Biology subjects

Hackley, R. K.

Publications and source records attributed to Hackley, R. K..

4 recordsLinked to original sources

bioRxiv2024

Genomic re-sequencing reveals substantial mutational divergence across genetically engineered strains of model archaea

Because archaea are the evolutionary ancestors of eukaryotes, archaeal molecular biology has been a topic of intense recent research. The hypersaline adapted archaeal species Halobacterium salinarum and Haloferax volcanii serve as important model organisms because facile tools enable genetic manipulation. As a result, the number of strains in circulation among the haloarchaeal research community has increased over the last few decades. However, the degree of genetic divergence and effects on genetic integrity during inter-lab transfers remain unclear. To address this question, we performed whole genome re-sequencing on a cross-section of wild-type, parental, and knockout strains in both model species. Integrating these data with existing repositories of re-sequencing data, we identify mutations that have arisen in a collection of 60 strains, sampled from 2 species across 8 different labs. Independent of sequencing, we construct strain lineages, identifying branch points and significant genetic effects in strain history. Combining this with our sequencing data, we identify small clusters of mutations that definitively separate lab strains. Additionally, an analysis of gene knockout strains suggests that roughly 1 in 3 strains currently in use harbors second-site mutations of potential phenotypic impact. Overall, we find that divergence among lab strains is thus far minimal, though as the archaeal research community continues to grow, careful strain provenance and genomic re-sequencing are required to keep inter-lab divergence to a minimum, prevent the compounding of mutations into fully independent lineages, and maintain the current high degree of reproducible research between lab groups in the haloarchaeal research community. Data SummaryNovel sequencing data for this project was submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) and can be found under bioproject accession PRJNA1120443. SRA accessions for previously published sequencing data are available in supplementary table 1. R code for performing analysis and generating figures is available at https://github.com/andrew-soborowski/halophile_genome_resequencing. Impact StatementArchaea are important due to their shared evolutionary history with eukaryotes. As the archaeal research community grows, keeping track of the genetic integrity of archaeal strains of interest is necessary. In particular, routine genetic manipulations and the common practice of sharing strains between labs allow mutations to arise in lab stocks. If these mutations affect cellular processes, they may jeopardize the reproducibility of work between research groups and confound the results of future studies. In this work, we examine DNA sequences from 60 strains across two species of archaea. We identify shared and unique mutations occurring between and within strains. Independently, we trace the lineage of each strain, identifying which genetic manipulations lead to observed off-target mutations. While overall divergence across labs is minimal so far, our work highlights the need for labs to continue proper strain husbandry.

genomics↗

bioRxiv2023

A conserved transcription factor controls gluconeogenesis via distinct targets in hypersaline-adapted archaea with diverse metabolic capabilities

Timely regulation of carbon metabolic pathways is essential for cellular processes and to prevent futile cycling of intracellular metabolites. In Halobacterium salinarum, a hypersaline adapted archaeon, a sugar-sensing TrmB family protein controls gluconeogenesis and other biosynthetic pathways. Notably, Hbt. salinarum does not utilize carbohydrates for energy, uncommon among Haloarchaea. We characterized a TrmB-family transcriptional regulator in a saccharolytic generalist, Haloarcula hispanica, to investigate whether the targets and function of TrmB, or its regulon, is conserved in related species with distinct metabolic capabilities. In Har. hispanica, TrmB binds to 15 sites across the genome and induces the expression of genes primarily involved in gluconeogenesis and tryptophan biosynthesis. An important regulatory control point in Hbt. salinarum, activation of ppsA and repression of pykA, is absent in Har. hispanica. Contrary to its role in Hbt. salinarum and saccharolytic hyperthermophiles, TrmB does not act as a global regulator: it does not directly repress the expression of glycolytic enzymes, peripheral pathways such as cofactor biosynthesis, or catabolism of other carbon sources in Har. hispanica. Cumulatively, these findings suggest re-wiring of the TrmB regulon alongside metabolic network evolution in Haloarchaea.

