Biology subjects

Hershey, D. M.

Publications and source records attributed to Hershey, D. M..

12 recordsLinked to original sources

bioRxiv2026

Disrupting chemotaxis stimulates adhesion by recruiting a putative c-di-GMP effector to the Caulobacter crescentus cell pole

Contact with solid surfaces activates signaling pathways that promote biofilm formation in many bacteria. The alphaproteobacterium Caulobacter crescentus uses its flagellum to sense surfaces and responds by synthesizing an adhesive called the holdfast. The C. crescentus surface sensing pathway can be activated by mutating genes required for the assembly of the flagellum or genes required for chemotaxis. However, flagellar assembly and chemotaxis mutations activate distinct surface sensing pathways that differ in the activation of the diguanylate cyclase PleD. Here, we used a genome-wide screen to identify cmrA (CCNA_02061) as a crucial determinant of hyperadhesion in the chemotaxis mutant {Delta}cheYII. Genetic analysis showed that cmrA is important for activation of PleD in a context-specific manner. It is dispensable in wild-type and late-stage flagellar ({Delta}flgH) mutant backgrounds but promotes adhesion in early-stage flagellar assembly ({Delta}fliF), chemotaxis ({Delta}cheYII) and stator ({Delta}motB) mutant backgrounds. Fluorescently tagged CmrA displays a mostly cytoplasmic localization in genetic backgrounds where cmrA is dispensable for adhesion but localizes to the cell pole in backgrounds where it regulates adhesion. Structural modeling indicates that CmrA is a degenerate, catalytically inactive GGDEF/EAL domain containing protein, but cmrA alleles with mutated conserved c-di-GMP coordinating residues are unable to support hyperadhesion. Our results indicate that altering the directional switching of MotAB stators recruits CmrA to the cell pole where it activates PleD to drive surface adaptation. Ultimately, this work underscores the complexity of flagellar surface sensing by highlighting how the many rotational states of the motor stimulate distinct but overlapping c-di-GMP signaling pathways.

microbiology↗

bioRxiv2026

The gac system integrates physical and chemical cues to promote plant root attachment

Plants host complex communities of microbes that are attached to root surfaces. While many studies have sampled mutant populations after prolonged incubation on roots to identify bacterial genes that enable long-term colonization, the molecular mechanisms governing the early stages root attachment remain less understood. Here, we developed an in vitro root culture system that enables controlled and scalable investigation of bacterial attachment to root tissue. We used this platform to perform a genome-wide screen for root attachment determinants in the plant-associated bacterium Pseudomonas protegens Pf-5. Our results reveal that the gacSA two-component system functions as a sensory integration hub for coordinating early root attachment. Mutations that disrupt gacS or gacA cause severe root attachment defects despite having no effect on abiotic surface attachment in standard biofilm assays. Mutation of flagellar assembly genes enhances root attachment by mimicking surface contact and activating the gac system. In parallel, chemical cues released by roots stimulate surface attachment in a gac dependent manner. By integrating these signals, the gac system activates cyclic di-GMP-mediated attachment programs that drive the transition from planktonic to sessile behavior required for root association. We build on this model to show that manipulating flagellar surface sensing enhances the competitive fitness of Pf-5 in the presence of a synthetic bacterial community, suggesting a strategy to improve the competitive fitness of beneficial microbes on crops. These findings establish a mechanistic framework linking surface sensing, global regulation, and root attachment in a beneficial rhizobacterium. ImportancePlant roots are covered with diverse microbes that strongly influence plant health. Growing in association with roots has many benefits, but how bacteria attach to root tissue remains poorly understood. We developed a system to study how a bacterium that improves plant growth called Pseudomonas protegens Pf-5 attaches to root tissue. We found that physical contact with the root surface and chemical cues released by roots both enhance attachment to root tissue. A sensory system called gacSA is responsible for integrating physical and chemical cues to activate a root attachment program. Variant bacteria that prematurely activate the gac system compete more effectively with other bacteria on roots, suggesting that the root attachment pathway we characterized could serve as a strategy to use beneficial bacteria in agriculture.

