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Transposon mutagenesis

Peng Tang, Jie Wu, Hou Liu, Youcai Liu, Xingding Zhou
One of the newly developed methods for Assimilable organic carbon (AOC) determination is leveraged on the cell enumeration by flow cytometry (FC) which could provide a rapid and automated solution for AOC measurement. However, cell samples staining with fluorescence dye is indispensable to reduce background and machine noise. This step would bring additional cost and time consuming for this method. In this study, a green fluorescence protein (GFP) tagged strain derived of AOC testing strain Pseudomonas fluorescens P-17 (GFP-P17) was generated using Tn5 transposon mutagenesis...
2018: PloS One
Benjamin O Fulton, Weina Sun, Nicholas S Heaton, Peter Palese
Influenza A and B viruses can continuously evade humoral immune responses by developing mutations in the globular head of the hemagglutinin (HA) that prevent antibody binding. However, the influenza B virus HA over time displays less antigenic variation despite being functionally and structurally similar to the influenza A virus HA. To determine if the influenza B virus HA is under constraints that limit its antigenic variation, we performed a transposon screen to compare the mutational tolerance of the currently circulating influenza A virus HAs (H1 and H3 subtypes) and influenza B virus HAs (B/Victoria87 and B/Yamagata88 antigenic lineages)...
June 13, 2018: Journal of Virology
Diane Mutumwinka, Shen-Bao Zhao, Yi-Shi Liu, Emmanuel Osei Mensah, Xiao-Dong Gao, Morihisa Fujita
Glycoengineered yeast cells, which express human-compatible glycan structures, are particularly attractive host cells to produce therapeutic glycoproteins. Disruption of OCH1 gene, which encodes an α-1,6-mannosyltransferase required for mannan-type N-glycan formation, is essential for the elimination of yeast-specific N-glycan structures. However, the gene disruption causes cell wall defects leading to growth defects. Here, we tried to identify factors to rescue the growth defects of och1Δ cells by in vivo mutagenesis using piggyBac (PB)-based transposon...
June 8, 2018: Bioscience, Biotechnology, and Biochemistry
Shuaixin Sun, Yunpeng Chen, Jiejie Cheng, Qiongjie Li, Zhenchuan Zhang, Zhengliang Lan
A gram-negative bacterium GXGL-4A was originally isolated from maize roots. It displayed nitrogen-fixing (NF) ability under nitrogen-free culture condition, and had a significant promotion effect on cucumber growth in the pot inoculation test. The preliminary physiological and biochemical traits of GXGL-4A were characterized. Furthermore, a phylogenetic tree was constructed based on 16S ribosomal DNA (rDNA) sequences of genetically related species. To determine the taxonomic status of GXGL-4A and further utilize its nitrogen-fixing potential, genome sequence was obtained using PacBio RS II technology...
June 6, 2018: Folia Microbiologica
Shuai Luo, Xin-Ai Chen, Xu-Ming Mao, Yong-Quan Li
Production of secondary metabolites in Streptomyces is regulated by a complex regulatory network precisely, elaborately, and hierarchically. One of the main reasons for the low yields of some high-value secondary metabolites is the repressed expression of their biosynthetic gene clusters, supposedly by some gene cluster out-situated negative regulators. Identification of these repressors and removal of the inhibitory effects based on the regulatory mechanisms will be an effective way to improve their yields...
June 6, 2018: Applied Microbiology and Biotechnology
Pang-Dian Fan, Giuseppe Narzisi, Anitha D Jayaprakash, Elisa Venturini, Nicolas Robine, Peter Smibert, Soren Germer, Helena A Yu, Emmet J Jordan, Paul K Paik, Yelena Y Janjigian, Jamie E Chaft, Lu Wang, Achim A Jungbluth, Sumit Middha, Lee Spraggon, Huan Qiao, Christine M Lovly, Mark G Kris, Gregory J Riely, Katerina Politi, Harold Varmus, Marc Ladanyi
In ∼30% of patients with EGFR -mutant lung adenocarcinomas whose disease progresses on EGFR inhibitors, the basis for acquired resistance remains unclear. We have integrated transposon mutagenesis screening in an EGFR -mutant cell line and clinical genomic sequencing in cases of acquired resistance to identify mechanisms of resistance to EGFR inhibitors. The most prominent candidate genes identified by insertions in or near the genes during the screen were MET , a gene whose amplification is known to mediate resistance to EGFR inhibitors, and the gene encoding the Src family kinase YES1...
