Biology subjects

Jacobs-Wagner, C.

Publications and source records attributed to Jacobs-Wagner, C..

At least 19 recordsLinked to original sources

bioRxiv2026

A cofactor-promiscuous HMGR from the Lyme disease pathogen illuminates diversity in bacterial isoprenoid biosynthesis

The Lyme disease pathogen Borrelia burgdorferi contains a highly reduced genome lacking many primary metabolic pathways. However, B. burgdorferi retains the mevalonate pathway that synthesizes isopentenyl pyrophosphate (IPP), the precursor to the peptidoglycan carrier lipid. While the mevalonate pathway and the enzyme that catalyzes its rate-limiting step (3-hydroxy-3-methyl glutaryl coenzyme A reductase, HMGR) are well studied in vertebrates, little is known about the pathway in B. burgdorferi and many pathogenic bacteria. In this work, we reveal that HMGR is a critical metabolic enzyme in B. burgdorferi. We demonstrate that loss of HMGR causes morphological defects and muted de novo synthesis of peptidoglycan; these defects are ameliorated by exogenous mevalonate and IPP. Biochemical characterization unveiled the HMGR as a highly unusual cofactor-promiscuous oxidoreductase that functions with both nicotinamide cofactors. Bioinformatics and biochemical characterization uncovered examples of similarly promiscuous HMGRs and revealed a previously unrecognized evolutionary link to cofactor choice. Moreover, structures of the enzyme reveal a highly divergent active site architecture. Together, these findings firmly establish HMGR as an opportunity target for the development of antibacterials for a diderm pathogen while highlighting cofactor promiscuity as an evolutionary acquired feature in HMGRs.

biochemistry↗

bioRxiv2026

A lipid compendium of a metabolically compromised bacterium provides insights into lipid acquisition, biosynthesis, and metabolism

The Lyme disease agent Borrelia burgdorferi belongs to a class of metabolically compromised bacteria that cannot survive without host-derived lipids. Survival of the agent in tick and vertebrate hosts requires substantial nutrient acquisition and potential cell envelope remodeling. While prior studies identified cholesterol, cholesterol glycolipids, and phosphatidylcholines as membrane lipids in B. burgdorferi, the identity of many other membrane lipids, their origin, and their physiological relevance remain unknown. Here, we used a suite of untargeted and targeted high-resolution mass spectrometry methods to reveal a complex lipid profile of the pathogen and to identify the origin of its lipids. The analysis detected more than 500 lipids in B. burgdorferi, the majority of which are sourced from the environment. However, the bacterium selectively accumulates certain lipids while excluding others, suggesting discriminatory uptake. These include cholesteryl esters and triglycerides that are organized in foci within the pathogen. Intriguingly, the pathogen also synthesizes predominantly eukaryotic lipids such as the lysosomal bis(monoacylglycerol)phosphate and the plant glycolipid sulfoquinovosyl diacylglycerol (SQDG). The biosynthesis of the latter is carried out by enzymes that exhibit structural homology to plant oxidoreductases and galactosyltransferases, yet their closest orthologs are found in bacteria. This hints that the capability of SQDG synthesis is more widespread in spirochaetes and other bacteria. Together, the comprehensive lipid profiling we report here uncovers novel aspects of the physiology of the metabolically challenged B. burgdorferi and highlights lipid acquisition and synthesis pathways as potentially critical for pathogen survival.

biochemistry↗

bioRxiv2025

Priority effects drive fungal and nematode emergence from insect larvae

Priority effects, in which species arrival history influences community assembly, are increasingly recognized to affect host-parasite systems. However, priority effects across disparate groups of parasitic organisms are poorly understood despite the wide range of taxonomic groups involved. In California oak woodland, we investigated how priority effects between two insect-parasitic fungi (Metarhizium and Beauveria) influenced emergence of nematodes from insect larvae. Field and laboratory results indicated that both fungi were common, but priority effects prevented them from co-emerging from the same larva. Metarhizium- and Beauveria-infected insects did not differ in the species composition of emerging nematodes, but larvae without fungal emergence had distinct nematode communities, with Oscheius almost always emerging without fungi. Experiments indicated that none of the commonly found nematodes (Acrobeloides, Mesorhabditis, Oscheius, and Rhabditis) were entomopathogenic, but that Oscheius could exclude Beauveria if arrived early. This time-dependent exclusion was likely caused by an Ochrobactrum bacterium that Oscheius nematodes carried. Together, these findings suggest that fungi enter insects as primary arrivers, while nematodes come as secondary arrivers to exploit fungus-killed insects, with priority effects influencing both groups. We suggest that this system is a promising natural microcosm for understanding priority effects across disparate groups in host-parasite systems.

