scholarly journals High-throughput discovery of phage receptors using transposon insertion sequencing of bacteria

2020 ◽  
Vol 117 (31) ◽  
pp. 18670-18679 ◽  
Author(s):  
Kaitlyn E. Kortright ◽  
Benjamin K. Chan ◽  
Paul E. Turner
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
Environmental Samples ◽  
Proof Of Concept ◽  
Cellular Receptors ◽  
Transposon Insertion ◽  
Genes Encoding ◽  
Phage Receptor ◽  
Bacterial Genes ◽  
Transposon Insertion Sequencing

As the most abundant microbes on Earth, novel bacteriophages (phages; bacteria-specific viruses) are readily isolated from environmental samples. However, it remains challenging to characterize phage–bacteria interactions, such as the host receptor(s) phages bind to initiate infection. Here, we tested whether transposon insertion sequencing (INSeq) could be used to identify bacterial genes involved in phage binding. As proof of concept, results showed that INSeq screens successfully identified genes encoding known receptors for previously characterized viruses ofEscherichia coli(phages T6, T2, T4, and T7). INSeq screens were then used to identify genes involved during infection of six newly isolated coliphages. Results showed that candidate receptors could be successfully identified for the majority (five of six) of the phages; furthermore, genes encoding the phage receptor(s) were the top hit(s) in the analyses of the successful screens. INSeq screens provide a generally useful method for high-throughput discovery of phage receptors. We discuss limitations of our approach when examining uncharacterized phages, as well as usefulness of the method for exploring the evolution of broad versus narrow use of cellular receptors among phages in the biosphere.

scholarly journals Identification of the Two Missing Bacterial Genes Involved in Thiamine Salvage: Thiamine Pyrophosphokinase and Thiamine Kinase

2004 ◽  
Vol 186 (11) ◽  
pp. 3660-3662 ◽  
Author(s):  
Jonathan Melnick ◽  
Ewa Lis ◽  
Joo-Heon Park ◽  
Cynthia Kinsland ◽  
Hirotada Mori ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Genes Encoding ◽  
Bacterial Genes

ABSTRACT The genes encoding thiamine kinase in Escherichia coli (ycfN) and thiamine pyrophosphokinase in Bacillus subtilis (yloS) have been identified. This study completes the identification of the thiamine salvage enzymes in bacteria.

scholarly journals Occurrence and Characteristics of Mobile Colistin Resistance (mcr) Gene-Containing Isolates from the Environment: A Review

2020 ◽  
Vol 17 (3) ◽  
pp. 1028 ◽  
Author(s):  
Madubuike Umunna Anyanwu ◽  
Ishmael Festus Jaja ◽  
Obichukwu Chisom Nwobi
Keyword(s):  
Escherichia Coli ◽  
Drug Resistance ◽  
Environmental Samples ◽  
Insertion Sequences ◽  
Environmental Isolates ◽  
Colistin Resistance ◽  
Public Places ◽  
Extensive Drug Resistance ◽  
Class 1 ◽  
Genes Encoding

The emergence and spread of mobile colistin (COL) resistance (mcr) genes jeopardize the efficacy of COL, a last resort antibiotic for treating deadly infections. COL has been used in livestock for decades globally. Bacteria have mobilized mcr genes (mcr-1 to mcr-9). Mcr-gene-containing bacteria (MGCB) have disseminated by horizontal/lateral transfer into diverse ecosystems, including aquatic, soil, botanical, wildlife, animal environment, and public places. The mcr-1, mcr-2, mcr-3, mcr-5, mcr-7, and mcr-8 have been detected in isolates from and/or directly in environmental samples. These genes are harboured by Escherichia coli, Enterobacter, Klebsiella, Proteus, Salmonella, Citrobacter, Pseudomonas, Acinetobacter, Kluyvera, Aeromonas, Providencia, and Raulotella isolates. Different conjugative and non-conjugative plasmids form the backbones for mcr in these isolates, but mcr have also been integrated into the chromosome of some strains. Insertion sequences (IS) (especially ISApl1) located upstream or downstream of mcr, class 1–3 integrons, and transposons are other drivers of mcr in the environment. Genes encoding multi-/extensive-drug resistance and virulence are often co-located with mcr on plasmids in environmental isolates. Transmission of mcr to/among environmental strains is clonally unrestricted. Contact with the mcr-containing reservoirs, consumption of contaminated animal-/plant-based foods or water, international animal-/plant-based food trades and travel, are routes for transmission of MGCB.