microbiology↗

bioRxiv2026

Diversification of biofilm architecture among freshwater Pararheinheimera isolates

Air-liquid interfaces (ALIs) at the upper layer of oceans, lakes and rivers cover the majority of the earths surface. Microbes are known to accumulate at these resource-rich boundaries, but the mechanisms of ALI colonization are often assumed to mirror the formation of pellicle biofilms by non-aquatic organisms. Here, we analyzed ALI colonization by natural aquatic bacteria. We used samples from a freshwater lake to enrich for microbes that colonize the ALI in liquid growth medium. Mixed-species pellicles formed rapidly in these enrichments, were structurally stable for weeks and displayed a pronounced ecological succession. We isolated 31 members of the genus Pararheinheimera from early stages of mixed-species pellicle maturation. Five phylogenetically distinct Pararheinheimera clades were identified, each with a shared colony morphology. We used representative isolates to show that only one Pararheinheimera clade formed thin, adherent films at the ALI resembling classical pellicles. Isolates from the four remaining clades formed floating structures that could be categorized either as non-adhesive films or large viscous masses. Viscous mass (VM) pellicle formation was a polyphyletic trait that correlated with a highly mucoid appearance on agar plates, suggesting that the process is driven by copious secretion of extracellular matrix. Matrices from VM biofilms were largely non-adhesive, contained a mixture of acidic polysaccharides and proteins and formed thermally stable, shear-thinning hydrogels. Our results demonstrate that ALI colonization strategies vary widely even among closely related aquatic bacteria and identify VM pellicles as a distinct biofilm architecture with unique mechanical properties. ImportanceLakes, rivers and oceans contain a boundary between the air and the waters surface known as the air-liquid interface (ALI). Microbial communities that populate the ALI play crucial roles in nutrient cycling, but how aquatic microbes partition to these sites remains poorly characterized. Our study investigated how bacteria from a freshwater lake accumulate at the ALI. Lake water samples incubated in nutrient medium formed a layer of cells known as a pellicle biofilm at the ALI, and we isolated 31 different bacteria from a genus (Pararheinheimera) that was abundant during the early stages of pellicle formation. Only a subset of Pararheinheimera isolates formed traditional pellicle biofilms. Most formed either thin, non-adhesive films or large, gelatinous aggregates that appeared to persist at the ALI due to buoyancy. These findings expand our understanding of biofilm diversity in aquatic systems and suggest that the production of buoyant hydrogels may play an important role in structuring microbial communities at air-water boundaries.

microbiology↗

bioRxiv2026

Rheinheimera sp. T2C2 Bacterial Biofilm for Bioremediation of Cobalt (II)

Toxic metals, including cobalt, are often the cause of contamination of rivers and lakes in mining regions. Heavy metal water pollution has been linked to numerous human health problems, prompting the need for environmental remediation. Existing techniques for removing heavy metals from water, such as chemical precipitation and filtration, produce toxic waste, are costly, or require high power consumption for pumping. Biosorption is a potential alternative strategy that is cost-effective and uses readily available and naturally produced biomass and living material to absorb pollutants. Engineering living materials, such as biofilms, which consist of living cells and a secreted polymer matrix, offer potential to integrate toxin sensing, sequestration, and metabolism capabilities of cells to improve pollution remediation strategies. New biofilm producing candidates need to be explored to implement these material capabilities. Previous biosorption studies have primarily used bacterial biofilms from known pathogens and/or generate toxic waste in the form of the absorbent material combined with the heavy metal. Here, we describe a newly isolated bacterium called Rheinheimera sp. T2C2 that forms biofilms with promising biosorption characteristics. T2C2 is a non-pathogenic, aquatic bacterium with low nutrient requirements and high biofilm production. We demonstrate 1) the efficacy of Rheinheimera sp. T2C2 as a biosorbent for cobalt bioremediation; 2) how biosorption is altered by water conditions to establish the efficacy of this strategy in different environments; and 3) how the metal can be released from the biofilm for metal recycling. Our findings will provide a living materials strategy that overcomes existing barriers for bioremediation, and improve the health of ecosystems and humans through heavy metal removal and recycling.

