Regulation of enterochelin synthetase in Escherichia coli K-12

Enterochelin synthetase activity is controlled by both repression and feed-back inhibition mechanisms. Inclusion of iron in growth media results in synthesis of all four (D, E, F and G) components of enterochelin synthetase being repressed. The specific inhibition of L-serine activation (partial reaction catalyzed by the F component) by the end products, ferric-enterochelin and 2,3-dihydroxybenzoylserine, is shown to inhibit overall enterochelin synthetase activity.     

Intron-derived small RNAs for silencing viral RNAs in mosquito cells

Aedes aegypti and Ae. albopictus are the main vectors of mosquito-borne viruses of medical and veterinary significance. Many of these viruses have RNA genomes. Exogenously provided, e.g. transgene encoded, small RNAs could be used to inhibit virus replication, breaking the transmission cycle. We tested, in Ae. aegypti and Ae. albopictus cell lines, reporter-based strategies for assessing the ability of two types of small RNAs to inhibit a chikungunya virus (CHIKV) derived target. Both types of small RNAs use a Drosophila melanogaster pre-miRNA-1 based hairpin for their expression, either with perfect base-pairing in the stem region (shRNA-like) or containing two mismatches (miRNA-like). The pre-miRNA-1 stem loop structure was encoded within an intron; this allows co-expression of one or more proteins, e.g. a fluorescent protein marker tracking the temporal and spatial expression of the small RNAs in vivo. Three reporter-based systems were used to assess the relative silencing efficiency of ten shRNA-like siRNAs and corresponding miRNA-like designs. Two systems used a luciferase reporter RNA with CHIKV RNA inserted either in the coding sequence or within the 3’ UTR. A third reporter used a CHIKV derived split replication system. All three reporters demonstrated that while silencing could be achieved with both miRNA-like and shRNA-like designs, the latter were substantially more effective. Dcr-2 was required for the shRNA-like siRNAs as demonstrated by loss of inhibition of the reporters in Dcr-2 deficient cell lines. These positive results in cell culture are encouraging for the potential use of this pre-miRNA-1-based system in transgenic mosquitoes.     

Cultivation of Mesophilic Soil Crenarchaeotes in Enrichment Cultures from Plant Roots

Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division Crenarchaeota over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two dominant phylotypes of Crenarchaeota that colonizes the roots of tomato plants grown in soil from a Wisconsin field is selectively enriched in mixed cultures amended with root extract. Clones recovered from enrichment cultures were found to group phylogenetically with sequences from clade C1b.A1. This work corroborates and extends our recent findings, indicating that the diversity of the crenarchaeal soil assemblage is influenced by the rhizosphere and that mesophilic soil crenarchaeotes are found associated with plant roots, and provides the first evidence for growth of nonthermophilic crenarchaeotes in culture.     

Effects of chemical and botanical insecticides used for locust control on Metarhizium anisopliae var. acridum conidia after short- to medium-term storage at 30°C

The short- to medium-term viability and growth of Metarhizium anisopliae var. acridum conidia were investigated when combined with six insecticides, at three different concentrations. All of the insecticides used in this study were suitable for immediate spraying with M. anisopliae var. acridum conidia except for fenitrothion. Fipronil, teflubenzuron, and fenitrothion formulations significantly reduced conidial viability over time. The 10% teflubenzuron treatment caused loss of viability relatively quickly with 9.9% germination after 28 days. Mycelial growth was affected by all the treatments except fenitrothion.     

Keeping it cool: Soil sample cold pack storage and DNA shipment up to 1 month does not impact metabarcoding results

With the advances of sequencing tools, the fields of environmental microbiology and soil ecology have been transformed. Today, the unculturable majority of soil microbes can be sequenced. Although these tools give us tremendous power and open many doors to answer important questions, we must understand how sample processing may impact our results and interpretations. Here, we test the impacts of four soil storage methods on downstream amplicon metabarcoding and qPCR analyses for fungi and bacteria. We further investigate the impact of thaw time on extracted DNA to determine a safe length of time during which this can occur with minimal impact on study results. Overall, we find that storage using standard cold packs with subsequent storage at ?20°C is little different than immediate storage in liquid nitrogen, suggesting that the historical and current method is adequate. We further find evidence that storage at room temperature or with aid of RNAlater can lead to changes in community composition and in the case of RNAlater, lower gene copies. We therefore advise against these storage methods for metabarcoding analyses. Finally, we show that over 1 month, DNA extract thaw time does not impact diversity or qPCR metrics. We hope that this work will help researchers working with soil bacteria and fungi make informed decisions about soil storage and transport to ensure repeatability and accuracy of results and interpretations.