Name: generation of marked and markerless mutants in model cyanobacterial species
Uploaded: 2016-05-29T15:00:00.000+00:00
Duration: 11 min 45 s
Description: Watch this Scientific Journal Video about Generation of Marked and Markerless Mutants in Model Cyanobacterial Species at JoVE.com

Estamos muy agradecidos a la Asociación de Servicios Medioambientales Fiduciario para la Educación, la biología sintética en el fondo de la Biología Sintética Cambridge y el Ministerio de Justicia Social y Empoderamiento, Gobierno de la India, por el apoyo financiero.

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The authors declare that they have no competing financial interests.

Los pasos más importantes en la generación de mutantes no marcados son: 1) Diseño de plásmido cuidado para asegurar que sólo se altera la región objetivo; 2) garantizar que las muestras permanezcan axénicos, especialmente cuando se cultivan en sacarosa; 3) placas células de marcado generación mutante transformada inicialmente en placas de agar BG11 que carecen de antibióticos, seguido de la adición de agar más antibióticos 24 horas más tarde; 4) cultivar marcado mutantes para 4 días completos antes de la ...

Las cianobacterias son un phylum antigua y diversa evolutivamente de bacterias que se encuentran en casi todos los medio natural en la Tierra. En los ecosistemas marinos son particularmente abundantes y juegan un papel clave en muchos ciclos de nutrientes, lo que representa aproximadamente la mitad de la fijación de carbono 1, la mayoría de la fijación de nitrógeno 2 y cientos de millones de toneladas de producción de hidrocarburos 3 en los océanos anualmente. Cloroplastos, el orgánulo responsable de la fotosíntesis de las algas y las plantas eucariota, es probable que hayan evolucionado a partir de una cianobacteria que fue engullido por un organismo huésped 4. Las cianobacterias han demostrado organismos modelo útil para el estudio de la fotosíntesis, transporte de electrones y 5 vías bioquímicas, muchos de los cuales se conservan en las plantas. Además cianobacterias cada vez se utilizan para la producción de alimentos, los biocombustibles 6, 7 y electricidad compuestos industriales 8, debido a su altaghly conversión eficiente del agua y CO 2 a la biomasa utilizando la energía solar 9. Muchas especies pueden cultivarse en tierras no cultivables con nutrientes mínimos y el agua de mar, lo que sugiere que las cianobacterias potencialmente podrían ser crecido a gran escala sin afectar la producción agrícola. Ciertas especies son también fuentes de productos naturales, incluidos los compuestos antifúngicos, antibacterianos y anti-cáncer 10,11. La capacidad de generar mutantes es clave para entender la fotosíntesis de cianobacterias, bioquímica y fisiología, y esencial para el desarrollo de cepas con fines industriales. La mayoría de los estudios publicados generate modificado genéticamente cepas mediante la inserción de un casete de resistencia a los antibióticos en el sitio de interés. Esto limita el número de mutaciones que pueden introducirse en una cepa, como sólo unos casetes de resistencia a antibióticos están disponibles para su uso en cianobacterias. Las cepas que contienen los genes que confieren re antibióticoresistencia no puede ser utilizado para la producción industrial en estanques abiertos, lo que es probable que sea el único medio rentables para la producción de biocombustibles y otros productos de bajo valor 12. La generación de mutantes no marcados supera estas limitaciones. mutantes no marcados no contienen ADN extraño, excepto si se incluye intencionalmente, y se pueden manipular varias veces. Por lo tanto es posible generar tantas alteraciones en una cepa como se desee. Además, los efectos polares de genes aguas abajo del sitio de modificación pueden minimizarse, lo que permite la modificación más preciso del organismo 13. Para generar cepas mutantes, plásmidos suicidas que contienen dos fragmentos de ADN idéntica a las regiones en el cromosoma de cianobacterias que flanquean el gen a eliminar (denominado el 5 &#39;y 3&#39; flanqueantes) se construyó primero. Dos genes se insertan entonces entre estas regiones flanqueantes. Uno de ellos codifica una proteína de resistencia a los antibióticos; la segunda codifica SacB, que produces levansucrasa, un compuesto que confiere sensibilidad a la sacarosa. En la primera etapa del proceso, los mutantes marcados, es decir, las cepas que contienen algo de ADN externa, se generan. La construcción de plásmido se mezcla con las células de cianobacterias y el ADN se recogió de forma natural por el organismo. Los transformantes se seleccionaron por crecimiento en placas de agar que contienen el antibiótico apropiado y el genotipo mutante verificada por PCR. plásmidos suicidas no pueden replicarse dentro de la cepa de interés. Por lo tanto cualquier colonias resistentes a los antibióticos serán el resultado de un evento de recombinación mediante el cual el gen de interés en insertado en el cromosoma. Para generar mutantes no marcados, el mutante marcada se mezcla a continuación con un segundo plásmido suicida que contiene sólo las regiones flanqueantes 5 &#39;y 3&#39;. Sin embargo, si se requiere la inserción de ADN extraño, un plásmido que consiste en el 5 &#39;y 3&#39; que flanquean las regiones con un casete que contiene los genes de interés insertado entre estos fragmentos de ADN, se puede utilizar. selección es a través del crecimiento en placas de agar que contienen sacarosa. Como la sacarosa es letal para las células cuando se expresa el producto del gen sacB, las únicas células que sobreviven son aquellos en los que se ha producido un segundo suceso de recombinación, con lo cual el gen de sensibilidad sacarosa, además de el gen de resistencia a los antibióticos, se ha recombinado de la cromosoma y en el plásmido. Como consecuencia del intercambio de recombinación, las regiones flanqueantes y cualquier ADN entre ellos se insertan en el cromosoma. Hemos utilizado con éxito estos métodos para generar múltiples mutaciones cromosómicas en la misma cepa de Synechocystis sp. PCC6803 (en adelante como Synechocystis) 13,14, para introducir mutaciones puntuales en un gen de interés y 13 para la expresión de cassettes de genes. Mientras que la generación de knockouts sin marcar se ha demostrado con anterioridad a nuestro trabajo en Synechocystis 15,16, un método detallado, con la ayuda deuna presentación visual de los pasos críticos, no está disponible públicamente. También hemos aplicado el mismo método para la generación de knockouts marcadas en otro modelo de cianobacteria, Synechococcus sp. PCC7002 (en lo sucesivo, Synechococcus). Este protocolo proporciona un método claro y sencillo para la generación de mutantes y un protocolo rápido para la validación y almacenamiento de estas cepas.

