 1). The sections dealing with each of the stress response systems begin with a description of the prokaryotic stress response because, in most instances, the prokaryotic systems are the best understood. This is followed by a discussion of the eukaryotic stress response systems, focusing on yeast and mammals. Finally, each section concludes with a discussion about what is known concerning the induction of these stress response systems in mammalian embryos, particularly postimplantation mammalian embryos. Normal embryonic development requires a precisely orchestrated chain of temporal and spatial events, and any alterations in this chain could lead to altered development and subsequent pathogenesis. Although the mammalian embryo develops within the protective environment of the uterus, this protection is not absolute and we now know that mammalian development can be perturbed by a wide variety of chemical and physical agents, many of which are known to induce one or more of these stress systems in nonembryonic systems. Thus understanding the embryo’s stress response capabilities is essential to the understanding of how developmental toxicants exert their toxicity.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been used as a complementary typing tool in diagnostic, epidemiologic, and evolutionary analyses of prokaryotic strains. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. Key elements of this unique prokaryotic defense system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading viruses and plasmids, generally followed by the degradation of the invader. In addition, several recent studies have pointed at direct links of CRISPR-Cas to regulation of a range of stress-related phenomena. An interesting example concerns a pathogenic bacterium that possesses a CRISPR-associated ribonucleoprotein complex that may play a dual role in defense and/or virulence. In this review, we describe recently reported cases of potential involvement of CRISPR-Cas systems in bacterial stress responses in general and bacterial virulence in particular.

Listeria spp. are pathogens widely distributed in the environment and Listeria monocytogenes is associated with food-borne illness in humans. Food facilities represent an adverse environment for this bacterium, mainly due to the disinfection and cleaning processes included in good hygiene practices, and its virulence is related to stress responses. One of the recently described stress-response systems is CRISPR-Cas. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (cas) genes have been found in several bacteria. CRISPR-Cas has revolutionized biotechnology since it acts as an adaptive immune system of bacteria, which also helps in the evasion of the host immune response. There are three CRISPR systems described on Listeria species. Type II is present in many pathogenic bacteria and characterized by the presence of cas9 that becomes the main target of some anti-CRISPR proteins, such as AcrIIA1, encoded on Listeria phages. The presence of Cas9, either alone or in combination with anti-CRISPR proteins, suggests having a main role on the virulence of bacteria. In this review, we describe the most recent information on CRISPR-Cas systems in Listeria spp., particularly in L. monocytogenes, and their relationship with the virulence and pathogenicity of those bacteria. Besides, some applications of CRISPR systems and future challenges in the food processing industry, bacterial vaccination, antimicrobial resistance, pathogens biocontrol by phage therapy, and regulation of gene expression have been explored.

Bacteria protect themselves from infection by bacteriophages (phages) using different defence systems, such as CRISPR-Cas. Although CRISPR-Cas provides phage resistance, fitness costs are incurred, such as through autoimmunity. CRISPR-Cas regulation can optimise defence and minimise these costs. We recently developed a genome-wide functional genomics approach (SorTn-seq) for high-throughput discovery of regulators of bacterial gene expression. Here, we applied SorTn-seq to identify loci influencing expression of the two type III-A Serratia CRISPR arrays. Multiple genes affected CRISPR expression, including those involved in outer membrane and lipopolysaccharide synthesis. By comparing loci affecting type III CRISPR arrays and cas operon expression, we identified PigU (LrhA) as a repressor that co-ordinately controls both arrays and cas genes. By repressing type III-A CRISPR-Cas expression, PigU shuts off CRISPR-Cas interference against plasmids and phages. PigU also represses interference and CRISPR adaptation by the type I-F system, which is also present in Serratia. RNA sequencing demonstrated that PigU is a global regulator that controls secondary metabolite production and motility, in addition to CRISPR-Cas immunity. Increased PigU also resulted in elevated expression of three Serratia prophages, indicating their likely induction upon sensing PigU-induced cellular changes. In summary, PigU is a major regulator of CRISPR-Cas immunity in Serratia.

Virulence of pathogenic bacteria is often determined by their ability to adapt to stress. The Brucella abortus general stress response (GSR) system is required for chronic mammalian infection and is regulated by phosphorylation and proteolysis. The B. abortus GSR signaling pathway has multiple layers of post-translational control and is a determinant of chronic infection. This study provides new, molecular level insight into chronic Brucella infection. Brucella spp. are adept at establishing a chronic infection in mammals. We demonstrate that core components of the α-proteobacterial general stress response (GSR) system, PhyR and σ(E1), are required for Brucella abortus stress survival in vitro and maintenance of chronic murine infection in vivo. ΔphyR and ΔrpoE1 null mutants exhibit decreased survival under acute oxidative and acid stress but are not defective in infection of primary murine macrophages or in initial colonization of BALB/c mouse spleens. However, ΔphyR and ΔrpoE1 mutants are attenuated in spleens beginning 1 month postinfection. Thus, the B. abortus GSR system is dispensable for colonization but is required to maintain chronic infection. A genome-scale analysis of the B. abortus GSR regulon identified stress response genes previously linked to virulence and genes that affect immunomodulatory components of the cell envelope. These data support a model in which the GSR system affects both stress survival and the interface between B. abortus and the host immune system. We further demonstrate that PhyR proteolysis is a unique feature of GSR control in B. abortus. Proteolysis of PhyR provides a mechanism to avoid spurious PhyR protein interactions that inappropriately activate GSR-dependent transcription. We conclude that the B. abortus GSR system regulates acute stress adaptation and long term survival within a mammalian host and that PhyR proteolysis is a novel regulatory feature in B. abortus that ensures proper control of GSR transcription.