ecology↗

bioRxiv2025

Cross-Molecular Active Learning for the Discovery of Antimicrobial Polyacrylamides

Antimicrobial resistance poses an urgent and increasing threat to global health. The development of new antimicrobials is crucial. Synthetic copolymers are attractive as a potential solution, because they can be produced at scale and designed to mimic antimicrobial peptides and act as broad-spectrum antimicrobials capable of evading resistance mechanisms. This work leverages a cross-molecular machine learning pipeline, trained on antimicrobial peptides, to develop potent antimicrobial polymers to combat Escherichia coli, which were then synthesized and validated experimentally. One candidate copolymer was further characterized and shown to permeabilize the bacterial membrane, which is associated with decreased resistance. Furthermore, this copolymer demonstrated remarkable synergy in eradicating biofilm-associated E. coli when combined with a first-line clinical drug regimen, reducing the amount needed to eradicate bacteria in biofilms by three orders of magnitude. These results demonstrate promise for potentiating antibacterial activity of currently available antibiotics, treating serious and complicated infections, and combatting antimicrobial resistance.

microbiology↗

bioRxiv2025

Time-resolved phenotyping at subcellular resolution reveals shared principles and key trade-offs across antimicrobial peptide activities

Cationic antimicrobial peptides are a broad family of host defense molecules that neutralize bacteria by permeabilizing one or more membranes and/or inhibiting intracellular targets. Here, we present a time-resolved single-cell pipeline for quantifying these effects in Escherichia coli. Applying this pipeline to 18 diverse natural peptides and synthetic peptidomimetics reveals shared core activities, but with different kinetics, defining two classes with opposite trade-offs. Class I peptides cause abrupt growth arrest, predominantly coupled with inner membrane permeabilization and ribosome/DNA reorganization, conferring fast, multipronged action. However, rapid intracellular absorption by the first permeabilized cells depletes the extracellular pool, rendering them ineffective against dense populations, including biofilms. Class II peptides act more gradually, with delayed or absent inner membrane permeabilization, limiting their speed of action. However, this results in slower intracellular absorption and greater efficacy at high cell densities and against biofilms. These opposing functional trade-offs point to important immunological and therapeutic implications.

microbiology↗

bioRxiv2025

Bactofilins are essential spatial organizers of peptidoglycan insertion in the Lyme disease spirochete Borrelia burgdorferi

The Lyme disease spirochete Borrelia burgdorferi has a distinctive pattern of growth. Newly-born cells elongate by primarily inserting peptidoglycan at mid-cell, while in longer cells, additional insertion sites form at the one-quarter and three-quarter positions along the cell length. It is not known how peptidoglycan insertion is concentrated at these locations in B. burgdorferi. In other bacteria, multi-protein complexes are known to synthesize new peptidoglycan and are often organized by cytoskeletal proteins. We show here that B. burgdorferis zonal concentration of peptidoglycan insertion requires BB0538 (BbbA) and BB0245 (BbbB), two members of the bactofilin class of cytoskeletal proteins. Bactofilin depletion redistributes peptidoglycan insertion along the cell length. Prolonged bactofilin depletion arrested growth in culture and induced extensive cell blebbing, indicating that B. burgdorferi bactofilins are essential for viability. Fluorescent protein fusions of BbbA and BbbB localized to areas of peptidoglycan insertion, with BbbB accumulation preceding peptidoglycan insertion at these sites. Similar to peptidoglycan insertion, BbbB localization was disrupted upon depletion of BbbA. Our results show that BbbB relies on BbbA for its localization, and that together, BbbA and BbbB direct the spatial patterning of new peptidoglycan insertion in B. burgdorferi. IMPORTANCEThe spirochetal bacterium Borrelia burgdorferi causes Lyme disease, the most prevalent vector-borne infection in North America and Europe. Cellular replication, which requires growth and division of the peptidoglycan cell wall, facilitates B. burgdorferi transmission to, and dissemination within, new hosts. Cellular replication is therefore essential for pathogenesis. Bactofilins regulate peptidoglycan-related processes in several bacteria. However, these functions are typically non-essential for cellular replication, as bactofilin-encoding genes can be readily deleted in multiple bacterial species. In contrast, we show that the B. burgdorferi bactofilins BbbA and BbbB are essential for cellular viability and direct zonal peptidoglycan insertion. Our findings broaden the spectrum of known bactofilin functions and advance our understanding of how peptidoglycan insertion is regulated in this unusual, medically important spirochete bacterium.

