Virulence studies on chromosomal α-toxin and Θ-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α-toxin in Clostridium perfringens-mediated gas gangrene

The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin-resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the‘chromosomal pfoA gene, which encodes an oxygen-labile haemolysin known as Θ-toxin or perfringolysin O. and in the chromosomal pic gene, which encodes the α-toxin or phospholipase C. The resultant mutants failed to produce detectable Θ-toxin or α-toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild-type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the pic mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of α-toxin in gas gangrene or clostridial myonecrosis.     

Mlc1p Is a Light Chain for the Unconventional Myosin Myo2p in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the unconventional myosin Myo2p is of fundamental importance in polarized growth. We explore the role of the neck region and its associated light chains in regulating Myo2p function. Surprisingly, we find that precise deletion of the six IQ sites in the neck region results in a myosin, Myo2-Δ6IQp, that can support the growth of a yeast strain at 90% the rate of a wild-type isogenic strain. We exploit this mutant in a characterization of the light chains of Myo2p. First, we demonstrate that the localization of calmodulin to sites of polarized growth largely depends on the IQ sites in the neck of Myo2p. Second, we demonstrate that a previously uncharacterized protein, Mlc1p, is a myosin light chain of Myo2p. MLC1 (YGL106w) is an essential gene that exhibits haploinsufficiency. Reduced levels of MYO2 overcome the haploinsufficiency of MLC1. The mutant MYO2-Δ6IQ is able to suppress haploinsufficiency but not deletion of MLC1. We used a modified gel overlay assay to demonstrate a direct interaction between Mlc1p and the neck of Myo2p. Overexpression of MYO2 is toxic, causing a severe decrease in growth rate. When MYO2 is overexpressed, Myo2p is fourfold less stable than in a wild-type strain. High copies of MLC1 completely overcome the growth defects and increase the stability of Myo2p. Our results suggest that Mlc1p is responsible for stabilizing this myosin by binding to the neck region.     

Retrograde Traffic Out of the Yeast Vacuole to the TGN Occurs via the Prevacuolar/Endosomal Compartment

A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid–containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.     

Unexpected Relationships and Inbreeding in HapMap Phase III Populations

Correct annotation of the genetic relationships between samples is essential for population genomic studies, which could be biased by errors or omissions. To this end, we used identity-by-state (IBS) and identity-by-descent (IBD) methods to assess genetic relatedness of individuals within HapMap phase III data. We analyzed data from 1,397 individuals across 11 ethnic populations. Our results support previous studies (Pemberton et al., 2010; Kyriazopoulou-Panagiotopoulou et al., 2011) assessing unknown relatedness present within this population. Additionally, we present evidence for 1,657 novel pairwise relationships across 9 populations. Surprisingly, significant Cotterman''s coefficients of relatedness K1 (IBD1) values were detected between pairs of known parents. Furthermore, significant K2 (IBD2) values were detected in 32 previously annotated parent-child relationships. Consistent with a hypothesis of inbreeding, regions of homozygosity (ROH) were identified in the offspring of related parents, of which a subset overlapped those reported in previous studies (Gibson et al. 2010; Johnson et al. 2011). In total, we inferred 28 inbred individuals with ROH that overlapped areas of relatedness between the parents and/or IBD2 sharing at a different genomic locus between a child and a parent. Finally, 8 previously annotated parent-child relationships had unexpected K0 (IBD0) values (resulting from a chromosomal abnormality or genotype error), and 10 previously annotated second-degree relationships along with 38 other novel pairwise relationships had unexpected IBD2 (indicating two separate paths of recent ancestry). These newly described types of relatedness may impact the outcome of previous studies and should inform the design of future studies relying on the HapMap Phase III resource.     

Variation within and between Closely Related Species Uncovers High Intra-Specific Variability in Dispersal

Mounting evidence shows that contrasting selection pressures generate variability in dispersal patterns among individuals or populations of the same species, with potential impacts on both species dynamics and evolution. However, this variability is hardly considered in empirical works, where a single dispersal function is considered to adequately reflect the species-specific dispersal ability, suggesting thereby that within-species variation is negligible as regard to inter-specific differences in dispersal abilities. We propose here an original method to make the comparison of intra- and inter-specific variability in dispersal, by decomposing the diversity of that trait along a phylogeny of closely related species. We used as test group European butterflies that are classic study organisms in spatial ecology. We apply the analysis separately to eight metrics that reflect the dispersal propensity, the dispersal ability or the dispersal efficiency of populations and species. At the inter-specific level, only the dispersal ability showed the signature of a phylogenetic signal while neither the dispersal propensity nor the dispersal efficiency did. At the within-species level, the partitioning of dispersal diversity showed that dispersal was variable or highly variable among populations: intra-specific variability represented from 11% to 133% of inter-specific variability in dispersal metrics. This finding shows that dispersal variation is far from negligible in the wild. Understanding the processes behind this high within-species variation should allow us to properly account for dispersal in demographic models. Accordingly, to encompass the within species variability in life histories the use of more than one value per trait per species should be encouraged in the construction of databases aiming at being sources for modelling purposes.     