<table><tbody><tr><td>NaNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5506</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>230391</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C1016</td><td></td></tr><tr><td>citric acid</td><td>Sigma</td><td>C0759</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;EDTA</td><td>Fisher</td><td>EDT002</td><td></td></tr><tr><td>H&lt;sub&gt;3&lt;/sub&gt;BO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>339067</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;.4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>M3634</td><td></td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>Z4750</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;MoO&lt;sub&gt;4&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>331058</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;.5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>209198</td><td></td></tr><tr><td>Co(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>239267</td><td></td></tr><tr><td>Ferric ammonium citrate</td><td>Sigma</td><td>F5879</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P3786</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher</td><td>SODC001</td><td></td></tr><tr><td>TES</td><td>Sigma</td><td>T1375</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher</td><td>SODH001</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>cyanocobalamin</td><td>Sigma</td><td>47869</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;S&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>72049</td><td></td></tr><tr><td>Bacto agar</td><td>BD</td><td>214010</td><td></td></tr><tr><td>Sucrose</td><td>Fisher</td><td>SUC001</td><td></td></tr><tr><td>Petri dish 90 mm triple vented</td><td>Greiner</td><td>633185</td><td></td></tr><tr><td>0.2 &amp;micro;m filters</td><td>Sartorius</td><td>16534</td><td></td></tr><tr><td>100 ml conical flasks</td><td>Pyrex</td><td>CON004</td><td></td></tr><tr><td>Parafilm M 100 mm x 38&amp;nbsp;m</td><td>Bemis</td><td>FIL003</td><td></td></tr><tr><td>Phusion high fidelity DNA polymerase&amp;nbsp;</td><td>Phusion</td><td>F-530</td><td></td></tr><tr><td>Agarose</td><td>Melford</td><td>MB1200</td><td></td></tr><tr><td>DNA purification kit&amp;nbsp;</td><td>MoBio</td><td>12100-300</td><td></td></tr><tr><td>Restriction endonucleases</td><td>NEB</td><td></td><td></td></tr><tr><td>T4 ligase</td><td>Thermo Scientific</td><td>EL0011</td><td></td></tr><tr><td>Luria Bertani broth</td><td>Invitrogen</td><td>12795-027</td><td></td></tr><tr><td>MES</td><td>Sigma</td><td>M8250</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>Sigma</td><td>60615</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A9518</td><td></td></tr><tr><td>GeneJET plasmid miniprep kit</td><td>Thermo Scientific</td><td>K0503</td><td></td></tr><tr><td>14 ml round-bottom tube</td><td>BD falcon</td><td>352059</td><td></td></tr><tr><td>GoTaq G2 Flexi DNA polymerase</td><td>Promega</td><td>M7805</td><td></td></tr><tr><td>425-600 &amp;micro;m glass beads</td><td>Sigma</td><td>G8772</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418</td><td></td></tr><tr><td>Fluorescent bulbs</td><td>Gro-Lux</td><td>69</td><td></td></tr><tr><td>HT multitron photobioreactor</td><td>Infors</td><td></td><td></td></tr></tbody></table>