June 6, 2018: Proceedings of the National Academy of Sciences of the United States of America
Hyun-Ji Choi, Han-Byul Lee, Sunyoung Jung, Hyun-Kyu Park, Woori Jo, Sung-Min Cho, Woo-Jin Kim, Woo-Chan Son
The Sleeping Beauty (SB) transposon system is non-viral and uses insertional mutagenesis, resulting in the permanent expression of transferred genes. Although the SB transposon is a useful method for establishing a mouse tumor model, there has been difficulty in using this method to generate tumors in the prostate. In the present study, electroporation was used to enhance the transfection efficiency of the SB transposon. To generate tumors, three constructs (a c-Myc expression cassette, a HRAS (HRas proto-oncogene, GTPase) expression cassette and a shRNA against p53 ) contained within the SB transposon plasmids were directly injected into the prostate...
June 5, 2018: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Liu Rui, Li Weiyi, Meng Yu, Zhou Hong, Yu Jiao, Ma Zhe, Fan Hongjie
Streptococcus suis serotype 2 (SS2) is a zoonotic agent that causes meningitis in humans and pigs. However, the mechanism whereby SS2 crosses the microvasculature endothelium of the brain is not understood. In this study, transposon (TnYLB-1) mutagenesis was used to identify virulence factors potentially associated with invasive ability in pathogenic SS2. A poorly invasive mutant was identified, and was found to contain a TnYLB-1 insertion in the serine/threonine kinase (stk) gene. Transwell chambers containing hBMECs were used to model the blood-brain barrier (BBB)...
May 24, 2018: Cellular Microbiology
Kurt Patterson, James Yu, Jenny Landberg, Ivan Chang, Farbod Shavarebi, Virginia Bilanchone, Suzanne Sandmeyer
Oleaginous yeasts are valuable systems for biosustainable production of hydrocarbon-based chemicals. Yarrowia lipolytica is one of the best characterized of these yeast with respect to genome annotation and flux analysis of metabolic processes. Nonetheless, progress is hampered by a dearth of genome-wide tools enabling functional genomics. In order to remedy this deficiency, we developed a library of Y. lipolytica insertion mutants via transposon mutagenesis. The Hermes DNA transposon was expressed to achieve saturation mutagenesis of the genome...
May 21, 2018: Metabolic Engineering
Mevlüt Arslan, Can Holyavkin, Halil İbrahim Kısakesen, Alican Topaloğlu, Yusuf Sürmeli, Zeynep Petek Çakar
High-throughput aging studies with yeast as a model organism involve transposon-mutagenesis and yeast knockout collection, which have been pivotal strategies for understanding the complex cellular aging process. In this study, a chronologically long-lived Saccharomyces cerevisiae mutant was successfully obtained by using another high-throughput approach, evolutionary engineering, based on systematic selection in successive batch cultures under gradually increasing levels of caloric restriction. Detailed comparative physiological and transcriptomic analyses of the chronologically long-lived mutant and the reference strain revealed enhanced levels of respiratory metabolism, upregulation of genes related to carbohydrate metabolic processes, glycogen-trehalose pathways, stress response, and repression of protein synthesis-related genes in the long-lived mutant SRM11, already in the absence of caloric restriction...
May 19, 2018: Molecular Biotechnology
Herman K Edskes, Maryam Mukhamedova, Bouke K Edskes, Reed B Wickner
[URE3] is an amyloid-based prion of Ure2p, a regulator of nitrogen catabolism. While most variants of the [URE3] prion are toxic, mild variants that only slightly slow growth are more widely studied. The existence of several anti-prion systems suggests that some components may be protecting cells from potential detrimental effects of mild [URE3] variants. Our extensive Hermes transposon mutagenesis showed that disruption of YLR352W dramatically slows growth of [URE3-1] strains. Ylr352wp is an F-box protein, directing selection of substrates for ubiquitination by a cullin-containing E-3 ligase...
May 16, 2018: Genetics
Min Zhang, Chengqi Wang, Thomas D Otto, Jenna Oberstaller, Xiangyun Liao, Swamy R Adapa, Kenneth Udenze, Iraad F Bronner, Deborah Casandra, Matthew Mayho, Jacqueline Brown, Suzanne Li, Justin Swanson, Julian C Rayner, Rays H Y Jiang, John H Adams
Severe malaria is caused by the apicomplexan parasite Plasmodium falciparum. Despite decades of research, the distinct biology of these parasites has made it challenging to establish high-throughput genetic approaches to identify and prioritize therapeutic targets. Using transposon mutagenesis of P. falciparum in an approach that exploited its AT-rich genome, we generated more than 38,000 mutants, saturating the genome and defining mutability and fitness costs for over 87% of genes. Of 5399 genes, our study defined 2680 genes as essential for optimal growth of asexual blood stages in vitro...
