Name: conjugative mating assays for sequence-specific analysis of transfer proteins involved in bacterial conjugation
Uploaded: 2017-01-04T12:30:00.000+00:00
Duration: 10 min 41 s
Description: Watch this Scientific Journal Video about Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation at JoVE.com

이 연구는 캐나다의 자연 과학 및 공학위원회에서 발견 그랜트 (NSERC)에 의해 지원되었다.

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저자는 더 경쟁 재정적 이해 관계가 없음을 선언합니다.

접합 공정은 박테리아와 같은 항생제 내성 마커의 확산 등의 어려운 환경에서 성장을 위해 진화하는 장점을 제공하는 유전자를 확산 할 수있는 수단을 제공한다. 공역 플라스미드 많은 너무 크기 때문에, 공역 플라스미드 자체의 표적 유전자의 돌연변이를 통해 전송 장치의 조립에 관여하는 단백질의 기능 연구 12 다루기이다. 본원에 설명 된 프로토콜을 하나 더 쉽게 더 작고 관리 발현 ?...

미세 유기체의 수준에서의 유전자 및 단백질의 전사는 박테리아의 생존과 발전뿐만 아니라 감염 과정에서 중요한 역할을한다. 박테리아 사이 또는 박테리아 및 세포 간 DNA의 교환은 변형, 결합, 벡터 전달을 통해 달성 될 수있다. 1,2- 활용은 대장균 등의 그람 음성균 간의 접합시 점에서 변환 및 전달에 비해 독특 DNA의 전사는 복합 고분자 시스템 공여자와 전지를 연결함으로써 도너 - 제어 방식으로 발생한다. 활용은 박테리아 세포는 호스트 시스템에서 유전자, 단백질 또는 화학 물질을 주입하는 숙주 세포와 상호 작용하는 가장 직접적인 방법입니다. 3 종종, 이러한 에이전트의 전송은 진화와 적응을 호스팅하는 기전과 발암에 이르기까지 호스트에 놀라운 효과를 가지고있다. 그것은 그 공역 recombinatio을 보였다n은 환경 스트레스 조건에서 높은 돌연변이 속도와 박테리아에 적응 3 배의 속도를 증가시킨다. (4) 또한, 접합은 균주의 항생제 내성 유전자가 확산되는을 통해 지금까지 가장 일반적인 경로입니다. 5,6 미생물 세포막 전체 거대 분자의 전달을 지원하기 위해 특수 분비 시스템 진화; 설명 된 그람 음성 세균에서 분비 시스템 (TSSs)의 9 종류의 현재 위치 : T1SS, T2SS, T3SS, T4SS, T5SS, T6SS, T7SS뿐만 아니라 초 (분비)과 문신이 (두 아르기닌 전위) 경로. 분비 시스템의 7,8 각 유형은 또한 다른 균주에 의한 단백질의 다양성과 관련된 경로의 특수성에, 다른 하위 유형으로 필요성을 분할된다. 예를 들어, 타입 IV 분비 시스템 (T4SS)에있어서, Ti 및 CAG 시스템은 F-플라스미드 반면 이펙터 전송을 용이 R27 A를차 pKM101 T4SSs는 공역 플라스미드의 이동을 용이하게한다. 생물받는 사람이나 자신의 주변 환경과 휴대 내용을 자신의 구성 요소 단백질에서 각각 분비 시스템을 조립하고 공유하는 메커니즘의 7,9,10 자세한 이해는 병원성 미생물의 프로세스에 대처하기 위해 대상 전략의 개발에 중요한 요소이다 세포 감염. Lederberg 및 테이텀 (11)에 의한 대장균의 초기 식별 접합 다음 모바일 공역 플라스미드 다수 확인되고 특성화되었다. (12) 이러한 모바일 플라스미드 그러나 모든 모바일 플라스미드 전송 (기술 Orit)하여 플라스미드의 전송을 가능하게하는 근원을 인식하는 relaxase를 포함, 상당한 범위 (1에서 200 킬로베이스 (킬로바이트)로) 크기가 표시됩니다. 공역 기능 T4SS 조립 용 플라스미드를 더 인코딩 유전자뿐만 아니라, 형IV 결합 단백질. 도 12는 예를 들어 대장균 100킬로바이트 F 플라스미드 33.3 kB의 전송 (TRA) 영역 내의 모든 공역 유전자를 암호화한다. 13 쌍의 형성 (MPF), DNA 전송 및 제외 기능을 짝짓기 pilus 형성을 촉진 모든 단백질 공역 플라스미드 전송시 F 플라스미드 인코딩의 TRA 지역에서 유전자. 10,14,15 지식의 중요한 몸은 공역 T4SSs 사용할 수 있습니다, 그러나 단지 최근에 사용 가능한되고있다 공역 단백질 복합체의 구조 연구를 설명. 16-28 공역 처리의 광범위한 관점을 조립하기 위해, 전사 공역 단백질의 돌연변이를 분석하는 상세한 구조 연구의 결합이 필요하다. 이 공역 정합 분석을 통해 달성 될 수있다. 은 F 플라스미드를 들어, TRA 영역 내에 인코딩 각 단백질은 F-CO 매개하는 역할njugation; 따라서, 전달 유전자의 녹아웃 / 삭제 셀 (도 1)의 공역 용량을 폐지 할 것이다. 작은 이동 플라스미드는 F와 같은 큰 공역 플라스미드위한 표준 삭제 절차에 더 도움이되는 동안 표적 유전자는 하나의 별개의 항생제 내성 유전자를 전달로 치환되는 경우, 유전자 녹아웃보다 쉽게 ​​상동 재조합을 통해 달성된다. 현재의 프로토콜에서, 우리는 55킬로바이트 F 플라스미드 유도체 pOX38-TC의 클로람페니콜 아세틸 트랜스퍼 라제 (CAT)와 관심 전사 유전자를 대체하는 상동 재조합을 사용; 29,30 얻어진 녹아웃 플라스미드 pOX38-TC Δgene :: 형상은 성장 배지에 클로람페니콜의 존재 (CM)로 저항을 용이하게한다. pOX38-TC Δgene :: CM 은닉 공여 세포는 결합 분석을 사용하여 관찰 공역 DNA 전달 / 결합에 영향을 줄 수 없다; pOX38-TC Δgene :: 형상 도너 셀의 결합 효율과 정상 RECIPient 더 자주, 폐지, 감소 또는 것입니다. pOX38-TC Δgene : CM 플라스미드의 공역 전송이 대상 전송 유전자를 품고 작은 복구 플라스미드를 통해 복원 할 수 있습니다. 이 복구 플라스미드는 플라스미드 pK184 (pK184 유전자), 31 또는 너무 오래 그 플라스미드가 제대로 셀 (세포질이나 주변 세포질) 내에서 올바른 위치에 유전자를 표적으로 유도 식을 제공 하나, 구성 적 발현을 제공 하나가 될 수 있습니다. 따라서,이 새로운 기증자 사이의 결합 분석에서 (pOX38-TC Δgene : CM + pK184 유전자 플라스미드를 은닉) 과받는 사람의 세포가 결합 효율은 거의 그 정상 기증자받는 사람 짝짓기 분석의 복원 것으로 예상된다. 이 시스템은 pK184 유전자 작 제물 (결실 또는 점 돌연변이)의 일련의 생성을 통해 기절 유전자의 기능을 검사하고 pOX38-TC Δgene :: 형상 은닉의 결합 용량을 복원하는 각 구조체의 능력을 테스트 한있게 기증자 세포에스.