microbiology↗

bioRxiv2025

Explosive cytotoxicity of 'ruptoblasts' bridges hormonal surveillance and immune defense

Current understanding of cytotoxic immunity is shaped by hematopoietic-derived cells - T cells, natural killer cells, and neutrophils. Here, we identify ruptoblasts, a previously unknown cytotoxic glandular cell type in regenerative planarian flatworms. Ruptoblasts undergo an explosive cell death, ruptosis, triggered by activin, a multifunctional hormone that also acts as an inflammatory cytokine. Excessive activin - induced through protein injection, genetic chimerism, or bacterial infection - initiates ruptosis, discharging potent diffusible cytotoxic agents capable of eliminating any nearby cells, bacteria, and even mammalian cells within minutes. Ruptoblast ablation suppresses inflammation but compromises bacterial clearance, highlighting their broad-spectrum immune functions. Mechanistically distinct from known cytotoxic mechanisms, the explosive nature of ruptosis relies on intracellular calcium and dynamic cytoskeletal reorganization. Ruptoblast-like cells appear conserved in diverse basal bilaterians, implying an ancient evolutionary origin. These findings unveil a widespread strategy coupling hormonal regulation with immune defense and expand the landscape of evolutionary immune innovations.

immunology↗

bioRxiv2025

Bacterial and host enzymes modulate the inflammatory response produced by the peptidoglycan of the Lyme disease agent

The spirochete Borrelia burgdorferi causes Lyme disease. In some patients, an excessive, dysregulated proinflammatory immune response can develop in joints leading to persistent arthritis even after antibiotic therapy. In such patients, persistence of antigenic B. burgdorferi peptidoglycan (PGBb) fragments within joint tissues may contribute to immunopathogenesis pre- and post-antibiotic treatment. In live B. burgdorferi cells, the outer membrane shields the polymeric PGBb sacculus from exposure to the immune system. However, unlike most diderm bacteria, B. burgdorferi releases PGBb turnover products into its environment due to the absence of recycling activity. In this study, we identified the released PGBb fragments using a mass spectrometry-based approach. By characterizing the L,D-carboxypeptidase activity of B. burgdorferi protein BB0605 (renamed DacA), we found that PGBb turnover largely occurs at sites of PGBb synthesis. In parallel, we demonstrated that the lytic transglycosylase activity associated with BB0259 (renamed MltS) releases PGBb fragments with 1,6-anhydro bond on their N-acetylmuramyl residues. Stimulation of human cell lines with various synthetic PGBb fragments revealed that 1,6-anhydromuramyl-containing PGBb fragments are poor inducers of a NOD2-dependent immune response relative to their hydrated counterparts found in the polymeric PGBb isolated from dead bacteria. We also showed that the activity of the human N-acetylmuramyl-L-alanine amidase PGLYRP2, which reduces the immunogenicity of PGBb material, is low in joint (synovial) fluids relative to serum. Altogether, our findings suggest that MltS activity helps B. burgdorferi evade PG-based immune detection by NOD2 during growth despite shedding PGBb fragments and that PGBb-induced immunopathology likely results from host sensing of PGBb material from dead (lysed) spirochetes. Additionally, our results suggest the possibility that natural variation in PGLYRP2 activity may contribute to differences in susceptibility to PG-induced inflammation across tissues and individuals. Author summaryDuring bacterial infection, the presence of peptidoglycan- a polymeric element of bacterial cell walls-triggers a host inflammatory response. Although generally protective during acute phases, inflammation, when chronic, can contribute to disease development. Recent work has suggested that the persistence of pro-inflammatory peptidoglycan derived from the Lyme disease spirochete Borrelia burgdorferi in joints may contribute to persistent arthritis in some patients despite appropriate antibiotic therapy. Interestingly, B. burgdorferi sheds peptidoglycan turnover products into the environment during growth. Here, we show that these shed products from live spirochetes are poor effectors of an immune response by the human NOD2 immune receptor due to the formation of an anhydro bond on the N-acetyl-muramic residue during peptidoglycan hydrolysis by a B. burgdorferi lytic transglycosylase. We also show that human N-acetylmuramyl-L-alanine amidase activity, which abrogates the NOD2-dependent response to the immunogenic peptidoglycan isolated from lysed B. burgdorferi cells, is low in human joint fluids relative to serum. Based on our findings, we propose that immunopathogenesis by peptidoglycan material more likely derives from lysed spirochetes (killed by an immune attack or antibiotics) than live ones and that the level of human peptidoglycan hydrolytic enzymes across tissues and individuals influences susceptibility to chronic inflammation.