May 4, 2018: Science
Rebecca L Lamason, Natasha M Kafai, Matthew D Welch
The rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular bacteria, rickettsiae are highly adapted to living inside a variety of host cells, including vascular endothelial cells during mammalian infection. Although it is assumed that the rickettsiae produce numerous virulence factors that usurp or disrupt various host cell pathways, they have been challenging to genetically manipulate to identify the key bacterial factors that contribute to infection...
2018: PloS One
Nina Jagmann, Bodo Philipp
The opportunistic pathogen Pseudomonas aeruginosa employs its complex quorum sensing (QS) network to regulate the expression of virulence factors such as pyocyanin. Besides cell density, QS in this bacterium is co-regulated by environmental cues. In this study, we employed a previously established co-culture model system to identify metabolic influences that are involved in the regulation of pyocyanin production in P. aeruginosa . In this co-culture consisting of P. aeruginosa and the chitinolytic bacterium Aeromonas hydrophila , parasitic growth of P...
2018: Frontiers in Microbiology
Imke Spöring, Sebastian Felgner, Matthias Preuße, Denitsa Eckweiler, Manfred Rohde, Susanne Häussler, Siegfried Weiss, Marc Erhardt
Flagellum-driven motility of Salmonella enterica serovar Typhimurium facilitates host colonization. However, the large extracellular flagellum is also a prime target for the immune system. As consequence, expression of flagella is bistable within a population of Salmonella , resulting in flagellated and nonflagellated subpopulations. This allows the bacteria to maximize fitness in hostile environments. The degenerate EAL domain protein RflP (formerly YdiV) is responsible for the bistable expression of flagella by directing the flagellar master regulatory complex FlhD4 C2 with respect to proteolytic degradation...
May 1, 2018: MBio
Edinson Puentes-Cala, Manuel Liebeke, Stephanie Markert, Jens Harder
The enzymatic functionalization of hydrocarbons is a central step in the global carbon cycle initiating the mineralization of methane, isoprene and monoterpenes, the most abundant biologically produced hydrocarbons. Also, terpene-modifying enzymes have found many applications in the energy-economic biotechnological production of fine chemicals. Here we describe a limonene dehydrogenase that was purified from the facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen grown on monoterpenes under denitrifying conditions in the absence of molecular oxygen...
May 1, 2018: Journal of Biological Chemistry
Masahiro Takeo, Kenta Yamamoto, Masashi Sonoyama, Kana Miyanaga, Nana Kanbara, Koichi Honda, Dai-Ichiro Kato, Seiji Negoro
3-Methyl-4-nitrophenol (3M4NP) is formed in soil as a hydrolysis product of fenitrothion, one of the major organophosphorus pesticides. A Pseudomonas strain was isolated as a 3M4NP degrader from a crop soil and designated TSN1. This strain utilized 3M4NP as a sole carbon and energy source. To elucidate the biodegradation pathway, we performed transposon mutagenesis with pCro2a (mini-Tn5495) and obtained three mutants accumulating a dark pink compound(s) from 3M4NP. Rescue cloning and sequence analysis revealed that in all mutants, the transposon disrupted an identical aromatic compound meta-cleaving dioxygenase gene, and a monooxygenase gene was located just downstream of the dioxygenase gene...
April 23, 2018: Journal of Bioscience and Bioengineering
Dieter Bulach, Ben Adler
Until about 15 years ago, the molecular and cellular basis for pathogenesis in leptospirosis was virtually unknown. The determination of the first full genome sequence in 2003 was followed rapidly by other whole genome sequences, whose availability facilitated the development of transposon mutagenesis and then directed mutagenesis of pathogenic Leptospira spp. The combination of genomics, transcriptomics and mutant construction and characterisation has resulted in major progress in our understanding of the taxonomy and biology of Leptospira...
April 26, 2018: Current Topics in Microbiology and Immunology
Chenggang Wu, Abu Amar Mohamed Al Mamun, Truc Thanh Luong, Bo Hu, Jianhua Gu, Ju Huck Lee, Melissa D'Amore, Asis Das, Hung Ton-That
Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions...
April 24, 2018: MBio
Ambika M V Murthy, Minh-Duy Phan, Kate M Peters, Nguyen Thi Khanh Nhu, Rodney A Welch, Glen C Ulett, Mark A Schembri, Matthew J Sweet
Uropathogenic E. coli (UPEC) causes the majority of urinary tract infections (UTIs), which are a major global public health concern. UPEC uses numerous mechanisms to subvert the innate immune system, including targeting macrophage functions. We recently showed that some UPEC strains rapidly kill human macrophages via an NLRP3-independent pathway, and also trigger NLRP3-dependent IL-1β processing. In this study, we used random transposon mutagenesis in the reference strain CFT073 to identify UPEC genes that mediate human macrophage cell death...
December 31, 2018: Virulence
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