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Maupin-Furlow, J. A.

Publications and source records attributed to Maupin-Furlow, J. A..

10 recordsLinked to original sources

A Bayesian Multi-Species Approach Infers Gene Regulatory Networks Across Non-Model Organisms

Control of gene expression by transcription factors (TFs) is a critical mechanism for cells to maintain homeostasis in response to environmental signals. Gene network models that predict regulatory interactions between transcription factors and the genes they control aid in understanding these complex processes. These models are useful as they provide testable hypotheses of regulatory interactions, transcription factor function, and accelerate the study of uncharacterized transcription factors. However, inference of these models is computationally challenging due to the vast quantity of data required given the many possible states of the regulatory network. Microbial genomes encode hundreds of transcription factors, with numerous interactions that require substantial functional genomics datasets to infer. This problem is accentuated in understudied organisms, species that would greatly benefit from an inferred network for biological discovery, where the lack of available data is particularly constraining for effective inference. To address this problem, we have developed GRN-BMuSeR (Gene Regulatory Networks from Bayesian MUlti-SpEcies Regression), a novel multitask approach to gene regulatory network inference that leverages gene orthology between closely related species to improve inference performance. We evaluate its performance on a dataset from the well-studied bacterial species Bacillus subtilis, demonstrating improved performance in multitask settings. Applying the model to simulated data reveals utility in multi-species contexts. Finally, we apply our models to infer GRNs and explore predictions for two hypersaline-adapted archaeal species. We leverage a rich dataset from Halobacterium salinarum to inform the inference of the gene regulatory network of Haloferax volcanii, for which a more limited genomics dataset was available. We generate a large compendium of gene expression data for Hfx.volcanii for GRN inference input. Through exploration of resultant network predictions, we show concordance with known TF functions and discover hundreds of novel TF functional predictions. Moving forward, our results provide a framework to generate testable hypotheses that will serve to guide experimental work and accelerate discovery in these understudied species.

systems biology↗

Mechanistic insights into redox activity and catalytic determinants of the haloarchaeal flavin-dependent oxidoreductase HvFdR

Members of the FAD-dependent oxidoreductase family (IPR050260) play diverse and key roles in maintaining cellular redox balance, yet the functions of many distinct subgroups within this family remain unknown. Here, we define the biochemical and physiological functions of the Haloferax volcanii flavin-dependent oxidoreductase HvFdR (HVO_2345; fdr), a haloarchaeal member of a previously uncharacterized IPR050260 subgroup. HvFdR binds FAD and catalyzes NAD(P)H oxidase, diaphorase and ferredoxin reductase activities, with a kinetic preference for NADPH over NADH and catalytic properties that are strongly influenced by oxygen availability. Under stoichiometric conditions, HvFdR mediates reverse electron transfer to NADP, suggesting that intracellular nicotinamide nucleotide pools regulate electron flow bidirectionally. Consistent with this reversibility, HvFdR bound-FAD exhibits a low midpoint redox potential (-413 mV), supporting its capacity to function as an electron donor. Deletion of fdr impairs growth and elevates intracellular NADPH levels, consistent with a role for HvFdR in maintaining NADP(H) homeostasis. Conserved residues K47 and Y323 are identified as determinants of HvFdR electron transfer activity and may function as a regulatory gate that modulates electron flow while limiting excessive H2O2 production under aerobic conditions. Together, these findings establish HvFdR as an oxygen-responsive flavin-dependent oxidoreductase that contributes to cellular redox homeostasis and provides functional insight into a previously uncharacterized subgroup of the IPR050260 family.

biochemistry↗

Lysine acetylation-mediated regulation of ferredoxin and ferredoxin reductase redox-active proteins in Haloferax volcanii