microbiology↗

bioRxiv2024

Borrelia burgdorferi loses essential genetic elements and cell proliferative potential during stationary phase in culture but not in the tick vector.

The Lyme disease agent Borrelia burgdorferi is a polyploid bacterium with a segmented genome in which both the chromosome and over 20 distinct plasmids are present in multiple copies per cell. This pathogen can survive at least nine months in its tick vector in an apparent dormant state between blood meals, without losing cell proliferative capability when re-exposed to nutrients. Cultivated B. burgdorferi cells grown to stationary phase or resuspended in nutrient-limited media are often used to study the effects of nutrient deprivation. However, a thorough assessment of the spirochetes ability to recover from nutrient depletion has been lacking. Our study shows that starved B. burgdorferi cultures rapidly lose cell proliferative. Loss of genetic elements essential for cell proliferation contributes to the observed proliferative defect in stationary phase. The gradual decline in copies of genetic elements is not perfectly synchronized between chromosomes and plasmids, generating cells that harbor one or more copies of the essential chromosome but lack all copies of one or more non-essential plasmids. This phenomenon likely contributes to the well-documented issue of plasmid loss during in vitro cultivation of B. burgdorferi. In contrast, B. burgdorferi cells from ticks starved for 14 months showed no evidence of reduced cell proliferative ability or plasmid loss. Beyond their practical implications for studying B. burgdorferi, these findings suggest that the midgut of the tick vector offers a unique environment that supports the maintenance of B. burgdorferis segmented genome and cell proliferative potential during periods of tick fasting. ImportanceBorrelia burgdorferi causes Lyme disease, a prevalent tick-borne illness. B. burgdorferi must survive long periods (months to a year) of apparent dormancy in the midgut of the tick vector between blood meals. Resilience to starvation is a common trait among bacteria. However, this study reveals that in laboratory cultures, B. burgdorferi poorly endures starvation and rapidly loses viability. This decline is linked to a gradual loss of genetic elements required for cell proliferation. These results suggest that the persistence of B. burgdorferi in nature is likely shaped more by unique environmental conditions in the midgut of the tick vector than by a general innate ability of this bacterium to endure nutrient deprivation.

microbiology↗

bioRxiv2024

The polyadenylase PAPI is required for virulence plasmid maintenance in pathogenic bacteria

