Biology subjects

Peters, J. M.

Publications and source records attributed to Peters, J. M..

At least 19 recordsLinked to original sources

bioRxiv2026

A Brain Circuit for Status Epilepticus

Status epilepticus (SE) is a life-threatening persistent epileptic seizure that can arise from various brain structures, leaving its brain circuit unknown. In this study, we utilize brain imaging changes during SE to reveal the brain architecture and circuit of persistent seizures. Multimodal lesion mapping identified that brain imaging changes during SE localize to a specific predisposed brain architecture characterized by increased metabolic rate, high synaptic and mitochondrial density, glutamate (mGLUR5 and NMDA) and GABA receptors. Gene expression patterns within lesion locations revealed a transcriptomic profile enriched for epilepsy pathologies (including SE), neuronal and synaptic processes, and glutamate signaling. Lesion network mapping demonstrated these same lesions map to a common brain circuit, unifying a traditionally heterogeneous patient population. Findings were validated in an independent cohort and the identified SE circuit distinguished brain imaging changes during SE from other lesion etiologies with excellent accuracy (91%), significantly outperforming all other tested maps. With this SE circuit, we identify therapeutic targets for precision therapy that could modulate this circuit. This study demonstrates brain imaging changes in SE converge on a unified brain circuit that could help diagnostic workup of patients in critical care and guide clinical trials of precision therapy for persistent seizures.

neuroscience↗

bioRxiv2026

Intracellular barriers and receptor masking limit success of a Pseudomonas aeruginosa clinical phage family

Bacteriophage therapy is needed to treat antibiotic resistant infections; however, when a clinical isolate resists a given phage, it is often unclear why. It is therefore currently unknown how to rationally fortify phage therapies to circumvent a priori resistance. Using a family of broad host range therapeutic Pseudomonas aeruginosa phages (Pbunaviruses), we show that cell surface receptor masking and intracellular defenses are both common barriers in distinct clinical isolates. In some cases these barriers can be bypassed by intrafamily phage engineering. Using unbiased genome-wide CRISPRi screens, we reveal that the broadly conserved L-Rhamnose in the core polysaccharide is the receptor for Pbunavirus family. This molecule is often masked by diverse O-antigen structures. In other isolates with the L-Rha receptor accessible, internal defense mechanisms commonly prevent Pbunavirus DNA replication. A single anti-defense locus often encoding 8-11 different genes within the Pbunavirus family is required for optimal host range, providing anti-defense genes that enable replication of both Pbunavirus phages and phages of other families. Our work demonstrates the importance of both internal and surface defense mechanisms in clinical isolates causally antagonizing a commonly used phage therapeutic and presents phage engineering strategies to circumvent a priori resistance.

microbiology↗

bioRxiv2025

PDS5 proteins control genome architecture by limiting the lifetime of cohesin-NIPBL complexes

Cohesin-NIPBL complexes extrude genomic DNA into loops that are constrained by CTCF boundaries. This process has important regulatory functions and weakens the separation between euchromatic and heterochromatic compartments. Cohesin can also bind PDS5A or PDS5B, which do not support loop extrusion but are required for the formation of CTCF boundaries. How PDS5 proteins perform this function is unknown. Here we show by in vitro single-molecule imaging that PDS5 proteins stop loop extrusion by facilitating the dissociation of NIPBL from cohesin. Hi-C experiments suggest that this function is required for the establishment of CTCF boundaries in cells. In silico modelling indicates that PDS5 proteins enable the separation between compartments by limiting cohesins velocity and chromatin-residence time. The degree of this compartmentalization depends on the frequency with which chromatin is extruded relative to the time it takes for compartments to form. These results identify PDS5 proteins as key regulators of genome organization. HighlightsO_LIPDS5 proteins stop loop extrusion by facilitating dissociation of NIPBL from cohesin. C_LIO_LIPDS5 proteins strengthen CTCF boundaries by limiting the lifetime of cohesin-NIPBL. C_LIO_LIPDS5 proteins regulate compartmentalization. C_LIO_LICompartmentalization is governed by polymer relaxation and loop extrusion dynamics. C_LI

molecular biology↗

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↗

bioRxiv2025

The immunometabolic topography of tuberculosis granulomas governs cellular organization and bacterial control

