Andrew I. Yao

A workflow for genome-wide mapping of archaeal transcription factors with ChIP-seq

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.

Promoter Element Arising from the Fusion of Standard BioBrick Parts

We characterize the appearance of a constitutive promoter element in the commonly used cI repressor-encoding BioBrick BBa_C0051. We have termed this promoter element pKAT. Full pKAT activity is created by the ordered assembly of sequences in BBa_C0051 downstream of the cI gene encoding the 11 amino acid LVA proteolytic degradation tag, a BioBrick standard double-TAA stop codon, a genetic barcode, and part of the RFC10 SpeI-XbaI BioBrick scar. Placing BBa_C0051 or other pKAT containing parts upstream of other functional RNA coding elements in a polycistronic context may therefore lead to the unintended transcription of the downstream elements. The frequent reuse of pKAT or pKAT-like containing basic parts in the Registry of Biological Parts has resulted in approximately 5% of registry parts encoding at least one instance of a predicted pKAT promoter located directly upstream of a ribosome binding site and ATG start codon. This example highlights that even seemingly simple modifications of a parts sequence (in this case addition of degradation tags and barcodes) may be sufficient to unexpectedly change the contextual behavior of a part and reaffirms the inherent challenge in carefully characterizing the behavior of standardized biological parts across a broad range of reasonable use scenarios.

Role of squalene in the organization of monolayers derived from lipid extracts of Halobacterium salinarum.

We have studied interfacial compressibility and lateral organization in monolayer configurations of total (squalene containing) and polar (squalene-devoid) lipid extracts of Halobacterium salinarum NRC-1, an extremely halophilic archaeon. Pressure-area isotherms derived from Langmuir experiments reveal that packing characteristics and elastic compressibility are strongly influenced by the presence of squalene in the total lipid extract. In conjunction with control experiments using mixtures of DPhPC and squalene, our results establish that the presence of squalene significantly extends elastic area compressibility of total lipid extracts, suggesting it has a role in facilitating tighter packing of archaeal lipid mixtures. Moreover, we find that squalene also influences spatial organization in archaeal membranes. Epifluorescence and atomic force microscopy characterization of Langmuir monolayers transferred onto solid hydrophilic substrates reveal an unusual domain morphology. Individual domains of microscopic dimensions (as well as their extended networks) exhibiting a peculiar bowl-like topography are evident in atomic force microscopy images. The tall rims outlining individual domains indicate that squalene accumulates at the domain periphery in a manner similar to the accumulation of cholesterol at domain boundaries in their mixtures with phospholipids. Taken together, the results presented here support the notion that squalene plays a role in modulating molecular packing and lateral organization (i.e., domain formation) in the membranes of archaea analogous to that of cholesterol in eukaryotic membranes.

Identification of an archaeal mercury regulon by chromatin immunoprecipitation

Mercury is a heavy metal and toxic to all forms of life. Metal exposure can invoke a response to improve survival. In archaea, several components of a mercury response system have been identified, but it is not known whether metal transport is a member of this system. To identify such missing components, a peptide-tagged MerR transcription factor was used to localize enriched chromosome regions by chromosome immunoprecipitation combined with DNA sequence analysis. Such regions could serve as secondary regulatory binding sites to control the expression of additional genes associated with mercury detoxification. Among the 31 highly enriched loci, a subset of five was pursued as potential candidates based on their current annotations. Quantitative reverse transcription-PCR analysis of these regions with and without mercury treatment in WT and mutant strains lacking merR indicated significant regulatory responses under these conditions. Of these, a Family 5 extracellular solute-binding protein and the MarR transcription factor shown previously to control responses to oxidation were most strongly affected. Inactivation of the solute-binding protein by gene disruption increased the resistance of mutant cells to mercury challenge. Inductively coupled plasma-MS analysis of the mutant cell line following metal challenge indicated there was less intracellular mercury compared with the isogenic WT strain. Together, these regulated genes comprise new members of the archaeal MerR regulon and reveal a cascade of transcriptional control not previously demonstrated in this model organism.

Regulatory Multidimensionality of Gas Vesicle Biogenesis in Halobacterium salinarum NRC-1

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.

RiboTALE: A modular, inducible system for accurate gene expression control

A limiting factor in synthetic gene circuit design is the number of independent control elements that can be combined together in a single system. Here, we present RiboTALEs, a new class of inducible repressors that combine the specificity of TALEs with the ability of riboswitches to recognize exogenous signals and differentially control protein abundance. We demonstrate the capacity of RiboTALEs, constructed through different combinations of TALE proteins and riboswitches, to rapidly and reproducibly control the expression of downstream targets with a dynamic range of 243.7 ± 17.6-fold, which is adequate for many biotechnological applications.

A Large and Phylogenetically Diverse Class of Type 1 Opsins Lacking a Canonical Retinal Binding Site

Opsins are photosensitive proteins catalyzing light-dependent processes across the tree of life. For both microbial (type 1) and metazoan (type 2) opsins, photosensing depends upon covalent interaction between a retinal chromophore and a conserved lysine residue. Despite recent discoveries of potential opsin homologs lacking this residue, phylogenetic dispersal and functional significance of these abnormal sequences have not yet been investigated. We report discovery of a large group of putatively non-retinal binding opsins, present in a number of fungal and microbial genomes and comprising nearly 30% of opsins in the Halobacteriacea, a model clade for opsin photobiology. We report phylogenetic analyses, structural modeling, genomic context analysis and biochemistry, to describe the evolutionary relationship of these recently described proteins with other opsins, show that they are expressed and do not bind retinal in a canonical manner. Given these data, we propose a hypothesis that these abnormal opsin homologs may represent a novel family of sensory opsins which may be involved in taxis response to one or more non-light stimuli. If true, this finding would challenge our current understanding of microbial opsins as a light-specific sensory family, and provides a potential analogy with the highly diverse signaling capabilities of the eukaryotic G-protein coupled receptors (GPCRs), of which metazoan type 2 opsins are a light-specific sub-clade.

A phylogenetically diverse class of blind type 1 opsins

Opsins are photosensitive proteins catalyzing light-dependent processes across the tree of life. For both microbial (type 1) and metazoan (type 2) opsins, photosensing depends upon covalent interaction between a retinal chromophore and a conserved lysine residue. Despite recent discoveries of potential opsin homologs lacking this residue, phylogenetic dispersal and functional significance of these abnormal sequences have not yet been investigated. We report discovery of a large group of putatively non-retinal binding opsins, present in a number of fungal and microbial genomes and comprising nearly 30% of opsins in the Halobacteriacea, a model clade for opsin photobiology. Based on phylogenetic analyses, structural modeling, genomic context and biochemistry, we propose that these abnormal opsin homologs represent a novel family of sensory opsins which may be involved in taxis response to one or more non-light stimuli. This finding challenges current understanding of microbial opsins as a light-specific sensory family, and provides a potential analogy with the highly diverse signaling capabilities of the eukaryotic G-protein coupled receptors (GPCRs), of which metazoan type 2 opsins are a light-specific sub-clade.