Assaying gene function by growth competition experiment

High-throughput screening and analysis is one of the emerging paradigms in biotechnology. In particular, high-throughput methods are essential in the field of functional genomics because of the vast amount of data generated in recent and ongoing genome sequencing efforts. In this report we discuss integrated functional analysis methodologies which incorporate both a growth competition component and a highly parallel assay used to quantify results of the growth competition. Several applications of the two most widely used technologies in the field, i.e., transposon mutagenesis and deletion strain library growth competition, and individual applications of several developing or less widely reported technologies are presented.     

Mycobacterium ulcerans Fails to Infect through Skin Abrasions in a Guinea Pig Infection Model: Implications for Transmission

Transmission of M. ulcerans, the etiological agent of Buruli ulcer, from the environment to humans remains an enigma despite decades of research. Major transmission hypotheses propose 1) that M. ulcerans is acquired through an insect bite or 2) that bacteria enter an existing wound through exposure to a contaminated environment. In studies reported here, a guinea pig infection model was developed to determine whether Buruli ulcer could be produced through passive inoculation of M. ulcerans onto a superficial abrasion. The choice of an abrasion model was based on the fact that most bacterial pathogens infecting the skin are able to infect an open lesion, and that abrasions are extremely common in children. Our studies show that after a 90d infection period, an ulcer was present at intra-dermal injection sites of all seven animals infected, whereas topical application of M. ulcerans failed to establish an infection. Mycobacterium ulcerans was cultured from all injection sites whereas infected abrasion sites healed and were culture negative. A 14d experiment was conducted to determine how long organisms persisted after inoculation. Mycobacterium ulcerans was isolated from abrasions at one hour and 24 hours post infection, but cultures from later time points were negative. Abrasion sites were qPCR positive up to seven days post infection, but negative at later timepoints. In contrast, M. ulcerans DNA was detected at intra-dermal injection sites throughout the study. M. ulcerans was cultured from injection sites at each time point. These results suggest that injection of M. ulcerans into the skin greatly facilitates infection and lends support for the role of an invertebrate vector or other route of entry such as a puncture wound or deep laceration where bacteria would be contained within the lesion. Infection through passive inoculation into an existing abrasion appears a less likely route of entry.     

AB5 toxins: structures and inhibitor design

High-resolution crystal structures of AB₅ toxins in their native form or in complex with a variety of ligands have led to the structure-based design and discovery of inhibitors targeting different areas of the toxins. The most significant progress is the development of highly potent multivalent ligands that block binding of the toxins to their receptors.     

Seed reproductive biology of the rare aquatic carnivorous plant Aldrovanda vesiculosa (Droseraceae)

Despite the unprecedented global decline in extant populations of Aldrovanda vesiculosa in the last century, little is known about the reproductive biology of this iconic aquatic carnivorous plant. This study aimed to investigate the role of seed‐based reproduction in the ecology of A. vesiculosa, with particular focus on the interplay between the regulation of seed dormancy by temperature cues and the efficacy of exogenous ethylene gas to act as a germination stimulant, the desiccation capacity and long‐term storage potential of seeds for conservation purposes. Sexual reproduction appears to be extremely limited in both natural and naturalized populations across three continents, with high variability in the success of flowering and seed set between sites and between seasons. Overall, flowering yielded few fertile fruit (6–19% of flowers producing fertile fruit) and seed viability was variable but generally low (29–88%). Fecundity appears to be influenced by seasonal climatic conditions and microhabitat characteristics. Aldrovanda vesiculosa possesses physiologically dormant seeds, with germination stimulated by exposure to ethylene gas (>90% germination) at 25 °C. Seeds appear sensitive to desiccation and sub‐zero temperature storage, with no germination and markedly reduced embryo growth after storage of seeds for >1 month at 15 °C and 15% relative humidity or after short‐term (24 h) storage at ?18 °C. In the absence of significant conservation and restoration initiatives, the continuing decline of dystrophic freshwater wetland habitats globally leaves A. vesiculosa facing extinction. As the successful long‐term storage of seeds appears unfeasible based on the approaches described in this study, other alternatives for germplasm conservation such as cryostorage of vegetative tissues or zygotic embryos must be considered for establishing long‐term ex situ collections of critical germplasm.     

Climate change decreases the cooling effect from postfire albedo in boreal North America

Fire is a primary disturbance in boreal forests and generates both positive and negative climate forcings. The influence of fire on surface albedo is a predominantly negative forcing in boreal forests, and one of the strongest overall, due to increased snow exposure in the winter and spring months. Albedo forcings are spatially and temporally heterogeneous and depend on a variety of factors related to soils, topography, climate, land cover/vegetation type, successional dynamics, time since fire, season, and fire severity. However, how these variables interact to influence albedo is not well understood, and quantifying these relationships and predicting postfire albedo becomes increasingly important as the climate changes and management frameworks evolve to consider climate impacts. Here we developed a MODIS‐derived ‘blue sky’ albedo product and a novel machine learning modeling framework to predict fire‐driven changes in albedo under historical and future climate scenarios across boreal North America. Converted to radiative forcing (RF), we estimated that fires generate an annual mean cooling of ?1.77 ± 1.35 W/m² from albedo under historical climate conditions (1971–2000) integrated over 70 years postfire. Increasing postfire albedo along a south–north climatic gradient was offset by a nearly opposite gradient in solar insolation, such that large‐scale spatial patterns in RF were minimal. Our models suggest that climate change will lead to decreases in mean annual postfire albedo, and hence a decreasing strength of the negative RF, a trend dominated by decreased snow cover in spring months. Considering the range of future climate scenarios and model uncertainties, we estimate that for fires burning in the current era (2016) the cooling effect from long‐term postfire albedo will be reduced by 15%–28% due to climate change.     

Natural and synthetic alleles provide complementary insights into the nature of selection acting on the Men polymorphism of Drosophila melanogaster

Two malic enzyme alleles, Men^113A and Men^113G, occur at approximately equal frequency in North American populations of Drosophila melanogaster, while only Men^113A occurs in African populations. We investigated the population genetics, biochemical characteristics, and selective potential of these alleles. Comparable levels of nucleotide polymorphism in both alleles suggest that the Men^113G allele is not recently derived, but we find no evidence in the DNA sequence data for selection maintaining the polymorphism. Interestingly, the alleles differ in both V_max and K_m for the substrate malate. Triglyceride concentration and isocitrate dehydrogenase (IDH) and glucose-6-phosphate dehydrogenase (G6PD) activities are negatively correlated with the in vivo activities of the Men alleles. We examined the causality of the observed correlations using P-element excision-derived knockout alleles of the Men gene and found significant changes in the maximum activities of both IDH and G6PD, but not in triglyceride concentration, suggesting compensatory interactions between MEN, IDH, and G6PD. Additionally, we found significantly higher than expected levels of MEN activity in knockout heterozygotes, which we attribute to transvection effects. The distinct differences in biochemistry and physiology between the naturally occurring alleles and between the engineered alleles suggest the potential for selection on the Men locus.     

Transplantation of neurons reveals processing areas and rules for synaptic connectivity in the cricket nervous system

In order to assess the nature of spatial cues in determining the characteristic projection sites of sensory neurons in the CNS, we have transplanted sensory neurons of the cricket Acheta domesticus to ectopic locations. Thoracic campaniform sensilla (CS) function as proprioceptors and project to an intermediate layer of neuropil in thoracic ganglia while cercal CS transduce tactile information and project into a ventral layer in the terminal abdominal ganglion (TAG). When transplanted to ectopic locations, these afferents retain their modality-specific projection in the host ganglion and terminate in the layer of neuropil homologous to that of their ganglion of origin. Thus, thoracic CS neurons project to intermediate neuropil when transplanted to the abdomen and cercal CS neurons project to a ventral layer of neuropil when transplanted to the thorax. We conclude that CS can be separated into two classes based on their characteristic axonal projections within each segmental ganglion. We also found that the sensory neurons innervating tactile hairs project to ventral neuropil in any ganglion they encounter after transplantation. Ectopic sensory neurons can form functional synaptic connections with identified interneurons located within the host ganglia. The new contacts formed by these ectopic sensory neurons can be with normal targets, which arborize within the same layer of neuropil in each segmental ganglion, or with novel targets, which lack dendrites in the normal ganglion and are thus normally unavailable for synaptogenesis. These observations suggest that a limited set of molecular markers are utilized for cell–cell recognition in each segmentally homologous ganglion. Regenerating sensory neurons can recognize novel postsynaptic neurons if they have dendrites in the appropriate layer of neuropil. We suggest that spatial constraints produced by the segmentation and the modality-specific layering of the nervous system have a pivotal role in determining synaptic specificity. © 1993 John Wiley & Sons, Inc.     

A Combination of Structural and Cis-Regulatory Factors Drives Biochemical Differences in Drosophila melanogaster Malic Enzyme

The evolutionary significance of molecular variation is still contentious, with much current interest focusing on the relative contribution of structural changes in proteins versus regulatory variation in gene expression. We present a population genetic and biochemical study of molecular variation at the malic enzyme locus (Men) in Drosophila melanogaster. Two amino acid polymorphisms appear to affect substrate-binding kinetics, while only one appears to affect thermal stability. Interestingly, we find that enzyme activity differences previously assigned to one of the polymorphisms may, instead, be a function of linked regulatory differences. These results suggest that both regulatory and structural changes contribute to differences in protein function. Our examination of the Men coding sequences reveals no evidence for selection acting on the polymorphisms, but earlier work on this enzyme indicates that the biochemical variation observed has physiological repercussions and therefore could potentially be under natural selection.