Despite being heavily infiltrated by immune cells, tuberculosis (TB) granulomas often subvert the host response to Mycobacterium tuberculosis (Mtb) infection and support bacterial persistence. We previously discovered that human TB granulomas are enriched for immunosuppressive factors typically associated with tumor-immune evasion, raising the intriguing possibility that they promote tolerance to infection. In this study, our goal was to identify the prime drivers for establishing this tolerogenic niche and to determine if the magnitude of this response correlates with bacterial persistence. To do this, we conducted a multimodal spatial analysis of 52 granulomas from 16 non-human primates (NHP) who were infected with low dose Mtb for 9-12 weeks. Notably, each granulomas bacterial burden was individually quantified allowing us to directly ask how granuloma spatial structure and function relate to infection control. We found that a universal feature of TB granulomas was partitioning of the myeloid core into two distinct metabolic environments, one of which is hypoxic. This hypoxic environment associated with pathologic immune cell states, dysfunctional cellular organization of the granuloma, and a near-complete blockade of lymphocyte infiltration that would be required for a successful host response. The extent of these hypoxia-associated features correlated with worsened bacterial burden. We conclude that hypoxia governs immune cell state and organization within granulomas and is a potent driver of subverted immunity during TB.

immunology↗

bioRxiv2024

Chemical genomics informs antibiotic and essential gene function in Acinetobacter baumannii

The Gram-negative pathogen, Acinetobacter baumannii, poses a serious threat to human health due to its role in nosocomial infections that are resistant to treatment with current antibiotics. Despite this, our understanding of fundamental A. baumannii biology remains limited, as many essential genes have not been experimentally characterized. These essential genes are critical for bacterial survival and, thus, represent promising targets for drug discovery. Here, we systematically probe the function of essential genes by screening a CRISPR interference knockdown library against a diverse panel of chemical inhibitors, including antibiotics. We find that most essential genes show chemical-gene interactions, allowing insights into both inhibitor and gene function. For instance, knockdown of lipooligosaccharide (LOS) transport genes increased sensitivity to a broad range of chemicals. Cells with defective LOS transport showed cell envelope hyper-permeability that was dependent on continued LOS synthesis. Using phenotypes across our chemical-gene interaction dataset, we constructed an essential gene network linking poorly understood genes to well-characterized genes in cell division and other processes. Finally, our phenotype-structure analysis identified structurally related antibiotics with distinct cellular impacts and suggested potential targets for underexplored inhibitors. This study advances our understanding of essential gene and inhibitor function, providing a valuable resource for mechanistic studies, therapeutic strategies, and future key targets for antibiotic development.

microbiology↗

bioRxiv2024

Pseudomonas aeruginosa essential gene perturbations that confer vulnerability to the mammalian host environment

Multidrug-resistant Pseudomonas aeruginosa causes highly morbid infections that are challenging to treat. While antibiotics reduce bacterial populations during infection, the host environment also plays a key role in inhibiting and eliminating pathogens. Identifying genetic targets that create vulnerabilities to the host environment may uncover strategies to synergize with nutrient limitation or inherent immune processes to clear bacterial infections. Here, we screened a partial knockdown library targeting P. aeruginosa essential and conditionally essential genes in a murine pneumonia model to identify genes with increased vulnerability in the host environment. We found that partial CRISPR interference (CRISPRi) knockdown of 178 genes showed significant fitness defects in mice relative to axenic culture. We validated two important outliers: ispD, encoding a key enzyme in isoprenoid precursor biosynthesis, and pgsA, encoding an enzyme involved in phospholipid synthesis that is strongly upregulated in human infections. Partial knockdown of both genes showed decreased virulence in a mouse survival assay but had little impact on in vitro growth. The use of CRISPRi screening to uncover genetic vulnerabilities represents a promising strategy to prioritize antibacterial targets that interact with the host environment.

