An unexpected role for leucyl aminopeptidase in UV tolerance revealed by a genome-wide fitness assessment in a model cyanobacterium.

UV radiation (UVR) has significant physiological effects on organisms living at or near the Earth's surface, yet the full suite of genes required for fitness of a photosynthetic organism in a UVR-rich environment remains unknown. This study reports a genome-wide fitness assessment of the genes that affect UVR tolerance under environmentally relevant UVR dosages in the model cyanobacterium Synechococcus elongatus PCC 7942. Our results highlight the importance of specific genes that encode proteins involved in DNA repair, glutathione synthesis, and the assembly and maintenance of photosystem II, as well as genes that encode hypothetical proteins and others without an obvious connection to canonical methods of UVR tolerance. Disruption of a gene that encodes a leucyl aminopeptidase (LAP) conferred the greatest UVR-specific decrease in fitness. Enzymatic assays demonstrated a strong pH-dependent affinity of the LAP for the dipeptide cysteinyl-glycine, suggesting an involvement in glutathione catabolism as a function of night-time cytosolic pH level. A low differential expression of the LAP gene under acute UVR exposure suggests that its relative importance would be overlooked in transcript-dependent screens. Subsequent experiments revealed a similar UVR-sensitivity phenotype in LAP knockouts of other organisms, indicating conservation of the functional role of LAPs in UVR tolerance.

Machine learning from Pseudomonas aeruginosa transcriptomes identifies independently modulated sets of genes associated with known transcriptional regulators.

The transcriptional regulatory network (TRN) of Pseudomonas aeruginosa coordinates cellular processes in response to stimuli. We used 364 transcriptomes (281 publicly available + 83 in-house generated) to reconstruct the TRN of P. aeruginosa using independent component analysis. We identified 104 independently modulated sets of genes (iModulons) among which 81 reflect the effects of known transcriptional regulators. We identified iModulons that (i) play an important role in defining the genomic boundaries of biosynthetic gene clusters (BGCs), (ii) show increased expression of the BGCs and associated secretion systems in nutrient conditions that are important in cystic fibrosis, (iii) show the presence of a novel ribosomally synthesized and post-translationally modified peptide (RiPP) BGC which might have a role in P. aeruginosa virulence, (iv) exhibit interplay of amino acid metabolism regulation and central metabolism across different carbon sources and (v) clustered according to their activity changes to define iron and sulfur stimulons. Finally, we compared the identified iModulons of P. aeruginosa with those previously described in Escherichia coli to observe conserved regulons across two Gram-negative species. This comprehensive TRN framework encompasses the majority of the transcriptional regulatory machinery in P. aeruginosa, and thus should prove foundational for future research into its physiological functions.

Advanced transcriptomic analysis reveals the role of efflux pumps and media composition in antibiotic responses of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen and major cause of hospital-acquired infections. The virulence of P. aeruginosa is largely determined by its transcriptional regulatory network (TRN). We used 411 transcription profiles of P. aeruginosa from diverse growth conditions to construct a quantitative TRN by identifying independently modulated sets of genes (called iModulons) and their condition-specific activity levels. The current study focused on the use of iModulons to analyze the biofilm production and antibiotic resistance of P. aeruginosa. Our analysis revealed: (i) 116 iModulons, 81 of which show strong association with known regulators; (ii) novel roles of regulators in modulating antibiotics efflux pumps; (iii) substrate-efflux pump associations; (iv) differential iModulon activity in response to beta-lactam antibiotics in bacteriological and physiological media; (v) differential activation of 'Cell Division' iModulon resulting from exposure to different beta-lactam antibiotics and (vi) a role of the PprB iModulon in the stress-induced transition from planktonic to biofilm lifestyle. In light of these results, the construction of an iModulon-based TRN provides a transcriptional regulatory basis for key aspects of P. aeruginosa infection, such as antibiotic stress responses and biofilm formation. Taken together, our results offer a novel mechanistic understanding of P. aeruginosa virulence.

RiboRid: A low cost, advanced, and ultra-efficient method to remove ribosomal RNA for bacterial transcriptomics.

