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Transposon mutagenesis

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https://www.readbyqxmd.com/read/28515302/transposon-mutagenesis-of-the-zika-virus-genome-highlights-regions-essential-for-rna-replication-and-restricted-for-immune-evasion
#1
Benjamin O Fulton, David Sachs, Megan C Schwarz, Peter Palese, Matthew J Evans
The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions throughout the MR766 ZIKV genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle...
May 17, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28512090/the-vibrio-cholerae-vexgh-rnd-efflux-system-maintains-cellular-homeostasis-by-effluxing-vibriobactin
#2
Dillon E Kunkle, X Renee Bina, James E Bina
Resistance-nodulation-division (RND) superfamily efflux systems have been widely studied for their role in antibiotic resistance, but their native biological functions remain poorly understood. We previously showed that loss of RND-mediated efflux in Vibrio cholerae resulted in activation of the Cpx two-component regulatory system, which mediates adaptation to stress resulting from misfolded membrane proteins. Here, we investigated the mechanism linking RND-mediated efflux to the Cpx response. We performed transposon mutagenesis screening of RND-deficient V...
May 16, 2017: MBio
https://www.readbyqxmd.com/read/28509393/nmea-a-novel-efflux-transporter-specific-for-nucleobases-and-nucleosides-contributes-to-metal-resistance-in-aspergillus-nidulans
#3
Sofia Balaska, Vassilios Myrianthopoulos, Martha Tselika, Dimitris G Hatzinikolaou, Emmanuel Mikros, George Diallinas
Through Minos transposon mutagenesis we obtained A. nidulans mutants resistant to 5-fluorouracil due to insertions into the upstream region of the uncharacterized gene nmeA, encoding a Major Facilitator Superfamily (MFS) transporter. Minos transpositions increased nmeA transcription, which is otherwise extremely low under all conditions tested. To dissect the function of NmeA we used strains overexpressing or genetically lacking the nmeA gene. Strains overexpressing NmeA are resistant to toxic purine analogues, but also, to cadmium, zinc and borate, whereas an isogenic nmeAΔ null mutant exhibits increased sensitivity to these compounds...
May 16, 2017: Molecular Microbiology
https://www.readbyqxmd.com/read/28507068/genome-wide-survey-of-pseudomonas-aeruginosa-pa14-reveals-a-role-for-the-glyoxylate-pathway-and-extracellular-proteases-in-the-utilization-of-mucin
#4
Jeffrey M Flynn, Chi Phan, Ryan C Hunter
Chronic airway infections by the opportunistic pathogen Pseudomonas aeruginosa are a major cause of mortality in cystic fibrosis (CF) patients. While this bacterium has been extensively studied for its virulence determinants, biofilm growth and immune evasion mechanisms, comparatively little is known about the nutrient sources that sustain its growth in vivo Respiratory mucins represent a potentially abundant bioavailable nutrient source, though we have recently shown that canonical pathogens inefficiently use these host glycoproteins as a growth substrate...
May 15, 2017: Infection and Immunity
https://www.readbyqxmd.com/read/28500044/rpon-%C3%AF-54-is-required-for-floc-formation-but-not-for-extracellular-polysaccharide-biosynthesis-in-a-floc-forming-aquincola-tertiaricarbonis-strain
#5
Dianzhen Yu, Ming Xia, Liping Zhang, Yulong Song, You Duan, Tong Yuan, Minjie Yao, Liyou Wu, Chunyuan Tian, Zhenbin Wu, Xiangzhen Li, Jizhong Zhou, Dongru Qiu
Some bacteria are capable of forming flocs in which bacterial cells are self-flocculated by secreted extracellular polysaccharides and other biopolymers. The floc-forming bacteria play a central role in activated sludge, which has been widely utilized for the treatment of municipal sewage and industrial wastewater. Here we use a floc-forming bacterium, Aquincola tertiaricarbonis RN12, as a model to explore the biosynthesis of EPS and the regulation of floc-formation. A large gene cluster for exopolysaccharide biosynthesis and a gene encoding the alternative sigma factor RpoN1, one of the four paralogues, have been identified in the floc-formation-deficient mutants generated by transposon mutagenesis and the gene functions have been further confirmed by genetic complementation analyses...
