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Fuqua, C.

Publications and source records attributed to Fuqua, C..

11 recordsLinked to original sources
bioRxiv2026

Complex roles for a conserved two-component system in a honey bee symbiosis

Elucidating how microbes associate with their hosts and respond to different environments is fundamental to our understanding of symbiosis. Here, we investigate the molecular and genetic mechanisms enabling the honey bee symbiont Bombella apis to colonize and persist in harsh honey bee microenvironments, like royal jelly and the larval gut. By using evolved B. apis strains, we demonstrate that the broadly conserved two-component system (TCS) ChvGI is integral for the bacteriums ability to withstand royal jelly and the larval gut. Furthermore, we show that in B. apis ChvGI also regulates critical bacterial behaviors such as motility, flagellar production, and biofilm formation. In other Alphaproteobacteria, ChvGI is known as a master regulator that mediates the bacterial responses to stressors and is also found to be involved in symbiosis, often through the production of EPS and biofilm formation. Our work underscores the broader significance of ChvGI as an important TCS in Alphaproteobacteria that regulates bacterial behavior in response to different types of stressors and uncovers a genetic mechanism that allows an important honey bee symbiont to survive in diverse host environments.

microbiology↗
bioRxiv2026

Lipopolysaccharide Precursor Mutants Disrupt Unipolar Polysaccharide Adhesin Synthesis and Cell Surface Functions in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a facultative phytopathogen and causative agent of crown gall disease, capable of attaching to surfaces via a polarly localized unipolar polysaccharide (UPP) adhesin. The UPP is composed of two distinct forms, one characterized by N-acetylglucosamine residues (UPPGlcN) and the other by N-acetylgalactosamine residues (UPPGalN). Two genes with presumptive roles in dTDP-L-rhamnose biosynthesis, rfbA and rfbD, were identified in a transposon screen to be involved specifically in UPPGalN biosynthesis. Examination of independent rfbA and rfbD mutations validates the transposon mutant phenotypes, with UPPGalN specific defects observed as well as general attachment defects and UPP-mediated cellular aggregation across all rfb mutants. Additionally, despite retaining flagella, mutations in the rfb genes impart flagellar motility defects. Suppressor mutations that rescue the non-motile {Delta}rfbD phenotype disrupt the remaining rfb genes (rfbA, rfbB, and rfbC), the phosphoglucomutase exoC and a putative glycosyl transferase ATU-RS21610. Single deletion mutants in the dTDP-L-rhamnose pathway also have hallmarks of compromised outer membrane integrity, such as increased sensitivity to high-molecular weight antibiotics and increased expression of target genes for the widely conserved ChvG-ChvI two component system outer membrane stress response. Thus, defects in the dTDP-L-rhamnose pathway cause multiple cell surface deficiencies, resulting in outer membrane stress, impaired flagellar motility, defective UPP production, and irregular adhesion. ImportssssanceBiofilms are clinically and industrially relevant in many different contexts, with substantial health impacts and monetary costs. Surface attachment and motility are essential components necessary for bacterial biofilm formation. Many microbes utilize specific surface appendages or structures to facilitate attachment and motility. This study probes the process of surface attachment and subsequent biofilm formation in A. tumefaciens and has revealed connections between the creation of polysaccharide precursors required for biofilm formation and outer membrane functions such as motility and membrane integrity. These findings broaden our understanding of the complex interactions between outer membrane surface functions and how disruption of relevant precursor pools can have multifaceted impacts that may represent useful targets for new antimicrobial approaches.

microbiology↗
bioRxiv2025

Linear dicentric chromosomes in bacterial natural isolates reveal common constraints for replicon fusion

Multipartite bacterial genome organization can confer advantages including coordinated gene regulation and faster genome replication but is challenging to maintain. Agrobacterium tumefaciens lineages often contain a circular chromosome (Ch1), a linear chromosome (Ch2), and multiple plasmids. We previously observed that in some stocks of the lab model strain C58, Ch1 and Ch2 were fused into a linear dicentric chromosome. Here we analyzed Agrobacterium natural isolates from the French Collection for Plant-Associated Bacteria (CFBP) and identified two strains with fused chromosomes. Chromosome conformation capture identified integration junctions that were different from the C58 fusion strain. Genome-wide DNA replication profiling showed both replication origins remain active. Transposon sequencing revealed that partitioning systems of both chromosome centromeres are essential. Importantly, the site-specific recombinases XerCD are required for the survival of the strains containing the fusion chromosome. Our findings show that replicon fusion occurs in natural environments and that balanced replication arm sizes and proper resolution systems enable the survival of such strains. ImportanceMost bacterial genomes are monopartite with a single, circular chromosome. But some species, like Agrobacterium tumefaciens, carry multiple chromosomes. Emergence of multipartite genomes is often related to adaptation to specific niches including pathogenesis or symbiosis. Multipartite genomes confer certain advantages, however, maintaining this complex structure can present significant challenges. We previously reported a laboratory-propagated lineage of A. tumefaciens strain C58 in which the circular and linear chromosomes fused to form a single dicentric chromosome. Here we discovered two environmental isolates of A. tumefaciens containing fused chromosomes derived from a different route, revealing the constraints and diversification of this process. We found that balanced replication arm sizes and the repurposing of multimer resolution systems enable the survival and stable maintenance of dicentric chromosomes. These findings help us better understand how multipartite genomes function across different bacterial species and the role of genomic plasticity in bacterial genetic diversification.

