Biology subjects

Myers, K. S.

Publications and source records attributed to Myers, K. S..

13 recordsLinked to original sources

bioRxiv2026

OxyR regulates the oxidative stress response in Zymomonas mobilis during oxic growth and anoxic biofuel fermentation

The bacterium Zymomonas mobilis is widely studied for its potential as an industrial biofuel producer. Anoxic fermentation by Z. mobilis in lignocellulosic hydrolysate can generate bioethanol from renewable plant biomass. In this study, we deleted a gene from the Z. mobilis genome encoding a homolog of OxyR, a transcription factor that activates an oxidative stress response in bacteria to reduce reactive oxygen species (ROS). Deletion of this transcription factor inhibited growth of Z. mobilis in oxic, but not anoxic, conditions in laboratory media. A ROS probe revealed that the oxyR response is required to reduce intracellular ROS during oxic growth. Importantly for biofuel production, the absence of oxyR inhibited growth and delayed ethanol production during anoxic hydrolysate fermentation. To determine the source of oxidative stress in hydrolysate, we grew{Delta} oxyR in a synthetic hydrolysate containing known inhibitors found in hydrolysate. There was no growth defect in{Delta} oxyR in the synthetic hydrolysate, indicating that known inhibitory compounds are not the source of anoxic oxidative stress. We determined that Ammonia-Fiber Expansion (AFEX) switchgrass hydrolysate contains significant peroxide concentrations. Addition of catalase to hydrolysate improves growth of both{Delta} oxyR and wild-type Z. mobilis in hydrolysate. This study uncovers an important source of stress to Z. mobilis during biofuel fermentation. ImportanceFermentation of non-food biomass is a promising avenue for sustainable production of fuels and chemicals, but several challenges currently limit applicability of this technology. One major hurdle is that when biomass is deconstructed into a fermentable form, many byproducts are generated that inhibit microbial fermentation. Here, we investigated how a fermentative bacterium, Zymomonas mobilis, experiences oxidative stress during anoxic biomass fermentation, and identified genes important in this response. These findings provide a better understanding of the stresses faced by Z. mobilis during biofuel production. Fully understanding the effects of hydrolysate on biofuel-producing microbes is crucial for optimizing production and making carbon-neutral fuel a reality.

microbiology↗

bioRxiv2025

Metagenomes and Metagenome-Assembled Genomes from Microbial Communities in a Biological Nutrient Removal Plant Operated at Hamptons Road Sanitation District (HRSD) with High and Low Dissolved Oxygen Conditions

Aeration in biological nutrient removal (BNR) systems constitutes one of the largest energy demands in water resource recovery facilities (WRRFs). Previous studies have shown that lowering dissolved oxygen (DO) concentrations can sustain effective nitrification and phosphorus removal while substantially reducing energy consumption. However, the microbial mechanisms enabling these low-DO processes remain poorly understood. In this study, we investigated microbial communities associated with reduced DO in a pilot-scale BNR system operated by the Hampton Roads Sanitation District (HRSD). DO was reduced over an 18-month period from 2.5 mg/L to 0.2 mg/L. Metagenomic DNA was obtained from samples from each DO condition then sequenced using PacBio HiFi technology. A total of 316 metagenome-assembled genomes were recovered and after dereplication, 207 were found to be unique. These data augment the metagenomic information related to wastewater treatment under low-DO conditions and provide valuable resources for understanding microbial adaptation to oxygen-limited BNR operation.

genomics↗

bioRxiv2025

Metagenomes and Metagenome-Assembled Genomes from Microbial Communities in a Biological Nutrient Removal Plant Operated at Los Angeles County Sanitation District (LACSD) with High and Low Dissolved Oxygen Conditions

Aeration represents one of the largest energy costs in water resource recovery facilities (WRRFs). Previous studies have shown that effective nitrification and phosphorus removal can be maintained in biological nutrient removal (BNR) systems operated under low dissolved oxygen (DO) conditions, significantly reducing energy use. To improve understanding of microbial community adaptation to reduced DO, we analyzed metagenomes and metagenome-assembled genomes (MAGs) from the Pomona WRRF (Los Angeles County Sanitation Districts) before and after a gradual decrease in operating DO from approximately 3.5 mg/L to 0.7 mg/L over 18 months. Metagenomic DNA was isolated from high and low DO samples, then sequenced using PacBio HiFi technology. A total of 492 MAGs were recovered of which 304 were unique after dereplication. These genomes expand current knowledge of microbial community dynamics in low-DO BNR systems and provide valuable genomic resources for understanding microbial adaptation to energy-efficient wastewater treatment processes.