microbiology↗

bioRxiv2024

A flagellar accessory protein links chemotaxis to surface sensing

Bacteria find suitable locations for colonization by sensing and responding to surfaces. Complex signaling repertoires control surface colonization, and surface contact sensing by the flagellum plays a central role in activating colonization programs. Caulobacter crescentus adheres to surfaces using a polysaccharide adhesin called the holdfast. In C. crescentus, disruption of the flagellum through interactions with a surface or mutation of flagellar genes increases holdfast production. Our group previously identified several C. crescentus genes involved in flagellar surface sensing. One of these, called fssF, codes for a protein with homology to the flagellar C-ring protein FliN. We show here that a fluorescently tagged FssF protein localizes to the flagellated pole of the cell and requires all components of the flagellar C-ring for proper localization, supporting the model that FssF associates with the C-ring. Deleting fssF results in a severe motility defect that we show is due to a disruption of chemotaxis. Epistasis experiments demonstrate that fssF promotes adhesion through a stator-dependent pathway when late-stage flagellar mutants are disrupted. Separately, we find that disruption of chemotaxis through deletion of fssF or other chemotaxis genes results in a hyperadhesion phenotype. Key genes in the surface sensing network (pleD, motB, and dgcB) contribute to both {Delta}flgH-dependent and {Delta}fssF- dependent hyperadhesion, but these genes affect adhesion differently in the two hyperadhesive backgrounds. Our results support a model in which the stator subunits of the flagella incorporate both mechanical and chemical signals to regulate adhesion. ImportanceBiofilms pose a threat in clinical and industrial settings. Surface sensing is an early step in biofilm formation. Studying surface sensing can help develop strategies for combating harmful biofilms. Here, we use the freshwater bacterium Caulobacter crescentus to study surface sensing. We characterize a previously unstudied gene, fssF, and find that it localizes to the cell pole in the presence of three proteins that make up a component of the flagellum called the C-ring. Additionally, we find that fssF is required for chemotaxis but dispensable for swimming motility. Lastly, our results show that mutating fssF and other genes required for chemotaxis causes a hyperadhesive phenotype. We propose that surface sensing requires chemotaxis for a robust response to a surface.

microbiology↗

bioRxiv2024

Engineering Bacterial Biomanufacturing: Characterization and Manipulation of Sphingomonas sp. LM7 Extracellular Polymers

Biologically produced materials are an attractive alternative to traditional materials such as metals and plastics and offer improved functionalities such as better biodegradability and biocompatibility. Polysaccharides are an example of a biologically produced materials that can have a range of chemical and physical properties including high stiffness to weight ratios and thermal stability. Biomanufactured bacterial polysaccharides can come with many advantages such as being non-toxic and are mechanically robust relative to proteins and lipids, which are also secreted by bacteria to generate a biofilm. One major goal in biomanufacturing is to produce quality material quickly and cost-effectively. Biomanufacturing offers additional benefits compared to traditional manufacturing including low resource investment and equipment requirements, providing an alternative to sourcing fossil fuel byproducts, and relatively low temperatures needed for production. However, many biologically produced materials require complex and lengthy purification processes before use. This paper 1) identifies the material properties of a novel polysaccharide, dubbed promonan, isolated from the extracellular polymeric substances of Sphingomonas sp. LM7; 2) demonstrates that these properties can be manipulated to suit specific applications; and 3) presents two alternative methods of processing to shorten purification time by more than 50% while maintaining comparable material.

biophysics↗

bioRxiv2024

A deoxynucleoside triphosphate triphosphohydrolase promotes cell cycle progression in Caulobacter crescentus

