scholarly journals The Expanding Molecular Genetics Tool Kit in Chlamydia

2018 ◽  
Vol 200 (24) ◽  
Author(s):  
Raphael H. Valdivia ◽  
Robert J. Bastidas
Keyword(s):  
Genetic Engineering ◽  
Molecular Genetics ◽  
Genetic Manipulation ◽  
Molecular Genetic ◽  
Model System ◽  
New Method ◽  
Content Type ◽  
Host Interactions ◽  
Additional Tool ◽  
Genomic Regions

ABSTRACT Chlamydia has emerged as an important model system for the study of host pathogen interactions, in part due to a resurgence in the development of tools for its molecular genetic manipulation. An additional tool, published by Keb et al. (G. Keb, R. Hayman, and K. A. Fields, J. Bacteriol. 200:e00479-18, 2018, https://doi.org/10.1128/JB.00479-18), now allows for custom genetic engineering of genomic regions that were traditionally recalcitrant to genetic manipulation, such as genes within operons. This new method will be an essential instrument for the elucidation of Chlamydia-host interactions.

scholarly journals A New Group of Phage Anti-CRISPR Genes Inhibits the Type I-E CRISPR-Cas System of Pseudomonas aeruginosa

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
April Pawluk ◽  
Joseph Bondy-Denomy ◽  
Vivian H. W. Cheung ◽  
Karen L. Maxwell ◽  
Alan R. Davidson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Genetic Manipulation ◽  
Sequence Similarity ◽  
Adaptive Immune System ◽  
Resistance Mechanisms ◽  
Type I ◽  
Content Type ◽  
Genetic Elements ◽  
Small Proteins ◽  
Genomic Regions

ABSTRACT CRISPR-Cas systems are one of the most widespread phage resistance mechanisms in prokaryotes. Our lab recently identified the first examples of phage-borne anti-CRISPR genes that encode protein inhibitors of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. A key question arising from this work was whether there are other types of anti-CRISPR genes. In the current work, we address this question by demonstrating that some of the same phages carrying type I-F anti-CRISPR genes also possess genes that mediate inhibition of the type I-E CRISPR-Cas system of P. aeruginosa. We have discovered four distinct families of these type I-E anti-CRISPR genes. These genes do not inhibit the type I-F CRISPR-Cas system of P. aeruginosa or the type I-E system of Escherichia coli. Type I-E and I-F anti-CRISPR genes are located at the same position in the genomes of a large group of related P. aeruginosa phages, yet they are found in a variety of combinations and arrangements. We have also identified functional anti-CRISPR genes within nonprophage Pseudomonas genomic regions that are likely mobile genetic elements. This work emphasizes the potential importance of anti-CRISPR genes in phage evolution and lateral gene transfer and supports the hypothesis that more undiscovered families of anti-CRISPR genes exist. Finally, we provide the first demonstration that the type I-E CRISPR-Cas system of P. aeruginosa is naturally active without genetic manipulation, which contrasts with E. coli and other previously characterized I-E systems. IMPORTANCE The CRISPR-Cas system is an adaptive immune system possessed by the majority of prokaryotic organisms to combat potentially harmful foreign genetic elements. This study reports the discovery of bacteriophage-encoded anti-CRISPR genes that mediate inhibition of a well-studied subtype of CRISPR-Cas system. The four families of anti-CRISPR genes described here, which comprise only the second group of anti-CRISPR genes to be identified, encode small proteins that bear no sequence similarity to previously studied phage or bacterial proteins. Anti-CRISPR genes represent a newly discovered and intriguing facet of the ongoing evolutionary competition between phages and their bacterial hosts.

scholarly journals Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium

2016 ◽  
Vol 82 (20) ◽  
pp. 6109-6119 ◽  
Author(s):  
Mark R. Bruder ◽  
Michael E. Pyne ◽  
Murray Moo-Young ◽  
Duane A. Chung ◽  
C. Perry Chou
Keyword(s):  
Genetic Engineering ◽  
Genome Editing ◽  
Transcriptional Repression ◽  
Clostridium Acetobutylicum ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Carbon Sources ◽  
Anaerobic Bacteria ◽  
Clostridium Pasteurianum ◽  
Content Type

ABSTRACTThe discovery and exploitation of the prokaryotic adaptive immunity system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized genetic engineering. CRISPR-Cas tools have enabled extensive genome editing as well as efficient modulation of the transcriptional program in a multitude of organisms. Progress in the development of genetic engineering tools for the genusClostridiumhas lagged behind that of many other prokaryotes, presenting the CRISPR-Cas technology an opportunity to resolve a long-existing issue. Here, we applied theStreptococcus pyogenestype II CRISPR-Cas9 (SpCRISPR-Cas9) system for genome editing inClostridium acetobutylicumDSM792. We further explored the utility of the SpCRISPR-Cas9 machinery for gene-specific transcriptional repression. For proof-of-concept demonstration, a plasmid-encoded fluorescent protein gene was used for transcriptional repression inC. acetobutylicum. Subsequently, we targeted the carbon catabolite repression (CCR) system ofC. acetobutylicumthrough transcriptional repression of thehprKgene encoding HPr kinase/phosphorylase, leading to the coutilization of glucose and xylose, which are two abundant carbon sources from lignocellulosic feedstocks. Similar approaches based on SpCRISPR-Cas9 for genome editing and transcriptional repression were also demonstrated inClostridium pasteurianumATCC 6013. As such, this work lays a foundation for the derivation of clostridial strains for industrial purposes.IMPORTANCEAfter recognizing the industrial potential ofClostridiumfor decades, methods for the genetic manipulation of these anaerobic bacteria are still underdeveloped. This study reports the implementation of CRISPR-Cas technology for genome editing and transcriptional regulation inClostridium acetobutylicum, which is arguably the most common industrial clostridial strain. The developed genetic tools enable simpler, more reliable, and more extensive derivation ofC. acetobutylicummutant strains for industrial purposes. Similar approaches were also demonstrated inClostridium pasteurianum, another clostridial strain that is capable of utilizing glycerol as the carbon source for butanol fermentation, and therefore can be arguably applied in other clostridial strains.

scholarly journals Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

2016 ◽  
Vol 80 (2) ◽  
pp. 411-427 ◽  
Author(s):  
Robert J. Bastidas ◽  
Raphael H. Valdivia
Keyword(s):  
Genetic Manipulation ◽  
Molecular Genetic ◽  
Transmitted Disease ◽  
Intracellular Pathogen ◽  
Molecular Tools ◽  
Content Type ◽  
Sexually Transmitted ◽  
Obligate Intracellular ◽  
Recent Developments ◽  
Etiological Agents

SUMMARYChlamydiaspecies infect millions of individuals worldwide and are important etiological agents of sexually transmitted disease, infertility, and blinding trachoma. Historically, the genetic intractability of this intracellular pathogen has hindered the molecular dissection of virulence factors contributing to its pathogenesis. The obligate intracellular life cycle ofChlamydiaand restrictions on the use of antibiotics as selectable markers have impeded the development of molecular tools to genetically manipulate these pathogens. However, recent developments in the field have resulted in significant gains in our ability to alter the genome ofChlamydia, which will expedite the elucidation of virulence mechanisms. In this review, we discuss the challenges affecting the development of molecular genetic tools forChlamydiaand the work that laid the foundation for recent advancements in the genetic analysis of this recalcitrant pathogen.

scholarly journals A Natural Mouse Model forNeisseriaColonization

2018 ◽  
Vol 86 (5) ◽  
Author(s):  
Mancheong Ma ◽  
Daniel A. Powell ◽  
Nathan J. Weyand ◽  
Katherine A. Rhodes ◽  
María A. Rendón ◽  
...  
Keyword(s):  
Animal Model ◽  
Genetic Manipulation ◽  
Laboratory Mouse ◽  
Small Animal ◽  
Wild Mice ◽  
Content Type ◽  
Host Restriction ◽  
Host Interactions ◽  
Host Interaction ◽  
Type Iv

ABSTRACTCommensals are important for the proper functioning of multicellular organisms. How a commensal establishes persistent colonization of its host is little understood. Studies of this aspect of microbe-host interactions are impeded by the absence of an animal model. We have developed a natural small animal model for identifying host and commensal determinants of colonization and of the elusive process of persistence. Our system couples a commensal bacterium of wild mice,Neisseria musculi, with the laboratory mouse. The pairing of a mouse commensal with its natural host circumvents issues of host restriction. Studies are performed in the absence of antibiotics, hormones, invasive procedures, or genetic manipulation of the host. A single dose ofN. musculi, administered orally, leads to long-term colonization of the oral cavity and gut. All mice are healthy. Susceptibility to colonization is determined by host genetics and innate immunity. ForN. musculi, colonization requires the type IV pilus. Reagents and powerful tools are readily available for manipulating the laboratory mouse, allowing easy dissection of host determinants controlling colonization resistance.N. musculiis genetically related to human-dwelling commensal and pathogenicNeisseriaand encodes host interaction factors and vaccine antigens of pathogenicNeisseria. Our system provides a natural approach for studyingNeisseria-host interactions and is potentially useful for vaccine efficacy studies.

Recent Updates on Molecular Genetic Engineering Approaches and Applications of Human Therapeutic Proteins

2017 ◽  
Vol 18 (3) ◽  
pp. 217-232 ◽  
Author(s):  
Asim Azhar ◽  
Ejaj Ahmad ◽  
Qamar Zia ◽  
Mohammad Owais ◽  
Ghulam Md Ashraf
Keyword(s):  
Genetic Engineering ◽  
Molecular Genetic ◽  
Therapeutic Proteins

scholarly journals Transfer of Plasmid DNA to Clinical Coagulase-Negative Staphylococcal Pathogens by Using a Unique Bacteriophage

2015 ◽  
Vol 81 (7) ◽  
pp. 2481-2488 ◽  
Author(s):  
Volker Winstel ◽  
Petra Kühner ◽  
Bernhard Krismer ◽  
Andreas Peschel ◽  
Holger Rohde
Keyword(s):  
Plasmid Dna ◽  
Genetic Manipulation ◽  
Current Knowledge ◽  
Virulence Determinants ◽  
Coagulase Negative Staphylococci ◽  
Reliable Technique ◽  
Content Type ◽  
Research Activities ◽  
Wide Range ◽  
Genetic Barriers

ABSTRACTGenetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a uniqueStaphylococcus aureusstrain via a specificS. aureusbacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinicalStaphylococcus epidermidisisolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

Lubricant compatibility of FKM seals in synthetic oils

2019 ◽  
Vol 72 (5) ◽  
pp. 557-565
Author(s):  
Dilek Bulut ◽  
Tatjana Krups ◽  
Gerhard Poll ◽  
Ulrich Giese
Keyword(s):  
Polyethylene Glycol ◽  
Design Methodology ◽  
Elevated Temperatures ◽  
Failure Mechanisms ◽  
Standard Test ◽  
New Method ◽  
Static Tests ◽  
Content Type ◽  
Lip Seals ◽  
First Results

Purpose Elastomer seals are used in many applications. They are exposed to lubricants and additives at elevated temperatures, as well as mechanical stresses. They can only provide good sealing function when they have resistance to those factors. There are many elastomer-lubricant compatibility tests based on DIN ISO 1817 in industry. However, they are insufficient and costly. Correlations between the tests and the applications are inadequate. The purpose of this study is investigating lubricant compatibility of fluoroelastomers (FKM) seals in polyethylene-glycol (PG)- and polyalphaolefin (PAO)- based synthetic oils and developing a methodology to predict seal service life. Design/methodology/approach A new compatibility test which is more sufficient in terms of time and cost was developed and compared with a standard test, currently used in industry. Compatibility of FKM radial lip seals with PG- and PAO-based synthetic oils with different additives was investigated chemically and dynamically. Failure mechanisms were examined. Findings The new method and the Freudenberg Flender Test FB 73 11 008 showed similar results concerning damages and similar tendencies regarding wear. The additive imidazole derivative was the most critical. Static tests give indications of possible chemically active additives, but alone they are insufficient to simulate the dynamic applications. Originality/value The paper describes a new method to investigate elastomer-lubricant compatibility and gives first results with a variety of lubricants.

scholarly journals Molecular genetics of bipolar disorder

2001 ◽  
Vol 178 (S41) ◽  
pp. s128-s133 ◽  
Author(s):  
Nick Craddock ◽  
Ian Jones
Keyword(s):  
Bipolar Disorder ◽  
Candidate Gene ◽  
Molecular Genetics ◽  
Ethical Issues ◽  
Association Studies ◽  
Single Gene ◽  
Molecular Genetic ◽  
Genetic Dissection ◽  
Adoption Studies ◽  
Candidate Gene Association

BackgroundA robust body of evidence from family, twin and adoption studies demonstrates the importance of genes in the pathogenesis of bipolar disorder. Recent advances in molecular genetics have made it possible to identify these susceptibility genes.AimsTo present an overview for clinical psychiatrists.MethodReview of current molecular genetics approaches and emerging findings.ResultsOccasional families may exist in which a single gene plays a major role in determining susceptibility, but the majority of bipolar disorder involves more complex genetic mechanisms such as the interaction of multiple genes and environmental factors. Molecular genetic positional and candidate gene approaches are being used for the genetic dissection of bipolar disorder. No gene has yet been identified but promising findings are emerging. Regions of interest include chromosomes 4p16, 12q23–q24, 16p13, 21q22, and Xq24–q26. Candidate gene association studies are in progress but no robust positive findings have yet emerged.ConclusionIt is almost certain that over the next few years the identification of bipolar susceptiblity genes will have a major impact on our understanding of disease pathophysiology. This is likely to lead to major improvements and treatment in patient care, but will also raise important ethical issues.

scholarly journals Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

2014 ◽  
Vol 80 (8) ◽  
pp. 2410-2416 ◽  
Author(s):  
Areen Banerjee ◽  
Ching Leang ◽  
Toshiyuki Ueki ◽  
Kelly P. Nevin ◽  
Derek R. Lovley
Keyword(s):  
Ethanol Production ◽  
Genetic Manipulation ◽  
Electron Flow ◽  
Model Organism ◽  
Inducible Expression ◽  
Carbon Flow ◽  
Wild Type ◽  
Content Type ◽  
Microbial Electrosynthesis ◽  
Clostridium Ljungdahlii

ABSTRACTThe development of tools for genetic manipulation ofClostridium ljungdahliihas increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox forC. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported forClostridium perfringens, was investigated. The plasmid pAH2, originally developed forC. perfringenswith agusAreporter gene, functioned as an effective lactose-inducible system inC. ljungdahlii. Lactose induction ofC. ljungdahliicontaining pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression ofadhE130-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression ofadhE1in a strain in whichadhE1and theadhE1homologadhE2had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression ofadhE2similarly failed to restore ethanol production, suggesting thatadhE1is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

scholarly journals Reconstruction ofmreBExpression in Staphylococcus aureus via a Collection of New Integrative Plasmids

2014 ◽  
Vol 80 (13) ◽  
pp. 3868-3878 ◽  
Author(s):  
Ana Yepes ◽  
Gudrun Koch ◽  
Andrea Waldvogel ◽  
Juan-Carlos Garcia-Betancur ◽  
Daniel Lopez
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Biology ◽  
Genetic Manipulation ◽  
Protein Localization ◽  
Antibiotic Resistance Genes ◽  
Wall Material ◽  
Content Type ◽  
Genetic Manipulations ◽  
Discrete Structures

ABSTRACTProtein localization has been traditionally explored in unicellular organisms, whose ease of genetic manipulation facilitates molecular characterization. The two rod-shaped bacterial modelsEscherichia coliandBacillus subtilishave been prominently used for this purpose and have displaced other bacteria whose challenges for genetic manipulation have complicated any study of cell biology. Among these bacteria is the spherical pathogenic bacteriumStaphylococcus aureus. In this report, we present a new molecular toolbox that facilitates gene deletion in staphylococci in a 1-step recombination process and additional vectors that facilitate the insertion of diverse reporter fusions into newly identified neutral loci of theS. aureuschromosome. Insertion of the reporters does not add any antibiotic resistance genes to the chromosomes of the resultant strains, thereby making them amenable for further genetic manipulations. We used this toolbox to reconstitute the expression ofmreBinS. aureus, a gene that encodes an actin-like cytoskeletal protein which is absent in coccal cells and is presumably lost during the course of speciation. We observed that inS. aureus, MreB is organized in discrete structures in association with the membrane, leading to an unusual redistribution of the cell wall material. The production of MreB also caused cell enlargement, but it did not revert staphylococcal shape. We present interactions of MreB with key staphylococcal cell wall-related proteins. This work facilitates the useS. aureusas a model system in exploring diverse aspects of cellular microbiology.

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