microbiology↗

bioRxiv2024

Antibody-Fab and -Fc features promote Mycobacterium tuberculosis restriction.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a leading cause of death by an infectious disease globally, with no efficacious vaccine. Antibodies are implicated in Mtb control, but the mechanisms of antibody action remain poorly understood. We assembled a library of TB monoclonal antibodies (mAb) and screened for the ability to restrict Mtb in mice, identifying protective antibodies targeting known and novel antigens. To dissect the mechanism of mAb-mediated Mtb restriction, we optimized a protective lipoarabinomannan-specific mAb through Fc-swapping. In vivo analysis of these Fc-variants revealed a critical role for Fc-effector function in Mtb restriction. Restrictive Fc-variants altered distribution of Mtb across innate immune cells. Single-cell transcriptomics highlighted distinctly activated molecular circuitry within innate immune cell subpopulations, highlighting early activation of neutrophils as a key signature of mAb-mediated Mtb restriction. Therefore, improved antibody-mediated restriction of Mtb is associated with reorganization of the tissue-level immune response to infection and depends on the collaboration of antibody Fab and Fc.

immunology↗

bioRxiv2024

Minocycline enhances antimicrobial activity of unpolarized macrophages against Acinetobacter baumannii while reducing the inflammatory response

Minocycline activity against Acinetobacter baumannii (AB) in vivo is underestimated by standard methods of susceptibility testing. We examined pharmacologic effects of minocycline on primary immunity that may be contributing to the in vivo vs. in vitro discrepancy of minocycline activity against AB. Minocycline MICs against 10 AB strains were compared in standard bacteriologic media (Mueller-Hinton broth, MHB) and physiologic (RPMI) media. Macrophages were pretreated with minocycline or comparator antibiotics before AB co-culture. Macrophage cytokine production and phagocytosis of AB were measured without and with pre-treatment with minocycline. Two to eight-fold reduction in minocycline MIC against 10 AB strains occurred in RPMI compared to MHB, which was more pronounced than other antibiotic classes. Macrophages pretreated with 1, 5, 10, 30, 50, and 100 g/mL minocycline before bacterial co-cultures significantly decreased AB inoculum at 6 hours of co-culture in a dose-dependent manner, with no bacterial colonies observed from co-cultures with macrophages pretreated with 30 g/mL or more of minocycline. Macrophages pretreated with minocycline for 24 hours before zymosan stimulation led to significantly higher levels of phagocytosis. Macrophages treated with minocycline for 24 hours significantly decreased production of IL-6, TNF-, and MCP-1 in a dose dependent manner. The minocycline in vivo efficacy may be attributed to enhanced activity in nutrient-limited, physiologic medium combined with increased macrophage phagocyte efficiency. Incorporating novel assays that recapitulate the in vivo environment will be important for understanding the host-pathogen-antibiotic relationship toward a goal of improved future drug discovery and overall treatment strategies against AB and other drug-resistant pathogens.

immunology↗

bioRxiv2024

Comprehensive double-mutant analysis of the Bacillus subtilis envelope using double-CRISPRi

Understanding bacterial gene function remains a major biological challenge. Double-mutant genetic interaction (GI) analysis addresses this challenge by uncovering the functional partners of targeted genes, allowing us to associate genes of unknown function with novel pathways and unravel connections between well-studied pathways, but is difficult to implement at the genome-scale. Here, we develop and use double-CRISPRi to systematically quantify genetic interactions at scale in the Bacillus subtilis envelope, including essential genes. We discover > 1000 known and novel genetic interactions. Our analysis pipeline and experimental follow-ups reveal the distinct roles of paralogous genes such as the mreB and mbl actin homologs, and identify new genes involved in the well-studied process of cell division. Overall, our study provides valuable insights into gene function and demonstrates the utility of double-CRISPRi for high-throughput dissection of bacterial gene networks, providing a blueprint for future studies in diverse bacterial species.