Intracellular pools of deoxynucleoside triphosphates (dNTPs) are strictly maintained throughout the cell cycle to ensure accurate and efficient DNA replication. DNA synthesis requires an abundance of dNTPs, but elevated dNTP concentrations in nonreplicating cells delay entry into S phase. Enzymes known as deoxyguanosine triphosphate triphosphohydrolases (Dgts) hydrolyze dNTPs into deoxynucleosides and triphosphates, and we propose that Dgts restrict dNTP concentrations to promote the G1 to S phase transition. We characterized a Dgt from the bacterium Caulobacter crescentus termed flagellar signaling suppressor C (fssC) to clarify the role of Dgts in cell cycle regulation. Deleting fssC increases dNTP levels and extends the G1 phase of the cell cycle. We determined that the segregation and duplication of the origin of replication (oriC) is delayed in {Delta}fssC, but the rate of replication elongation is unchanged. We conclude that dNTP hydrolysis by FssC promotes the initiation of DNA replication through a novel nucleotide signaling pathway. This work further establishes Dgts as important regulators of the G1 to S phase transition, and the high conservation of Dgts across all domains of life implies that Dgt-dependent cell cycle control may be widespread in both prokaryotic and eukaryotic organisms. ImportanceCells must faithfully replicate their genetic material in order to proliferate. Studying the regulatory pathways that determine when a cell initiates DNA replication is important for understanding fundamental biological processes, and it can also improve the strategies used to treat diseases that affect the cell cycle. Here, we describe a nucleotide signaling pathway that regulates when cells will begin DNA replication. We show that this pathway promotes the transition from the G1 to the S phase of the cell cycle in the bacterium Caulobacter crescentus and propose that this pathway is prevalent in all domains of life.

microbiology↗

bioRxiv2023

A novel exopolysaccharide pathway from a freshwater Sphingomonas isolate

Bacteria embellish their cell envelopes with a variety of specialized polysaccharides. Biosynthesis pathways for these glycans are complex, and final products vary greatly in their chemical structures, physical properties and biological activities. This tremendous diversity comes from the ability to arrange complex pools of monosaccharide building blocks into polymers with many possible linkage configurations. Due to the complex chemistry of bacterial glycans, very few biosynthetic pathways have been defined in detail. To better understand the breadth of polysaccharide production in nature we isolated a bacterium from Lake Michigan called Sphingomonas sp. LM7 that is proficient in exopolysaccharide (EPS) production. We identified genes that contribute to EPS biosynthesis in LM7 by screening a transposon mutant library for colonies displaying altered colony morphology. A gene cluster was identified that appears to encode a complete wzy/wzx-dependent polysaccharide assembly pathway. Deleting individual genes in this cluster caused a non-mucoid phenotype and a corresponding loss of EPS secretion, confirming that LM7 assembles a novel wzy/wzx-dependent polysaccharide. We extracted EPS from LM7 cultures and showed that it contains a linear chain of 3- and 4-linked glucose, galactose, and glucuronic acid residues. Finally, we found that the EPS pathway we identified diverges from those of adhesive polysaccharides such as the holdfast that are conserved in higher Alphaproteobacteria. Our approach of characterizing complete biosynthetic pathways holds promise for engineering of polysaccharides with valuable properties. ImportanceBacteria produce complex polysaccharides that serve a range of biological functions. These polymers often have properties that make them attractive for industrial applications, but they remain woefully underutilized. In this work, we studied a novel polysaccharide called promonan that is produced by Sphingomonas sp. LM7, a bacterium we isolated from Lake Michigan. We extracted promonan from LM7 cultures and identified which sugars are present in the polymer. We also identified the genes responsible for polysaccharide production. Comparing the promonan genes to those of other bacteria showed that promonan is distinct from previously characterized polysaccharides. We conclude by discussing how the promonan pathway could be used to produce new polysaccharides through genetic engineering.