Lysine acetylation is an evolutionarily conserved, post-translational modification that regulates metabolism and protein function, yet its role in archaeal electron transfer systems remains poorly understood. Here, we investigated lysine acetylation of the 2Fe-2S ferredoxin HvFdx (HVO_2995) and its flavin-dependent oxidoreductase HvFdR (HVO_2345) partner in the halophilic archaeon Haloferax volcanii. Genetic and biochemical analyses established HvFdx as an essential 2Fe-2S ferredoxin with a midpoint redox potential of -385 mV. Lysine acetylation of HvFdx was found to occur primarily at K119, a residue positioned near the [Fe-S] cluster interface, and to modulate electron transfer capacity without impacting Fe-S cluster incorporation, midpoint potential, or protein abundance. In contrast, HvFdR was found lysine acetylated at multiple sites in a manner consistent with a non-enzymatic mechanism that resulted in altered flavin binding, enzymatic activity, and thermal stability. Lysine acetylation of HvFdx was found to stimulate electron flow from HvFdR as measured by an anaerobic NADPH [->] HvFdR [->] HvFdx [->] DCIP assay. 3D structural modeling, proteomic, biochemical, and genetic assays suggest the haloarchaeal GNAT-family acetyltransferase homolog HVO_2874 as a candidate enzyme associated with HvFdx lysine acetylation and optimal growth of H. volcanii. Together, these findings demonstrate that lysine acetylation differentially regulates archaeal redox-active proteins and functions as an important mechanism coordinating redox metabolism in H. volcanii.

microbiology↗

Dynamic acetylation of a conserved lysine impacts glycerol kinase activity and abundance in the haloarchaeon Haloferax volcanii

Haloferax volcanii is a halophilic archaeon that preferentially utilizes glycerol as a carbon source, placing glycerol kinase (GK, glpK) at the center of its metabolism. In contrast to bacterial GKs, which are often regulated by allosteric inhibition, H. volcanii GK lacks this mode of control, indicating alternative regulatory mechanisms. Here, we show that lysine acetylation of H. volcanii GK enhances its activity and abundance during growth on glycerol, with K153 identified as the primary site of modification. Structural modeling and comparative genomics revealed that K153 resides in a conserved flexible loop common to haloarchaeal GKs. Carbon shifts from glucose to glycerol led to increased activity and enrichment of the K153-acetylated form, as determined by AQUA-MS. GK and the acetylation mimic K153Q supported growth on glycerol, while the non-acetylatable K153R variant did not. Thermal shift analysis showed that the K153R substitution reduced GK stability, while K153Q had no effect. Size exclusion chromatography indicated that GK is predominantly dimeric but forms a tetramer when purified from glycerol-grown cells and assayed with glycerol - coinciding with the highest K153 acetylation levels. Kinetic analysis revealed that K153 acetylation is required to maintain cooperative substrate binding, with the non-acetylatable K153R variant exhibiting a loss of allosteric behavior. The GNAT-family acetyltransferase Pat2 was found to acetylate GK at K153, and{Delta} pat2 mutants exhibited reduced GK protein abundance, linking Pat2 to regulation of GK. These results identify a dynamic, carbon source-responsive lysine acetylation mechanism that modulates GK, highlighting lysine acetylation as a key component of haloarchaeal metabolic regulation. IMPORTANCEPost-translational modifications allow microorganisms to rapidly adapt their metabolism to changing environmental conditions. Here, we uncover a carbon source-dependent acetylation mechanism that regulates GK activity and abundance in the halophilic archaeon H. volcanii. Unlike bacterial systems, where allosteric inhibitors control GK, H. volcanii relies on lysine acetylation to fine-tune and enhance enzymatic function based on nutrient availability. Our findings highlight acetylation at a conserved lysine as a key modulator of archaeal carbon metabolism, linking environmental signals directly to enzymatic activity and cellular fitness. This work expands our understanding of extremophile metabolic regulation and reveals how archaea deploy unique strategies to survive and thrive in environments that shift in carbon availability.

microbiology↗

GNAT family Pat2 lysine acetylation of glycerol kinase and its key role in glycerol metabolism in hypersaline-adapted archaea

