RESUMO
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.
Assuntos
Mycoplasma genitalium/genética , Mycoplasma genitalium/fisiologia , Organelas/genética , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Aderência Bacteriana/genética , Proteínas de Bactérias/metabolismo , Adesão Celular , Tomografia com Microscopia Eletrônica/métodos , Elétrons , Mutação , Mycoplasma pneumoniae/genética , Organelas/metabolismoRESUMO
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.
Assuntos
Regulação Bacteriana da Expressão Gênica , Engenharia Genética/métodos , Mycoplasma/genética , Aminoglicosídeos/farmacologia , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Redes Reguladoras de Genes , Proteínas Luminescentes/genética , Metiltransferases/genética , Microrganismos Geneticamente Modificados , Mycoplasma/efeitos dos fármacos , Riboswitch/genética , Tetraciclina/farmacologia , Proteína Vermelha FluorescenteRESUMO
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.