microbiology↗

bioRxiv2020

Flagellar perturbations activate adhesion through two distinct pathways in Caulobacter crescentus

Bacteria carry out sophisticated developmental programs to colonize exogenous surfaces. The rotary flagellum, a dynamic machine that drives motility, is a key regulator of surface colonization. The specific signals recognized by flagella and the pathways by which those signals are transduced to coordinate adhesion remain subjects of debate. Mutations that disrupt flagellar assembly in the dimorphic bacterium Caulobacter crescentus stimulate the production of a polysaccharide adhesin called the holdfast. Using a genome-wide phenotyping approach, we compared surface adhesion profiles in wild-type and flagellar mutant backgrounds of C. crescentus. We identified a diverse set of flagellar mutations that enhance adhesion by inducing a hyper-holdfast phenotype and discovered a second set of mutations that suppress this phenotype. Epistasis analysis of the flagellar signaling suppressor (fss) mutations demonstrated that the flagellum stimulates holdfast production via two genetically distinct pathways. The developmental regulator PleD contributes to holdfast induction in mutants disrupted at both early and late stages of flagellar assembly. Mutants disrupted at late stages of flagellar assembly, which assemble an intact rotor complex, induce holdfast production through an additional process that requires the MotAB stator and its associated diguanylate cyclase, DgcB. We have assigned a subset of the fss genes to either the stator- or pleD-dependent networks and characterized two previously unidentified motility genes that regulate holdfast production via the stator complex. We propose a model through which the flagellum integrates mechanical stimuli into the C. crescentus developmental program to coordinate adhesion. ImportanceUnderstanding how bacteria colonize solid surfaces is of significant clinical, industrial and ecological importance. In this study, we identified genes that are required for Caulobacter crescentus to activate surface attachment in response to signals from a macromolecular machine called the flagellum. Genes involved in transmitting information from the flagellum can be grouped into separate pathways, those that control the C. crescentus morphogenic program and those that are required for flagellar motility. Our results support a model in which a developmental and a mechanical signaling pathway operate in parallel downstream of the flagellum and converge to regulate adhesion. We conclude that the flagellum serves as a signaling hub by integrating internal and external cues to coordinate surface colonization and emphasize the role of signal integration in linking complex sets of environmental stimuli to individual behaviors.

microbiology↗

bioRxiv2020

Discovering the Molecular Determinants of Phaeobacter inhibens susceptibility to Phaeobacter phage MD18

Bacteriophage technologies have immense potential as antibiotic therapies and in genetic engineering. Understanding the mechanisms that bacteriophages implement to infect their hosts will allow researchers to manipulate these systems and adapt them to specific bacterial targets. Here, we isolated a bacteriophage capable of infecting the marine alphaproteobacterium Phaeobacter inhibens and dissected its mechanism of infection. Phaeobacter phage MD18, a novel species of bacteriophage isolated in Woods Hole, MA, exhibits potent lytic ability against P. inhibens and appears to be of the Siphoviridae morphotype. Consistent with this finding, the sequence of the MD18 revealed significant similarity to another siphophage, the recently discovered Roseobacter phage DSS3P8. We incubated MD18 with a library of barcoded P. inhibens transposon insertion mutants and identified 22 genes that appear to be required for phage predation of this host. Network analysis of these genes using genomic position, GO term enrichment, and protein associations reveals that these genes are enriched for roles in assembly of a type IV pilus (T4P) and regulators of cellular morphology. Our results suggest that T4P serve as receptors for a novel marine virus that targets P. inhibens. ImportanceBacteriophages are useful non-antibiotic therapeutics for bacterial infections as well as threats to industries utilizing bacterial agents. This study identifies Phaeobacter phage MD18, the first documented phage of Phaeobacter inhibens, a bacterium with promising use as a probiotic for aquatic farming industries. Genomic analysis suggests that the P haeobacter phage MD18 has evolved to enhance its replication in P. inhibens by adopting favorable tRNA genes as well as through genomic sequence adaptation to resemble host codon usage. Lastly, a high-throughput analysis of P. inhibens transposon insertion mutants identifies genes that modulate host susceptibility to phage MD18 and implicates the type IV pilus as the likely receptor recognized for adsorption. This study marks the first characterization of the relationship between P. inhibens and an environmentally sampled phage, which informs our understanding of natural threats to the bacterium and may promote the development of novel phage technologies for genetic manipulation of this host.

