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Colin W Russell, Amanda C Richards, Alexander S Chang, Matthew A Mulvey
Extraintestinal pathogenic Escherichia coli (ExPEC) strains are typically benign within the mammalian gut, but can disperse to extraintestinal sites to cause disease. As occupation of the intestinal tract is often a prerequisite for ExPEC-mediated pathogenesis, we set out to understand how ExPEC colonizes this niche. A screen using transposon sequencing (Tn-seq) was performed to search for genes within the ExPEC isolate F11 that are important for growth in intestinal mucus, which is thought to be a major source of nutrients for E...
April 3, 2017: Infection and Immunity
Michael A DeJesus, Subhalaxmi Nambi, Clare M Smith, Richard E Baker, Christopher M Sassetti, Thomas R Ioerger
Tn-Seq is an experimental method for probing the functions of genes through construction of complex random transposon insertion libraries and quantification of each mutant's abundance using next-generation sequencing. An important emerging application of Tn-Seq is for identifying genetic interactions, which involves comparing Tn mutant libraries generated in different genetic backgrounds (e.g. wild-type strain versus knockout strain). Several analytical methods have been proposed for analyzing Tn-Seq data to identify genetic interactions, including estimating relative fitness ratios and fitting a generalized linear model...
February 22, 2017: Nucleic Acids Research
Meghan E Ramsey, Jenny A Hyde, Diana N Medina-Perez, Tao Lin, Lihui Gao, Maureen E Lundt, Xin Li, Steven J Norris, Jon T Skare, Linden T Hu
Borrelia burgdorferi, the causative agent of Lyme disease in humans, is exposed to reactive oxygen and nitrogen species (ROS and RNS) in both the tick vector and vertebrate reservoir hosts. B. burgdorferi contains a limited repertoire of canonical oxidative stress response genes, suggesting that novel gene functions may be important for protection of B. burgdorferi against ROS or RNS exposure. Here, we use transposon insertion sequencing (Tn-seq) to conduct an unbiased search for genes involved in resistance to nitric oxide, hydrogen peroxide, and tertiary-butyl hydroperoxide in vitro...
February 2017: PLoS Pathogens
Andrew K Fenton, Lamya El Mortaji, Derek T C Lau, David Z Rudner, Thomas G Bernhardt
Most bacterial cells are surrounded by a peptidoglycan cell wall that is essential for their integrity. The major synthases of this exoskeleton are called penicillin-binding proteins (PBPs)(1,2). Surprisingly little is known about how cells control these enzymes, given their importance as drug targets. In the model Gram-negative bacterium Escherichia coli, outer membrane lipoproteins are critical activators of the class A PBPs (aPBPs)(3,4), bifunctional synthases capable of polymerizing and crosslinking peptidoglycan to build the exoskeletal matrix(1)...
December 12, 2016: Nature Microbiology
J Elijah Powell, Sean P Leonard, Waldan K Kwong, Philipp Engel, Nancy A Moran
Animal guts are often colonized by host-specialized bacterial species to the exclusion of other transient microorganisms, but the genetic basis of colonization ability is largely unknown. The bacterium Snodgrassella alvi is a dominant gut symbiont in honey bees, specialized in colonizing the hindgut epithelium. We developed methods for transposon-based mutagenesis in S. alvi and, using high-throughput DNA sequencing, screened genome-wide transposon insertion (Tn-seq) and transcriptome (RNA-seq) libraries to characterize both the essential genome and the genes facilitating host colonization...
November 29, 2016: Proceedings of the National Academy of Sciences of the United States of America
Kristel Lourdault, James Matsunaga, David A Haake
Pathogenic species of Leptospira are the causative agents of leptospirosis, a zoonotic disease that causes mortality and morbidity worldwide. The understanding of the virulence mechanisms of Leptospira spp is still at an early stage due to the limited number of genetic tools available for this microorganism. The development of random transposon mutagenesis in pathogenic strains a decade ago has contributed to the identification of several virulence factors. In this study, we used the transposon sequencing (Tn-Seq) technique, which combines transposon mutagenesis with massive parallel sequencing, to study the in vivo fitness of a pool of Leptospira interrogans mutants...
