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Functional analysis of the catalytic triad of the hAT-family transposase TcBuster hat家族转座酶TcBuster催化三联体的功能分析
IF 2.6 4区 生物学 Q3 GENETICS & HEREDITY Pub Date : 2021-03-01 DOI: 10.1016/j.plasmid.2021.102554
Lauren E. Woodard , Felisha M. Williams , Isria C. Jarrett , Matthew H. Wilson

TcBuster is a hAT-family DNA transposon from the red flour beetle, Tribolium castaneum. The TcBuster transposase is of interest for genome engineering as it is highly active in insect and mammalian cells. To test the predicted catalytic triad of TcBuster, each residue of the catalytic triad of a haemagglutinin-tagged TcBuster transposase was individually mutated to a structurally conserved amino acid. Using a drug-resistant colony assay for transposon integration, we found that the D223N, D289N, and E589Q mutants of TcBuster transposase were inactive in human cells. We used a modified chromatin immunoprecipitation assay to determine that each mutant maintained binding to TcBuster transposon inverted repeat elements. Although the catalytic mutants retained their transposon binding properties, mutants displayed altered expression and localization in human cells. None of the catalytic mutants formed characteristic TcBuster transposase rodlet structures, and the D223N and D289N mutants were not able to be detected by immunofluorescence microscopy. Immunoblot analysis demonstrated that the E589Q mutant is less abundant than wild-type TcBuster transposase. Cells transfected with either TcBuster or TcBuster-E589Q transposase were imaged by structured illumination microscopy to quantify differences in the length of the transposase rodlets. The average length of the TcBuster transposase rodlets (N = 39) was 3.284 μm while the E589Q rodlets (N = 33) averaged 1.157 μm (p < 0.0001; t-test). The catalytic triad mutations decreased overall protein levels and disrupted transposase rodlet formation while nuclear localization and DNA binding to the inverted repeat elements were maintained. Our results may have broader implications for the overproduction inhibition phenomenon observed for DNA transposons.

TcBuster是一种hat家族DNA转座子,来自红粉甲虫Tribolium castaneum。TcBuster转座酶在昆虫和哺乳动物细胞中高度活跃,是基因组工程研究的热点。为了测试预测的TcBuster催化三联体,将血凝素标记的TcBuster转座酶的催化三联体的每个残基单独突变为一个结构保守的氨基酸。通过对转座子整合的耐药集落试验,我们发现TcBuster转座酶的D223N、D289N和E589Q突变体在人细胞中无活性。我们使用改良的染色质免疫沉淀法来确定每个突变体与TcBuster转座子反向重复元件的结合。虽然催化突变体保留了转座子结合特性,但突变体在人类细胞中的表达和定位发生了改变。没有一个催化突变体形成典型的TcBuster转座酶小棒状结构,D223N和D289N突变体无法通过免疫荧光显微镜检测到。免疫印迹分析表明,E589Q突变体的丰度低于野生型TcBuster转座酶。转染TcBuster或TcBuster- e589q转座酶的细胞通过结构照明显微镜成像,以量化转座酶小棒长度的差异。TcBuster转座酶(N = 39)的平均长度为3.284 μm, E589Q转座酶(N = 33)的平均长度为1.157 μm。0.0001;t检验)。催化三联体突变降低了总蛋白质水平,破坏了转座酶小棒的形成,同时维持了核定位和DNA与倒置重复元件的结合。我们的结果可能对观察到的DNA转座子的过度生产抑制现象具有更广泛的意义。
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引用次数: 0
Insights into the individual evolutionary origins of Yersinia virulence factor effector proteins 耶尔森氏菌毒力因子效应蛋白的个体进化起源
IF 2.6 4区 生物学 Q3 GENETICS & HEREDITY Pub Date : 2021-03-01 DOI: 10.1016/j.plasmid.2021.102562
Veronica R. Moorman , James I. Cohen

Pathogenic Yersinia bacteria, including Y. pseudotubuclosis Y. enterocolitica, and Y. pestis, contain the mosaic plasmid pYV that encodes for, among other things, a number of proteinaceous virulence factors. While the evolutionary histories of many of the biovars and strains of pathogenic Yersinia species are well documented, the origins of many of the individual virulence factors have not been comprehensively examined. Here, the evolutionary origins of the genes coding for a set of Yersinia outer protein (Yop) virulence factors were investigated through phylogenetic reconstruction and subsequence analysis. It was found that many of these genes had only a few sequenced homologs and none of the resolved phylogenies recovered the same relationships as was resolved from chromosomal analyses. Many of the evolutionary relationships differ greatly among genes on the plasmid, and variation is also found across different domains of the same gene, which provides evidence of the mosaic nature of the plasmid as well as multiple genes on the plasmid. This mosaic aspect also relates to patterns of selection, which vary among the studied domains.