generation of marked and markerless mutants in model cyanobacterial species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Diseño plásmido es crítica para la generación exitosa de ambos mutantes marcados y sin marcar. Figura 1 da un ejemplo del plásmido A y B utiliza para generar un mutante de deleción en los genes Synechocystis cpcC1 y cpcC2 13. En cada caso, las regiones flanqueantes 5 &#39;y 3&#39; son de aproximadamente 900-1.000 pb. regiones que flanquean reducidos se pueden utilizar aunque el más pequeño que hemos ensayado con éxito ha sid...

Watch this Scientific Journal Video about Generation of Marked and Markerless Mutants in Model Cyanobacterial Species at JoVE.com

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

Generación de mutantes sin marcadores marcados y en el modelo de especies de cianobacterias

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

generation-marked-markerless-mutants-model-cyanobacterial

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.9K vistas.  University of Cambridge. Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

Video: Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Optogenética aleatoria de mutagénesis, utilizando la histona-miniSOG en<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Investigación

Genética

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the conventional approaches of permanent gene deletion cannot be applied to essential genes. We have pioneered a unique collection of ~70 temperature-sensitive (ts) lethal mutants for studying cell cycle regulation in the unicellular green algae Chlamydomonas reinhardtii1. These mutations identify essential genes, and the ts alleles can be conditionally inactivated by temperature shift, providing valuable tools to identify and analyze essential functions. Mutant collections are much more valuable if they are close to comprehensive, since scattershot collections can miss important components. However, this requires the efficient collection of a large number of mutants, especially in a wide-target screen. Here, we describe a robotics-based pipeline for generating ts lethal mutants and analyzing their phenotype in Chlamydomonas. This technique can be applied to any microorganism that grows on agar. We have collected over 3000 ts mutants, probably including mutations in most or all cell-essential pathways, including about 200 new candidate cell cycle mutations. Subsequent molecular and cellular characterization of these mutants should provide new insights in plant cell biology; a comprehensive mutant collection is an essential prerequisite to ensure coverage of a broad range of biological pathways. These methods are integrated with downstream genetics and bioinformatics procedures for efficient mapping and identification of the causative mutations that are beyond the scope of this manuscript.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

De alto rendimiento de aislamiento robótica asistida sensible a la temperatura del letal mutantes en<em&gt; Chlamydomonas reinhardtii</em

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally identified by harnessing the competitions between amino acids and their analogs. However, this analog-based method is of low accuracy, and proper analogs for specific amino acids are limited. Here, we present an alternative strategy that enables an accurate, sensitive, and high-throughput screening of amino acid overproducers using rare-codon-rich markers. This strategy is inspired by the phenomenon of codon usage bias in protein translation, for which codons are categorized into common or rare ones based on their frequencies of occurrence in the coding DNA. The translation of rare codons depends on their corresponding rare transfer RNAs (tRNAs), which cannot be fully charged by the cognate amino acids under starvation. Theoretically, the rare tRNAs can be charged if there is a surplus of the amino acids after charging the synonymous common isoacceptors. Therefore, retarded translations caused by rare codons could be restored by feeding or intracellular overproductions of the corresponding amino acids. Under this assumption, a selection or screening system for identifying amino acid overproducers is established by replacing the common codons of the targeted amino acids with their synonymous rare alternatives in the antibiotic resistance genes or the genes encoding fluorescent or chromogenic proteins. We show that the protein expressions can be greatly hindered by the incorporation of rare codons and that the levels of proteins correlate positively with the amino acid concentrations. Using this system, overproducers of multiple amino acids can be readily screened out from mutation libraries. This rare-codon-based strategy only requires a single modified gene, and the host is less likely to escape the selection than in other methods. It offers an alternative approach for obtaining amino acid overproducers.

This study presents an alternative strategy to the conventional toxic analog-based method in identifying amino acid overproducers by using rare-codon-rich markers to achieve accuracy, sensitivity, and high-throughput simultaneously.

Identificación de los sobreproductores de aminoácidos utilizando marcadores ricos en codón raros

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally ...

Bioquímica

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

Evaluación visual y microscópica de mutantes desarrollo de Streptomyces

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

Inmunología e Infección

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. The non-replicative elementary body (EB) initiates infection of the host. Once inside, the EB differentiates into the reticulate body (RB). The RB then undergoes multiple rounds of replication, before differentiating back to the infectious EB form. This cycle is essential for chlamydial survival as failure to switch between cell types prevents either host invasion or replication.
Limitations in genetic techniques due to the obligate intracellular nature of Chlamydia have hampered identification of the molecular mechanisms involved in the cell-type development. We designed a novel dual promoter-reporter plasmid system that, in conjunction with live-cell microscopy, allows for the visualization of cell type switching in real time. To identify genes involved in the regulation of cell-type development, the live-cell promoter-reporter system was leveraged for the development of a forward genetic approach by combining chemical mutagenesis of the dual reporter strain, imaging and tracking of Chlamydia with altered developmental kinetics, followed by clonal isolation of mutants. This forward genetic workflow is a flexible tool that can be modified for directed interrogation into a wide range of genetic pathways.

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

This protocol utilizes fluorescent promoter-reporters, live-cell microscopy, and individual inclusion extraction in a directed forward genetic approach to identify and isolate developmental mutants of Chlamydia trachomatis.

Enfoque genético de células vivas para identificar y aislar mutantes del desarrollo en Chlamydia trachomatis

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. ...

Integración de genes estables en Chlorella vulgaris utilizando Agrobacterium tumefaciens

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

Autor destacado: Protocolo de transformación optimizado para Chlorella vulgaris utilizando Agrobacterium tumefaciens 

This protocol outlines the utilization of Agrobacterium tumefaciens-mediated transformation (AMT) for integrating gene(s) of interest into the nuclear genome of the green microalgae Chlorella vulgaris, leading to the production of stable transformants.

Agrobacterium tumefaciens-Ingeniería genética mediada por microalgas verdes, Chlorella vulgaris

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit ...

Bioingeniería

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

Generación de mutantes nulos para dilucidar el papel de las glicosiltransferasas bacterianas en la motilidad bacteriana

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans ...

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.&#160;

Generación de mutantes sin marcadores marcados y en el modelo de especies de cianobacterias

Resumen

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