November 2016: PLoS Neglected Tropical Diseases
Angela M Mitchell, Wei Wang, Thomas J Silhavy
Gram-negative bacteria have effective methods of excluding toxic compounds, including a largely impermeable outer membrane (OM) and a range of efflux pumps. Furthermore, when cells become nutrient limited, RpoS enacts a global expression change providing cross-protection against many stresses. Here, we utilized sensitivity to an anionic detergent (sodium dodecyl sulfate [SDS]) to probe changes occurring to the cell's permeability barrier during nutrient limitation. Escherichia coli is resistant to SDS whether cells are actively growing, carbon limited, or nitrogen limited...
January 15, 2017: Journal of Bacteriology
Martin Holm Rau, Patricia Calero, Rebecca M Lennen, Katherine S Long, Alex T Nielsen
BACKGROUND: Economically viable biobased production of bulk chemicals and biofuels typically requires high product titers. During microbial bioconversion this often leads to product toxicity, and tolerance is therefore a critical element in the engineering of production strains. RESULTS: Here, a systems biology approach was employed to understand the chemical stress response of Escherichia coli, including a genome-wide screen for mutants with increased fitness during chemical stress...
October 13, 2016: Microbial Cell Factories
Peter E Burby, Taylor M Nye, Jeremy W Schroeder, Lyle A Simmons
Few discoveries have been more transformative to the biological sciences than the development of DNA sequencing technologies. The rapid advancement of sequencing and bioinformatics tools has revolutionized bacterial genetics, deepening our understanding of model and clinically relevant organisms. Although application of newer sequencing technologies to studies in bacterial genetics is increasing, the implementation of DNA sequencing technologies and development of the bioinformatics tools required for analyzing the large data sets generated remain a challenge for many...
January 1, 2017: Journal of Bacteriology
Tim van Opijnen, Sandra Dedrick, José Bento
The interaction between an antibiotic and bacterium is not merely restricted to the drug and its direct target, rather antibiotic induced stress seems to resonate through the bacterium, creating selective pressures that drive the emergence of adaptive mutations not only in the direct target, but in genes involved in many different fundamental processes as well. Surprisingly, it has been shown that adaptive mutations do not necessarily have the same effect in all species, indicating that the genetic background influences how phenotypes are manifested...
September 2016: PLoS Pathogens
Nikki E Freed, Dirk Bumann, Olin K Silander
BACKGROUND: Gene essentiality - whether or not a gene is necessary for cell growth - is a fundamental component of gene function. It is not well established how quickly gene essentiality can change, as few studies have compared empirical measures of essentiality between closely related organisms. RESULTS: Here we present the results of a Tn-seq experiment designed to detect essential protein coding genes in the bacterial pathogen Shigella flexneri 2a 2457T on a genome-wide scale...
2016: BMC Microbiology
Federico Rosconi, Stefan P W de Vries, Abiyad Baig, Elena Fabiano, Andrew J Grant
The interior of plants contains microorganisms (referred to as endophytes) distinct to those present in the root surface or in the surrounding soil. Herbaspirillum seropedicae strain SmR1, belonging to the β-Proteobacterium, is an endophyte that colonizes crops, including rice, maize, sugarcane and sorghum. Different approaches have revealed genes and pathways regulated during the interaction of H. seropedicae with its plant hosts. However, functional genomic analysis of transposon (Tn) mutants has been hampered by the lack of genetic tools...
September 2, 2016: Applied and Environmental Microbiology
Roberto C Molina-Quiroz, David W Lazinski, Andrew Camilli, Stuart B Levy
Persister cells are highly tolerant to different antibiotics and are associated with relapsing infections. In order to understand this phenomenon further, we exposed a transposon library to a lethal concentration of ampicillin, and mutants that survived were identified by transposon sequencing (Tn-Seq). We determined that mutations related to carbon metabolism, cell envelope (cell wall generation and membrane proteins), and stress response have a role in persister cell generation.
November 2016: Antimicrobial Agents and Chemotherapy
Elizabeth M Selleck, Michael S Gilmore
Infections caused by multiple organisms, or polymicrobial infections, are likely more common than is broadly appreciated. Interaction among microbial communities (and with their host) can change the infection landscape by subverting immunity, providing nutrients and inhibiting competing microbes. Stacy et al. (A. Stacy, D. Fleming, R. J. Lamont, K. P. Rumbaugh, and M. Whiteley, mBio 7:e00782-16, 2016, described a novel mechanism that results in synergistic growth of oral microbes Aggregatibacter actinomycetemcomitans and Streptococcus gordonii The authors used whole-genome fitness profiling by transposon sequencing (Tn-seq) to identify genes differentially required for growth in vitro versus in a mono- or coinfection in a thigh abscess model...