microbiology↗
bioRxiv2023

Control of Biofilm Formation by an Agrobacterium tumefaciens Pterin-Binding Periplasmic Protein Conserved Among Pathogenic Bacteria

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (cdGMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase (DGC-PDE), is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are formed via a non-essential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering cdGMP breakdown and dampening its synthesis. Pterins are excreted and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon that is conserved amongst multiple Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a new pterin-responsive regulatory mechanism that controls biofilm formation and related cdGMP-dependent phenotypes in A. tumefaciens and is found in multiple additional bacterial pathogens. SIGNIFICANCEBiofilms are bacterial communities attached to surfaces, physiologically distinct from free-living cells, and a common cause of persistent infections. Here we define the mechanism of a novel biofilm regulatory system based on excreted metabolites called pterins, that is conserved within a wide range of Gram-negative bacteria, including multiple pathogens of animals and plants. The molecular mechanism of pterin-dependent regulation is reported including structural determination of several members of a new family of pterin-binding proteins. Pterins are produced across all domains of life and mechanistic insights into this regulatory circuit could lead to new advances in antibiofilm treatments.

microbiology↗
bioRxiv2021

Motility control through an anti-activation mechanism in Agrobacterium tumefaciens

Many bacteria can migrate from a free-living, planktonic state to an attached, biofilm existence. One factor regulating this transition in the facultative plant pathogen Agrobacterium tumefaciens is the ExoR-ChvG-ChvI system. Periplasmic ExoR regulates activity of the ChvG-ChvI two-component system in response to environmental stress, most notably low pH. ChvI impacts hundreds of genes, including those required for type VI secretion, virulence, biofilm formation, and flagellar motility. Previous studies revealed that activated ChvG-ChvI represses expression of most of class II and class III flagellar biogenesis genes, but not the master motility regulators visN, visR, and rem. In this study, we characterized the integration of the ExoR-ChvG-ChvI and VisNR-Rem pathways. We isolated motile suppressors of the non-motile {Delta}exoR mutant and thereby identified the previously unannotated mirA gene encoding a 76 amino acid protein. We report that the MirA protein interacts directly with the Rem DNA-binding domain, sequestering Rem and preventing motility gene activation. The ChvG-ChvI pathway activates mirA expression and elevated mirA is sufficient to block motility. This study reveals how the ExoR-ChvG-ChvI pathway prevents flagellar motility in A. tumefaciens. MirA is also conserved among other members of the Rhizobiales suggesting similar mechanisms of motility regulation. Plain language summaryAgrobacterium tumefaciens is a plant pathogen that responds to low pH, stressful environments via a complex two-component regulatory system. This regulatory system can completely inhibit bacterial locomotion by flagella, and in this study we define the mechanism for this inhibition and identify a novel small protein regulator that blocks flagellar gene expression.

microbiology↗
bioRxiv2021

Dual adhesive unipolar polysaccharides synthesized by overlapping biosynthetic pathways in Agrobacterium tumefaciens

Agrobacterium tumefaciens, is a member of the Alphaproteobacteria that pathogenizes plants, and associates with biotic and abiotic surfaces via a single cellular pole. A. tumefaciens produces the unipolar polysaccharide (UPP) at the site of surface contact. UPP production is normally surface-contact inducible, but elevated levels of the second messenger cyclic diguanylate monophosphate (cdGMP) bypass this requirement. Multiple lines of evidence suggest that the UPP has a central polysaccharide component. Using an A. tumefaciens derivative with elevated cdGMP and mutationally disabled for other dispensable polysaccharides, a series of related genetic screens have identified a large number of genes involved in UPP biosynthesis, most of which are Wzx-Wzy-type polysaccharide biosynthetic components. Extensive analyses of UPP production in these mutants have revealed that the UPP is comprised of two genetically, chemically and spatially discrete forms of polysaccharide, and that each requires a specific Wzy-type polymerase. Other important biosynthetic, processing and regulatory functions for UPP production are also revealed, some of which are common to both polysaccharides, and a subset of which are specific to each species. Many of the UPP genes identified are conserved among diverse rhizobia, whereas others are more lineage specific. Plain language summaryBacteria attach to a wide variety of surfaces including host tissues, via externalized structures described as adhesins. We define a large set of genes involved in synthesis of a complex unipolar adhesin comprised of two distinct polysaccharides, that is required for surface attachment by the plant-associated pathogen Agrobacterium tumefaciens.