Many species of pathogenic bacteria harbor critical plasmid-encoded virulence factors, and yet the regulation of plasmid replication is often poorly understood despite playing a critical role in plasmid-encoded gene expression. Human pathogenic Yersinia, including the plague agent Y. pestis and its close relative Y. pseudotuberculosis, require the type III secretion system (T3SS) virulence factor to subvert host defense mechanisms and colonize host tissues. The Yersinia T3SS is encoded on the IncFII plasmid for Yersinia virulence (pYV). Several layers of gene regulation enables a large increase in expression of Yersinia T3SS genes at mammalian body temperature. Surprisingly, T3SS expression is also controlled at the level of gene dosage. The number of pYV molecules relative to the number of chromosomes per cell, referred to as plasmid copy number, increases with temperature. The ability to increase and maintain elevated pYV plasmid copy number, and therefore T3SS gene dosage, at 37{degrees}C is important for Yersinia virulence. In addition, pYV is highly stable in Yersinia at all temperatures, despite being dispensable for growth outside the host. Yet how Yersinia reinforces elevated plasmid replication and plasmid stability remains unclear. In this study, we show that the chromosomal gene pcnB encoding the polyadenylase PAP I is required for regulation of pYV plasmid copy number (PCN), maintenance of pYV in the bacterial population outside the host, robust T3SS activity, and Yersinia virulence in a mouse infection model. Likewise, pcnB/PAP I is also required for robust expression of the Shigella flexneri virulence plasmid-encoded T3SS. Furthermore, Yersinia and Shigella pcnB/PAP I is required for maintaining normal PCN of model antimicrobial resistance (AMR) plasmids whose replication is regulated by sRNA, thereby increasing antibiotic resistance by ten-fold. These data suggest that pcnB/PAP I contributes to the spread and stabilization of virulence and AMR plasmids in bacterial pathogens, and is essential in maintaining the gene dosage required to mediate plasmid-encoded traits. Importantly pcnB/PAP I has been bioinformatically identified in many species of bacteria despite being studied in only a few species to date. Our work highlights the potential importance of pcnB/PAP I in antibiotic resistance, and shows for the first time that pcnB/PAP I reinforces PCN and virulence plasmid stability in natural pathogenic hosts with a direct impact on bacterial virulence. Author SummaryMany pathogens carry extrachromosomal DNA elements known as plasmids, which encode genes that confer bacterial virulence or antimicrobial resistance (AMR). Acquisition of these plasmids by bacteria can lead to the emergence of new pathogenic traits and the spread of AMR, yet the mechanisms by which plasmids are retained in bacterial populations particularly in the absence of selective pressure remain incompletely understood. Here we show that the major bacterial polyadenylase enzyme PAP I, encoded by the pcnB gene, is critical for the human pathogen Yersinia pseudotuberculosis to maintain its native virulence plasmid as well as AMR plasmids. Very little is known about the process of polyadenylation in bacteria, or the post-transcriptional addition of adenosine residues to the 3 end of transcripts. This study represents the first demonstration that PAP I-mediated polyadenylation contributes to bacterial pathogenesis.

microbiology↗

bioRxiv2024

DNA/polysome phase separation and cell width confinement couple nucleoid segregation to cell growth in Escherichia coli

Chromosome segregation is essential for cellular proliferation. Unlike eukaryotes, bacteria lack cytoskeleton-based machinery to segregate their chromosomal DNA (nucleoid). The bacterial ParABS system segregates the duplicated chromosomal regions near the origin of replication. However, this function does not explain how bacterial cells partition the rest (bulk) of the chromosomal material. Furthermore, some bacteria, including Escherichia coli, lack a ParABS system. Yet, E. coli faithfully segregates nucleoids across various growth rates. Here, we provide theoretical and experimental evidence that polysome production during chromosomal gene expression helps compact, split, segregate, and position nucleoids in E. coli through out-of-equilibrium dynamics and polysome exclusion from the DNA meshwork, inherently coupling these processes to biomass growth across nutritional conditions. Halting chromosomal gene expression and thus polysome production immediately stops sister nucleoid migration while ensuing polysome depletion gradually reverses nucleoid segregation. Redirecting gene expression away from the chromosome and toward plasmids causes ectopic polysome accumulations that are sufficient to drive aberrant nucleoid dynamics. Cell width enlargement suggest that the proximity of the DNA to the membrane along the radial axis is important to limit the exchange of polysomes across DNA-free regions, ensuring nucleoid segregation along the cell length. Our findings suggest a self-organizing mechanism for coupling nucleoid segregation to cell growth.

microbiology↗

bioRxiv2024

Glycogen phase separation drives macromolecular rearrangement and asymmetric division in E. coli

Bacteria often experience nutrient limitation. While the exponential and stationary growth phases have been characterized in the model bacterium Escherichia coli, little is known about what transpires inside individual cells during the transition between these two phases. Through quantitative cell imaging, we found that the positions of nucleoids and cell division sites become increasingly asymmetric during transition phase. These asymmetries were coupled with asymmetric reorganization of protein, ribosome, and RNA probes in the cytoplasm. Results from live-cell imaging experiments, complemented with genetic and 13C whole-cell nuclear magnetic resonance spectroscopy studies, show that preferential accumulation of the storage polymer glycogen at the old cell pole leads to the observed rearrangements and asymmetric divisions. Live-cell atomic force microscopy analysis, combined with in vitro biochemical experiments, suggests that these phenotypes are due to the propensity of glycogen to phase separate into soft condensates in the crowded cytoplasm. Glycogen-associated differences in cell sizes between strains and future daughter cells suggest that glycogen phase separation allows cells to store large glucose reserves without counting them as cytoplasmic space.