<table><tbody><tr><td>GeneJet Plasmid Mini-Prep Kit</td><td>Fisher Scientific</td><td>K0503</td><td></td></tr><tr><td>GeneJet Gel Extraction Kit</td><td>Fisher Scientific</td><td>K0692</td><td></td></tr><tr><td>GeneJet PCR Purification Kit</td><td>Fisher Scientific</td><td>K0702</td><td></td></tr><tr><td>Q5 Site-Directed Mutagenesis Kit</td><td>New England Biolabs</td><td>E0554S</td><td></td></tr><tr><td>Broad Range DNA Ladder</td><td>New England Biolabs</td><td>N0303A</td><td></td></tr><tr><td>Petri Dishes</td><td>Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>Electroporator</td><td>Eppendorf</td><td>4309000027</td><td></td></tr><tr><td>Electroporation cuvettes</td><td>Fisher Scientific</td><td>FB101</td><td>Cuvettes have a 1 mm gap.</td></tr><tr><td>&lt;strong&gt;Enzymes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AvaI</td><td>New England Biolabs</td><td>R0152S</td><td></td></tr><tr><td>EcoRI</td><td>New England Biolabs</td><td>R0101S</td><td></td></tr><tr><td>HindIII</td><td>New England Biolabs</td><td>R0104L</td><td></td></tr><tr><td>NdeI</td><td>New England Biolabs</td><td>R0111S</td><td></td></tr><tr><td>Phusion DNA Polymerase</td><td>New England Biolabs</td><td>M0530L</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>DpnI</td><td>New England Biolabs</td><td>R0176S</td><td></td></tr><tr><td>&lt;strong&gt;Antibiotics&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Final Concentrations&lt;/strong&gt;</td></tr><tr><td>Chloramphenicol (Cm)</td><td>Fisher Scientific</td><td>BP904-100</td><td>20 &amp;micro;g/mL</td></tr><tr><td>Kanamycin (Km)</td><td>BioBasic Inc.</td><td>DB0286</td><td>50 &amp;micro;g/mL</td></tr><tr><td>Nalidixic acid (Nal)</td><td>Sigma-Aldrich</td><td>N8878-25G</td><td>10 &amp;micro;g/mL</td></tr><tr><td>Rifampicin (Rif)</td><td>Calbiochem</td><td>557303</td><td>20 &amp;micro;g/mL</td></tr><tr><td>Tetracycline (Tc)</td><td>Fisher Scientific</td><td>BP912-100</td><td>10 &amp;micro;g/mL</td></tr><tr><td>Streptomycin (Sm)</td><td>Fisher Scientific</td><td>BP910-50</td><td>50 &amp;micro;g/mL</td></tr></tbody></table>

