Trade-offs Between Light and Nutrient Availability Across Gradients of Dissolved Organic Carbon Lead to Spatially and Temporally Variable Responses of Lake Phytoplankton Biomass to Browning

Ecosystems - Northern lakes are experiencing widespread increases in dissolved organic carbon (DOC) that are likely to lead to changes in pelagic phytoplankton biomass. Pelagic phytoplankton...     

Cytotoxicity of the Vibrio vulnificus MARTX toxin Effector DUF5 is linked to the C2A Subdomain

The multifunctional‐autoprocessing repeats‐in‐toxin (MARTX) toxins are bacterial protein toxins that serve as delivery platforms for cytotoxic effector domains. The domain of unknown function in position 5 (DUF5) effector domain is present in at least six different species' MARTX toxins and as a hypothetical protein in Photorhabdus spp. Its presence increases the potency of the Vibrio vulnificus MARTX toxin in mouse virulence studies, indicating DUF5 directly contributes to pathogenesis. In this work, DUF5 is shown to be cytotoxic when transiently expressed in HeLa cells. DUF5 localized to the plasma membrane dependent upon its C1 domain and the cells become rounded dependent upon its C2 domain. Both full‐length DUF5 and the C2 domain caused growth inhibition when expressed in Saccharomyces cerevisiae. A structural model of DUF5 was generated based on the structure of Pasteurella multocida toxin facilitating localization of the cytotoxic activity to a 186 amino acid subdomain termed C2A. Within this subdomain, an alanine scanning mutagenesis revealed aspartate‐3721 and arginine‐3841 as residues critical for cytotoxicity. These residues were also essential for HeLa cell intoxication when purified DUF5 fused to anthrax toxin lethal factor was delivered cytosolically. Thermal shift experiments indicated that these conserved residues are important to maintain protein structure, rather than for catalysis. The Aeromonas hydrophila MARTX toxin DUF5_Ah domain was also cytotoxic, while the weakly conserved C1–C2 domains from P. multocida toxin were not. Overall, this study is the first demonstration that DUF5 as found in MARTX toxins has cytotoxic activity that depends on conserved residues in the C2A subdomain. Proteins 2014; 82:2643–2656. © 2014 Wiley Periodicals, Inc.     

Comparative analysis of CRISPR cassettes from the human gut metagenomic contigs

Background

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a prokaryotic adaptive defence system that provides resistance against alien replicons such as viruses and plasmids. Spacers in a CRISPR cassette confer immunity against viruses and plasmids containing regions complementary to the spacers and hence they retain a footprint of interactions between prokaryotes and their viruses in individual strains and ecosystems. The human gut is a rich habitat populated by numerous microorganisms, but a large fraction of these are unculturable and little is known about them in general and their CRISPR systems in particular.

Results

We used human gut metagenomic data from three open projects in order to characterize the composition and dynamics of CRISPR cassettes in the human-associated microbiota. Applying available CRISPR-identification algorithms and a previously designed filtering procedure to the assembled human gut metagenomic contigs, we found 388 CRISPR cassettes, 373 of which had repeats not observed previously in complete genomes or other datasets. Only 171 of 3,545 identified spacers were coupled with protospacers from the human gut metagenomic contigs. The number of matches to GenBank sequences was negligible, providing protospacers for 26 spacers.Reconstruction of CRISPR cassettes allowed us to track the dynamics of spacer content. In agreement with other published observations we show that spacers shared by different cassettes (and hence likely older ones) tend to the trailer ends, whereas spacers with matches in the metagenomes are distributed unevenly across cassettes, demonstrating a preference to form clusters closer to the active end of a CRISPR cassette, adjacent to the leader, and hence suggesting dynamical interactions between prokaryotes and viruses in the human gut. Remarkably, spacers match protospacers in the metagenome of the same individual with frequency comparable to a random control, but may match protospacers from metagenomes of other individuals.

Conclusions

The analysis of assembled contigs is complementary to the approach based on the analysis of original reads and hence provides additional data about composition and evolution of CRISPR cassettes, revealing the dynamics of CRISPR-phage interactions in metagenomes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-202) contains supplementary material, which is available to authorized users.     