RNA sequencing techniques have enabled the systematic elucidation of gene expression (RNA-Seq), transcription start sites (differential RNA-Seq), transcript 3' ends (Term-Seq), and post-transcriptional processes (ribosome profiling). The main challenge of transcriptomic studies is to remove ribosomal RNAs (rRNAs), which comprise more than 90% of the total RNA in a cell. Here, we report a low-cost and robust bacterial rRNA depletion method, RiboRid, based on the enzymatic degradation of rRNA by thermostable RNase H. This method implemented experimental considerations to minimize nonspecific degradation of mRNA and is capable of depleting pre-rRNAs that often comprise a large portion of RNA, even after rRNA depletion. We demonstrated the highly efficient removal of rRNA up to a removal efficiency of 99.99% for various transcriptome studies, including RNA-Seq, Term-Seq, and ribosome profiling, with a cost of approximately $10 per sample. This method is expected to be a robust method for large-scale high-throughput bacterial transcriptomic studies.

Unraveling the functions of uncharacterized transcription factors in Escherichia coli using ChIP-exo.

Bacteria regulate gene expression to adapt to changing environments through transcriptional regulatory networks (TRNs). Although extensively studied, no TRN is fully characterized since the identity and activity of all the transcriptional regulators comprising a TRN are not known. Here, we experimentally evaluate 40 uncharacterized proteins in Escherichia coli K-12 MG1655, which were computationally predicted to be transcription factors (TFs). First, we used a multiplexed chromatin immunoprecipitation method combined with lambda exonuclease digestion (multiplexed ChIP-exo) assay to characterize binding sites for these candidate TFs; 34 of them were found to be DNA-binding proteins. We then compared the relative location between binding sites and RNA polymerase (RNAP). We found 48% (283/588) overlap between the TFs and RNAP. Finally, we used these data to infer potential functions for 10 of the 34 TFs with validated DNA binding sites and consensus binding motifs. Taken together, this study: (i) significantly expands the number of confirmed TFs to 276, close to the estimated total of about 280 TFs; (ii) provides putative functions for the newly discovered TFs and (iii) confirms the functions of four representative TFs through mutant phenotypes.

Revealing 29 sets of independently modulated genes in Staphylococcus aureus, their regulators, and role in key physiological response.

The ability of Staphylococcus aureus to infect many different tissue sites is enabled, in part, by its transcriptional regulatory network (TRN) that coordinates its gene expression to respond to different environments. We elucidated the organization and activity of this TRN by applying independent component analysis to a compendium of 108 RNA-sequencing expression profiles from two S. aureus clinical strains (TCH1516 and LAC). ICA decomposed the S. aureus transcriptome into 29 independently modulated sets of genes (i-modulons) that revealed: 1) High confidence associations between 21 i-modulons and known regulators; 2) an association between an i-modulon and σS, whose regulatory role was previously undefined; 3) the regulatory organization of 65 virulence factors in the form of three i-modulons associated with AgrR, SaeR, and Vim-3; 4) the roles of three key transcription factors (CodY, Fur, and CcpA) in coordinating the metabolic and regulatory networks; and 5) a low-dimensional representation, involving the function of few transcription factors of changes in gene expression between two laboratory media (RPMI, cation adjust Mueller Hinton broth) and two physiological media (blood and serum). This representation of the TRN covers 842 genes representing 76% of the variance in gene expression that provides a quantitative reconstruction of transcriptional modules in S. aureus, and a platform enabling its full elucidation.

Adaptive evolution reveals a tradeoff between growth rate and oxidative stress during naphthoquinone-based aerobic respiration.

Evolution fine-tunes biological pathways to achieve a robust cellular physiology. Two and a half billion years ago, rapidly rising levels of oxygen as a byproduct of blooming cyanobacterial photosynthesis resulted in a redox upshift in microbial energetics. The appearance of higher-redox-potential respiratory quinone, ubiquinone (UQ), is believed to be an adaptive response to this environmental transition. However, the majority of bacterial species are still dependent on the ancient respiratory quinone, naphthoquinone (NQ). Gammaproteobacteria can biosynthesize both of these respiratory quinones, where UQ has been associated with aerobic lifestyle and NQ with anaerobic lifestyle. We engineered an obligate NQ-dependent γ-proteobacterium, Escherichia coli ΔubiC, and performed adaptive laboratory evolution to understand the selection against the use of NQ in an oxic environment and also the adaptation required to support the NQ-driven aerobic electron transport chain. A comparative systems-level analysis of pre- and postevolved NQ-dependent strains revealed a clear shift from fermentative to oxidative metabolism enabled by higher periplasmic superoxide defense. This metabolic shift was driven by the concerted activity of 3 transcriptional regulators (PdhR, RpoS, and Fur). Analysis of these findings using a genome-scale model suggested that resource allocation to reactive oxygen species (ROS) mitigation results in lower growth rates. These results provide a direct elucidation of a resource allocation tradeoff between growth rate and ROS mitigation costs associated with NQ usage under oxygen-replete condition.