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

Systematic deconstruction of myeloid cell signaling in tuberculosis granulomas reveals IFN-γ, TGF-β, and time are associated with conserved myeloid diversity

Myeloid cells are key constituents of tuberculosis (TB) granulomas. They are the major target of pathogen infection and play central roles in pathogen control, antigen presentation, adaptive immune cell recruitment, and tissue homeostasis. However, the role of myeloid cells in TB has been studied largely through ex vivo experimental approaches that do not capture the dynamic phenotypic and functional states of these cells in the disease environment. To address this gap, we used a combination of bulk and single-cell RNA sequencing (scRNA-seq), computational modeling, and imaging to define the molecular diversity of myeloid cells in granulomas from Mycobacterium tuberculosis-infected nonhuman primates. We observed an increase in myeloid cell diversity in granulomas compared to non-granulomatous lung tissue. This increased transcriptional diversity is defined by a continuum of macrophage differentiation-, metabolism-, and cytokine-regulated transcriptional programs. In vitro experimental modeling of monocyte-to-macrophage differentiation in defined cytokine environments implicates differentiation time, IFN-{gamma}, and TGF-{beta} signaling as candidate drivers of macrophage diversity. We next examined the conservation of these populations across additional experimental models of Mtb infection and found myeloid cell subsets enriched across the TB disease spectrum. To further contextualize these responses, we constructed an atlas of myeloid cells across diverse human lung pathologies, finding myeloid cell subpopulations that were similar between TB and other lung pathologies as well as subpopulations that distinguish between diseases. Collectively, this study identifies points of integration between myeloid cell biology in TB granulomas and other lung diseases that can be used for defining the signals that instruct myeloid cell behavior in TB and other diseases, as well as advance myeloid cell-targeted therapies.

immunology↗

bioRxiv2024

Modular, inducible, and titratable expression systems for Escherichia coli and Acinetobacter baumannii

Gene expression systems that transcend species barriers are needed for cross-species analysis of gene function. In particular, expression systems that can be utilized in both model and pathogenic bacteria underpin comparative functional approaches that inform conserved and variable features of bacterial physiology. Here, we develop replicative and integrative vectors alongside a novel, IPTG-inducible promoter that can be used in the model bacterium Escherichia coli K-12 as well as strains of the antibiotic-resistant pathogen, Acinetobacter baumannii. We generate modular vectors that transfer by conjugation at high efficiency and either replicate or integrate into the genome, depending on design. Embedded in these vectors, we also developed a synthetic, IPTG-inducible promoter, PabstBR, that induces to a high level, but is less leaky than the commonly used trc promoter. We show that PabstBR is titratable at both the population and single cell level, regardless of species, highlighting the utility of our expression systems for cross-species functional studies. Finally, as a proof of principle, we use our integrating vector to develop a reporter for the E. coli envelope stress {sigma} factor, RpoE, and deploy the reporter in E. coli and A. baumannii, finding that A. baumannii does not recognize RpoE-dependent promoters unless RpoE is heterologously expressed. We envision that these vector and promoter tools will be valuable for the community of researchers that study fundamental biology of E. coli and A. baumannii. ImportanceAcinetobacter baumannii is a multidrug-resistant, hospital-acquired pathogen with the ability to cause severe infections. Understanding the unique biology of this non-model bacterium may lead to the discovery of new weaknesses that can be targeted to treat antibiotic-resistant infections. Here, we provide expression tools that can be used to study gene function in A. baumannii, including in drug-resistant clinical isolates. These tools are also compatible with the model bacterium, Escherichia coli, enabling cross-species comparisons of gene function. We anticipate that the use of these tools by the scientific community will accelerate our understanding of Acinetobacter biology.

microbiology↗

bioRxiv2024

Systematic genome-wide discovery of host factors governing bacteriophage infectivity

