FISH-TAMB, a Fixation-Free mRNA Fluorescent Labeling Technique to Target Transcriptionally Active Members in Microbial Communities.

Keystone species or ecological engineers are vital to the health of an ecosystem; however, often, their low abundance or biomass present challenges for their discovery, identification, visualization and selection. We report the development of fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol that is applicable to archaea and bacteria. The FISH-TAMB method differs from existing FISH methods by the absence of fixatives or surfactants in buffers, the fast hybridization time of as short as 15 min at target cells' growth temperature, and the omission of washing steps. Polyarginine cell-penetrating peptides are employed to deliver molecular beacons (MBs) across prokaryotic cell walls and membranes, fluorescently labeling cells when MBs hybridize to target mRNA sequences. Here, the detailed protocol of the preparation and application of FISH-TAMB is presented. To demonstrate FISH-TAMB's ability to label intracellular mRNA targets, differentiate transcriptional states, detect active and rare taxa, and keep cell viability, labeling experiments were performed that targeted the messenger RNA (mRNA) of methyl-coenzyme M reductase A (mcrA) expressed in (1) Escherichia coli containing a plasmid with a partial mcrA gene of the methanogen Methanosarcina barkeri (E. coli mcrA+); (2) M. barkeri; and (3) an anaerobic methanotrophic (ANME) enrichment from a deep continental borehole. Although FISH-TAMB was initially envisioned for mRNA of any functional gene of interest without a requirement of prior knowledge of 16S ribosomal RNA (rRNA)-based taxonomy, FISH-TAMB has the potential for multiplexing and going beyond mRNA and thus is a versatile addition to the molecular ecologist's toolkit, with potentially widespread application in the field of environmental microbiology.

Counting Chromosomes in Individual Bacteria to Quantify Their Impacts on Persistence.

Persisters are phenotypic variants within bacterial populations that tolerate antibiotic treatments considerably better than the majority of cells. A phenotypic quality that varies within bacterial populations is the chromosome number of individual cells. One, two, four, or more chromosomes per cell have been observed previously, and the impact of genome copy number can range from gene dosage effects to an inability to perform specific DNA repair functions, such as homologous recombination. We hypothesize that chromosome abundance is an underappreciated phenotypic variable that could impact persistence to antibiotics. Here, we describe methodologies to segregate bacterial populations based on chromosome number, assess the purity of those subpopulations, and suggest assays that could be used to quantify the impacts of genome abundance on persistence.

TGF-ß-induced DACT1 biomolecular condensates repress Wnt signalling to promote bone metastasis.

The complexity of intracellular signalling requires both a diversity of molecular players and the sequestration of activity to unique compartments within the cell. Recent findings on the role of liquid-liquid phase separation provide a distinct mechanism for the spatial segregation of proteins to regulate signalling pathway crosstalk. Here, we discover that DACT1 is induced by TGFß and forms protein condensates in the cytoplasm to repress Wnt signalling. These condensates do not localize to any known organelles but, rather, exist as phase-separated proteinaceous cytoplasmic bodies. The deletion of intrinsically disordered domains within the DACT1 protein eliminates its ability to both form protein condensates and suppress Wnt signalling. Isolation and mass spectrometry analysis of these particles revealed a complex of protein machinery that sequesters casein kinase 2-a Wnt pathway activator. We further demonstrate that DACT1 condensates are maintained in vivo and that DACT1 is critical to breast and prostate cancer bone metastasis.

Analyzing Persister Physiology with Fluorescence-Activated Cell Sorting.

Bacterial persisters are phenotypic variants that exhibit an impressive ability to tolerate antibiotics. Persisters are hypothesized to cause relapse infections, and therefore, understanding their physiology may lead to novel therapeutics to treat recalcitrant infections. However, persisters have yet to be isolated due to their low abundance, transient nature, and similarity to the more highly abundant viable but non-culturable cells (VBNCs), resulting in limited knowledge of their phenotypic state. This technical hurdle has been addressed through the use of fluorescence-activated cell sorting (FACS) and quantification of persister levels in the resulting sorted fractions. These assays provide persister phenotype distributions, which can be compared to the phenotype distributions of the entire population, and can also be used to examine persister heterogeneity. Here, we describe two detailed protocols for analysis of persister physiology with FACS. One protocol assays the metabolic state of persisters using a fluorescent metabolic stain, whereas the other assays the growth state of persisters with use of a fluorescent protein.

Strong correlation between meiotic crossovers and haplotype structure in a 2.5-Mb region on the long arm of chromosome 21.

Although the haplotype structure of the human genome has been studied in great detail, very little is known about the mechanisms underlying its formation. To investigate the role of meiotic recombination on haplotype block formation, single nucleotide polymorphisms were selected at a high density from a 2.5-Mb region of human chromosome 21. Direct analysis of meiotic recombination by high-throughput multiplex genotyping of 662 single sperm identifies 41 recombinants. The crossovers were nonrandomly distributed within 16 small areas. All, except one, of these crossovers fall in areas where the haplotype structure exhibits breakdown, displaying a strong statistically positive association between crossovers and haplotype block breaks. The data also indicate a particular clustered distribution of recombination hotspots within the region. This finding supports the hypothesis that meiotic recombination makes a primary contribution to haplotype block formation in the human genome.