genetics↗

bioRxiv2025

Orthogonal chemical genomics approaches reveal genomic targets for increasing anaerobic chemical tolerance in Zymomonas mobilis

Genetically-engineered microbes have the potential to increase efficiency in the bioeconomy by overcoming growth-limiting production stress. Screens of gene perturbation libraries against production stressors can identify high-value engineering targets, but follow-up experiments needed to guard against false positives are slow and resource-intensive. In principle, the use of orthogonal gene perturbation approaches could increase recovery of true positives over false positives because the strengths of one technique compensate for the weaknesses of the other, but, in practice, two parallel screens are rarely performed at the genome-scale. Here, we screen genome-scale CRISPRi (CRISPR interference) knockdown and TnSeq (transposon insertion sequencing) libraries of the bioenergy-relevant Alphaproteobacterium, Zymomonas mobilis, against growth inhibitors commonly found in deconstructed plant material. Integrating data from the two gene perturbation techniques, we established an approach for defining engineering targets with high specificity. This allowed us to identify all known genes in the cytochrome bc1 and cytochrome c synthesis pathway as potential targets for engineering resistance to phenolic acids under anaerobic conditions, a subset of which we validated using precise gene deletions. Strikingly, this finding is specific to the cytochrome bc1 and cytochrome c pathway and does not extend to other branches of the electron transport chain. We further show that exposure of Z. mobilis to ferulic acid causes substantial remodeling of the cell envelope proteome, as well as the downregulation of TonB-dependent transporters. Our work provides a generalizable strategy for identifying high-value engineering targets from gene perturbation screens that is broadly applicable. IMPORTANCEEngineering microorganisms to tolerate harsh production conditions stands to increase bioproduct yields of engineered microbes. In this study, we systematically identified Z. mobilis genes that confer resistance or susceptibility to chemical stressors found in deconstructed plant material. We used complementary genetic techniques to cross-validate these genes at scale, providing a widely applicable method for precisely identifying genetic alterations that increase chemical resilience. We discovered genetic modifications that improve anaerobic growth of Z. mobilis in the presence of inhibitory chemicals found in renewable plant-based feedstocks. These results have implications in engineering robust production strains to support efficient and resilient bioproduction. Our methodologies can be broadly applied to understand microbial responses to chemicals across systems, paving the way for developments in biomanufacturing, therapeutics, and agriculture.

genomics↗

bioRxiv2024

Altering translation allows E. coli to overcome chemically stabilized G-quadruplexes

G-quadruplex (G4) structures can form in guanine-rich DNA or RNA and have been found to modulate cellular processes including replication, transcription, and translation. Many studies on the cellular roles of G4s have focused on eukaryotic systems, with far fewer probing bacterial G4s. Using a chemical-genetic approach, we identified genes in Escherichia coli that are important for growth in G4-stabilizing conditions. Reducing levels of elongation factor Tu or slowing translation elongation with chloramphenicol suppress the effects of G4 stabilization. In contrast, reducing expression of certain translation termination or ribosome recycling proteins is detrimental to growth in G4-stabilizing conditions. Proteomic and transcriptomic analyses demonstrate that ribosome assembly factors and other proteins involved in translation are less abundant in G4-stabilizing conditions. Our integrated systems approach allowed us to propose a model for how RNA G4s can present barriers to E. coli growth and that reducing the rate of translation can compensate for G4-related stress.