Bacterial host factors regulate the infection cycle of bacteriophages. Except for some well-studied host factors (e.g., receptors or restriction-modification systems), the contribution of the rest of the host genome on phage infection remains poorly understood. We developed PHAGEPACK, a pooled assay that systematically and comprehensively measures each host genes impact on phage fitness. PHAGEPACK combines CRISPR interference with phage packaging to link host perturbation to phage fitness during active infection. Using PHAGEPACK, we constructed a genome-wide map of genes impacting T7 phage fitness in permissive E. coli, revealing pathways previously unknown to affect phage packaging. When applied to the non-permissive E. coli O121, PHAGEPACK identified pathways leading to host resistance; their removal increased phage susceptibility up to a billion-fold. Bioinformatic analysis indicates phage genomes carry homologs or truncations of key host factors, potentially for fitness advantage. In summary, PHAGEPACK offers valuable insights into phage-host interactions, phage evolution, and bacterial resistance.

synthetic biology↗

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

A Small Multidrug Resistance Transporter in Pseudomonas aeruginosa Confers Substrate-Specific Resistance or Susceptibility

Small Multidrug Resistance (SMR) transporters are key players in the defense of multidrug-resistant pathogens to toxins and other homeostasis-perturbing compounds. However, recent evidence demonstrates that EmrE, an SMR from Escherichia coli and a model for understanding transport, can also induce susceptibility to some compounds by drug-gated proton leak. This runs down the {Delta}pH component of the Proton Motive Force (PMF), reducing viability of the affected bacteria. Proton leak may provide an unexplored drug target distinct from the targets of most known antibiotics. Activating proton leak requires an SMR to be merely present, rather than be the primary resistance mechanism, and dissipates the energy source for many other efflux pumps. PAsmr, an EmrE homolog from P. aeruginosa, transports many EmrE substrates in cells and purified systems. We hypothesized that PAsmr, like EmrE, may confer susceptibility to some compounds via drug-gated proton leak. Growth assays of E. coli expressing PAsmr displayed substrate-dependent resistance and susceptibility phenotypes, and in vitro solid-supported membrane electrophysiology experiments revealed that PAsmr performs both antiport and substrate-gated proton uniport, demonstrating the same functional promiscuity observed in EmrE. Growth assays of P. aeruginosa strain PA14 demonstrated that PAsmr contributes resistance to some antimicrobial compounds, but no growth defect is observed with susceptibility substrates, suggesting P. aeruginosa can compensate for the proton leak occurring through PAsmr. These phenotypic differences between P. aeruginosa and E. coli advance our understanding of underlying resistance mechanisms in P. aeruginosa and prompt further investigation into the role that SMRs play in antibiotic resistance in pathogens. IMPORTANCESmall multidrug resistance transporters are a class of efflux pumps found in many pathogens, but whose contributions to antibiotic resistance are not fully understood. We hypothesize that these transporters may confer not only resistance, but also susceptibility, by dissipating the proton-motive force. This means to use an SMR transporter as a target, it merely needs to be present (as opposed to being the primary resistance mechanism). Here, we test this hypothesis with an SMR transporter found in Pseudomonas aeruginosa and find that it can perform both antiport (conferring resistance) and substrate-gated proton leak. Proton leak is detrimental to growth in E. coli but not P. aeruginosa, suggesting that P. aeruginosa responds differently to or can altogether prevent {Delta}pH dissipation.

biochemistry↗

bioRxiv2023

Tools for Genetic Engineering and Gene Expression Control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides

Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts have the potential to form the backbone of the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans, Rhodobacter sphaeroides, and Zymomonas mobilis, show particular promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis, N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides. We screen a synthetic promoter library to identify inducible promoters with strong, regulated activity in both organisms. Combining Tn7 integration with promoters from our library, we establish CRISPR interference systems for N. aromaticivorans and R. sphaeroides that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these industrially important species and other Alphaproteobacteria. IMPORTANCEIt is important to increase our understanding of the microbial world to improve health, agriculture, the environment and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise excellent properties for conversion are challenging to genetically engineer. Here, we report systems to overcome that barrier in the Alphaproteobacteria, Novosphingobium aromaticivorans and Rhodobacter sphaeroides, by producing genetic tools that allow easy insertion of engineered pathways into their genomes and to precisely control gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to optimize yield of biofuels and bioproducts.

microbiology↗