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ABSTRACT Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multilevel regulation. Compared to established knockout methodologies, CRISPR interference (CRISPRi) approaches enable the rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here, we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both nontemplate and template strand targeting. Since the nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication. IMPORTANCE Enterococcus faecalis causes multidrug-resistant life-threatening infections and is often coisolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labor-intensive allelic-exchange methods. Built upon streptococcal dCas9, we developed an easily modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation and antibiotic resistance and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance and can be used to perturb preformed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.

Primers that contain portions noncomplementary to the target region are usually used to add to the PCR product a utility sequence such as a restriction site or a universal probe binding site. We have demonstrated that primers with short 5′ AT-rich overhangs increase real-time PCR fluorescent signal. The improvement is particularly significant for difficult to amplify templates, such as highly variable viral sequences or bisulfite-treated DNA.

The title compound, C18H12S, comprises five fused rings forming an essentially planar molecule, with a total puckering amplitude (Q) of 0.032 Å and a maximum deviation from the mean plane of 0.014 (4) Å for the C atoms of the methylene groups. A crystallographic mirror plane orthogonal to the molecular plane passes through the S atom and the midpoint of the opposite C—C bond within the central five-membered ring. The molecules lie in layers, forming edge-to-face C—H...π interactions, with a separation of 2.66 Å between one H atom of the methylene group and the centroid of an adjacent indene ring.

Three new diindenodithienothiophene (DITT) based materials were synthesised and their electrochemical properties investigated. The HOMO–LUMO gaps were observed to be 3.33, 3.48 and 2.81 eV, respectively. Cyclic voltammetry results indicate increased stability for the alkylated derivatives. The dioxide exhibits strong photoluminescence, giving a photoluminescence quantum yield of 0.72 in solution and 0.14 in the solid state. Hole mobility measurements were carried out on the non-alkylated derivative and the corresponding values were ∼10−4cm2V−1s−1. [ABSTRACT FROM AUTHOR]

The analysis of microorganism communities in uncultured environmental samples requires laborious and cumbersome techniques such as denaturing gradient gel electrophoresis of amplicons generated with 16S rRNA generic primers with subsequent fragment sequencing. We have developed a simple method for genus identification of methanogen archaea and sulfate-reducing bacteria based on a real-time PCR hybridization probe melting curve analysis. The method takes advantage of a recent explosion of microorganism sequencing data conveniently packaged in the Ribosomal Database Project. Specificity of detection is based on a genus-specific real-time PCR fluorescent 5′-MGB-probe melt. As the probes are designed to have destabilizing mismatches with undesired genera, only samples with a proper melting temperature are called positive. [ABSTRACT FROM AUTHOR]