BONCAT-FACS-Seq reveals the active fraction of a biocrust community undergoing a wet-up event.

Determining which microorganisms are active within soil communities remains a major technical endeavor in microbial ecology research. One promising method to accomplish this is coupling bioorthogonal non-canonical amino acid tagging (BONCAT) with fluorescence activated cell sorting (FACS) which sorts cells based on whether or not they are producing new proteins. Combined with shotgun metagenomic sequencing (Seq), we apply this method to profile the diversity and potential functional capabilities of both active and inactive microorganisms in a biocrust community after being resuscitated by a simulated rain event.

A centimeter-long bacterium with DNA contained in metabolically active, membrane-bound organelles.

Cells of most bacterial species are around 2 micrometers in length, with some of the largest specimens reaching 750 micrometers. Using fluorescence, x-ray, and electron microscopy in conjunction with genome sequencing, we characterized () Thiomargarita magnifica, a bacterium that has an average cell length greater than 9000 micrometers and is visible to the naked eye. These cells grow orders of magnitude over theoretical limits for bacterial cell size, display unprecedented polyploidy of more than half a million copies of a very large genome, and undergo a dimorphic life cycle with asymmetric segregation of chromosomes into daughter cells.

Probing the active fraction of soil microbiomes using BONCAT-FACS.

The ability to link soil microbial diversity to soil processes requires technologies that differentiate active microbes from extracellular DNA and dormant cells. Here, we use BONCAT (bioorthogonal non-canonical amino acid tagging) to measure translationally active cells in soils. We compare the active population of two soil depths from Oak Ridge (Tennessee, USA) and find that a maximum of 25-70% of the extractable cells are active.

Optimizing genome assembly from PCR-amplified metagenomes.

Background: Metagenomics has transformed our understanding of microbial diversity across ecosystems, with recent advances enabling assembly of genomes from metagenomes. These metagenome-assembled genomes are critical to provide ecological, evolutionary, and metabolic context for all the microbes and viruses yet to be cultivated. Metagenomes can now be generated from nanogram to subnanogram amounts of DNA.

Preparing Mate-Paired Illumina Libraries Using Cre Recombinase.

Large insert mate pair reads have been used in de novo assembly and discovery of structural variants. We developed a new approach, Cre-LoxP inverse PCR paired end (CLIP-PE), which exploits the advantages of (1) Cre-LoxP recombination system to efficiently circularize large DNA fragments, (2) inverse PCR to enrich for the desired products that contain both ends of the large DNA fragments, and (3) use of restriction enzymes to introduce a recognizable junction site between ligated fragment ends. We have successfully created CLIP-PE libraries of up to 22 kb jumping pairs and demonstrated their ability to improve genome assemblies.

Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics.

Single-cell genomics is a powerful tool for exploring the genetic makeup of environmental microorganisms, the vast majority of which are difficult, if not impossible, to cultivate with current approaches. Here we present a comprehensive protocol for obtaining genomes from uncultivated environmental microbes via high-throughput single-cell isolation by FACS. The protocol encompasses the preservation and pretreatment of differing environmental samples, followed by the physical separation, lysis, whole-genome amplification and 16S rRNA-based identification of individual bacterial and archaeal cells.

Identification of a protein mediating respiratory supercomplex stability.

The complexes of the electron transport chain associate into large macromolecular assemblies, which are believed to facilitate efficient electron flow. We have identified a conserved mitochondrial protein, named respiratory supercomplex factor 1 (Rcf1-Yml030w), that is required for the normal assembly of respiratory supercomplexes. We demonstrate that Rcf1 stably and independently associates with both Complex III and Complex IV of the electron transport chain.

Generation of long insert pairs using a Cre-LoxP Inverse PCR approach.

Large insert mate pair reads have a major impact on the overall success of de novo assembly and the discovery of inherited and acquired structural variants. The positional information of mate pair reads generally improves genome assembly by resolving repeat elements and/or ordering contigs. Currently available methods for building such libraries have one or more of limitations, such as relatively small insert size; unable to distinguish the junction of two ends; and/or low throughput.

Functional characterization of human proapoptotic molecules in yeast S. cerevisiae.

The presence of a complete (BH1-3) proapoptotic molecule is necessary for the induction of the intrinsic apoptotic cascade in mammalian cells. It is unclear, however, what distinct roles the members of the large family of BH3-only proapoptotic molecules play in apoptosis. Although biochemical analysis of these molecules can characterize binding efficiencies of BH3 family members, the biologic consequences of these interactions are difficult to predict.

Yeast Pak1 kinase associates with and activates Snf1.

Members of the Snf1/AMP-activated protein kinase family are activated under conditions of nutrient stress by a distinct upstream kinase. Here we present evidence that the yeast Pak1 kinase functions as a Snf1-activating kinase. Pak1 associates with the Snf1 kinase in vivo, and the association is greatly enhanced under glucose-limiting conditions when Snf1 is active.

Isolation of mutations in the catalytic domain of the snf1 kinase that render its activity independent of the snf4 subunit.

Activation of the Snf1 kinase requires at least two events, phosphorylation of the activation loop on threonine 210 and an Snf4-dependent process that is not completely defined. Snf4 directly interacts with a region of the regulatory domain of Snf1 that may otherwise act as an autoinhibitory domain. In order to gain insight into the regulation of Snf1 kinase by Snf4, deletions in the regulatory domain of the catalytic subunit were engineered and tested for their effect on Snf1 function in the absence of Snf4.

Purification and characterization of Snf1 kinase complexes containing a defined Beta subunit composition.

The Snf1 kinase complex of Saccharomyces cerevisiae contains one of three possible beta subunits encoded by either SIP1, SIP2, or GAL83. Snf1 kinase complexes were purified from cells expressing only one of the three beta subunits using a tandem affinity purification tag on the C terminus of the Snf1 protein. The purified kinase complexes were enzymatically active as judged by their ability to phosphorylate a recombinant protein containing the Snf1-responsive domain of the Mig1 protein.