A genetic assay for gene essentiality in Clostridium

Essential genes of pathogens are potential therapeutic targets, but are difficult to verify. Here, gene essentiality was determined by targeted knockout following engineered gene duplication. Null mutants of candidate essential genes of Clostridium difficile were viable only in the presence of a stable second copy of the gene

Fitness Ranking of Individual Mutants Drives Patterns of Epistatic Interactions in HIV-1

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Adaptation of HIV-1 Depends on the Host-Cell Environment

Many viruses have the ability to rapidly develop resistance against antiviral drugs and escape from the host immune system. To which extent the host environment affects this adaptive potential of viruses is largely unknown. Here we show that for HIV-1, the host-cell environment is key to the adaptive potential of the virus. We performed a large-scale selection experiment with two HIV-1 strains in two different T-cell lines (MT4 and C8166). Over 110 days of culture, both virus strains adapted rapidly to the MT4 T-cell line. In contrast, when cultured on the C8166 T-cell line, the same strains did not show any increase in fitness. By sequence analyses and infections with viruses expressing either yellow or cyan fluorescent protein, we were able to show that the absence of adaptation was linked to a lower recombination rate in the C8166 T-cell line. Our findings suggest that if we can manipulate the host-cellular factors that mediate viral evolution, we may be able to significantly retard viral adaptability

Extreme genetic fragility of the HIV-1 capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

Global gene disruption in human cells to assign genes to phenotypes

Insertional mutagenesis in a haploid background can disrupt gene function[superscript 1]. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones[superscript 1] enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites[superscript 2]. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT

Wolbachia in butterflies and moths: geographic structure in infection frequency.

INTRODUCTION: Butterflies and moths (Lepidoptera) constitute one of the most diverse insect orders, and play an important role in ecosystem function. However, little is known in terms of their bacterial communities. Wolbachia, perhaps the most common and widespread intracellular bacterium on Earth, can manipulate the physiology and reproduction of its hosts, and is transmitted vertically from mother to offspring, or sometimes horizontally between species. While its role in some hosts has been studied extensively, its incidence across Lepidoptera is poorly understood. A recent analysis using a beta-binomial model to infer the between-species distribution of prevalence estimated that approximately 40 % of arthropod species are infected with Wolbachia, but particular taxonomic groups and ecological niches seem to display substantially higher or lower incidences. In this study, we took an initial step and applied a similar, maximum likelihood approach to 300 species of Lepidoptera (7604 individuals from 660 populations) belonging to 17 families and 10 superfamilies, and sampled from 36 countries, representing all continents excluding Antarctica. RESULTS: Approximately a quarter to a third of individuals appear to be infected with Wolbachia, and around 80 % of Lepidoptera species are infected at a non-negligible frequency. This incidence estimate is very high compared to arthropods in general. Wolbachia infection in Lepidoptera is shown to vary between families, but there is no evidence for closely related groups to show similar infection levels. True butterflies (Papilionoidea) are overrepresented in our data, however, our estimates show this group can be taken as a representative for the other major lepidopteran superfamilies. We also show substantial variation in infection level according to geography - closer locations tend to show similar infection levels. We further show that variation in geography is due to a latitudinal gradient in Wolbachia infection, with lower frequencies towards higher latitudes. CONCLUSIONS: Our comprehensive survey of Wolbachia infection in Lepidoptera suggests that infection incidence is very high, and provides evidence that climate and geography are strong predictors of infection frequency.We thank the McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History for their continued support. This study was supported by the University of Florida Research Opportunity Seed Fund (ROSF) and the National Science Foundation grant number DEB-1354585 to AYK.This is the final published version. It first appeared at http://link.springer.com/article/10.1186%2Fs12983-015-0107-z

Genome-wide analysis of Sphingomonas wittichii RW1 behaviour during inoculation and growth in contaminated sand.

The efficacy of inoculation of single pure bacterial cultures into complex microbiomes, for example, in order to achieve increased pollutant degradation rates in contaminated material (that is, bioaugmentation), has been frustrated by insufficient knowledge on the behaviour of the inoculated bacteria under the specific abiotic and biotic boundary conditions. Here we present a comprehensive analysis of genome-wide gene expression of the bacterium Sphingomonas wittichii RW1 in contaminated non-sterile sand, compared with regular suspended batch growth in liquid culture. RW1 is a well-known bacterium capable of mineralizing dibenzodioxins and dibenzofurans. We tested the reactions of the cells both during the immediate transition phase from liquid culture to sand with or without dibenzofuran, as well as during growth and stationary phase in sand. Cells during transition show stationary phase characteristics, evidence for stress and for nutrient scavenging, and adjust their primary metabolism if they were not precultured on the same contaminant as found in the soil. Cells growing and surviving in sand degrade dibenzofuran but display a very different transcriptome signature as in liquid or in liquid culture exposed to chemicals inducing drought stress, and we obtain evidence for numerous 'soil-specific' expressed genes. Studies focusing on inoculation efficacy should test behaviour under conditions as closely as possible mimicking the intended microbiome conditions

Identification of genes required for soil survival in Burkholderia thailandensis by transposon-directed insertion site sequencing.

Transposon-directed insertion site sequencing was used to identify genes required by Burkholderia thailandensis to survive in plant/soil microcosms. A total of 1,153 genetic loci fulfilled the criteria as being likely to encode survival characteristics. Of these, 203 (17.6 %) were associated with uptake and transport systems; 463 loci (40.1 %) coded for enzymatic properties, 99 of these (21.4 %) had reduction/oxidation functions; 117 (10.1 %) were gene regulation or sensory loci; 61 (5.3 %) encoded structural proteins found in the cell envelope or with enzymatic activities related to it, distinct from these, 46 (4.0 %) were involved in chemotaxis and flagellum, or pilus synthesis; 39 (3.4 %) were transposase enzymes or were bacteriophage-derived; and 30 (2.6 %) were involved in the production of antibiotics or siderophores. Two hundred and twenty genes (19.1 %) encoded hypothetical proteins or those of unknown function. Given the importance of motility and pilus formation in microcosm persistence the nature of the colonization of the rhizosphere was examined by confocal microscopy. Wild type B. thailandensis expressing red fluorescent protein was inoculated into microcosms. Even though the roots had been washed, the bacteria were still present but they were motile with no attachment having taken place, perhaps being retained in a biofilm