致病性耶尔森氏菌,包括假管耶尔森氏菌、小肠结肠炎耶尔森氏菌和鼠疫耶尔森氏菌,含有编码多种蛋白毒力因子的镶嵌质粒pYV。虽然许多致病性耶尔森菌物种的生物变种和菌株的进化历史已被很好地记录下来,但许多单个毒力因素的起源尚未得到全面检查。本文通过系统发育重建和子序列分析,研究了一组耶尔森菌外蛋白(Yop)毒力因子编码基因的进化起源。结果发现,其中许多基因只有少数同源序列,没有一个已确定的系统发育恢复了从染色体分析中确定的相同关系。许多进化关系在质粒上的基因之间差异很大,并且在同一基因的不同结构域之间也发现了变异,这为质粒以及质粒上的多个基因的马赛克性质提供了证据。这个镶嵌的方面也涉及到选择的模式,在不同的研究领域。
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引用次数: 3
The ever-expanding tcp conjugation locus of pCW3 from Clostridium perfringens 产气荚膜梭菌pCW3不断扩大的tcp偶联位点
IF 2.6 4区 生物学 Q3 GENETICS & HEREDITY Pub Date : 2021-01-01 DOI: 10.1016/j.plasmid.2020.102516
Sarah A. Revitt-Mills, Thomas D. Watts, Dena Lyras, Vicki Adams, Julian I. Rood

The spore-forming, anaerobic Gram positive pathogen Clostridium perfringens encodes many of its disease-causing toxins on closely related conjugative plasmids. Studies of the tetracycline resistance plasmid pCW3 have identified many of the genes involved in conjugative transfer, which are located in the tcp conjugation locus. Upstream of this locus is an uncharacterised region (the cnaC region) that is highly conserved. This study examined the importance in pCW3 conjugation of several highly conserved proteins encoded in the cnaC region. Conjugative mating studies suggested that the SrtD, TcpN and Dam proteins were required for efficient pCW3 transfer between C. perfringens cells from the same strain background. The requirement of these proteins for conjugation was amplified in matings between C. perfringens cells of different strain backgrounds. Additionally, the putative collagen adhesin protein, CnaC, was only required for the optimal transfer of pCW3 between cells of different strain backgrounds. Based on these studies we postulate that CnaC, SrtD, TcpN and Dam are involved in enhancing the transfer frequency of pCW3. These studies have led to a significant expansion of the tcp conjugation locus, which now encompasses a 19 kb region.

产芽孢的厌氧革兰氏阳性病原体产气荚膜梭菌在密切相关的共轭质粒上编码其许多致病毒素。对四环素耐药质粒pCW3的研究已经鉴定出许多参与接合转移的基因,这些基因位于tcp接合位点。该位点的上游是一个高度保守的未表征区域(cnaC区域)。本研究考察了cnaC区域编码的几种高度保守蛋白在pCW3偶联中的重要性。结合研究表明,SrtD、TcpN和Dam蛋白是产气荚膜荚膜菌在相同菌株背景下细胞间高效转移pCW3所需的蛋白。在不同菌株背景的产气荚膜荚膜杆菌细胞间的交配中,这些蛋白的偶联需求被扩增。此外,只有在不同菌株背景的细胞之间,pCW3的最佳转移才需要推定的胶原粘附蛋白CnaC。基于这些研究,我们推测CnaC、SrtD、TcpN和Dam参与了pCW3转移频率的增强。这些研究导致了tcp偶联位点的显著扩展,现在包含了一个19 kb的区域。
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引用次数: 8
Evolution and dissemination of L and M plasmid lineages carrying antibiotic resistance genes in diverse Gram-negative bacteria 不同革兰氏阴性菌中携带抗生素抗性基因的L和M质粒谱系的进化和传播
IF 2.6 4区 生物学 Q3 GENETICS & HEREDITY Pub Date : 2021-01-01 DOI: 10.1016/j.plasmid.2020.102528
Grace A. Blackwell , Emma L. Doughty , Robert A. Moran

