Biology subjects

Sakrikar, S.

Publications and source records attributed to Sakrikar, S..

5 recordsLinked to original sources

bioRxiv2026

A high aneuploidy supply rate underlies parallel evolution of fluconazole resistance in Saccharomyces cerevisiae

Copy number variants (CNVs) of ERG11 play a major role in acquisition of resistance to the commonly used azole family of antifungal drugs in many pathogenic yeast species. However, the conditions in which these CNVs arise, and the factors that underlie their emergence and selection in evolving populations, remain poorly understood. Here, we studied the dynamics of de novo CNVs at the ERG11 locus in Saccharomyces cerevisiae to determine the effect of different drug concentrations and temperatures on CNV formation and selection. We found that ERG11 CNVs emerge reproducibly at fluconazole concentrations around the IC50. With increases in temperature, CNVs emerged more rapidly and had a higher tendency towards fixation. Evolved ERG11 CNV strains provided a significant growth advantage across a narrow range of fluconazole concentrations near the IC50 of the wildtype strain. Whole genome sequencing of 35 independent evolved lineages revealed that all ERG11 CNVs are aneuploidies of chromosome VIII. Relocation of ERG11 to chromosome XI also results exclusively in selection for chromosome XI aneuploids. We show that fluconazole does not increase the frequency at which aneuploids are generated. Therefore, we conclude that a high spontaneous rate of aneuploidy formation underlies recurrent acquisition of resistance to fluconazole in a temperature and drug concentration dependent manner. Author SummaryCopy number variations (CNV), defined as duplications or deletions of genomic regions, are pervasive throughout all forms of life. Although CNVs can provide a route toward rapid adaptation, they can also be associated with high fitness costs. In pathogenic yeast species, CNVs with diverse genetic structures are known to play a role in antifungal drug resistance. However, the conditions that favor CNV formation and selection are unclear. We applied a fluorescent CNV reporter system to track newly formed CNVs and found that they arise reproducibly at a narrow range of drug concentrations, and that increases in temperature generally lead to more rapid emergence of CNVs. DNA sequencing revealed that the evolved CNVs contained duplications of the entire chromosome, rather than just the region surrounding the gene under selection. Thus, aneuploidy - gain or loss of an entire chromosome - is a mechanism for rapid evolution of drug resistance, potentially without the fitness costs thought to be associated with this mechanism.

evolutionary biology↗

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 rRNA removal methods for RNA-seq Differential Expression in Halophilic Archaea

Despite intense recent research interest in archaea, the scientific community has experienced a bottleneck in the study of genome-scale gene expression experiments by RNA-seq due to the lack of commercial and specifically designed rRNA depletion kits. The high ratio rRNA:mRNA (80-90%: [~]10%) in prokaryotes hampers global transcriptomic analysis. Insufficient ribodepletion results in low sequence coverage of mRNA and therefore requires a substantially higher number of replicate samples and/or sequencing reads to achieve statistically reliable conclusions regarding the significance of differential gene expression between case and control samples. Here we show that after the discontinuation of the previous version of RiboZero (Illumina) that was useful to partially deplete rRNA from halophilic archaea, archaeal transcriptomics studies have experienced a standstill. To overcome this limitation, here we analyze the efficiency for four different hybridization-based kits from three different commercial suppliers, each with two sets of sequence-specific probes to remove rRNA from four different species of halophilic archaea. We conclude that the key for transcriptomic success with the currently available tools is the probe-specificity for the rRNA sequence hybridization. With this paper we provide insights to the archaeal community for selecting certain reagents and strategies over others depending on the archaeal species of interest. These methods yield improved RNA-seq sensitivity and enhanced detection of low abundance transcripts.

genomics↗

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↗

bioRxiv2021

An archaeal histone-like protein regulates gene expression in response to salt stress

Histones, ubiquitous in eukaryotes as DNA-packing proteins, find their evolutionary origins in archaea. Unlike the characterized histone proteins of a number of methanogenic and themophilic archaea, previous research indicated that HpyA, the sole histone encoded in the model halophile Halobacterium salinarum, is not involved in DNA packaging. Instead, it was found to have widespread but subtle effects on gene expression and to maintain wild type cell morphology; however, its precise function remains unclear. Here we use quantitative phenotyping, genetics, and functional genomic to investigate HpyA function. These experiments revealed that HpyA is important for growth and rod-shaped morphology in reduced salinity. HpyA preferentially binds DNA at discrete genomic sites under low salt to regulate expression of ion uptake, particularly iron. HpyA also globally but indirectly activates other ion uptake and nucleotide biosynthesis pathways in a salt-dependent manner. Taken together, these results demonstrate an alternative function for an archaeal histone-like protein as a transcriptional regulator, with its function tuned to the physiological stressors of the hypersaline environment.

genomics↗