conjugative mating assays for sequence-specific analysis of transfer proteins involved in bacterial conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

F 플라스미드 구동 접합 과정은 pilus 합성과 공역 DNA 전달을 용이하게하기 T4SS을 어셈블 F-플라스미드 TRA 영역 내에 전달 단백질을 포함하는 조정 방법이다. 단백질 트래픽 (GenBank 액 # BAA97961, UniProt ID P14497)을 공역 F-pilus 형성을 위해 필요합니다. 10,14,35 -이 촉매 CXXC 모티브가 없습니다 불구하고 37 단백질은 C 말단 티 오레 독신과 같은 도메인?...

Watch this Scientific Journal Video about Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation at JoVE.com

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

접합에 참여 전송 단백질의 서열 별 분석을위한 공역 결합 분석 실험

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

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JoVE Journal

Genetics

13.7K 조회수.  York University. Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations. 

비디오: Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

시험 관내 전사 분석 실험 및 약물 발견에의 응용

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

연구

유전학

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree&#39;s lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant&#39;s secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

나무 형성 및 보조 스템 개발에 관여하는 유전자 및 발기인 유학을위한 유도 체세포 업종 분석 (ISSA)의 사용

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

생리 산소 조건에 노출 된 인간 세포의 캡 결합 단백질의 분석

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

사이트 특정 박테리아 염색체 공학 : ΦC31 Integrase 중재 카세트 거래소 (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

생체공학

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

세균성 활용 주파수의 고해상도 비교

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the study of the mobilization of naturally occurring conjugative plasmids, using two naturally-occurring plasmids as an example. These protocols rely on the differential presence of selectable markers in donor, recipient, and conjugative plasmid. Specifically, the methods described include 1) the identification of natural conjugative plasmids, 2) the quantification of conjugation rates in solid culture, and 3) the diagnostic detection of the antibiotic resistance genes and plasmid replicon types in transconjugant recipients by polymerase chain reaction (PCR). The protocols described here have been developed in the context of studying the evolutionary ecology of horizontal gene transfer, to screen for the presence of conjugative plasmids carrying antibiotic-resistance genes in bacteria found in the environment. The efficient transfer of conjugative plasmids observed in these experiments in culture highlights the biological relevance of conjugation as a mechanism promoting horizontal gene transfer in general and the spread of antibiotic resistance in particular.

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation mediates horizontal gene transfer by mobilizing plasmid DNA across two different cells, facilitating the spread of beneficial genes. This work describes a widely-used method for the efficient detection of conjugative plasmid transfer, based on the use of differential markers in the conjugative plasmid, donor, and recipient to detect transconjugation.

대장균에서 천연 접합 플라스미드에 의해 매개되는 수평 유전자 전달의 검출

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

수평 유전자 전달의 연구에 대한 방법론<em&gt; 황색 포도상 구균</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

면역학 및 감염병학

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

시안게이트 모듈형 복제 툴킷을 사용하여 컨쥬게이션에 의한 시아노박테리아의 유전자 변형

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful ...

생물학

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The &#8220;last-line&#8221; antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

고처리량 시퀀싱을 위한 그람 음성 박테리아에 트랜스포슨 삽입 라이브러리 생성

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways ...