biochemistry↗

bioRxiv2024

Physiological Roles of an Acinetobacter-specific σ Factor

The Gram-negative pathogen Acinetobacter baumannii is considered an "urgent threat" to human health due to its propensity to become antibiotic resistant. Understanding the distinct regulatory paradigms used by A. baumannii to mitigate cellular stresses may uncover new therapeutic targets. Many {gamma}-proteobacteria use the extracytoplasmic function (ECF) {sigma} factor, RpoE, to invoke envelope homeostasis networks in response to stress. Acinetobacter species contain the poorly characterized ECF "SigAb;" however, it is unclear if SigAb has the same physiological role as RpoE. Here, we show that SigAb is a metal stress-responsive ECF that appears unique to Acinetobacter species and distinct from RpoE. We combine promoter mutagenesis, motif scanning, and ChIP-seq to define the direct SigAb regulon, which consists of sigAb itself, the stringent response mediator, relA, and the uncharacterized small RNA, "sabS." However, RNA-seq of strains overexpressing SigAb revealed a large, indirect regulon containing hundreds of genes. Metal resistance genes are key elements of the indirect regulon, as CRISPRi knockdown of sigAb or sabS resulted in increased copper sensitivity and excess copper induced SigAb-dependent transcription. Further, we found that two uncharacterized genes in the sigAb operon, "aabA" and "aabB", have anti-SigAb activity. Finally, employing a targeted Tn-seq approach that uses CRISPR-associated transposons, we show that sigAb, aabA, and aabB are important for fitness even during optimal growth conditions. Our work reveals new physiological roles for SigAb and SabS, provides a novel approach for assessing gene fitness, and highlights the distinct regulatory architecture of A. baumannii. ImportanceAcinetobacter baumannii is a hospital-acquired pathogen, and many strains are resistant to multiple antibiotics. Understanding how A. baumannii senses and responds to stress may uncover novel routes to treat infections. Here, we examine how the Acinetobacter-specific transcription factor, SigAb, mitigates stress. We find that SigAb directly regulates only a small number of genes, but indirectly controls hundreds of genes that have substantial impacts on cell physiology. We show that SigAb is required for maximal growth, even during optimal conditions, and is acutely required during growth in the presence of elevated copper. Given that copper toxicity plays roles in pathogenesis and on copper-containing surfaces in hospitals, we speculate that SigAb function may be important in clinically-relevant contexts.

microbiology↗

bioRxiv2024

A Targeted Genome-scale Overexpression Platform for Proteobacteria

Targeted, genome-scale gene perturbation screens using Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) and activation (CRISPRa) have revolutionized eukaryotic genetics, advancing medical, industrial, and basic research. Although CRISPRi knockdowns have been broadly applied in bacteria, options for genome-scale gene overexpression face key limitations. Here, we develop a facile approach for genome-scale overexpression in bacteria we call, "CRISPRtOE" (CRISPR transposition and OverExpression). We first create a platform for comprehensive gene targeting using CRISPR-associated transposons (CAST) and show that transposition occurs at a higher frequency in non-transcribed DNA. We then demonstrate that CRISPRtOE can upregulate gene expression in Proteobacteria with medical and industrial relevance by integrating synthetic promoters of varying strength upstream of target genes. Finally, we employ CRISPRtOE screening at the genome-scale in the model bacterium Escherichia coli and the non-model biofuel producer Zymomonas mobilis, recovering known and novel antibiotic and engineering targets. We envision that CRISPRtOE will be a valuable overexpression tool for antibiotic mode of action, industrial strain optimization, and gene function discovery in bacteria. ImportanceSystematic alteration of bacterial gene expression enables identification of genes relevant to diverse fields of study and practical applications. Although many targeted, genome-scale genetic tools exist for reducing or eliminating gene expression, there are few facile approaches for systematic gene overexpression in bacteria. Here, we develop a targeted overexpression approach for Proteobacteria of medical and industrial importance that precisely inserts strong promoters upstream of genes using CRISPR-associated transposons. We demonstrate that this approach can be used to systematically overexpress genes in both model (Escherichia coli K-12) and non-model (Zymomonas mobilis) Proteobacteria for the purpose of understanding antibiotic resistance mechanisms and improving strain resilience in biofuel production conditions, respectively.

systems biology↗

bioRxiv2023

Refinement of the "Candidatus Accumulibacter" Genus Based on a Metagenomic Analysis of Biological Nutrient Removal (BNR) Pilot-Scale Plants Operated with Reduced Aeration