Lysine acetylation is a widespread post-translational modification (PTM) involved in regulating key biological processes including central metabolism and chromatin dynamics, yet its roles in archaea remain poorly understood. Here, we investigated two GNAT (Gcn5-related N-acetyltransferase) family homologs, pat1 and pat2, in the halophilic archaeon Haloferax volcanii (Hv). We found that a pat2 mutant exhibited impaired growth and premature cell death on glycerol, a phenotype not observed in the parent strain, pat1 mutant, or during growth on glucose. Complementation with plasmid-expressed pat2 restored growth on glycerol, confirming this biological role. In vitro assays demonstrated that HvPat2 catalyzes the lysine acetylation of HvGlpK, a glycerol kinase essential for glycerol metabolism. Computational modeling predicted that HvPat2 residues E105, Y154, V110, and N147 may form hydrogen bonds with acetyl-CoA. To assess the functional importance of these residues, alanine substitutions were introduced at each site. Growth assays revealed that E105A and Y154A variants failed to restore growth on glycerol, while V110A and N147A had no significant effect. In vitro, HvPat2 Y154A, E105A, and V110A lacked acetyltransferase activity toward GlpK, whereas N147A retained partial activity. HvPat2 Y154A co-purified with a protein partner, potentially explaining the discrepancy between in vivo and in vitro results. These findings highlight the critical role of the GNAT HvPat2 in mediating lysine acetylation in regulating glycerol metabolism in archaea and offer mechanistic insight into GNAT family acetyltransferases. IMPORTANCEGNAT family homologs are widespread and diverse in their use of acyl-CoAs to acylate small molecules and proteins. These functions can be difficult to predict based on in silico analysis alone. Here we reveal a critical role for lysine acetylation in archaeal central carbon metabolism, identifying the GNAT family homolog Pat2 as an essential regulator of glycerol utilization in Haloferax volcanii. The findings expand our understanding of GNAT family acetyltransferases and highlight conserved mechanisms of metabolic control by PTM across domains of life.

microbiology↗

Revisiting synthetic lethality of Gcn5-related N-acetyltransferase (GNAT) family mutations in Haloferax volcanii

Lysine acetylation is a post-translational modification that occurs in all domains of life, highlighting its evolutionary significance. Previous genome comparison identified three Gcn5-related N-acetyltransferase (GNAT) family members as lysine acetyltransferase homologs (Pat1, Pat2, and Elp3) and two deacetylase homologs (Sir2 and HdaI) in the halophilic archaeon Haloferax volcanii, with elp3 and pat2 proposed as a synthetic lethal gene pair. Here we advance these findings by performing single and double mutagenesis of elp3 with the pat1 and pat2 lysine acetyltransferase gene homologs. Genome sequencing and PCR screens of these strains reveal successful generation of{Delta} elp3, {Delta}pat1{Delta}elp3, and{Delta} pat2{Delta}elp3 mutant strains. Although these mutant strains exhibited a reduced growth rate compared to the parent, they remained viable. Overall, this study provides genetic evidence that elp3 and pat2, while impacting cell growth, are not a synthetic lethal gene pair as previously reported. IMPORTANCEHere we reveal by whole genome sequencing that the GNAT family gene homologs elp3 and pat2 can be deleted in the same H. volcanii strain. Beyond the targeted deletions, minimal differences between the parent and{Delta} elp3 {Delta}pat2 mutant were observed suggesting that suppressor mutations are not responsible for our ability to generate this double mutant strain. Elp3 and Pat2, thus, may not share as close a functional relationship as implied by earlier study. Our finding is significant as Elp3 is thought to function in acetylation in tRNA modification, while Pat2 likely functions in the lysine acetylation of proteins.

genetics↗

Biochemical properties of glycerol kinase from the hypersaline-adapted archaeon Haloferax volcanii