Conjugative, broad host-range plasmids of the L/M complex have been associated with antibiotic resistance since the 1970s. They are found in Gram-negative bacterial genera that cause human infections and persist in hospital environments. It is crucial that these plasmids are typed accurately so that their clinical and global dissemination can be traced in epidemiological studies. The L/M complex has previously been divided into L, M1 and M2 subtypes. However, those types do not encompass all diversity seen in the group. Here, we have examined 148 complete L/M plasmid sequences in order to understand the diversity of the complex and trace the evolution of distinct lineages. The backbone sequence of each plasmid was determined by removing translocatable genetic elements and reversing their effects in silico. The sequence identities of replication regions and complete backbones were then considered for typing. This supported the distinction of L and M plasmids and revealed that there are five L and eight M types, where each type is comprised of further sub-lineages that are distinguished by variation in their backbone and translocatable element content. Regions containing antibiotic resistance genes in L and M sub-lineages have often formed by initial rare insertion events, followed by insertion of other translocatable elements within the inceptive element. As such, islands evolve in situ to contain genes conferring resistance to multiple antibiotics. In some cases, different plasmid sub-lineages have acquired the same or related resistance genes independently. This highlights the importance of these plasmids in acting as vehicles for the dissemination of emerging resistance genes. Materials are provided here for typing plasmids of the L/M complex from complete sequences or draft genomes. This should enable rapid identification of novel types and facilitate tracking the evolution of existing lineages.

自20世纪70年代以来,L/M复合体的共轭广泛宿主质粒与抗生素耐药性有关。它们存在于引起人类感染并在医院环境中持续存在的革兰氏阴性细菌属中。至关重要的是,这些质粒必须准确分型,以便在流行病学研究中追踪它们的临床和全球传播。L/M复合体以前被分为L、M1和M2亚型。然而,这些类型并不能涵盖群体中所有的多样性。在这里,我们检查了148个完整的L/M质粒序列,以了解该复合体的多样性并追踪不同谱系的进化。每个质粒的主干序列是通过去除可易位的遗传元件并在硅中逆转它们的作用来确定的。然后考虑复制区域和完整主干的序列身份进行分型。这支持了L和M质粒的区分,并揭示了有5个L型和8个M型,其中每个型都由进一步的子谱系组成,这些子谱系通过其主干和可易位元素含量的变化来区分。L和M亚系中含有抗生素耐药基因的区域通常是由最初罕见的插入事件形成的,随后在初始元件中插入其他可易位元件。因此,岛屿在原地进化,包含对多种抗生素具有抗性的基因。在某些情况下,不同的质粒亚系独立获得相同或相关的抗性基因。这突出了这些质粒作为新出现的抗性基因传播载体的重要性。这里提供了从完整序列或草图基因组中分型L/M复合物质粒的材料。这将使新的类型的快速识别和促进跟踪现有谱系的演变成为可能。
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引用次数: 7
Bacteriophages as sources of small non-coding RNA molecules 噬菌体是小的非编码RNA分子的来源
IF 2.6 4区 生物学 Q3 GENETICS & HEREDITY Pub Date : 2021-01-01 DOI: 10.1016/j.plasmid.2020.102527
Sylwia Bloch , Natalia Lewandowska , Grzegorz Węgrzyn , Bożena Nejman-Faleńczyk

Bacteriophages play an essential role in the transferring of genes that contribute to the bacterial virulence and whose products are dangerous to human health. Interestingly, phages carrying virulence genes are mostly temperate and in contrast to lytic phages undergo both lysogenic and lytic cycles. Importantly, expression of the majority of phage genes and subsequent production of phage encoded proteins is suppressed during lysogeny. The expression of the majority of phage genes is tightly linked to lytic development. Among others, small non-coding RNAs (sRNAs) of phage origin are involved in the regulation of phage gene expression and thus play an important role in both phage and host development. In the case of bacteria, sRNAs affect processes such as virulence, colonization ability, motility and cell growth or death. In turn, in the case of phages, they play essential roles during the early stage of infection, maintaining the state of lysogeny and silencing the expression of late structural genes, thereby regulating the transition between phage life cycles. Interestingly, sRNAs have been identified in both lytic and temperate phages and they have been discussed in this work according to this classification. Particular attention was paid to viral sRNAs resembling eukaryotic microRNAs.