Members of the "Candidatus Accumulibacter" genus are widely studied as key polyphosphate-accumulating organisms (PAOs) in biological nutrient removal (BNR) facilities performing enhanced biological phosphorus removal (EBPR). This diverse lineage includes 18 "Ca. Accumulibacter" species, which have been proposed based on the phylogenetic divergence of the polyphosphate kinase 1 (ppk1) gene and genome-scale comparisons of metagenome-assembled genomes (MAGs). Phylogenetic classification based on the 16S rRNA genetic marker has been difficult to attain because most "Ca. Accumulibacter" MAGs are incomplete and often do not include the rRNA operon. Here, we investigate the "Ca. Accumulibacter" diversity in pilot-scale treatment trains performing BNR under low dissolved oxygen (DO) conditions using genome-resolved metagenomics. Using long-read sequencing, we recovered medium and high-quality MAGs for 5 of the 18 "Ca. Accumulibacter" species, all with rRNA operons assembled, which allowed a reassessment of the 16S rRNA-based phylogeny of this genus and an analysis of phylogeny based on the 23S rRNA gene. In addition, we recovered a cluster of MAGs that based on 16S rRNA, 23S rRNA, ppk1, and genome-scale phylogenetic analyses do not belong to any of the currently recognized "Ca. Accumulibacter" species for which we propose the new species designation "Ca. Accumulibacter jenkinsii" sp. nov. Relative abundance evaluations of the genus across all pilot plant operations revealed that regardless of the operational mode, "Ca. A. necessarius" and "Ca. A. propinquus" accounted for more than 40% of the "Ca. Accumulibacter" community, whereas the newly proposed "Ca. A. jenkinsii" accounted for about 5% of the "Ca. Accumulibacter" community. IMPORTANCEOne of the main drivers of energy use and operational costs in activated sludge processes is the amount of oxygen provided to enable biological phosphorus and nitrogen removal. Wastewater treatment facilities are increasingly considering reduced aeration to decrease energy consumption, and whereas successful BNR has been demonstrated in systems with minimal aeration, an adequate understanding of the microbial communities that facilitate nutrient removal under these conditions is still lacking. In this study, we used genome-resolved metagenomics to evaluate the diversity of the "Candidatus Accumulibacter" genus in pilot-scale plants operating with minimal aeration. We identified the "Ca. Accumulibacter" species enriched under these conditions, including one novel species for which we propose "Ca. Accumulibacter jenkinsii" sp. nov. as its designation. Furthermore, the MAGs obtained for 5 additional "Ca. Accumulibacter" species further refine the phylogeny of the "Ca. Accumulibacter" genus and provide new insight into its diversity within unconventional biological nutrient removal systems.

microbiology↗

bioRxiv2023

A hybrid nitrogenase with regulatory elasticity in Azotobacter vinelandii

Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs restrict the available sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred, ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to nitrogenase sequence-level perturbations. Rather, physiological compatibility with the ancestral nitrogenase variant is restored by reducing trace metal and electron resource allocation to nitrogenase. Our results spotlight cellular processes adjacent to nitrogen fixation as productive engineering targets to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCEAzotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and remodeled nitrogenase variants can be modulated at the regulatory level. Engineered cells respond by adjusting expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.

microbiology↗

bioRxiv2023

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

genetics↗

bioRxiv2022

Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli

Collisions between DNA replication complexes (replisomes) and impediments such as damaged DNA or proteins tightly bound to the chromosome lead to premature dissociation of replisomes at least once per cell cycle in Escherichia coli. Left unrepaired, these events produce incompletely replicated chromosomes that cannot be properly partitioned into daughter cells. DNA replication restart, the process that reloads replisomes at prematurely terminated sites, is therefore essential in E. coli and other bacteria. Three replication restart pathways have been identified in E. coli: PriA/PriB, PriA/PriC, and PriC/Rep. A limited number of genetic interactions between replication restart and other genome maintenance pathways have been defined, but a systematic study placing replication restart reactions in a broader cellular context has not been performed. We have utilized transposon insertion sequencing to identify new genetic interactions between DNA replication restart pathways and other cellular systems. Known genetic interactors with the priB replication restart gene (uniquely involved in the PriA/PriB pathway) were confirmed and several novel priB interactions were discovered. Far fewer connections were found with the PriA/PriC or PriC/Rep pathways, suggesting a primacy role for the PriA/PriB pathway in E. coli. Targeted genetic and imaging-based experiments with priB and its genetic partners revealed significant double-strand DNA break (DSB) accumulation in strains with mutations in dam, rep, rdgC, lexA, or polA. Modulating the activity of the RecA recombinase partially suppressed the detrimental effects of rdgC or lexA mutations in {Delta}priB cells. Taken together, our results highlight roles for several genes in DSB homeostasis and define a genetic network that facilitates DNA repair/processing upstream of PriA/PriB-mediated DNA replication restart in E. coli. Author SummaryAll organisms rely on DNA replication to grow, develop, and reproduce. In bacteria, the cellular machinery that carries out DNA replication is estimated to fail and prematurely dissociate from the genome at least once per cell cycle. As a result, bacteria have evolved "DNA replication restart" mechanisms that resuscitate failed replication reactions. To probe the function and context of DNA replication restart in the bacterium Escherichia coli, we employed a genetic screen to identify genes that were conditionally important in mutant E. coli strains compromised in their ability to perform DNA replication restart. Identification of genes with previously known relationships with DNA replication restart confirmed the robustness of our screen, while additional findings implicated novel genetic relationships. Targeted experiments validated the importance of these genes and provided an explanation for their significance in preventing double-strand DNA breaks in cells, a severe form of DNA damage. Our results help to define specific roles for the genes identified by our screen and elucidate the contextual environment of DNA repair upstream of DNA replication restart in E. coli.