May 12, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28499399/identification-of-potential-campylobacter-jejuni-genes-involved-in-biofilm-formation-by-ez-tn5-transposome-mutagenesis
#6
Amy Huei Teen Teh, Sui Mae Lee, Gary A Dykes
BACKGROUND: Biofilm formation has been suggested to play a role in the survival of Campylobacter jejuni in the environment and contribute to the high incidence of human campylobacteriosis. Molecular studies of biofilm formation by Campylobacter are sparse. RESULTS: We attempted to identify genes that may be involved in biofilm formation in seven C. jejuni strains through construction of mutants using the EZ-Tn5 Transposome system. Only 14 mutants with reduced biofilm formation were obtained, all from one strain of C...
May 12, 2017: BMC Research Notes
https://www.readbyqxmd.com/read/28489862/novel-genes-associated-with-enhanced-motility-of-escherichia-coli-st131
#7
Asha Kakkanat, Minh-Duy Phan, Alvin W Lo, Scott A Beatson, Mark A Schembri
Uropathogenic Escherichia coli (UPEC) is the cause of ~75% of all urinary tract infections (UTIs) and is increasingly associated with multidrug resistance. This includes UPEC strains from the recently emerged and globally disseminated sequence type 131 (ST131), which is now the dominant fluoroquinolone-resistant UPEC clone worldwide. Most ST131 strains are motile and produce H4-type flagella. Here, we applied a combination of saturated Tn5 mutagenesis and transposon directed insertion site sequencing (TraDIS) as a high throughput genetic screen and identified 30 genes associated with enhanced motility of the reference ST131 strain EC958...
2017: PloS One
https://www.readbyqxmd.com/read/28487432/deciphering-mcr-2-colistin-resistance
#8
Jian Sun, Yongchang Xu, Rongsui Gao, Jingxia Lin, Wenhui Wei, Swaminath Srinivas, Defeng Li, Run-Shi Yang, Xing-Ping Li, Xiao-Ping Liao, Ya-Hong Liu, Youjun Feng
Antibiotic resistance is a prevalent problem in public health worldwide. In general, the carbapenem β-lactam antibiotics are considered a final resort against lethal infections by multidrug-resistant bacteria. Colistin is a cationic polypeptide antibiotic and acts as the last line of defense for treatment of carbapenem-resistant bacteria. Very recently, a new plasmid-borne colistin resistance gene, mcr-2, was revealed soon after the discovery of the paradigm gene mcr-1, which has disseminated globally. However, the molecular mechanisms for MCR-2 colistin resistance are poorly understood...
May 9, 2017: MBio
https://www.readbyqxmd.com/read/28483441/the-cytidine-repressor-participates-in-the-regulatory-pathway-of-indole-in-pantoea-agglomerans
#9
Mengqi Jia, Xuemei Yu, Jing Jiang, Zihua Li, Yongjun Feng
Indole, an important signal molecule in both intraspecies and interspecies, regulates a variety of bacterial behaviors, but its regulatory mechanism is still unknown. Pantoea agglomerans YS19, a preponderant endophytic bacterium isolated from rice, does not produce indole, yet it senses exogenous indole. In this study, a mutant of YS19-Rp(r) whose target gene expression was downregulated by indole was selected through mTn5 transposon mutagenesis. Using the TAIL-PCR technique, the mutation gene was identified as a cytR homologue, which encodes a cytidine repressor (CytR) protein, a bacterial transcription factor involved in a complex regulation scheme...
May 5, 2017: Research in Microbiology
https://www.readbyqxmd.com/read/28481201/functional-mapping-of-yeast-genomes-by-saturated-transposition
#10
Agnès Henria Michel, Riko Hatakeyama, Philipp Kimmig, Meret Arter, Matthias Peter, Joao Matos, Claudio De Virgilio, Benoît Kornmann
Yeast is a powerful model for systems genetics. We present a versatile, time- and labor-efficient method to functionally explore the Saccharomyces cerevisiae genome using saturated transposon mutagenesis coupled to high-throughput sequencing. SAturated Transposon Analysis in Yeast (SATAY) allows one-step mapping of all genetic loci in which transposons can insert without disrupting essential functions. SATAY is particularly suited to discover loci important for growth under various conditions. SATAY (1) reveals positive and negative genetic interactions in single and multiple mutant strains, (2) can identify drug targets, (3) detects not only essential genes, but also essential protein domains, (4) generates both null and other informative alleles...
May 8, 2017: ELife
https://www.readbyqxmd.com/read/28461449/linearmycins-activate-a-two-component-signaling-system-involved-in-bacterial-competition-and-biofilm-morphology
#11
Reed M Stubbendieck, Paul D Straight
Bacteria use two-component signaling systems to adapt and respond to their competitors and changing environments. For instance, competitor bacteria may produce antibiotics and other bioactive metabolites and sequester nutrients. To survive, some species of bacteria escape competition through antibiotic production, biofilm formation or motility. Specialized metabolite production and biofilm formation are relatively well understood for bacterial species in isolation. How bacteria control these functions when competitors are present is not well studied...