噬菌体在促进细菌毒力的基因转移中起着重要作用,其产物对人体健康有害。有趣的是,携带毒力基因的噬菌体大多是温和的,与裂解噬菌体相反,它们经历溶原和裂解周期。重要的是,大多数噬菌体基因的表达和随后的噬菌体编码蛋白的产生在溶原过程中受到抑制。大多数噬菌体基因的表达与裂解发育密切相关。其中,噬菌体来源的小非编码rna (sRNAs)参与噬菌体基因表达的调控,因此在噬菌体和宿主发育中都起着重要作用。就细菌而言,sRNAs影响诸如毒力、定植能力、运动性和细胞生长或死亡等过程。反过来,就噬菌体而言,它们在感染的早期阶段发挥着至关重要的作用,维持溶原状态,沉默晚期结构基因的表达,从而调节噬菌体生命周期之间的过渡。有趣的是,在裂解性和温带噬菌体中都发现了sRNAs,并根据这种分类进行了讨论。特别关注的是类似真核microrna的病毒sRNAs。
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引用次数: 14
Incorporating the plasmidome into antibiotic resistance surveillance in animal agriculture 将质粒纳入动物农业抗生素耐药性监测
IF 2.6 4区 生物学 Q3 GENETICS & HEREDITY Pub Date : 2021-01-01 DOI: 10.1016/j.plasmid.2020.102529
N. Ricker , B.S. Spoja , N. May , G. Chalmers

Mobile genetic elements (MGE) carrying resistance genes represent a unique challenge to risk assessment and surveillance of antimicrobial resistance (AMR). Yet determining the mobility of resistance genes within animal microbiomes is essential to evaluating the potential dissemination from livestock to potential human pathogens, as well as evaluating co-selection mechanisms that may impact persistence of resistance genes with changing antibiotic use patterns. Current surveillance efforts utilize phenotypic testing and sequencing of individual isolates for tracking of AMR in livestock. In this work, we investigated the utility of using long-read sequencing of the plasmids from mixed Enterobacterales enrichments of swine fecal samples as a surveillance strategy for AMR plasmids. Enrichments were performed in either MacConkey broth without selection or with selection by addition of tetracycline or ceftriaxone, and plasmids were extracted and sequenced in order to evaluate the diversity of plasmids enriched by each method. Intact resistance plasmids were successfully assembled, as well as complex resistance transposons carrying multiple repeated elements that would interfere with assembly by short read sequencing technologies. Comparison of the assembled plasmids with representatives from public databases confirmed the quality of the assemblies and also revealed the occurrence of IncI2 plasmids carrying blaCMY-2 in Ontario swine samples, which have not been found in previous studies.

携带耐药基因的移动遗传元件(MGE)对抗菌素耐药性(AMR)的风险评估和监测提出了独特的挑战。然而,确定动物微生物组内耐药基因的流动性对于评估从牲畜到潜在人类病原体的潜在传播以及评估可能随着抗生素使用模式的改变而影响耐药基因持久性的共选择机制至关重要。目前的监测工作利用表型检测和单个分离株测序来跟踪牲畜抗菌素耐药性。在这项工作中,我们研究了利用猪粪便混合肠杆菌富集质粒的长读序列作为AMR质粒监测策略的实用性。在不加选择或加入四环素或头孢曲松选择的MacConkey肉汤中进行富集,提取质粒并测序,以评估每种方法富集的质粒的多样性。通过短读测序技术,成功组装了完整的抗性质粒,以及携带多个干扰组装的重复元件的复杂抗性转座子。将组装的质粒与公共数据库中的代表进行比较,证实了组装的质量,并发现安大略省猪样本中存在携带blaCMY-2的IncI2质粒,这在以前的研究中未发现。
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引用次数: 1
Quantifying plasmid dynamics using single-cell microfluidics and image bioinformatics 利用单细胞微流体和图像生物信息学定量质粒动力学
IF 2.6 4区 生物学 Q3 GENETICS & HEREDITY Pub Date : 2021-01-01 DOI: 10.1016/j.plasmid.2020.102517
J.C.R. Hernandez-Beltran , J. Rodríguez-Beltrán , A. San Millán , R. Peña-Miller , A. Fuentes-Hernández