scholarly journals Molecular Cloning of Cyanobacterial Pteridine Glycosyltransferases That Catalyze the Transfer of either Glucose or Xylose to Tetrahydrobiopterin

2010 ◽  
Vol 76 (22) ◽  
pp. 7658-7661 ◽  
Author(s):  
Yeol Gyun Lee ◽  
Ae Hyun Kim ◽  
Mi Bi Park ◽  
Hye-Lim Kim ◽  
Kon Ho Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Cloning ◽  
Environmental Samples ◽  
Genomic Dna ◽  
Pcr Amplification ◽  
Activity Assay ◽  
Genes Encoding ◽  
Cyanobacterial Genes

ABSTRACT Here, we report cloning of cyanobacterial genes encoding pteridine glycosyltransferases that catalyze glucosyl or xylosyl transfer from UDP-sugars to tetrahydrobiopterin. The genes were cloned by PCR amplification from genomic DNA which was isolated from culture and environmental samples and overexpressed in Escherichia coli for an in vitro activity assay.

scholarly journals Osmosensitivity Associated with Insertions in argP (iciA) or glnE in Glutamate Synthase-Deficient Mutants of Escherichia coli

2004 ◽  
Vol 186 (19) ◽  
pp. 6391-6399 ◽  
Author(s):  
Madhusudan R. Nandineni ◽  
Rakesh S. Laishram ◽  
J. Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Glutamate Synthase ◽  
Insertion Mutagenesis ◽  
Inhibitory Effects ◽  
Small Perturbations ◽  
Transposon Insertion ◽  
Genes Encoding ◽  
Glutamate Dehydrogenase Activity ◽  
Derivatives Of ◽  
Enrichment Strategy

ABSTRACT An ampicillin enrichment strategy following transposon insertion mutagenesis was employed to obtain NaCl-sensitive mutants of a gltBD (glutamate synthase [GOGAT]-deficient) strain of Escherichia coli. It was reasoned that the gltBD mutation would sensitize the parental strain even to small perturbations affecting osmotolerance. Insertions conferring an osmosensitive phenotype were identified in the proU, argP (formerly iciA), and glnE genes encoding a glycine betaine/proline transporter, a LysR-type transcriptional regulator, and the adenylyltransferase for glutamine synthetase, respectively. The gltBD+ derivatives of the strains were not osmosensitive. The argP mutation, but not the glnE mutation, was associated with reduced glutamate dehydrogenase activity and a concomitant NH4 + assimilation defect in the gltBD strain. Supplementation of the medium with lysine or a lysine-containing dipeptide phenocopied the argP null mutation for both osmosensitivity and NH4 + assimilation deficiency in a gltBD background, and a dominant gain-of-function mutation in argP was associated with suppression of these lysine inhibitory effects. Osmosensitivity in the gltBD strains, elicited either by lysine supplementation or by introduction of the argP or glnE mutations (but not proU mutations), was also correlated with a reduction in cytoplasmic glutamate pools in cultures grown at elevated osmolarity. We propose that an inability to accumulate intracellular glutamate at high osmolarity underlies the osmosensitive phenotype of both the argP gltBD and glnE gltBD mutants, the former because of a reduction in the capacity for NH4 + assimilation into glutamate and the latter because of increased channeling of glutamate into glutamine.