Extremophilic microorganisms hold promise to serve as robust biocatalysts in the conversion of glycerol waste into high value products. Haloferax volcanii is a hypersaline-adapted archaeon that prefers glycerol over glucose and channels this carbon source into central metabolism through glycerol kinase (GK). Here we report the biochemical properties of the H. volcanii GK and evaluated its potential for biotechnological applications. The N-terminal His-tagged form of GK was found functional in vivo and was readily purified to homogeneity at 4.5-fold higher yield (3 mg/L culture) than GK fused to a C-terminal StrepII tag. Further analysis of His-GK by size exclusion chromatography revealed the enzyme exhibited a glycerol-induced shift from a homodimer to a homodimer-homotetramer equilibrium. Purified His-GK demonstrated robust activity over a broad pH and salinity range, with optimal activity at 100 mM NaCl and 50-60 {degrees}C. The enzyme was catalytically active in organic solvent (5-10 % DMSO) and crude glycerol containing methanol. His-GK was also found to exhibit full activity after freeze-thaw, showed prolonged thermotolerance in 2 M NaCl supplemented buffers, and had a melting temperature (Tm) in the range of 83-84 {degrees}C. Kinetic analysis using the Hill equation indicated His-GK displayed positive cooperativity for glycerol, ATP, and magnesium, with manganese and cobalt also found to serve as divalent cation cofactors. These findings underscore the unique and robust enzymatic properties of H. volcanii GK, representing the first known GK to exhibit positive cooperativity with glycerol and ATP, and highlighting its potential for biotechnological applications in glycerol conversion. IMPORTANCEThis study provides new insight into glycerol kinase kinetics through the discovery of an enzyme exhibiting positive cooperativity for glycerol and ATP. By purifying and characterizing H. volcanii glycerol kinase, this work marks the first isolation of a glycerol kinase from a halophilic archaeon. The enzyme displays unique properties, including resilience to organic solvents, high temperatures, and extreme salinity. It also exhibits sigmoidal kinetics, with Hill coefficients averaging n = 2 for glycerol, ATP, and magnesium, indicating positive cooperativity. This behavior, previously unobserved in other glycerol kinases, aligns with the preference of H. volcanii for glycerol over glucose. Given the role of glycerol kinases in converting glycerol waste into value-added products, their efficiency is often a bottleneck in bacterial systems that prefer glucose and struggle under extreme conditions. The distinctive properties of H. volcanii glycerol kinase suggest potential for biotechnological applications in harsh environments.

biochemistry↗

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↗

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↗

TrmB family transcription factor as a thiol-based regulator of oxidative stress response

Oxidative stress causes cellular damage including DNA mutations, protein dysfunction and loss of membrane integrity. Here we discovered TrmB (transcription regulator of mal operon) family proteins (Pfam PF01978) composed of a single winged-helix DNA binding domain (InterPro IPR002831) can function as thiol-based transcriptional regulators of oxidative stress responses. Using the archaeon Haloferax volcanii as a model system, we demonstrate that the TrmB-like OxsR is important for recovery of cells from hypochlorite stress. OxsR is shown to bind specific regions of genomic DNA, particularly during hypochlorite stress. OxsR-bound intergenic regions were found proximal to oxidative stress operons including genes associated with thiol relay and low molecular weight thiol biosynthesis. Further analysis of a subset of these sites, revealed OxsR to function during hypochlorite stress as a transcriptional activator and repressor. OxsR was shown to require a conserved cysteine (C24) for function and to use a CG-rich motif upstream of conserved BRE/TATA box promoter elements for transcriptional activation. Protein modeling suggested the C24 is located at a homodimer interface formed by antiparallel helices, and that oxidation of this cysteine would result in the formation of an intersubunit disulfide bond. This covalent linkage may promote stabilization of an OxsR homodimer with the enhanced DNA binding properties observed in the presence of hypochlorite stress. The phylogenetic distribution TrmB family proteins, like OxsR, that have a single winged-helix DNA binding domain and conserved cysteine residue suggests this type of redox signaling mechanism is widespread in Archaea. ImportanceTrmB-like proteins, while not yet correlated with redox stress, are found in bacteria and widespread in archaea. Here we expand annotation of a large group of TrmB-like single winged-helix DNA binding domain proteins from diverse archaea to function as thiol-based transcriptional regulators of oxidative stress response. Using Haloferax volcanii as a model, we reveal the TrmB-like OxsR functions during hypochlorite stress as a transcriptional activator and repressor of an extensive gene co-expression network associated with thiol relay and other related activities. A conserved cysteine residue of OxxR serves as the thiol-based sensor for this function and likely forms an intersubunit disulfide bond during hypochlorite stress that stabilizes a homodimeric configuration with enhanced DNA binding properties. A CG-rich DNA motif in the promoter region of a subset of sites identified to be OxsR-bound is required for regulation; however, not all sites have this motif suggesting added complexity to the regulatory network.

microbiology↗