Conjugation: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

Source: Alexander S. Gold1, Tonya M. Colpitts1 
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases Laboratories, Boston, MA
First discovered by Lederberg and Tatum in 1946, conjugation is a form of horizontal gene transfer between bacteria that relies on direct physical contact between two bacterial cells (1). Unlike other forms of gene transfer, such as transformation or transduction, conjugation is a naturally occurring process in which DNA is secreted from a donor cell to a recipient cell in a unidirectional manner. This directionality and the ability for this process to increase the genetic diversity of bacteria has given conjugation the reputation as a form of bacterial &#34;mating,&#34; which is believed to have contributed greatly to the recent rise in antibiotic resistant bacteria (2, 3). By using selective pressures, for example the use of antibiotics, conjugation has been manipulated for use in the laboratory setting, making it a powerful tool for horizontal gene transfer between bacteria, and in some cases from bacteria to yeast, plant, and animal cells (4). Apart from applications in the laboratory, bacterium-eukaryote gene transfer by conjugation is an exciting avenue of DNA transfer with a multitude of possible biotechnical applications and naturally occurring implications (5).
Conjugation is thought to work by a &#34;two-step mechanism&#34; (6). First, before any DNA can be transferred, the donor cell must make direct cell-to-cell contact with the recipient. This process has been characterized best in gram-negative bacteria, the most studied of which is Escherichia coli. Cell-to-cell contact is established by the presence of a complex network of extracellular filaments on the donor known as the sex pilus, a conjugative element encoded for by the transferable gene known as the F (fertility) factor (7, 8). In addition to establishing contact between donor and recipient, several proteins are transported via the sex pilus to the recipient cytoplasm, forming a type IV secretion system (T4SS) conduit between the two cells, a necessary structure for the second step of conjugation, DNA transfer (6). By combining this function of the sex pilus with rolling circle replication of DNA, the donor cell is able to transfer DNA in the form of a transposable element, such as a plasmid or transposon, to the recipient by a &#34;shoot and pump&#34; model (6). In this case, the &#34;shooting&#34; is the transport of the pilot protein, with linked DNA, by the T4SS into the recipient cell, and the &#34;pumping&#34; is the active transport of DNA to the recipient, a process reliant on the T4SS and catalyzed by coupling proteins (6). The machinery used in this this process is composed of an origin of transfer sequence (oriT), which must be provided by the DNA in cis and trans genes, which encode a relaxase, mate pair formation complex, and type IV coupling protein, and can be present in cis or trans (9). This relaxase cleaves the nic site within the oriT sequence and covalently attaches to the 5&#39; end of the transferred strand to produce the relaxosome, a single-stranded DNA-relaxase complex with other auxiliary proteins (9). Once formed, the relaxosome connects to the mating pair formation complex, via the type IV coupling protein, which allows for the transfer of the ssDNA-relaxase complex into recipient cells by the T4SS (10). Once in the cytoplasm of the recipient, the DNA can integrate into the recipient genome or exist separately in the form of a plasmid, either of which allow for the expression of its genes.
In this experiment, the widely used conjugation donor strain E. coli WM3064 was used to transfer the gene encoding for ampicillin resistance to the recipient strain E. coli J53. While both strains of the gram-negative bacteria were resistant to tetracycline, only the donor strain WM3064 had the gene for ampicillin resistance, encoded for in the pWD2-oriT shuttle vector, and was auxotrophic to diaminopimelic acid (DAP) (11-13). This experiment consisted of two main steps, the preparation of donor and recipient strains, followed by the transfer of the ampicillin resistance gene from donor to recipient by conjugation (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10516/10516fig1.jpg" /> 
Figure 1: Conjugation schematic. This schematic shows the successful transfer of a plasmid, only one example of a transposable DNA element, from a donor cell to a recipient cell using conjugation. Upon contact with the recipient cell by the donor cell via the sex pilus, the plasmid replicates by rolling circle replication, moves through the multiprotein complex joining the two cells, and forms a new full-length plasmid in the recipient cell.
By incubating a mixture of donor and recipient cells, then successively plating these cells in the presence of tetracycline and DAP, this allowed for the successful transfer of the ampicillin resistance gene. Successively plating cells grown from this mixture in the presence of tetracycline and ampicillin, removed all donor cells due to the lack of DAP and any recipient cells that may not have gained the ampicillin resistance gene, yielding strictly recipient J53 strain bacteria that acquired ampicillin resistance (Figure 2). Once carried out, the successful transfer of the ampicillin resistance gene was confirmed by PCR. Since conjugation was successful, the J53 strain of E. coli contained pWD2-oriT and was resistant to ampicillin, and the gene encoding for this resistance is detectable by PCR. However, if unsuccessful there would have been no detection of the ampicillin resistance gene and ampicillin would still function as an effective antibiotic against the J53 strain.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10516/10516fig2.jpg" /> 
Figure 2: Protocol schematic. This schematic shows an overview of the presented protocol.
<img alt="Figure 3A" class="xfigimg" src="/files/ftp_upload/10516/10516fig3a.jpg" /> 
Figure 3A: The confirmation of successful conjugation by PCR. A) Freezer stocks of the conjugated and negative control samples were streaked out on agar plates and a colony was selected (red) for DNA isolation.

Conjugation: Transferring Ampicillin Resistance in E. coli

접합: 공여 E.coli에서 수용 E.coli로 암피실린 내성을 전달하는 방법

Source: Alexander S. Gold1, Tonya M. Colpitts1
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases ...