私たちは、財政支援のため、インド政府環境サービス協会教育トラスト、ケンブリッジシンバイオファンドにおける合成生物学と社会正義とエンパワーメント省に感謝しています。

<ol>
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The authors declare that they have no competing financial interests.

マークされていない変異体の世代の中で最も重要な手順は次のとおりです。1）慎重なプラスミドのデザインのみ標的領域が改変されていることを確認します。 2）特に、培養された場合、スクロース上のサンプルは、無菌のままであることを保証します。 3）24時間後に寒天を加えた抗生物質を添加した抗生物質を欠くBG11寒天プレート上に最初にマークされた変異体の生成のためのめっき形?...

シアノバクテリアは、地球上のほぼすべての自然環境の中で見つかった細菌の進化的に古代と多様な門です。海洋生態系において、それらは特に豊富であり、炭素固定1の約半分を、海洋における窒素固定2と炭化水素生産3のトン数百万の大半は毎年を占め、多くの栄養素のサイクルで重要な役割を果たしています。葉緑体は、真核藻類や植物の光合成を担う細胞小器官は、宿主生物4に包まれたシアノバクテリアから進化した可能性があります。シアノバクテリアは、その多くが植物に保存されている、光合成、電子輸送5および生化学的経路の研究のための有用なモデル生物を証明しています。付加シアノバクテリアにおいてますます食品、バイオ6、電気7及びそれらのハイによる産業用化合物8の製造に使用されています太陽エネルギー9を用いて、バイオマスへの水とCO 2のghly効率的に変換します。多くの種はシアノバクテリアが潜在的に農業生産に影響を与えることなく、大規模に成長させることができたことを示唆し、最小限の栄養素と海水と非耕地で栽培することができます。特定の種も抗真菌、抗菌性及び抗癌化合物10,11を含む天然物の源です。 変異体を生成する能力は、シアノバクテリアの光合成、生化学および生理学、および産業用菌株の開発に不可欠なを理解するための鍵です。公表された研究の大部分は、目的の部位に抗生物質耐性カセットの挿入によって遺伝的に改変された株を生成します。これは、いくつかの抗生物質耐性カセットは、シアノバクテリアにおける使用のために利用可能であるように、株に導入され得る変異の数を制限します。抗生物質の再付与する遺伝子を含む株sistanceは、バイオ燃料および他の低価値製品12を製造するための唯一の費用効果的な手段である可能性が高い開放池の工業生産のために使用することができません。マークされていない変異体の生成は、これらの制限を克服します。マークされていない変異体は、意図的に含まれない限り、外来DNAを含有しない、複数回操作することができます。したがって、所望のように株のように多くの変更を生成することができます。また、修飾部位の下流の遺伝子上の極性効果は、生体13のより正確な修正を可能にする、最小限にすることができます。 変異株、削除対象の遺伝子に隣接するシアノバクテリアの染色体における領域と同一の2つのDNA断片を含有する自殺プラスミド（隣接領域の5 &#39;および3&#39;と呼ばれる）を生成するために最初に構成されています。二つの遺伝子は、これらの隣接領域間に挿入されます。これらの一つは、抗生物質耐性タンパク質をコードします。第二は、SacBを、PRODを符号化レバンスクラーゼuces、化合物は、スクロースに対する感受性を付与します。プロセスの最初の段階では、マークされた変異体は、いくつかの外来DNAを含む、すなわち株は、生成されます。プラスミド構築物は、シアノバクテリア細胞と混合し、DNAは、生物によって天然に取り込まれます。形質転換体は、適切な抗生物質およびPCRにより確認変異体の遺伝子型を含有する寒天プレート上での増殖によって選択されます。自殺プラスミドは、目的の菌株内で複製することはできません。したがって、任意の抗生物質耐性コロニーは、目的の遺伝子が染色体に挿入することにより組換え事象から生じます。マークされていない変異体を生成するために、マークされた変異体は、その後、ちょうど5 &#39;および3&#39;フランキング領域を含む第二の自殺プラスミドと混合されます。外来DNAの挿入が必要な場合は、これらのDNA断片の間に挿入された目的の遺伝子を含有するカセットを有する領域に隣接する5 &#39;および3&#39;からなるプラスミドを用いることができます。セレctionは、ショ糖を含有する寒天プレート上での成長によるものです。 sacB遺伝子産物が発現されたときに、スクロースを細胞に致死的であるように、生存細胞のみがショ糖感受性遺伝子は、抗生物質耐性遺伝子に加えて、外に再結合されていることにより第2の組換え事象が発生したものであり、染色体とプラスミド上に。組換え交換の結果として、隣接する領域と、それらの間のいずれかのDNAが染色体に挿入されています。 我々は成功しシネコシスティスの同じ系統で複数の染色体の変異を生成するために、これらのメソッドを使用しています。 （以下、 シネコシスティスという）PCC6803 13,14は 、関心対象13の遺伝子に、遺伝子カセットの発現のための単一点突然変異を導入します。マークされていないノックアウトの生成はシネコシスティス 15,16、詳細な方法で私たちの仕事の前に実証されてきたが、によって支援重要なステップのビジュアルプレゼンテーションは、公開されていません。また、別のモデルのラン藻、 シネココッカス属にマークされたノックアウトを生成するために同じ方法を適用しました。 PCC7002（以下、 シネココッカスと呼ばれます）。このプロトコルは、変異体およびこれらの株を検証し、保存するための迅速なプロトコルを生成するための明確な、簡単な方法を提供します。

<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.

プラスミドのデザインは、両方のマークされ、マークされていない変異体の生成に成功するために重要である。 図1は 、プラスミドAの例を示し、Bはシネコシスティス遺伝子 cpcC1とcpcC2 13に欠失変異体を生成するために使用されます。それぞれの場合において、5 &#39;及び3&#39;フランキング領域は約900〜1000塩基対です。我々は成...

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.

モデルシアノバクテリア種のマークとマーカーレス変異体の生成

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 ビュー.  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.

ビデオ: 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.

でヒストンminiSOGを使用した光遺伝学ランダム突然変異誘発<em&gt; C。エレガンス</em

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

遺伝学

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.

ハイスループットは、ロボットに温度に敏感な致死変異体の単離を支援します<em&gt;クラミドモナス</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.

希少コドンリッチマーカーを用いてアミノ酸過剰生産者を同定

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

生化学

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.

放線菌突然変異体のビジュアルと顕微鏡的評価

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

免疫・感染学

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.

クラミジア・トラコマティスにおける発生変異体の同定と分離に対する生細胞前方遺伝的アプローチ

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

Agrobacterium tumefaciensを用いたChlorella vulgarisの安定的な遺伝子統合

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.

著者スポットライト:Agrobacterium tumefaciensを使用したChlorella vulgarisの最適化された形質転換プロトコル 

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-緑色微細藻類Chlorella vulgarisの遺伝子工学

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

生物工学

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.

細菌の運動性における細菌の糖転移酵素の役割を解明するためのヌル変異体の生成

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;

モデルシアノバクテリア種のマークとマーカーレス変異体の生成

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