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1.  Phylogenetically Driven Sequencing of Extremely Halophilic Archaea Reveals Strategies for Static and Dynamic Osmo-response 
PLoS Genetics  2014;10(11):e1004784.
Organisms across the tree of life use a variety of mechanisms to respond to stress-inducing fluctuations in osmotic conditions. Cellular response mechanisms and phenotypes associated with osmoadaptation also play important roles in bacterial virulence, human health, agricultural production and many other biological systems. To improve understanding of osmoadaptive strategies, we have generated 59 high-quality draft genomes for the haloarchaea (a euryarchaeal clade whose members thrive in hypersaline environments and routinely experience drastic changes in environmental salinity) and analyzed these new genomes in combination with those from 21 previously sequenced haloarchaeal isolates. We propose a generalized model for haloarchaeal management of cytoplasmic osmolarity in response to osmotic shifts, where potassium accumulation and sodium expulsion during osmotic upshock are accomplished via secondary transport using the proton gradient as an energy source, and potassium loss during downshock is via a combination of secondary transport and non-specific ion loss through mechanosensitive channels. We also propose new mechanisms for magnesium and chloride accumulation. We describe the expansion and differentiation of haloarchaeal general transcription factor families, including two novel expansions of the TATA-binding protein family, and discuss their potential for enabling rapid adaptation to environmental fluxes. We challenge a recent high-profile proposal regarding the evolutionary origins of the haloarchaea by showing that inclusion of additional genomes significantly reduces support for a proposed large-scale horizontal gene transfer into the ancestral haloarchaeon from the bacterial domain. The combination of broad (17 genera) and deep (≥5 species in four genera) sampling of a phenotypically unified clade has enabled us to uncover both highly conserved and specialized features of osmoadaptation. Finally, we demonstrate the broad utility of such datasets, for metagenomics, improvements to automated gene annotation and investigations of evolutionary processes.
Author Summary
The ability to adjust to changing osmotic conditions (osmoadaptation) is crucial to the survival of organisms across the tree of life. However, significant gaps still exist in our understanding of this important phenomenon. To help fill some of these gaps, we have produced high-quality draft genomes for 59 osmoadaptation “experts” (extreme halophiles of the euryarchaeal family Halobacteriaceae). We describe the dispersal of osmoadaptive protein families across the haloarchaeal evolutionary tree. We use this data to suggest a generalized model for haloarchaeal ion transport in response to changing osmotic conditions, including proposed new mechanisms for magnesium and chloride accumulation. We describe the evolutionary expansion and differentiation of haloarchaeal general transcription factor families and discuss their potential for enabling rapid adaptation to environmental fluxes. Lastly, we challenge a recent high-profile proposal regarding the evolutionary origins of the haloarchaea by showing that inclusion of additional genomes significantly reduces support for a proposed large-scale horizontal gene transfer into the ancestral haloarchaeon from the bacterial domain. This result highlights the power of our dataset for making evolutionary inferences, a feature which will make it useful to the broader evolutionary community. We distribute our genomic dataset through a user-friendly graphical interface.
doi:10.1371/journal.pgen.1004784
PMCID: PMC4230888  PMID: 25393412
2.  A workflow for genome-wide mapping of archaeal transcription factors with ChIP-seq 
Nucleic Acids Research  2012;40(10):e74.
Deciphering the structure of gene regulatory networks across the tree of life remains one of the major challenges in postgenomic biology. We present a novel ChIP-seq workflow for the archaea using the model organism Halobacterium salinarum sp. NRC-1 and demonstrate its application for mapping the genome-wide binding sites of natively expressed transcription factors. This end-to-end pipeline is the first protocol for ChIP-seq in archaea, with methods and tools for each stage from gene tagging to data analysis and biological discovery. Genome-wide binding sites for transcription factors with many binding sites (TfbD) are identified with sensitivity, while retaining specificity in the identification the smaller regulons (bacteriorhodopsin-activator protein). Chromosomal tagging of target proteins with a compact epitope facilitates a standardized and cost-effective workflow that is compatible with high-throughput immunoprecipitation of natively expressed transcription factors. The Pique package, an open-source bioinformatics method, is presented for identification of binding events. Relative to ChIP-Chip and qPCR, this workflow offers a robust catalog of protein–DNA binding events with improved spatial resolution and significantly decreased cost. While this study focuses on the application of ChIP-seq in H. salinarum sp. NRC-1, our workflow can also be adapted for use in other archaea and bacteria with basic genetic tools.
doi:10.1093/nar/gks063
PMCID: PMC3378898  PMID: 22323522
3.  Regulatory Multidimensionality of Gas Vesicle Biogenesis in Halobacterium salinarum NRC-1 
Archaea  2011;2011:716456.
It is becoming clear that the regulation of gas vesicle biogenesis in Halobacterium salinarum NRC-1 is multifaceted and appears to integrate environmental and metabolic cues at both the transcriptional and posttranscriptional levels. The mechanistic details underlying this process, however, remain unclear. In this manuscript, we quantify the contribution of light scattering made by both intracellular and released gas vesicles isolated from Halobacterium salinarum NRC-1, demonstrating that each form can lead to distinct features in growth curves determined by optical density measured at 600 nm (OD600). In the course of the study, we also demonstrate the sensitivity of gas vesicle accumulation in Halobacterium salinarum NRC-1 on small differences in growth conditions and reevaluate published works in the context of our results to present a hypothesis regarding the roles of the general transcription factor tbpD and the TCA cycle enzyme aconitase on the regulation of gas vesicle biogenesis.
doi:10.1155/2011/716456
PMCID: PMC3202098  PMID: 22110395

Results 1-3 (3)