genetics↗

bioRxiv2020

Rewiring the specificity of extra-cytoplasmic function sigma factors

SUMMARYBacterial genomes are being sequenced at an exponentially increasing rate, but our inability to decipher their transcriptional wiring limits our ability to derive new biology from these sequences. De novo determination of regulatory interactions requires accurate prediction of regulators’ DNA binding and precise determination of biologically significant binding sites. Here, we address these challenges by solving the DNA-specificity code of extra-cytoplasmic function sigma factors (ECF σs), a major family of bacterial regulators, and determining their regulons. We generated an aligned collection of ECF σs and their promoters by leveraging the auto-regulatory nature of ECF σs as a means of promoter discovery and analyzed it to identify and characterize the conserved amino acid – nucleotide interactions that determine promoter specificity. This enabled de novo prediction of ECF σ specificity, which we combined with a statistically rigorous phylogenetic foot-printing pipeline based on precomputed orthologs to predict the direct targets of ∼67% of ECF σs. This global survey indicated that ECF σs play varied roles: some are global regulators controlling many genes throughout the genome that are important under many conditions, while others are local regulators, controlling few closely linked genes in response to specific stimuli. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

bioRxiv2019

PKA and HOG signaling contribute separable roles to anaerobic xylose fermentation in yeast engineered for biofuel production

Lignocellulosic biomass offers a sustainable source for biofuel production that does not compete with food-based cropping systems. Importantly, two critical bottlenecks prevent economic adoption: many industrially relevant microorganisms cannot ferment pentose sugars prevalent in lignocellulosic medium, leaving a significant amount of carbon unutilized. Furthermore, chemical biomass pretreatment required to release fermentable sugars generates a variety of toxins, which inhibit microbial growth and metabolism, specifically limiting pentose utilization in engineered strains. Here we dissected genetic determinants of anaerobic xylose fermentation and stress tolerance in chemically pretreated corn stover biomass, called hydrolysate. We previously revealed that loss-of-function mutations in the stress-responsive MAP kinase HOG1 and negative regulator of the RAS/Protein Kinase A (PKA) pathway, IRA2, enhances anaerobic xylose fermentation. However, these mutations likely reduce cells ability to tolerate the toxins present in lignocellulosic hydrolysate, making the strain especially vulnerable to it. We tested the contributions of Hog1 and PKA signaling via IRA2 or PKA negative regulatory subunit BCY1 to metabolism, growth, and stress tolerance in corn stover hydrolysate and laboratory medium with mixed sugars. We found mutations causing upregulated PKA activity increase growth rate and glucose consumption in various media but do not have a specific impact on xylose fermentation. In contrast, mutation of HOG1 specifically increased xylose usage. We hypothesized improving stress tolerance would enhance the rate of xylose consumption in hydrolysate. Surprisingly, increasing stress tolerance did not augment xylose fermentation in lignocellulosic medium in this strain background, suggesting other mechanisms besides cellular stress limit this strains ability for anaerobic xylose fermentation in hydrolysate.

genetics↗