Transposon Insertion Sequencing, a Global Measure of Gene Function

2020 ◽  
Vol 54 (1) ◽  
pp. 337-365
Author(s):  
Tim van Opijnen ◽  
Henry L. Levin
Keyword(s):  
Dna Sequencing ◽  
High Throughput ◽  
Bacterial Infections ◽  
Single Experiment ◽  
Transposon Insertion ◽  
High Throughput Method ◽  
High Throughput Dna Sequencing ◽  
Complete Set ◽  
Living Organisms ◽  
Transposon Insertion Sequencing

The goal of genomics and systems biology is to understand how complex systems of factors assemble into pathways and structures that combine to form living organisms. Great advances in understanding biological processes result from determining the function of individual genes, a process that has classically relied on characterizing single mutations. Advances in DNA sequencing has made available the complete set of genetic instructions for an astonishing and growing number of species. To understand the function of this ever-increasing number of genes, a high-throughput method was developed that in a single experiment can measure the function of genes across the genome of an organism. This occurred approximately 10 years ago, when high-throughput DNA sequencing was combined with advances in transposon-mediated mutagenesis in a method termed transposon insertion sequencing (TIS). In the subsequent years, TIS succeeded in addressing fundamental questions regarding the genes of bacteria, many of which have been shown to play central roles in bacterial infections that result in major human diseases. The field of TIS has matured and resulted in studies of hundreds of species that include significant innovations with a number of transposons. Here, we summarize a number of TIS experiments to provide an understanding of the method and explanation of approaches that are instructive when designing a study. Importantly, we emphasize critical aspects of a TIS experiment and highlight the extension and applicability of TIS into nonbacterial species such as yeast.

scholarly journals Plant Lectin-Like Bacteriocin from a Rhizosphere-Colonizing Pseudomonas Isolate

2003 ◽  
Vol 185 (3) ◽  
pp. 897-908 ◽  
Author(s):  
Annabel H. A. Parret ◽  
Geert Schoofs ◽  
Paul Proost ◽  
René De Mot
Keyword(s):  
Signal Sequence ◽  
Insertion Site ◽  
Acid Product ◽  
E Coli ◽  
Transposon Insertion ◽  
Mannose Binding ◽  
Genes Encoding ◽  
Bacterial Genes ◽  
Binding Lectin ◽  
Minimal Region

ABSTRACT Rhizosphere isolate Pseudomonas sp. strain BW11M1, which belongs to the Pseudomonas putida cluster, secretes a heat- and protease-sensitive bacteriocin which kills P. putida GR12-2R3. The production of this bacteriocin is enhanced by DNA-damaging treatment of producer cells. We isolated a TnMod mutant of strain BW11M1 that had lost the capacity to inhibit the growth of strain GR12-2R3. A wild-type genomic fragment encompassing the transposon insertion site was shown to confer the bacteriocin phenotype when it was introduced into Escherichia coli cells. The bacteriocin structural gene was identified by defining the minimal region required for expression in E. coli. This gene was designated llpA (lectin-like putidacin) on the basis of significant homology of its 276-amino-acid product with mannose-binding lectins from monocotyledonous plants. LlpA is composed of two monocot mannose-binding lectin (MMBL) domains. Several uncharacterized bacterial genes encoding diverse proteins containing one or two MMBL domains were identified. A phylogenetic analysis of the MMBL domains present in eukaryotic and prokaryotic proteins assigned the putidacin domains to a new bacterial clade within the MMBL-containing protein family. Heterologous expression of the llpA gene also conveyed bacteriocin production to several Pseudomonas fluorescens strains. In addition, we demonstrated that strain BW11M1 and heterologous hosts secrete LlpA into the growth medium without requiring a cleavable signal sequence. Most likely, the mode of action of this lectin-like bacteriocin is different from the modes of action of previously described Pseudomonas bacteriocins.

scholarly journals ESSENTIALS: Software for Rapid Analysis of High Throughput Transposon Insertion Sequencing Data