Random-phase calculation of the low-energy circular dichroism in glucans

The CD of the long-wavelength electronic transition in the α-(1 → 4)-linked glucan dimer (maltose) and polymer (amylose), and in the β-(1 → 6)-linked dimer (gentiobiose) and polymer (pustulan), are calculated in the random-phase approximation using time-dependent Hartree theory. This long-wavelength transition (180–190 nm) is assumed to be localized on the linkage oxygen atom and to be of a σ*/3 s ← n character. The zerothorder σ*/3s ← n magnetic-dipole transition moment is coupled to the CC, CO, and CH bond density of states via a polarizability approximation. We assume this coupling is dominated energetically by the electric-dipole, electric-dipole interaction terms in the context of Schellman's μ_e-μ_m coupling mechanism of rotatory power. The CD is calculated as a function of rotation about the two single bonds of the linkage oxygen atom and also as a function of rotation about the C(5)-C(6) bond. For maltose, four rotational isomers are considered, resulting from combinations of the gg and gt C(6)H₂OH group rotational isomers. The calculated CD values were then Boltzmann-averaged over an empirical potential, and the resulting CD was found to compare satisfactorily with experiment. In the case of the polymers, only structures having periodicity (helicity) were examined.     

Plasticity of monkey triceps muscle fibers in microgravity conditions.

We examined the changes in functional properties of triceps brachii skinned fibers from monkeys flown aboard the BION 11 satellite for 14 days and after ground-based arm immobilization. The composition of myosin heavy chain (MHC) isoforms allowed the identification of pure fibers containing type I (slow) or type IIa (fast) MHC isoforms or hybrid fibers coexpressing predominantly slow (hybrid slow; HS) or fast (hybrid fast) MHC isoforms. The ratio of HS fibers to the whole slow population was higher after flight (28%) than in the control population (7%), and the number of fast fibers was increased (up to 86% in flight vs. 12% in control). Diameters and maximal tensions of slow fibers were decreased after flight. The tension-pCa curves of slow and fast fibers were modified, with a decrease in pCa threshold and an increase in steepness. The proper effect of microgravity was distinguishable from that of immobilization, which induced less marked slow-to-fast transitions (only 59% of fast fibers) and changed the tension-pCa relationships.     

The toad fly Lucilia bufonivora: its evolutionary status and molecular identification

The blow fly genus Lucilia is composed largely of saprophages and facultative myasis agents, including the economically important species Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae) and Lucilia sericata (Meigen). Only one species is generally recognized as an obligate agent of myiasis, Lucilia bufonivora Moniez, and this is an obligate parasite of toads. Lucilia silvarum (Meigen), a sister species, behaves mainly as a carrion breeder; however, it has also been reported as a facultative parasite of amphibians. Morphologically, these species are almost identical, and historically this has led to misidentification, taxonomic ambiguity and a paucity of studies of L. bufonivora. In this study, dipterous larvae were analysed from toad myiasis cases from the U.K., The Netherlands and Switzerland, together with adult specimens of fly species implicated in amphibian parasitism: L. bufonivora, L. silvarum and Lucilia elongata Shannon (from North America). Partial sequences of two genes, cox1 and ef1α, were amplified. Seven additional blow fly species were analysed as outgroups. Bayesian inference trees of cox1, ef1α and a combined‐gene dataset were constructed. All larvae isolated from toads were identified as L. bufonivora and no specimens of L. silvarum were implicated in amphibian myiasis. This study confirms L. silvarum and L. bufonivora as distinct sister species and provides unambiguous molecular identification of L. bufonivora.     

Spatial and temporal habitat partitioning by calliphorid blowflies

Calliphorid blowflies perform an essential ecosystem service in the consumption, recycling and dispersion of carrion nutrients and are considered amongst the most important functional groups in an ecosystem. Some species are of economic importance as facultative agents of livestock myiasis. The interspecific ecological differences that facilitate coexistence within the blowfly community are not fully understood. The aim of this work was to quantify differences in habitat use by calliphorid species. Thirty traps were distributed among three habitats at two sites in southwest England for collections made during March–August 2016. A total of 17 246 specimens were caught, of which 2427 were Lucilia sericata, 51 Lucilia richardsi, 6580 Lucilia caesar, 307 Lucilia ampullacea, 4881 Calliphora vicina and 2959 Calliphora vomitoria (all: Diptera: Calliphoridae). Lucilia sericata was the dominant species in open habitats, whereas L. caesar was the most abundant species in shaded habitats. Calliphora specimens were more abundant in the cooler months. These findings suggest that Calliphora and Lucilia species show strong temporal segregation mediated by temperature, and that species of the genus Lucilia show differences in the use of habitats that are likely to be driven by differences in humidity tolerance and light intensity. These factors in combination result in effective niche partitioning.