Multicopy plasmids play an important role in bacterial ecology and evolution by accelerating the rate of adaptation and providing a platform for rapid gene amplification and evolutionary rescue. Despite the relevance of plasmids in bacterial evolutionary dynamics, evaluating the population-level consequences of randomly segregating and replicating plasmids in individual cells remains a challenging problem, both in theory and experimentally. In recent years, technological advances in fluorescence microscopy and microfluidics have allowed studying temporal changes in gene expression by quantifying the fluorescent intensity of individual cells under controlled environmental conditions. In this paper, we will describe the manufacture, experimental setup, and data analysis pipeline of different microfluidic systems that can be used to study plasmid dynamics, both in single-cells and in populations. To illustrate the benefits and limitations of microfluidics to study multicopy plasmid dynamics, we will use an experimental model system consisting on Escherichia coli K12 carrying non-conjugative, multicopy plasmids (19 copies per cell, in average) encoding different fluorescent markers and β-lactam resistance genes. First, we will use an image-based flow cytometer to estimate changes in the allele distribution of a heterogeneous population under different selection regimes. Then we will use a mothermachine microfluidic device to obtain time-series of fluorescent intensity of individual cells to argue that plasmid segregation and replication dynamics are inherently stochastic processes. Finally, using a microchemostat, we track thousands of cells in time to reconstruct bacterial lineages and evaluate the allele frequency distributions that emerge in response to a range of selective pressures.

多拷贝质粒通过加快细菌的适应速度,为基因快速扩增和进化救援提供平台,在细菌的生态和进化中发挥着重要作用。尽管质粒在细菌进化动力学中具有相关性,但评估单个细胞中随机分离和复制质粒的群体水平后果仍然是一个具有挑战性的问题,无论是在理论上还是在实验上。近年来,荧光显微镜和微流体技术的进步,使得在受控环境条件下,通过量化单个细胞的荧光强度来研究基因表达的时间变化成为可能。在本文中,我们将描述不同的微流体系统的制造,实验设置和数据分析管道,可用于研究质粒动力学,在单细胞和群体。为了说明微流体技术在研究多拷贝质粒动力学方面的优势和局限性,我们将使用一个实验模型系统,该系统由大肠杆菌K12组成,该大肠杆菌K12携带非共轭多拷贝质粒(平均每个细胞19个拷贝),编码不同的荧光标记和β-内酰胺抗性基因。首先,我们将使用基于图像的流式细胞仪来估计不同选择制度下异质群体等位基因分布的变化。然后,我们将使用母机微流控装置获得单个细胞荧光强度的时间序列,以证明质粒分离和复制动力学本质上是随机过程。最后,我们使用一个微化恒温器,及时跟踪数千个细胞,重建细菌谱系,并评估在一系列选择压力下出现的等位基因频率分布。
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引用次数: 4
Evolution of IS26-bounded pseudo-compound transposons carrying the tet(C) tetracycline resistance determinant 携带tet(C)四环素抗性决定因子的is26结合伪复合转座子的进化
IF 2.6 4区 生物学 Q3 GENETICS & HEREDITY Pub Date : 2020-11-01 DOI: 10.1016/j.plasmid.2020.102541
Carol H. Pong, Robert A. Moran , Ruth M. Hall

A large plasmid, pCERC14, found in an antibiotic resistant commensal Escherichia coli isolate recovered from a healthy adult was sequenced. pCERC14 was 162,926 bp and carried FII-18 and FIB-1 replicons and an F-like transfer region as well as several virulence determinants, some of which are involved in the uptake of iron which would be advantageous for the commensal lifestyle. The plasmid backbone is interrupted in 11 places by complete IS (IS1 (4 copies), IS2 (2), IS629 (2) and single IS100, IS186, ISEc33) and in three places by partial IS copies. The antibiotic resistance genes were found in two IS26-bounded pseudo-compound transposons (PCT). One contained a remnant of a class 1 integron that includes a dfrA5 gene cassette and the sul1 gene conferring resistance to trimethoprim and sulphonamides, respectively. The second, named PTntet(C)-var, contained a 4828 bp DNA segment that includes the tet(C) tetracycline resistance determinant. As tet(C) is relatively rare in E. coli and other Gram-negative bacterial isolates, the structure and evolution of tet(C)-containing PCT in available sequences was examined. The largest identified was PTntet(C), a close relative of PTntet(C)-var, and a potential progenitor for these PCT. Most PCT shared one internal boundary with PTntet(C) but the length of the central tet(C)-containing segment was shorter due to IS26-mediated deletions. The most abundant variant form, previously named Tn6309, was widely distributed and, in a derivative of it, most of the tetA(C) gene has been replaced by the tetA(A) gene presumably by homologous recombination.