2016: MBio
Mithila Rajagopal, Melissa J Martin, Marina Santiago, Wonsik Lee, Veronica N Kos, Tim Meredith, Michael S Gilmore, Suzanne Walker
UNLABELLED: Staphylococcus aureus is a leading cause of life-threatening infections worldwide. The MIC of an antibiotic against S. aureus, as well as other microbes, is determined by the affinity of the antibiotic for its target in addition to a complex interplay of many other cellular factors. Identifying nontarget factors impacting resistance to multiple antibiotics could inform the design of new compounds and lead to more-effective antimicrobial strategies. We examined large collections of transposon insertion mutants in S...
2016: MBio
Elena Capel, Aldert L Zomer, Thomas Nussbaumer, Christine Bole, Brigitte Izac, Eric Frapy, Julie Meyer, Haniaa Bouzinba-Ségard, Emmanuelle Bille, Anne Jamet, Anne Cavau, Franck Letourneur, Sandrine Bourdoulous, Thomas Rattei, Xavier Nassif, Mathieu Coureuil
UNLABELLED: Neisseria meningitidis is a leading cause of bacterial meningitis and septicemia, affecting infants and adults worldwide. N. meningitidis is also a common inhabitant of the human nasopharynx and, as such, is highly adapted to its niche. During bacteremia, N. meningitidis gains access to the blood compartment, where it adheres to endothelial cells of blood vessels and causes dramatic vascular damage. Colonization of the nasopharyngeal niche and communication with the different human cell types is a major issue of the N...
2016: MBio
Apollo Stacy, Derek Fleming, Richard J Lamont, Kendra P Rumbaugh, Marvin Whiteley
UNLABELLED: Bacteria rarely inhabit infection sites alone, instead residing in diverse, multispecies communities. Despite this fact, bacterial pathogenesis studies primarily focus on monoculture infections, overlooking how community interactions influence the course of disease. In this study, we used global mutant fitness profiling (transposon sequencing [Tn-seq]) to determine the genetic requirements for the pathogenic bacterium Aggregatibacter actinomycetemcomitans to cause disease when coinfecting with the commensal bacterium Streptococcus gordonii Our results show that S...
June 28, 2016: MBio
Erin B Troy, Tao Lin, Lihui Gao, David W Lazinski, Maureen Lundt, Andrew Camilli, Steven J Norris, Linden T Hu
Borrelia burgdorferi maintains a complex life cycle between tick and vertebrate hosts. Although some genes have been identified as contributing to bacterial adaptation in the different hosts, the list is incomplete. In this manuscript, we report the first use of transposon mutagenesis combined with high-throughput sequencing (Tn-seq) in B. burgdorferi. We utilize the technique to investigate mechanisms of carbohydrate utilization in B. burgdorferi and the role of carbohydrate metabolism during mouse infection...
September 2016: Molecular Microbiology
Thomas A Hooven, Andrew J Catomeris, Leor H Akabas, Tara M Randis, Duncan J Maskell, Sarah E Peters, Sandra Ott, Ivette Santana-Cruz, Luke J Tallon, Hervé Tettelin, Adam J Ratner
BACKGROUND: Next-generation sequencing of transposon-genome junctions from a saturated bacterial mutant library (Tn-seq) is a powerful tool that permits genome-wide determination of the contribution of genes to fitness of the organism under a wide range of experimental conditions. We report development, testing, and results from a Tn-seq system for use in Streptococcus agalactiae (group B Streptococcus; GBS), an important cause of neonatal sepsis. METHODS: Our method uses a Himar1 mini-transposon that inserts at genomic TA dinucleotide sites, delivered to GBS on a temperature-sensitive plasmid that is subsequently cured from the bacterial population...
2016: BMC Genomics
Fangfang Liu, Chong Wang, Zuowei Wu, Qijing Zhang, Peng Liu
MOTIVATION: Transposon insertion sequencing (Tn-seq) is an emerging technology that combines transposon mutagenesis with next-generation sequencing technologies for the identification of genes related to bacterial survival. The resulting data from Tn-seq experiments consist of sequence reads mapped to millions of potential transposon insertion sites and a large portion of insertion sites have zero mapped reads. Novel statistical method for Tn-seq data analysis is needed to infer functions of genes on bacterial growth...
June 1, 2016: Bioinformatics
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