ABSTRACT
Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies. In the synthetic cell, we also demonstrate inducible gene expression for the first time. Time-course data suggest rapid kinetics and reversible engagement of CRISPRi. Targeting of six selected endogenous genes with this system results in lowered transcript levels or reduced growth rates that agree with lack or shortage of data in previous transposon bombardment studies, and now produces actual cells to analyze. The ksgA gene encodes a methylase that modifies 16S rRNA, rendering it vulnerable to inhibition by the antibiotic kasugamycin. Targeting the ksgA gene with CRISPRi removes the lethal effect of kasugamycin and enables cell growth, thereby establishing specific and effective gene modulation with our system. The facile methods for conditional gene activation and inactivation in mycoplasmas open the door to systematic dissection of genetic programs at the core of cellular life.
Subject(s)
Gene Expression Regulation, Bacterial , Genetic Engineering/methods , Mycoplasma/genetics , Aminoglycosides/pharmacology , Bacterial Proteins/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Regulatory Networks , Luminescent Proteins/genetics , Methyltransferases/genetics , Microorganisms, Genetically-Modified , Mycoplasma/drug effects , Riboswitch/genetics , Tetracycline/pharmacology , Red Fluorescent ProteinABSTRACT
The terminal organelle of Mycoplasma genitalium is responsible for bacterial adhesion, motility and pathogenicity. Localized at the cell tip, it comprises an electron-dense core that is anchored to the cell membrane at its distal end and to the cytoplasm at its proximal end. The surface of the terminal organelle is also covered with adhesion proteins. We performed cellular cryoelectron tomography on deletion mutants of eleven proteins that are implicated in building the terminal organelle, to systematically analyze the ultrastructural effects. These data were correlated with microcinematographies, from which the motility patterns can be quantitatively assessed. We visualized diverse phenotypes, ranging from mild to severe cell adhesion, motility and segregation defects. Based on our observations, we propose a double-spring ratchet model for the motility mechanism that explains our current and previous observations. Our model, which expands and integrates the previously suggested inchworm model, allocates specific functions to each of the essential components of this unique bacterial motility system.
Subject(s)
Mycoplasma genitalium/genetics , Mycoplasma genitalium/physiology , Organelles/genetics , Adhesins, Bacterial/genetics , Adhesins, Bacterial/metabolism , Bacterial Adhesion/genetics , Bacterial Proteins/metabolism , Cell Adhesion , Electron Microscope Tomography/methods , Electrons , Mutation , Mycoplasma pneumoniae/genetics , Organelles/metabolismABSTRACT
Mycoplasma genitalium is an appealing model of a minimal cell and synthetic biology study, and it was one of the first organisms whose genome was fully sequenced and chemically synthesized. Despite its usefulness as a model organism, many genetic tools well established for other microorganisms are not currently available in mycoplasmas. We have developed several vectors to adapt the Cre-lox technology for genome engineering in M. genitalium, providing an all-in-one construct that could be also useful to obtain unmarked genetic modifications in many other slow growing microorganisms. This construct contains a modified promoter sequence based in TetR system that exhibits an enhanced control on Cre recombinase expression, virtually abolishing the presence of this recombinase in the absence of inducer. This allows to introduce the Cre recombinase gene and the desired genetic modification in a single transformation step. In addition, this inducible promoter may be a very promising tool for a wide range of molecular applications.
Subject(s)
Gene Targeting/methods , Genetic Engineering/methods , Genome, Bacterial , Integrases/genetics , Mycoplasma genitalium/genetics , Genetic Vectors/genetics , Integrases/metabolism , Promoter Regions, Genetic , Recombination, GeneticABSTRACT
Una de las características singulares de la enfermedad de Parkinson (EP) es la gran variabilidad clínica en relación con el tratamiento que puede acontecer en un mismo paciente. Esto sucede tanto con el tratamiento específico para la EP como con otra serie de fármacos que pueden empeorar la función motora. Por esta razón, el manejo perioperatorio de la EP requiere experiencia y sobre todo una planificación adecuada. En este artículo se revisan las peculiaridades de la EP y su tratamiento, y se plantea una estrategia para el perioperatorio de estos pacientes (AU)
One of the particular characteristics of Parkinsons disease (PD) is the wide clinical variation as regards the treatment that can be found in the same patient. This occurs with specific treatment for PD, as well as with other drug groups that can make motor function worse. For this reason, the perioperative management of PD requires experience and above all appropriate planning. In this article, the peculiarities of PD and its treatment are reviewed, and astrategy is set out for the perioperative management of these patients (AU)
Subject(s)
Humans , Parkinson Disease/surgery , Intraoperative Care/methods , /methods , Risk Factors , Antiparkinson Agents/administration & dosage , Dyskinesias/prevention & control , Diet Therapy/methodsABSTRACT
One of the particular characteristics of Parkinson's disease (PD) is the wide clinical variation as regards the treatment that can be found in the same patient. This occurs with specific treatment for PD, as well as with other drug groups that can make motor function worse. For this reason, the perioperative management of PD requires experience and above all appropriate planning. In this article, the peculiarities of PD and its treatment are reviewed, and a strategy is set out for the perioperative management of these patients.
Subject(s)
Parkinson Disease/surgery , Perioperative Care , HumansABSTRACT
The Moches were a pre-Columbian culture from Peru, who had a fine ceramic technique and used to represent diseases. One example is the potter presented here which represents a man with a probable Meige's syndrome and may be the first artistic representation of this disease.
Subject(s)
Culture , Ethnicity , Medicine in the Arts , Meige Syndrome/history , Meige Syndrome/physiopathology , Blepharospasm/history , Blepharospasm/physiopathology , Ceramics/history , History, Ancient , Humans , PeruABSTRACT
Reportamos un caso de hepatocarcinoma asociado a degeneración grasa de higado, el cual se presentó con clínica compatible con un cuadro de abdomen agudo quirúrgico, intervenido en el Hospital "Victorino Santaella", siendo el primer caso observado en nuestra institución y el único reportado en la literatura nacional e internacionaal revisada desde 1971 a 1995