对从健康成人中回收的耐抗生素共生大肠杆菌分离物中发现的大质粒pCERC14进行了测序。pCERC14全长162,926 bp,携带FII-18和FIB-1复制子、f样转移区以及几个毒力决定因子,其中一些与铁的摄取有关,这对共生生活方式是有利的。质粒主干在11个地方被完整的is (IS1(4拷贝)、IS2(2)、IS629(2)和单个的IS100、IS186、ISEc33)中断,在3个地方被部分is拷贝中断。在两个is26结合的伪复合转座子(PCT)中发现了耐药基因。其中一个含有1类整合子的残余物,其中包括一个dfrA5基因盒和sul1基因,分别赋予对甲氧苄啶和磺胺类药物的抗性。第二个基因名为PTntet(C)-var,包含一个4828bp的DNA片段,其中包含tet(C)四环素抗性决定因子。由于tet(C)在大肠杆菌和其他革兰氏阴性细菌分离株中相对罕见,因此对现有序列中含有tet(C)的PCT的结构和进化进行了研究。鉴定到的最大的是PTntet(C),它是PTntet(C)-var的近亲,是这些PCT的潜在祖先。大多数PCT与PTntet(C)共享一个内部边界,但由于is26介导的缺失,中心包含tet(C)的片段长度较短。最丰富的变异形式,以前被命名为Tn6309,广泛分布,在它的衍生物中,大部分tetA(C)基因被tetA(a)基因所取代,可能是通过同源重组。
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引用次数: 2
Improved pEKEx2-derived expression vectors for tightly controlled production of recombinant proteins in Corynebacterium glutamicum 改良的pekex2衍生表达载体在谷氨酸棒状杆菌中严格控制重组蛋白的产生
IF 2.6 4区 生物学 Q3 GENETICS & HEREDITY Pub Date : 2020-11-01 DOI: 10.1016/j.plasmid.2020.102540
Patrick J. Bakkes, Paul Ramp, Astrid Bida, Doris Dohmen-Olma, Michael Bott, Roland Freudl

The Escherichia coli/Corynebacterium glutamicum shuttle vector pEKEx2 is an IPTG-inducible expression vector that has been used successfully for the synthesis of numerous proteins in C. glutamicum. We discovered that the leaky gene expression observed for pEKEx2-derived plasmids relates to reduced functionality of the plasmid-encoded repressor LacI carrying a modified C-terminus, while duplicate DNA sequences in the pEKEx2 backbone contribute to plasmid instability. We constructed the pEKEx2-derivatives pPBEx2 and pPREx2, which harbor a restored lacI gene and which lack the unnecessary duplicate DNA sequences. pPREx2 in addition enables fusion of target genes to a C-terminal Strep-tag II coding region for easy protein detection and purification. In the absence of inducer, the novel vectors exhibit tight gene repression in C. glutamicum, as shown for the secretory production of Fusarium solani pisi cutinase and the cytosolic production of green fluorescent protein and C. glutamicum myo-inositol dehydrogenase. Undesired heterogeneity amongst clones expressing cutinase from pEKEx2 was attributed to the loss of a vector fragment containing the cutinase gene, which likely occurred via homologous recombination of the identical flanking DNA sequences. Such loss was not observed for pPBEx2. Using pPREx2, IolG-Strep was successfully produced and purified to homogeneity by Strep-Tactin affinity chromatography, obtaining 1.5 mg IolG with a specific activity of 27 μmol·min−1·(mg protein)−1 from 100 mL culture. The tight gene repression in the absence of inducer and the improved plasmid stability make expression vectors pPBEx2/pPREx2 attractive alternatives to the available molecular tools for genetic manipulation and high-level production of recombinant proteins in C. glutamicum.