Acaricidal activity and sublethal effects of an oxymatrine-based biopesticide on two-spotted spider mite (Acari: Tetranychidae)

Lethal and sublethal effects of the biopesticide Kingbo (oxymatrine 0.2 % + psoralen 0.4 %) on the two-spotted spider mite (Tetranychus urticae Koch) were investigated in laboratory bioassays. The biopesticide was applied to bean leaf discs or primary leaves by using a Potter spray tower. Acute toxicity tests showed no significant ovicidal action: toxic effect (LC₅₀ = 55.49 μl/l) was the result of a residual activity against larvae that hatched from the treated eggs. Preovipositional females and female teleiochrysales showed similar susceptibility (LC₅₀ = 52.68 and 59.03 μl/l, respectively), whereas larvae, protonymphs and female deutonymphs were the most susceptible stages (LC₅₀ = 6.88, 13.03, and 8.80 μl/l, respectively). In a choice test, females preferred the untreated halves of leaves over the halves treated with 2,000, 1,000, and 500 μl/l in the first 24 h, and their oviposition in those treatments was significantly greater on the untreated halves after 24 and 48 h, as well as the summed oviposition over 72 h. Viability and reproduction of survivors, as well as population growth, were strongly affected after the treatments of preovipositional females and female teleiochrysales with 100, 50 and 25 μl/l. On the other hand, sublethal effects on the females that survived treatment at the egg stage or reached adulthood from the eggs laid on the treated surface (treatments with 50 and 25 μl/l) were significantly weaker. Acaricidal and sublethal effects of the biopesticide Kingbo were discussed as a starting point for further research aimed to improve management of T. urticae populations. Regulatory issues and safety concerns regarding further commercialization of this biopesticide are addressed as well.     

Hybrid Sterility Locus on Chromosome X Controls Meiotic Recombination Rate in Mouse

Meiotic recombination safeguards proper segregation of homologous chromosomes into gametes, affects genetic variation within species, and contributes to meiotic chromosome recognition, pairing and synapsis. The Prdm9 gene has a dual role, it controls meiotic recombination by determining the genomic position of crossover hotspots and, in infertile hybrids of house mouse subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd), it further functions as the major hybrid sterility gene. In the latter role Prdm9 interacts with the hybrid sterility X 2 (Hstx2) genomic locus on Chromosome X (Chr X) by a still unknown mechanism. Here we investigated the meiotic recombination rate at the genome-wide level and its possible relation to hybrid sterility. Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, yet the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. The male-limited transgressive effect of Meir1 on recombination rate parallels the male-limited transgressive role of Hstx2 in hybrid male sterility. Thus, both genetic factors, the Prdm9 gene and the Hstx2/Meir1 genomic locus, indicate a link between meiotic recombination and hybrid sterility. A strong female-specific modifier of meiotic recombination rate with the effect opposite to Meir1 was localized on Chr X, distally to Meir1. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene(s) responsible for variation in the global recombination rate between closely related mouse subspecies.     

Endothelial actin and cell stiffness is modulated by substrate stiffness in 2D and 3D

There is a growing appreciation of the profound effects that passive mechanical properties, especially the stiffness of the local environment, can have on cellular functions. Many experiments are conducted in a 2D geometry (i.e., cells grown on top of substrates of varying stiffness), which is a simplification of the 3D environment often experienced by cells in vivo. To determine how matrix dimensionality might modulate the effect of matrix stiffness on actin and cell stiffness, endothelial cells were cultured on top of and within substrates of various stiffnesses. Endothelial cells were cultured within compliant (1.0–1.5 mg/ml, 124±8 to 202±27 Pa) and stiff (3.0 mg/ml, 502±48 Pa) type-I collagen gels. Cells elongated and formed microvascular-like networks in both sets of gels as seen in previous studies. Cells in stiffer gels exhibited more pronounced stress fibers and ～1.5-fold greater staining for actin. As actin is a major determinant of a cell's mechanical properties, we hypothesized that cells in stiff gels will themselves be stiffer. To test this hypothesis, cells were isolated from the gels and their stiffness was assessed using micropipette aspiration. Cells isolated from relatively compliant gels were 1.9-fold more compliant than cells isolated from relatively stiff gels (p<0.05). Similarly, cells cultured on top of 1700 Pa polyacrylamide gels were 2.0-fold more compliant that those cultured on 9000 Pa (p<0.05). These data demonstrate that extracellular substrate stiffness regulates endothelial stiffness in both three- and two-dimensional environments, though the range of stiffnesses that cells respond to vary significantly in different environments.     

The absence of a DNA replication checkpoint in porcine zygotes

It has been demonstrated that in the zygotes of some mammals a unique checkpoint controls the onset of DNA replication. Thus, DNA replication begins in the maternal pronucleus only after the paternal pronucleus is fully formed. In our experiments we have investigated whether this checkpoint also operates in porcine zygotes produced either by in vitro fertilization (IVF) or by intracytoplasmic sperm injection (ICSI). Our results show that the onset of DNA replication occurs in the maternal pronucleus even in the presence of an intact sperm head in zygotes produced by ICSI, as well as in polyspermic eggs where some sperm heads are intact or male pronuclei are not yet fully developed. We conclude that in porcine zygotes there is an absence of the DNA replication checkpoint that is typical for some other mammals.