Cellular responses to reactive oxygen species are predicted from molecular mechanisms.

Catalysis using iron-sulfur clusters and transition metals can be traced back to the last universal common ancestor. The damage to metalloproteins caused by reactive oxygen species (ROS) can prevent cell growth and survival when unmanaged, thus eliciting an essential stress response that is universal and fundamental in biology. Here we develop a computable multiscale description of the ROS stress response in Escherichia coli, called OxidizeME. We use OxidizeME to explain four key responses to oxidative stress: 1) ROS-induced auxotrophy for branched-chain, aromatic, and sulfurous amino acids; 2) nutrient-dependent sensitivity of growth rate to ROS; 3) ROS-specific differential gene expression separate from global growth-associated differential expression; and 4) coordinated expression of iron-sulfur cluster (ISC) and sulfur assimilation (SUF) systems for iron-sulfur cluster biosynthesis. These results show that we can now develop fundamental and quantitative genotype-phenotype relationships for stress responses on a genome-wide basis.

OxyR Is a Convergent Target for Mutations Acquired during Adaptation to Oxidative Stress-Prone Metabolic States.

Oxidative stress is concomitant with aerobic metabolism. Thus, bacterial genomes encode elaborate mechanisms to achieve redox homeostasis. Here we report that the peroxide-sensing transcription factor, oxyR, is a common mutational target using bacterial species belonging to two genera, Escherichia coli and Vibrio natriegens, in separate growth conditions implemented during laboratory evolution. The mutations clustered in the redox active site, dimer interface, and flexible redox loop of the protein. These mutations favor the oxidized conformation of OxyR that results in constitutive expression of the genes it regulates. Independent component analysis of the transcriptome revealed that the constitutive activity of OxyR reduces DNA damage from reactive oxygen species, as inferred from the activity of the SOS response regulator LexA. This adaptation to peroxide stress came at a cost of lower growth, as revealed by calculations of proteome allocation using genome-scale models of metabolism and macromolecular expression. Further, identification of similar sequence changes in natural isolates of E. coli indicates that adaptation to oxidative stress through genetic changes in oxyR can be a common occurrence.

Distinct Subpopulations of Intravalvular Methicillin-Resistant Staphylococcus aureus with Variable Susceptibility to Daptomycin in Tricuspid Valve Endocarditis.

We present a case of endocarditis wherein organisms cultured from different valve leaflets yielded different daptomycin susceptibilities from each other and from organisms obtained from peripheral blood culture. Genomic analyses showed mutations in mprF, purR, and agrA Pharmacokinetic simulations showed consistent activity of daptomycin plus beta-lactam against all subpopulations. This represents an opportunity to understand S. aureus evolution and fitness in vivo on daptomycin therapy and the role of beta-lactams to prevent the selection of daptomycin-resistant subpopulations.

Adaptive laboratory evolution of Escherichia coli under acid stress.

The ability of Escherichia coli to tolerate acid stress is important for its survival and colonization in the human digestive tract. Here, we performed adaptive laboratory evolution of the laboratory strain E. coli K-12 MG1655 at pH 5.5 in glucose minimal medium. After 800 generations, six independent populations under evolution had reached 18.0 % higher growth rates than their starting strain at pH 5.5, while maintaining comparable growth rates to the starting strain at pH 7. We characterized the evolved strains and found that: (1) whole genome sequencing of isolated clones from each evolved population revealed mutations in rpoC appearing in five of six sequenced clones; and (2) gene expression profiles revealed different strategies to mitigate acid stress, which are related to amino acid metabolism and energy production and conversion. Thus, a combination of adaptive laboratory evolution, genome resequencing and expression profiling revealed, on a genome scale, the strategies that E. coli uses to mitigate acid stress.

Adaptation to the coupling of glycolysis to toxic methylglyoxal production in tpiA deletion strains of Escherichia coli requires synchronized and counterintuitive genetic changes.