microbiology↗

bioRxiv2024

Coupling of cell growth modulation to asymmetric division and cell cycle regulation in Caulobacter crescentus

In proliferating bacteria, growth rate is often assumed to be similar between daughter cells. However, most of our knowledge of cell growth derives from studies on symmetrically dividing bacteria. In many -proteobacteria, asymmetric division is a normal part of the life cycle, with each division producing daughter cells with different sizes and fates. Here, we demonstrate that the functionally distinct swarmer and stalked daughter cells produced by the model -proteobacterium Caulobacter crescentus have different average growth rates despite sharing an identical genome and environment. The discrepancy in growth rate is due to a growth slowdown during the cell cycle stage preceding DNA replication (the G1 phase), which is predominantly associated with swarmer cell functionality. We also provide evidence that the second messenger (p)ppGpp extends the timing of the G1/swarmer cell stage by slowing growth specifically during the beginning of the cell cycle. Our data further show that cells factor the amount and rate of their growth to control the G1/S transition, allowing them to adjust the time they spend with ecologically important G1-specific traits. Significance statementBacterial growth rate modulation is generally associated with changes in genetic make-up or environmental condition. This study demonstrates that the rate of cell growth can also vary between daughter cells and across cell cycle stages under invariant and unstressed environmental conditions. This is illustrated by the asymmetrically dividing -proteobacterium Caulobacter crescentus, which, at each division, produces two functionally distinct daughter cells that differ in average growth rate. This growth rate difference arises from a G1 phase-specific growth slowdown mediated, in part, by the (p)ppGpp alarmone. Altogether, this study showcases the coupling of cell growth modulation to asymmetric division and cell cycle regulation, which may have implications for other -proteobacteria given their cell cycle similarities with C. crescentus.

microbiology↗

bioRxiv2024

Genome concentration limits cell growth and modulates proteome composition in Escherichia coli

Defining the cellular factors that drive growth rate and proteome composition is essential for understanding and manipulating cellular systems. In bacteria, ribosome concentration is known to be a constraining factor of cell growth rate, while gene concentration is usually assumed not to be limiting. Here, using single-molecule tracking, quantitative single-cell microscopy, and modeling, we show that genome dilution in Escherichia coli cells arrested for DNA replication limits total RNA polymerase activity within physiological cell sizes across tested nutrient conditions. This rapid-onset limitation on bulk transcription results in sub-linear scaling of total active ribosomes with cell size and sub-exponential growth. Such downstream effects on bulk translation and cell growth are near-immediately detectable in a nutrient-rich medium, but delayed in nutrient-poor conditions, presumably due to cellular buffering activities. RNA sequencing and tandem-mass-tag mass spectrometry experiments further reveal that genome dilution remodels the relative abundance of mRNAs and proteins with cell size at a global level. Altogether, our findings indicate that chromosome concentration is a limiting factor of transcription and a global modulator of the transcriptome and proteome composition in E. coli. Experiments in Caulobacter crescentus and comparison with eukaryotic cell studies identify broadly conserved DNA concentration-dependent scaling principles of gene expression.

cell biology↗

bioRxiv2023

Organization and replicon interactions within the highly segmented genome of Borrelia burgdorferi