May 1, 2017: Journal of Bacteriology
https://www.readbyqxmd.com/read/28439038/pseudomonas-aeruginosa-derived-rhamnolipids-and-other-detergents-modulate-colony-morphotype-and-motility-in-the-burkholderia-cepacia-complex
#12
Steve P Bernier, Courtney Hum, Xiang Li, George A O'Toole, Nathan Magarvey, Michael G Surette
Competitive interactions mediated by released chemicals (e.g. toxins) are prominent in multispecies communities, but their effect at subinhibitory concentrations on susceptible bacteria is poorly understood. Although Pseudomonas aeruginosa and species of the Burkholderia cepacia complex (Bcc) can exist together as a co-infection in cystic fibrosis airways, P. aeruginosa toxins can kill Bcc species in vitro Consequently, these bacteria become an ideal in vitro model system to study the impact of sublethal levels of toxins on the biology of typical susceptible bacteria such as the Bcc when exposed to P...
April 24, 2017: Journal of Bacteriology
https://www.readbyqxmd.com/read/28438927/the-role-of-psl-genes-in-antibiotic-tolerance-of-adherent-pseudomonas-aeruginosa
#13
Keiji Murakami, Tsuneko Ono, Darija Viducic, Yoko Somiya, Reiko Kariyama, Kenji Hori, Takashi Amoh, Katsuhiko Hirota, Hiromi Kumon, Matthew R Parsek, Yoichiro Miyake
Bacteria attached to a surface are generally more tolerant to antibiotics than their planktonic counterparts, even without the formation of a biofilm. The mechanism of antibiotic tolerance in biofilm communities is multifactorial, and the genetic backgound underlying this antibiotic tolerance has not yet been fully elucidated. Using transposon mutagenesis, we isolated a mutant with reduced tolerance to biapenem (relative to the wild-type) from adherent cells. Sequencing analysis revealed a mutation in the pslL gene, which is a part of the polysaccharide biosynthesis operon...
April 24, 2017: Antimicrobial Agents and Chemotherapy
https://www.readbyqxmd.com/read/28432092/a-genome-wide-investigation-on-biofilm-formation-in-bacillus-cereus
#14
Fang Yan, Yiyang Yu, Kevin Gozzi, Yun Chen, Jian-Hua Guo, Yunrong Chai
Bacillus cereus is a soil-dwelling Gram-positive bacterium capable of forming structured multicellular communities, or biofilms. However, the regulatory pathways controlling biofilm formation are less well understood in B. cereus In this work, we developed a method to study B. cereus biofilms formed at the air-liquid interface. We applied two genome-wide approaches, random transposon insertion mutagenesis to identify genes that are potentially important for biofilm formation, and transcriptome analyses by RNA-seq to characterize genes that are differentially expressed in B...
April 21, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/28371510/acuc-a-histone-deacetylase-contributes-to-the-pathogenicity-of-aeromonas-hydrophila
#15
Qingling Jiang, Wenbo Chen, Yingxue Qin, Lixing Huang, Xiaojin Xu, Lingmin Zhao, Qingpi Yan
The interactions of pathogens and phagocytes are complex. Our study demonstrated that Aeromonas hydrophila B11 can survive in the macrophagocytes of Tilapia mossambica. To explore the regulatory processes of A. hydrophila survival in the macrophagocytes, we used the mini-Tn10 transposon mutagenesis system to build a mutant library by mixing Escherichia coli Sm10 (pLOFKm) and A. hydrophila B11. In total, 102 mutant colonies were detected, and 11 of them showed reduced survival in macrophagocytes. The mutant with the most severe phenotype, AM73, was chosen for further research...
March 30, 2017: MicrobiologyOpen
https://www.readbyqxmd.com/read/28340408/ongoing-evolution-of-pseudomonas-aeruginosa-pao1-sublines-complicates-studies-of-dna-damage-repair-and-tolerance
#16
Julia Sidorenko, Tatjana Jatsenko, Maia Kivisaar
Sublines of the major P. aeruginosa reference strain PAO1 are derivatives of the original PAO1 isolate, which are maintained in laboratories worldwide. These sublines display substantial genomic and phenotypic variation due to ongoing microevolution. Here, we examined four sublines, MPAO1, PAO1-L, PAO1-DSM and PAO1-UT, originated from different laboratories, and six DNA polymerase-deficient mutants from the P. aeruginosa MPAO1 transposon library for their employment in elucidation of DNA damage repair and tolerance mechanisms in P...