Methylglyoxal is a highly toxic metabolite that can be produced in all living organisms. Methylglyoxal was artificially elevated by removal of the tpiA gene from a growth optimized Escherichia coli strain. The initial response to elevated methylglyoxal and its toxicity was characterized, and detoxification mechanisms were studied using adaptive laboratory evolution. We found that: 1) Multi-omics analysis revealed biological consequences of methylglyoxal toxicity, which included attack on macromolecules including DNA and RNA and perturbation of nucleotide levels; 2) Counter-intuitive cross-talk between carbon starvation and inorganic phosphate signalling was revealed in the tpiA deletion strain that required mutations in inorganic phosphate signalling mechanisms to alleviate; and 3) The split flux through lower glycolysis depleted glycolytic intermediates requiring a host of synchronized and coordinated mutations in non-intuitive network locations in order to re-adjust the metabolic flux map to achieve optimal growth. Such mutations included a systematic inactivation of the Phosphotransferase System (PTS) and alterations in cell wall biosynthesis enzyme activity. This study demonstrated that deletion of major metabolic genes followed by ALE was a productive approach to gain novel insight into the systems biology underlying optimal phenotypic states.

Adaptive laboratory evolution resolves energy depletion to maintain high aromatic metabolite phenotypes in Escherichia coli strains lacking the Phosphotransferase System.

Aromatic metabolites provide the backbone for numerous industrial and pharmaceutical compounds of high value. The Phosphotransferase System (PTS) is common to many bacteria, and is the primary mechanism for glucose uptake by Escherichia coli. The PTS was removed to conserve phosphoenolpyruvate (pep), which is a precursor for aromatic metabolites and consumed by the PTS, for aromatic metabolite production. Replicate adaptive laboratory evolution (ALE) of PTS and detailed omics data sets collected revealed that the PTS bridged the gap between respiration and fermentation, leading to distinct high fermentative and high respiratory rate phenotypes. It was also found that while all strains retained high levels of aromatic amino acid (AAA) biosynthetic precursors, only one replicate from the high glycolytic clade retained high levels of intracellular AAAs. The fast growth and high AAA precursor phenotypes could provide a starting host for cell factories targeting the overproduction aromatic metabolites.

Multiple Optimal Phenotypes Overcome Redox and Glycolytic Intermediate Metabolite Imbalances in Escherichia coli pgi Knockout Evolutions.

A mechanistic understanding of how new phenotypes develop to overcome the loss of a gene product provides valuable insight on both the metabolic and regulatory functions of the lost gene. The pgi gene, whose product catalyzes the second step in glycolysis, was deleted in a growth-optimized Escherichia coli K-12 MG1655 strain. The initial knockout (KO) strain exhibited an 80% drop in growth rate that was largely recovered in eight replicate, but phenotypically distinct, cultures after undergoing adaptive laboratory evolution (ALE). Multi-omic data sets showed that the loss of pgi substantially shifted pathway usage, leading to a redox and sugar phosphate stress response. These stress responses were overcome by unique combinations of innovative mutations selected for by ALE. Thus, the coordinated mechanisms from genome to metabolome that lead to multiple optimal phenotypes after the loss of a major gene product were revealed.IMPORTANCE A mechanistic understanding of how microbes are able to overcome the loss of a gene through regulatory and metabolic changes is not well understood. Eight independent adaptive laboratory evolution (ALE) experiments with pgi knockout strains resulted in eight phenotypically distinct endpoints that were able to overcome the gene loss. Utilizing multi-omics analysis, the coordinated mechanisms from genome to metabolome that lead to multiple optimal phenotypes after the loss of a major gene product were revealed.

Use of adaptive laboratory evolution to discover key mutations enabling rapid growth of Escherichia coli K-12 MG1655 on glucose minimal medium.