microbiology↗

bioRxiv2019

Composition of the holdfast polysaccharide from Caulobacter crescentus

Surface colonization is central to the lifestyles of many bacteria. Exploiting surface niches requires sophisticated systems for sensing and attaching to solid materials. Caulobacter crescentus synthesizes a polysaccharide-based adhesin known as the holdfast at one of its cell poles, which enables tight attachment to exogenous surfaces. The genes required for holdfast biosynthesis have been analyzed in detail, but an inability to isolate sufficient quantities of holdfast has limited efforts to characterize its composition and structure. In this report we describe a method to extract the holdfast from C. crescentus cultures and present a survey of its carbohydrate content. Glucose, 3-O-methylglucose, mannose, N-acetylglucosamine and xylose were detected in our extracts. Our results suggest that the holdfast contains a 1,4-linked backbone of glucose, mannose, N-acetylglucosamine and xylose that is decorated with branches at the C-6 positions of glucose and mannose. By defining the monosaccharide components in the polysaccharide, our work establishes a framework for characterizing enzymes in the holdfast pathway and provides a broader understanding of how polysaccharide adhesins are built.

microbiology↗

bioRxiv2018

A genome-wide analysis of adhesion in Caulobacter crescentus identifies new regulatory and biosynthetic components for holdfast assembly

Due to their intimate physical interactions with the environment, surface polysaccharides are critical determinants of fitness for bacteria. Caulobacter crescentus produces a specialized structure at one of its cell poles called the holdfast that enables attachment to surfaces. Previous studies have shown that the holdfast is a carbohydrate-based material and identified a number of genes required for holdfast development. However, incomplete information about its chemical structure, biosynthetic genes and regulatory principles has limited progress in understanding the mechanism of holdfast synthesis. We have leveraged the adhesive properties of the holdfast to perform a saturating screen for genes affecting attachment to cheesecloth over a multi-day time course. Using similarities in the temporal profiles of mutants in a transposon library, we defined discrete clusters of genes with related effects on cheesecloth colonization. Holdfast synthesis, flagellar motility, type IV pilus assembly and smooth lipopolysaccharide (SLPS) production represented key classes of adhesion determinants. Examining these clusters in detail allowed us to predict and experimentally define the functions of multiple uncharacterized genes in both the holdfast and SLPS pathways. In addition, we showed that the pilus and flagellum control holdfast synthesis separately by modulating the holdfast inhibitor hfiA. This study defines a set of genes contributing to adhesion that includes newly discovered genes required for holdfast biosynthesis and attachment. Our data provide evidence that the holdfast contains a complex polysaccharide with at least four monosaccharides in the repeating unit and underscore the central role of cell polarity in mediating attachment of C. crescentus to surfaces.\n\nImportanceBacteria routinely encounter biotic and abiotic materials in their surrounding environments, and they often enlist specific behavioral programs to colonize these materials. Adhesion is an early step in colonizing a surface. Caulobacter crescentus produces a structure called the holdfast, which allows this organism to attach to and colonize surfaces. To understand how the holdfast is produced, we performed a genome-wide search for genes that contribute to adhesion by selecting for mutants that could not attach to cheesecloth. We discovered complex interactions between genes that mediate surface contact and genes that contribute to holdfast development. Our genetic selection identified what likely represents a comprehensive set of genes required to generate a holdfast, laying the groundwork for a detailed characterization of the enzymes that build this specialized adhesin.

microbiology↗