Borrelia burgdorferi, a causative agent of Lyme disease, contains the most segmented bacterial genome known to date, with one linear chromosome and over twenty plasmids. How this unusually complex genome is organized, and whether and how the different replicons interact are unclear. We recently demonstrated that B. burgdorferi is polyploid and that the copies of the chromosome and plasmids are regularly spaced in each cell, which is critical for faithful segregation of the genome to daughter cells. Regular spacing of the chromosome is controlled by two separate partitioning systems that involve the protein pairs ParA/ParZ and ParB/SMC. Here, using chromosome conformation capture (Hi-C), we characterized the organization of the B. burgdorferi genome and the interactions between the replicons. We uncovered that although the linear chromosome lacks contacts between the two replication arms, the two telomeres are in frequent contact. Moreover, several plasmids specifically interact with the chromosome oriC region, and a subset of plasmids interact with each other more than with others. We found that SMC and the SMC-like MksB protein mediate long-range interactions on the chromosome, but they minimally affect plasmid-chromosome or plasmid-plasmid interactions. Finally, we found that disruption of the two partition systems leads to chromosome restructuring, correlating with the mis-positioning of chromosome oriC. Altogether, this study revealed the conformation of a complex genome and analyzed the contribution of the partition systems and SMC family proteins to this organization. This work expands the understanding of the organization and maintenance of multipartite bacterial genomes.

molecular biology↗

bioRxiv2023

Apparent simplicity and emergent robustness in bacterial cell cycle control

To examine how bacteria achieve robust cell proliferation across diverse conditions, we developed a method that quantifies 77 cell morphological, cell cycle and growth phenotypes of a fluorescently-labeled Escherichia coli strain and >800 gene deletion derivatives under multiple nutrient conditions. This approach revealed extensive phenotypic plasticity and deviating mutant phenotypes were often found to be nutrient-dependent. From this broad phenotypic landscape emerged simple and robust unifying rules (laws) that connect DNA replication initiation, nucleoid segregation, FtsZ-ring formation, and cell constriction to specific aspects of cell size (volume, length, or added length). Furthermore, completion of cell division followed the initiation of cell constriction after a constant time delay across strains and nutrient conditions, identifying cell constriction as a key control point for cell size determination. Our work provides a systems-level understanding of the design principles by which E. coli integrates cell cycle processes and growth rate with cell size to achieve its robust proliferative capability.

systems biology↗

bioRxiv2022

Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi

The spirochete Borrelia burgdorferi, which causes Lyme disease, has the most segmented genome among known bacteria. In addition to a linear chromosome, the B. burgdorferi genome contains over 20 linear and circular endogenous plasmids. While many of these plasmids are dispensable under in vitro culture conditions, they are maintained during the natural life cycle of the pathogen. Plasmid-encoded functions are required for colonization of the tick vector, transmission to the vertebrate host, and evasion of host immune defenses. Different Borrelia strains can vary substantially in the type of plasmids they carry. The gene composition within the same type of plasmid can also differ from strain to strain, impeding the inference of plasmid function from one strain to another. To facilitate the investigation of the role of specific B. burgdorferi plasmids, we developed a Cas9-based approach that targets a plasmid for removal. As a proof-of-principle, we showed that targeting wild-type Cas9 to several loci on the endogenous plasmids lp25 or lp28-1 of the B. burgdorferi type strain B31 results in sgRNA-specific plasmid loss even when homologous sequences (i.e., potential sequence donors for DNA recombination) are present nearby. Cas9 nickase versions, Cas9D10A or Cas9H840A, also cause plasmid loss, though not as robustly. Thus, sgRNA-directed Cas9 DNA cleavage provides a highly efficient way to eliminate B. burgdorferi endogenous plasmids that are non-essential in axenic culture.

microbiology↗

bioRxiv2022

Polyploidy, regular patterning of genome copies, and unusual control of DNA partitioning in the Lyme disease spirochete

Borrelia burgdorferi, the tick-transmitted spirochete agent of Lyme disease, has a highly segmented genome with a linear chromosome and various linear or circular plasmids. Here, by imaging several chromosomal loci and 16 distinct plasmids, we show that B. burgdorferi is polyploid during growth in culture and that the number of genome copies decreases during stationary phase. B. burgdorferi is also polyploid inside fed ticks and chromosome copies are regularly spaced along the spirochetes length in both growing cultures and ticks. This patterning involves the conserved DNA partitioning protein ParA whose localization is controlled by a potentially phage-derived protein, ParZ, instead of its usual partner ParB. ParZ binds its own coding region and acts as a centromere-binding protein. While ParA works with ParZ, ParB controls the localization of the condensin, SMC. Together, the ParA/ParZ and ParB/SMC pairs ensure faithful chromosome inheritance. Our findings underscore the plasticity of cellular functions, even those as fundamental as chromosome segregation.

microbiology↗