Adaptive laboratory evolution (ALE) has emerged as an effective tool for scientific discovery and addressing biotechnological needs. Much of ALE's utility is derived from reproducibly obtained fitness increases. Identifying causal genetic changes and their combinatorial effects is challenging and time-consuming. Understanding how these genetic changes enable increased fitness can be difficult. A series of approaches that address these challenges was developed and demonstrated using Escherichia coli K-12 MG1655 on glucose minimal media at 37°C. By keeping E. coli in constant substrate excess and exponential growth, fitness increases up to 1.6-fold were obtained compared to the wild type. These increases are comparable to previously reported maximum growth rates in similar conditions but were obtained over a shorter time frame. Across the eight replicate ALE experiments performed, causal mutations were identified using three approaches: identifying mutations in the same gene/region across replicate experiments, sequencing strains before and after computationally determined fitness jumps, and allelic replacement coupled with targeted ALE of reconstructed strains. Three genetic regions were most often mutated: the global transcription gene rpoB, an 82-bp deletion between the metabolic pyrE gene and rph, and an IS element between the DNA structural gene hns and tdk. Model-derived classification of gene expression revealed a number of processes important for increased growth that were missed using a gene classification system alone. The methods described here represent a powerful combination of technologies to increase the speed and efficiency of ALE studies. The identified mutations can be examined as genetic parts for increasing growth rate in a desired strain and for understanding rapid growth phenotypes.

The hallmarks of a tradeoff in transcriptomes that balances stress and growth functions.

Fast growth phenotypes are achieved through optimal transcriptomic allocation, in which cells must balance tradeoffs in resource allocation between diverse functions. One such balance between stress readiness and unbridled growth in E. coli has been termed the fear versus greed (f/g) tradeoff. Two specific RNA polymerase (RNAP) mutations observed in adaptation to fast growth have been previously shown to affect the f/g tradeoff, suggesting that genetic adaptations may be primed to control f/g resource allocation. Here, we conduct a greatly expanded study of the genetic control of the f/g tradeoff across diverse conditions. We introduced 12 RNA polymerase (RNAP) mutations commonly acquired during adaptive laboratory evolution (ALE) and obtained expression profiles of each. We found that these single RNAP mutation strains resulted in large shifts in the f/g tradeoff primarily in the RpoS regulon and ribosomal genes, likely through modifying RNAP-DNA interactions. Two of these mutations additionally caused condition-specific transcriptional adaptations. While this tradeoff was previously characterized by the RpoS regulon and ribosomal expression, we find that the GAD regulon plays an important role in stress readiness and ppGpp in translation activity, expanding the scope of the tradeoff. A phylogenetic analysis found the greed-related genes of the tradeoff present in numerous bacterial species. The results suggest that the f/g tradeoff represents a general principle of transcriptome allocation in bacteria where small genetic changes can result in large phenotypic adaptations to growth conditions.IMPORTANCETo increase growth, E. coli must raise ribosomal content at the expense of non-growth functions. Previous studies have linked RNAP mutations to this transcriptional shift and increased growth but were focused on only two mutations found in the protein's central region. RNAP mutations, however, commonly occur over a large structural range. To explore RNAP mutations' impact, we have introduced 12 RNAP mutations found in laboratory evolution experiments and obtained expression profiles of each. The mutations nearly universally increased growth rates by adjusting said tradeoff away from non-growth functions. In addition to this shift, a few caused condition-specific adaptations. We explored the prevalence of this tradeoff across phylogeny and found it to be a widespread and conserved trend among bacteria.

Advancing the scale of synthetic biology via cross-species transfer of cellular functions enabled by iModulon engraftment.

Machine learning applied to large compendia of transcriptomic data has enabled the decomposition of bacterial transcriptomes to identify independently modulated sets of genes, such iModulons represent specific cellular functions. The identification of iModulons enables accurate identification of genes necessary and sufficient for cross-species transfer of cellular functions. We demonstrate cross-species transfer of: 1) the biotransformation of vanillate to protocatechuate, 2) a malonate catabolic pathway, 3) a catabolic pathway for 2,3-butanediol, and 4) an antimicrobial resistance to ampicillin found in multiple Pseudomonas species to Escherichia coli. iModulon-based engineering is a transformative strategy as it includes all genes comprising the transferred cellular function, including genes without functional annotation. Adaptive laboratory evolution was deployed to optimize the cellular function transferred, revealing mutations in the host. Combining big data analytics and laboratory evolution thus enhances the level of understanding of systems biology, and synthetic biology for strain design and development.

Reconstructing the transcriptional regulatory network of probiotic L. reuteri is enabled by transcriptomics and machine learning.