March 2017: Mutation Research
https://www.readbyqxmd.com/read/28331161/isolation-of-cyanobacterial-mutants-exhibiting-growth-defects-under-microoxic-conditions-by-transposon-tagging-mutagenesis-of-synechocystis-sp-pcc-6803
#17
Kazuki Terauchi, Riho Sobue, Yuho Furutani, Rina Aoki, Yuichi Fujita
Cyanobacteria are photosynthetic prokaryotes that perform oxygenic photosynthesis by extracting electrons from water, with the generation of oxygen as a byproduct. Cyanobacteria use oxygen not only for respiration to produce energy in the dark but also for biosynthesis of various metabolites, such as heme and chlorophyll. Oxygen levels dynamically fluctuate in the field environments, from hyperoxic at daytime to almost anaerobic at night. Thus, adaptation to anaerobiosis should be important for cyanobacteria to survive in low-oxygen and anaerobic environments...
March 22, 2017: Journal of General and Applied Microbiology
https://www.readbyqxmd.com/read/28321208/detection-of-a-putative-tetr-like-gene-related-to-mycobacterium-bovis-bcg-growth-in-cholesterol-using-a-gfp-transposon-mutagenesis-system
#18
Isabel Otal, Esther Pérez-Herrán, Lazaro Garcia-Morales, María C Menéndez, Jorge A Gonzalez-Y-Merchand, Carlos Martín, María J García
In vitro transposition is a powerful genetic tool for identifying mycobacterial virulence genes and studying virulence factors in relation to the host. Transposon shuttle mutagenesis is a method for constructing stable insertions in the genome of different microorganisms including mycobacteria. Using an IS1096 derivative, we have constructed the Tngfp, a transposon containing a promoterless green fluorescent protein (gfp) gene. This transposon was able to transpose randomly in Mycobacterium bovis BCG. Bacteria with a single copy of the gfp gene per chromosome from an M...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28319090/a-single-copy-sleeping-beauty-transposon-mutagenesis-screen-identifies-new-pten-cooperating-tumor-suppressor-genes
#19
Jorge de la Rosa, Julia Weber, Mathias Josef Friedrich, Yilong Li, Lena Rad, Hannes Ponstingl, Qi Liang, Sandra Bernaldo de Quirós, Imran Noorani, Emmanouil Metzakopian, Alexander Strong, Meng Amy Li, Aurora Astudillo, María Teresa Fernández-García, María Soledad Fernández-García, Gary J Hoffman, Rocío Fuente, George S Vassiliou, Roland Rad, Carlos López-Otín, Allan Bradley, Juan Cadiñanos
The overwhelming number of genetic alterations identified through cancer genome sequencing requires complementary approaches to interpret their significance and interactions. Here we developed a novel whole-body insertional mutagenesis screen in mice, which was designed for the discovery of Pten-cooperating tumor suppressors. Toward this aim, we coupled mobilization of a single-copy inactivating Sleeping Beauty transposon to Pten disruption within the same genome. The analysis of 278 transposition-induced prostate, breast and skin tumors detected tissue-specific and shared data sets of known and candidate genes involved in cancer...
May 2017: Nature Genetics
https://www.readbyqxmd.com/read/28295042/generation-of-non-viral-transgene-free-hepatocyte-like-cells-with-piggybac-transposon
#20
Hokahiro Katayama, Kentaro Yasuchika, Yuya Miyauchi, Hidenobu Kojima, Ryoya Yamaoka, Takayuki Kawai, Elena Yukie Yoshitoshi, Satoshi Ogiso, Sadahiko Kita, Katsutaro Yasuda, Naoya Sasaki, Ken Fukumitsu, Junji Komori, Takamichi Ishii, Shinji Uemoto
Somatic cells can be reprogrammed to induced hepatocyte-like cells (iHeps) by overexpressing certain defined factors in direct reprogramming techniques. Of the various methods to deliver genes into cells, typically used genome-integrating viral vectors are associated with integration-related adverse events such as mutagenesis, whereas non-integrating viral vectors have low efficiency, making viral vectors unsuitable for clinical application. Therefore, we focused on developing a transposon system to establish a non-viral reprogramming method...
March 15, 2017: Scientific Reports
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