PLoS ONE ◽  
2012 ◽  
Vol 7 (8) ◽  
pp. e43012 ◽  
Author(s):  
Aldert Zomer ◽  
Peter Burghout ◽  
Hester J. Bootsma ◽  
Peter W. M. Hermans ◽  
Sacha A. F. T. van Hijum
Keyword(s):  
High Throughput ◽  
Rapid Analysis ◽  
Sequencing Data ◽  
Transposon Insertion ◽  
Transposon Insertion Sequencing

scholarly journals Genome-wide identification of sexual-reproduction genes in fission yeast via transposon-insertion sequencing

2021 ◽  
Author(s):  
R. Blake Billmyre ◽  
Michael T. Eickbush ◽  
Caroline J. Craig ◽  
Jeffrey J. Lange ◽  
Christopher Wood ◽  
...  
Keyword(s):  
Sexual Reproduction ◽  
Fission Yeast ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Spore Viability ◽  
Transposon Insertion ◽  
Uncharacterized Gene ◽  
Genome Wide ◽  
Cell Densities ◽  
Transposon Insertion Sequencing

AbstractMany genes required for sexual reproduction remain to be identified. Moreover, many of the genes that are known have been characterized in distinct experiments using different conditions, which complicates understanding the relative contributions of genes to sex. To address these challenges, we developed an assay in Schizosaccharomyces pombe that couples transposon mutagenesis with high-throughput sequencing (TN-seq) to quantitatively measure the fitness contribution of nonessential genes across the genome to sexual reproduction. This approach identified 532 genes that contribute to sex, including more than 200 that were not previously annotated to be involved in the process, of which more than 150 have orthologs in vertebrates. Among our verified hits was an uncharacterized gene, ifs1 (important for sex), that is required for spore viability. In two other hits, plb1 and alg9, we observed a novel mutant phenotype of poor spore health wherein viable spores are produced, but the spores exhibit low fitness and are rapidly outcompeted by wildtype. Finally, we fortuitously discovered that a gene previously thought to be essential, sdg1 (social distancing gene), is instead required for growth at low cell densities. Our assay will be valuable in further studies of sexual reproduction in S. pombe and identifies multiple candidate genes that could contribute to sexual reproduction in other eukaryotes, including humans.

scholarly journals Genes Involved in Intrinsic Antibiotic Resistance of Acinetobacter baylyi

2006 ◽  
Vol 50 (11) ◽  
pp. 3562-3567 ◽  
Author(s):  
Maria J. Gomez ◽  
Alexander A. Neyfakh
Keyword(s):  
Intrinsic Resistance ◽  
Acinetobacter Baylyi ◽  
Glutathione Biosynthesis ◽  
Transposon Insertion ◽  
Homologous Genes ◽  
Knockout Mutants ◽  
Genes Encoding ◽  
Bacterial Genes ◽  
Species Specific ◽  
Transposon Insertions

ABSTRACT Bacterial genes defining intrinsic resistance to antibiotics encode proteins that can be targeted by antibiotic potentiators. To find such genes, a transposon insertion library of Acinetobacter baylyi was screened with subinhibitory concentrations of various antibiotics to find supersusceptible mutants. A DNA microarray printer was used to replica plate 10,000 individual library clones to select mutants unable to grow at 1/10 the MICs of 12 different antibiotics. Transposon insertions in 11 genes were found to cause an eightfold or higher hypersusceptibility to at least one antibiotic. Most of the mutants identified exhibited hypersusceptibility to β-lactam antibiotics. These included mutants with disruptions of genes encoding proteins involved in efflux (acrB and oprM) as well as genes pertaining to peptidoglycan synthesis and modification (ampD, mpl, and pbpG). However, disruptions of genes encoding proteins with seemingly unrelated functions (gph, argH, hisF, and ACIAD0795) can also render cells hypersusceptible to β-lactam antibiotics. A knockout of gshA, involved in glutathione biosynthesis, enhanced the susceptibility to metronidazole, while a knockout of recD, involved in recombination and repair, made the bacteria hypersusceptible to ciprofloxacin. Disruption of acrB in Escherichia coli rendered the cells hypersusceptible to several antibiotics. However, knockout mutants of other homologous genes in E. coli showed no significant changes in antibiotic MICs, indicating that the intrinsic resistance genes are species specific.

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