Limosilactobacillus reuteri, a probiotic microbe instrumental to human health and sustainable food production, adapts to diverse environmental shifts via dynamic gene expression. We applied the independent component analysis (ICA) to 117 RNA-seq data sets to decode its transcriptional regulatory network (TRN), identifying 35 distinct signals that modulate specific gene sets. Our findings indicate that the ICA provides a qualitative advancement and captures nuanced relationships within gene clusters that other methods may miss. This study uncovers the fundamental properties of L. reuteri's TRN and deepens our understanding of its arginine metabolism and the co-regulation of riboflavin metabolism and fatty acid conversion. It also sheds light on conditions that regulate genes within a specific biosynthetic gene cluster and allows for the speculation of the potential role of isoprenoid biosynthesis in L. reuteri's adaptive response to environmental changes. By integrating transcriptomics and machine learning, we provide a system-level understanding of L. reuteri's response mechanism to environmental fluctuations, thus setting the stage for modeling the probiotic transcriptome for applications in microbial food production. IMPORTANCE: We have studied Limosilactobacillus reuteri, a beneficial probiotic microbe that plays a significant role in our health and production of sustainable foods, a type of foods that are nutritionally dense and healthier and have low-carbon emissions compared to traditional foods. Similar to how humans adapt their lifestyles to different environments, this microbe adjusts its behavior by modulating the expression of genes. We applied machine learning to analyze large-scale data sets on how these genes behave across diverse conditions. From this, we identified 35 unique patterns demonstrating how L. reuteri adjusts its genes based on 50 unique environmental conditions (such as various sugars, salts, microbial cocultures, human milk, and fruit juice). This research helps us understand better how L. reuteri functions, especially in processes like breaking down certain nutrients and adapting to stressful changes. More importantly, with our findings, we become closer to using this knowledge to improve how we produce more sustainable and healthier foods with the help of microbes.

Independent component analysis reveals 49 independently modulated gene sets within the global transcriptional regulatory architecture of multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii causes severe infections in humans, resists multiple antibiotics, and survives in stressful environmental conditions due to modulations of its complex transcriptional regulatory network (TRN). Unfortunately, our global understanding of the TRN in this emerging opportunistic pathogen is limited. Here, we apply independent component analysis, an unsupervised machine learning method, to a compendium of 139 RNA-seq data sets of three multidrug-resistant A. baumannii international clonal complex I strains (AB5075, AYE, and AB0057). This analysis allows us to define 49 independently modulated gene sets, which we call iModulons. Analysis of the identified A. baumannii iModulons reveals validating parallels to previously defined biological operons/regulons and provides a framework for defining unknown regulons. By utilizing the iModulons, we uncover potential mechanisms for a RpoS-independent general stress response, define global stress-virulence trade-offs, and identify conditions that may induce plasmid-borne multidrug resistance. The iModulons provide a model of the TRN that emphasizes the importance of transcriptional regulation of virulence phenotypes in A. baumannii. Furthermore, they suggest the possibility of future interventions to guide gene expression toward diminished pathogenic potential.IMPORTANCEThe rise in hospital outbreaks of multidrug-resistant Acinetobacter baumannii infections underscores the urgent need for alternatives to traditional broad-spectrum antibiotic therapies. The success of A. baumannii as a significant nosocomial pathogen is largely attributed to its ability to resist antibiotics and survive environmental stressors. However, there is limited literature available on the global, complex regulatory circuitry that shapes these phenotypes. Computational tools that can assist in the elucidation of A. baumannii's transcriptional regulatory network architecture can provide much-needed context for a comprehensive understanding of pathogenesis and virulence, as well as for the development of targeted therapies that modulate these pathways.

Comprehensive whole genome sequencing with hybrid assembly of multi-drug resistant Candida albicans isolate causing cerebral abscess.

Comprehensive whole genome sequencing (WGS) with hybrid assembly of a multi-drug resistant (MDR) Candida albicans (CA) isolate causing cerebral abscess was performed using Illumina paired end and Oxford Nanopore long read technologies. The innovative technologies utilized here enabled us to resolve fragmented assemblies, and implement comprehensive and detailed genomic analyses involved in antifungal resistance of Candida spp. Functionally important genes (MDR1, CDR2 and SQN2) involved in antifungal resistance were identified and a phylogenetic analysis of the clinical isolate was performed. Additionally, our clinical isolate was found to share 4 single nucleotide polymorphisms with two other sequenced strains of MDR C. auris (381 and 386) including translation elongation factor EF1α and EF3, ATPase activity associated proteins, and the lysine tRNA ligase.