大肠杆菌/谷氨酸棒状杆菌穿梭载体pEKEx2是iptg诱导的表达载体,已成功用于谷氨酸棒状杆菌多种蛋白的合成。我们发现,pEKEx2衍生质粒的基因表达泄漏与质粒编码抑制因子LacI携带修饰的c端功能降低有关,而pEKEx2主干中的重复DNA序列导致质粒不稳定。我们构建了pekex2衍生物pPBEx2和pPREx2,它们含有一个修复的lacI基因,并且缺少不必要的重复DNA序列。此外,pPREx2还能将靶基因融合到c端Strep-tag II编码区,便于蛋白检测和纯化。在缺乏诱导物的情况下,新载体在谷氨酰胺镰刀菌中表现出紧密的基因抑制,如分泌生产茄灰镰刀菌角质酶和胞质生产绿色荧光蛋白和谷氨酰胺肌醇脱氢酶。表达pEKEx2角质酶的克隆之间不希望的异质性归因于含有角质酶基因的载体片段的丢失,这可能是通过相同的侧翼DNA序列的同源重组发生的。pPBEx2没有观察到这种损失。利用pPREx2成功制备IolG- strep,并通过strep - actin亲和层析纯化到均匀性,从100 mL培养物中获得1.5 mg IolG,比活性为27 μmol·min−1·(mg protein)−1。在没有诱导剂的情况下,pPBEx2/pPREx2表达载体具有较强的基因抑制作用,且质粒稳定性较好,这使得pPBEx2/pPREx2表达载体成为谷氨酸酵母基因操作和高水平重组蛋白生产的有效分子工具。
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引用次数: 20
Antimicrobial resistance gene shuffling and a three-element mobilisation system in the monophasic Salmonella typhimurium strain ST1030 单相鼠伤寒沙门菌ST1030耐药基因重组及三要素动员系统研究
IF 2.6 4区 生物学 Q3 GENETICS & HEREDITY Pub Date : 2020-09-01 DOI: 10.1016/j.plasmid.2020.102532
M. Oliva , C. Calia , M. Ferrara , P. D'Addabbo , M. Scrascia , G. Mulè , R. Monno , C. Pazzani

In this study we describe the genetic elements and the antimicrobial resistance units (RUs) harboured by the Salmonella Typhimurium monophasic variant 1,4,[5],12:i:- strain ST1030. Of the three identified RUs two were chromosomal, RU1 (IS26-blaTEM-1-IS26-strAB-sul2- IS26) and RU2 (IS26-tetR(B)-tetA(B)-ΔIS26), and one, RU3 (a sul3-associated class 1 integron with cassette array dfrA12-orfF-aadA2-cmlA1-aadA1), was embedded in a Tn21-derived element harboured by the conjugative I1 plasmid pST1030-1A. IS26 elements mediated the antimicrobial resistance gene (ARG) shuffling and this gave rise to pST1030-1A derivatives with different sets of ARGs. ST1030 also harboured two ColE1-like plasmids of which one, pST1030-2A, was mobilisable and the target of an intracellular translocation of the Tn21-derived element; the second (pST1030–3) was an orphan mob-associated oriT plasmid co-transferred with pST1030-1A and pST1030-2A. pST1030-2A and pST1030-3 also carried a parA gene and a type III restriction modification system, respectively. Overall analysis of our data reinforces the role played by IS26, Tn21-derived elements and non-conjugative plasmids in the spread of ARGs and supplies the first evidence, at least in Salmonella, for the identification of a natural isolate harbouring a three-element mobilisation system in the same cell.

本文描述了鼠伤寒沙门菌单相变异株ST1030的遗传因子和耐药单位(RUs)。在鉴定的三个RUs中,两个是染色体,RU1 (IS26- blem -1-IS26- strab -sul2- IS26)和RU2 (IS26- tetr (B)- teta (B)-ΔIS26),一个RU3 (sul3相关的1类整合子,盒阵列dfrA12-orfF-aadA2-cmlA1-aadA1),嵌入由共轭I1质粒pST1030-1A携带的tn21衍生元件中。IS26元件介导抗菌素耐药基因(ARG)洗牌,从而产生具有不同ARG组的pST1030-1A衍生物。ST1030还含有两个cole1样质粒,其中一个是可移动的,是tn21衍生元件的细胞内易位的目标;第二个(pST1030-3)是与pST1030-1A和pST1030-2A共转移的孤儿暴发菌相关oriT质粒。pST1030-2A和pST1030-3也分别携带一个parA基因和一个III型限制性修饰系统。我们数据的整体分析强化了IS26、tn21衍生元素和非共轭质粒在ARGs传播中的作用,并提供了第一个证据,至少在沙门氏菌中,为鉴定在同一细胞中具有三元素动员系统的天然分离物提供了第一个证据。
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引用次数: 4
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