microbiology↗

bioRxiv2023

TroR is the primary regulator of the iron homeostasis transcription network in the halophilic archaeon Haloferax volcanii

Maintaining intracellular iron concentration within the homeostatic range is vital to meet cellular metabolic needs and reduce oxidative stress. Previous research revealed that the haloarchaeon Halobacterium salinarum encodes four diphtheria toxin repressor (DtxR) family transcription factors (TFs) that together regulate the iron response through an interconnected transcriptional regulatory network (TRN). However, the metal specificity of DtxR TFs and the conservation of the TRN remained poorly understood. Here we identified and characterized the TRN of Haloferax volcanii for comparison. Genetic analysis demonstrated that Hfx. volcanii relies on three DtxR transcriptional regulators (Idr, SirR, and TroR), with TroR as the primary regulator of iron homeostasis. Bioinformatics and molecular approaches revealed that TroR binds a conserved cis-regulatory motif located [~]100 nt upstream of the start codon of iron-related target genes. Transcriptomics analysis demonstrated that, under conditions of iron sufficiency, TroR repressed iron uptake and induced iron storage mechanisms. TroR repressed the expression of one other DtxR TF, Idr. This reduced DtxR TRN complexity relative to that of Hbt. salinarum appeared correlated with natural variations in iron availability. Based on these data, we hypothesize that increasing TRN complexity appears selected for under variable environmental conditions such as iron availability.

microbiology↗

bioRxiv2022

Comparative analysis of genome-wide protein-DNA interactions across domains of life reveals unique binding patterns for hypersaline archaeal histones

DNA-binding proteins with roles in chromatin architecture and transcriptional regulation are present in all three domains of life. Histones package DNA and regulate gene expression in eukaryotes, and find their evolutionary origin in the domain of life Archaea. Previously characterised archaeal histones have a somewhat conserved functional role in nucleosome formation and DNA packaging. However, previous research has indicated that the histone-like proteins of high salt-adapted archaea, or halophiles, appear to function differently. The sole histone protein encoded by the model halophilic species Halobacterium salinarum is non-essential, is involved in direct and indirect transcriptional regulation, and does not appear to package DNA. Here we use protein-DNA binding assays, computational analysis, and quantitative phenotyping to compare DNA binding patterns across halophilic histone proteins, bacterial and archaeal TFs, NAPs, and eukaryotic histones. Like TFs, halophilic histones bind the genome too sparsely to compact the genome. However, unlike TFs, binding occurs in both coding and intergenic regions. Unlike histones, halophilic histone occupancy is not depleted at the start sites of genes, and halophilic genomes lack the dinucleotide periodicity known to facilitate histone binding. We detect unique sequence preferences for histone binding in halophiles. Together these data suggest that the non-essentiality and genome-wide binding features of halophilic histone-like proteins are conserved across halophiles; they bind DNA in ways resembling both TFs and chromatin proteins, but do not appear to play a role in forming chromatin. IMPORTANCEMost cells in eukaryotic species - from yeast to humans- possess histone proteins that pack and unpack DNA in response to environmental cues. These essential proteins regulate the genes necessary for important cellular processes, including development and stress protection. The domain of life Archaea represent the evolutionary progenitors of eukaryotes. The universal conservation of the primary sequences of histone proteins across archaeal lineages suggests that eukaryotic histones originated in the Archaea. However, archaeal histones lack N-terminal tails and, in some species, package DNA in a continuous helix with no linker DNA between nucleosomes. We recently discovered that histones in hypersaline adapted archaeal species do not package DNA, and can act like transcription factors (TFs) to regulate stress response gene expression. Here we compare hypersaline histone function to a variety of DNA binding proteins across the tree of life, revealing a mosaic of functions for hypersaline-adapted histones.

microbiology↗