Dual Predation by Bacteriophage and Bdellovibrio bacteriovorus Can Eradicate Escherichia coli Prey in Situations where Single Predation Cannot.

Bacteria are preyed upon by diverse microbial predators, including bacteriophage and predatory bacteria, such as Bdellovibrio bacteriovorus While bacteriophage are used as antimicrobial therapies in Eastern Europe and are being applied for compassionate use in the United States, predatory bacteria are only just beginning to reveal their potential therapeutic uses. However, predation by either predator type can falter due to different adaptations arising in the prey bacteria. When testing poultry farm wastewater for novel Bdellovibrio isolates on Escherichia coli prey lawns, individual composite plaques were isolated containing both an RTP (rosette-tailed-phage)-like-phage and a B. bacteriovorus strain and showing central prey lysis and halos of extra lysis. Combining the purified phage with a lab strain of B. bacteriovorus HD100 recapitulated haloed plaques and increased killing of the E. coli prey in liquid culture, showing an effective side-by-side action of these predators compared to their actions alone. Using approximate Bayesian computation to select the best fitting from a variety of different mathematical models demonstrated that the experimental data could be explained only by assuming the existence of three prey phenotypes: (i) sensitive to both predators, (ii) genetically resistant to phage only, and (iii) plastic resistant to B. bacteriovorus only. Although each predator reduces prey availability for the other, high phage numbers did not abolish B. bacteriovorus predation, so both predators are competent to coexist and are causing different selective pressures on the bacterial surface while, in tandem, controlling prey bacterial numbers efficiently. This suggests that combinatorial predator therapy could overcome problems of phage resistance.IMPORTANCE With increasing levels of antibiotic resistance, the development of alternative antibacterial therapies is urgently needed. Two potential alternatives are bacteriophage and predatory bacteria. Bacteriophage therapy has been used, but prey/host specificity and the rapid acquisition of bacterial resistance to bacteriophage are practical considerations. Predatory bacteria are of interest due to their broad Gram-negative bacterial prey range and the lack of simple resistance mechanisms. Here, a bacteriophage and a strain of Bdellovibrio bacteriovorus, preyed side by side on a population of E. coli, causing a significantly greater decrease in prey numbers than either alone. Such combinatorial predator therapy may have greater potential than individual predators since prey surface changes selected for by each predator do not protect prey against the other predator.

Evolution of DUX gene macrosatellites in placental mammals.

Macrosatellites are large polymorphic tandem arrays. The human subtelomeric macrosatellite D4Z4 has 11-150 repeats, each containing a copy of the intronless DUX4 gene. DUX4 is linked to facioscapulohumeral muscular dystrophy, but its normal function is unknown. The DUX gene family includes DUX4, the intronless Dux macrosatellites in rat and mouse, as well as several intron-containing members (DUXA, DUXB, Duxbl, and DUXC). Here, we report that the genomic organization (though not the syntenic location) of primate DUX4 is conserved in the Afrotheria. In primates and Afrotheria, DUX4 arose by retrotransposition of an ancestral intron-containing DUXC, which is itself not found in these species. Surprisingly, we discovered a similar macrosatellite organization for DUXC in cow and other Laurasiatheria (dog, alpaca, dolphin, pig, and horse), and in Xenarthra (sloth). Therefore, DUX4 and Dux are not the only DUX gene macrosatellites. Our data suggest a new retrotransposition-displacement model for the evolution of intronless DUX macrosatellites.

Diagnosis by sequencing: correction of misdiagnosis from FSHD2 to LGMD2A by whole-exome analysis.

We studied and validated facioscapulohumeral muscular dystrophy (FSHD) samples from patients without a D4Z4 contraction (FSHD2 or 'phenotypic FSHD'). For this, we developed non-radioactive protocols to test D4Z4 allele constitution and DNA methylation, and applied these to samples from the Coriell Institute Cell Repository. The D4Z4 sizing showed two related subjects to have classic chromosome 4 contraction-dependent FSHD1. A third sample (GM17726) did not have a short chromosome 4 fragment, and had been assigned as non-4q FSHD (FSHD2). We tested D4Z4 haplotype and methylation for this individual but found both to be inconsistent with this diagnosis. Using exome sequencing, we identified two known pathogenic mutations in CAPN3 (Arg490Gln and Thr184Argfs(*)36), indicating a case of LGMD2A rather than FSHD. Our study shows how a wrong diagnosis can easily be corrected by whole-exome sequencing by constraining the variant analysis to candidate genes after the data have been generated. This new way of 'diagnosis by sequencing' is likely to become common place in genetic diagnostic laboratories. We also publish a digoxigenin-labeled Southern protocol to test D4Z4 methylation. Our data supports hypomethylation as a good epigenetic predictor for FSHD2. The non-radioactive protocol will help to make this assay more accessible to clinical diagnostic laboratories and the wider FSHD research community.

A family history of DUX4: phylogenetic analysis of DUXA, B, C and Duxbl reveals the ancestral DUX gene.

BACKGROUND: DUX4 is causally involved in the molecular pathogenesis of the neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD). It has previously been proposed to have arisen by retrotransposition of DUXC, one of four known intron-containing DUX genes. Here, we investigate the evolutionary history of this multi-member double-homeobox gene family in eutherian mammals. RESULTS: Our analysis of the DUX family shows the distribution of different homologues across the mammalian class, including events of secondary loss. Phylogenetic comparison, analysis of gene structures and information from syntenic regions confirm the paralogous relationship of Duxbl and DUXB and characterize their relationship with DUXA and DUXC. We further identify Duxbl pseudogene orthologues in primates. A survey of non-mammalian genomes identified a single-homeobox gene (sDUX) as a likely representative homologue of the mammalian DUX ancestor before the homeobox duplication. Based on the gene structure maps, we suggest a possible mechanism for the generation of the DUX gene structure. CONCLUSIONS: Our study underlines how secondary loss of orthologues can obscure the true ancestry of individual gene family members. Their relationships should be considered when interpreting the relevance of functional data from DUX4 homologues such as Dux and Duxbl to FSHD.