Pseudomonas aeruginosa has abundant signaling systems that exquisitely control its antibiotic resistance in response to different environmental cues. Understanding the regulation of antibiotic resistance will provide important implications for precise antimicrobial interventions. However, efficient genetic tools for functional gene characterizations are sometimes not available, particularly, in clinically isolated strains. Here, we established a type I-F CRISPRi (CSYi) system for programmable gene silencing. By incorporating anti-CRISPR proteins, this system was even applicable to bacterial hosts encoding a native type I-F CRISPR-Cas system. With the newly developed gene-silencing system, we revealed that the response regulator CzcR from the zinc (Zn2+)-responsive two-component system CzcS/CzcR is a repressor of efflux pumps MexAB-OprM and MexGHI-OpmD, which inhibits the expression of both operons by directly interacting with their promoters. Repression of MexAB-OprM consequently increases the susceptibility of P. aeruginosa to multiple antibiotics such as levofloxacin and amikacin. Together, this study provided a simple approach to study gene functions, which enabled us to unveil the novel role of CzcR in modulating efflux pump genes and multidrug resistance in P. aeruginosa. IMPORTANCE P. aeruginosa is a ubiquitous opportunistic pathogen frequently causing chronic infections. In addition to being an important model organism for antibiotic-resistant research, this species is also important for understanding and exploiting CRISPR-Cas systems. In this study, we established a gene-silencing system based on the most abundant type I-F CRISPR-Cas system in this species, which can be readily employed to achieve targeted gene repression in multiple bacterial species. Using this gene-silencing system, the physiological role of Zn2+ and its responsive regulator CzcR in modulating multidrug resistance was unveiled with great convenience. This study not only displayed a new framework to expand the abundant CRISPR-Cas and anti-CRISPR systems for functional gene characterizations but also provided new insights into the regulation of multidrug resistance in P. aeruginosa and important clues for precise anti-pseudomonal therapies.

Identifying and validating targets that underlie disease mechanisms and can be addressed to provide efficacious therapies remains a significant challenge in the drug discovery and development process. Use of siRNA and shRNA to knock-down RNA and suppress gene function, have provided insights into mechanism of action, but depending on the nature of the targets, cells, biology and end-point assays these approaches may suffer variously from their transient nature, design complexity, incomplete knock-down or off-target effects. The use of CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease and guide RNA (gRNA) provides a strong alternative that can produce long-lasting impact, straightforward design, knock-out of genes and increased specificity. A number of laboratories have already published reports demonstrating how pools of gRNA can be delivered to cells and “hits” can be established through enrichment or depletion of cells following a “survival” assay and identified by sequencing the introduced gRNAs in the remaining cell population. Here we demonstrate a knock-out screening approach that utilizes the Invitrogen™ LentiArray™ CRISPR library to interrogate the impact of individual gene knock-outs on the NFκB pathway as measured by a functional cell-based assay. We describe the library design concepts, the assay development, initial screening results and validation of specific identified hits. The gRNAs are designed to primarily 5’ coding exons of a target gene using our CRISPR design tool to maximize knock-out efficiency and minimize off-target effects. Each gRNA is delivered as a separate lentiviral particle including an antibiotic-resistant marker and each gene is targeted by 4 gRNAs per well, delivered in a 96-well plate. We tested the approach using a library that targets the human kinome and developed a loss-of-function assay using our CellSensor® NF-κB-bla ME180 cell line, which is based on the ratiometric blue/green reporter assay and easily enables identification of genomic targets associated with the NF-κB pathway. We elucidate the key factors in developing a robust assay including both transduction and assay optimization to achieve the highest levels of transduction efficiency and assay window. Using these optimized parameters, we screened the Invitrogen™ LentiArray™ CRISPR kinome library that targets &amp;gt;800 kinases and demonstrate how we followed-up on and validated a subset of the identified hits. We expect these approaches to be scalable to the entire human genome and portable to multiple cell types and end-point assays including both high-throughput plate-based assays and high-content imaging based assays. Citation Format: Chetana M. Revankar, Justin Wetter, Julia Braun, Natasha Roark, Veronica Magnon, LaiYee Wong, Yanfei Zou, Namritha Ravinder, Jian-Ping Yang, Jonathan Chesnut, David Piper. Functional genomics screening using LentiArray™ CRISPR libraries and CellSensor™ assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-116. doi:10.1158/1538-7445.AM2017-LB-116