microbiology↗
bioRxiv2020

Co-Dependent and Interdigitated: Dual Quorum Sensing Systems Regulate Conjugative Transfer of the Ti Plasmid and the At Megaplasmid in Agrobacterium tumefaciens 15955

Members of the Rhizobiaceae, often carry multiple secondary replicons in addition to the primary chromosome with compatible repABC-based replication systems. Unlike secondary chromosomes and chromids, repABC-based megaplasmids and plasmids can undergo copy number fluctuations and are capable of conjugative transfer in response to environmental signals. Several Agrobacterium tumefaciens lineages harbor three secondary repABC-based replicons, including a secondary chromosome (often linear), the Ti (tumor-inducing) plasmid and the At megaplasmid. The Ti plasmid is required for virulence and encodes a conjugative transfer (tra) system that is strictly regulated by a subset of plant-tumor released opines and a well-described acyl-homoserine lactone (AHL)-based quorum-sensing mechanism. At plasmids are generally not required for virulence, but carry genes that enhance rhizosphere survival, and these plasmids are often conjugatively proficient. We report that the At megaplasmid of the octopine-type strain A. tumefaciens 15955 encodes a quorum-controlled conjugation system that directly interacts with the paralogous quorum sensing system on the co-resident Ti plasmid. Both the pAt15955 and pTi15955 plasmids carry homologues of a TraI-type AHL synthase, a TraR-type AHL-responsive transcription activator, and a TraM-type anti-activator. The traI genes from both pTi15955 and pAt15955 can direct production of the inducing AHL (3-octanoyl-L-homoserine lactone) and together contribute to the overall AHL pool. The TraR protein encoded on each plasmid activates AHL-responsive transcription of target tra gene promoters. The pAt15955 TraR can cross-activate tra genes on the Ti plasmid as strongly as its cognate tra genes, whereas the pTi15955 TraR preferentially biased towards its own tra genes. Putative tra box elements are located upstream of target promoters, and comparing between plasmids, they are in similar locations and share an inverted repeat structure, but have distinct consensus sequences. The two AHL quorum sensing systems have a combinatorial effect on conjugative transfer of both plasmids. Overall, the interactions described here have implications for the horizontal transfer and evolutionary stability of both plasmids and, in a broad sense, are consistent with other repABC systems that often have multiple quorum-sensing controlled secondary replicons. CONTRIBUTION TO THE FIELDMany bacteria harbor multiple types of plasmids, and in a fraction of these the plasmids encode independent conjugative transfer systems. In the plant pathogen Agrobacterium tumefaciens, conjugation of the virulence plasmid, called the Ti (tumor-inducing) plasmid, is regulated by plant released signals called opines, and by a well characterized quorum sensing mechanism also encoded on the plasmid. The co-resident At megaplasmid also has its own conjugative transfer system. In certain lineages of A. tumefaciens the At plasmid carries a discrete quorum sensing system. This study aimed to determine the extent to which these plasmid-borne quorum sensing systems overlap to co-regulate conjugative transfer of both plasmids. Co-resident plasmids can impact each other, and the host bacterium at multiple levels, and for the Ti plasmid and At plasmid, we find that each plasmid clearly can influence the conjugative transfer systems of the other plasmid. The findings reported also shed light on how bacteria with multiple quorum sensing pathways have evolved to integrate these systems, in this case to control horizontal gene transfer of two different plasmids. Overall, the interactions described here have broader implications for the horizontal transfer and evolutionary stability of co-resident plasmids, and the degree to which their regulatory systems are coordinated.

microbiology↗
bioRxiv2019

Destabilization of the tumor-inducing plasmid from an octopine-type Agrobacterium tumefaciens lineage drives a large deletion in the co-resident At megaplasmid

Bacteria with multi-replicon genome organizations, including members of the family Rhizobiaceae, often carry a variety of niche-associated functions on large plasmids. While evidence exists for cross-replicon interactions and co-evolution between replicons in many of these systems, remarkable strain-to-strain variation is also observed for extrachromosomal elements, suggesting increased genetic plasticity. Here, we show that curing of the tumor-inducing virulence plasmid (pTi) of an octopine-type Agrobacterium tumefaciens lineage leads to a large deletion in the co-resident At megaplasmid (pAt). The deletion event is mediated by a repetitive IS-element, IS66, and results in a variety of environment-dependent fitness consequences, including loss of independent conjugal transfer of the plasmid. Interestingly, a related and otherwise wild-type A. tumefaciens strain is missing exactly the same large pAt segment as the pAt deletion derivatives, suggesting a similar event over its natural history. Overall, the findings presented here uncover a novel genetic interaction between the two large plasmids of A. tumefaciens and provide evidence for cross-replicon integration and co-evolution of these plasmids.

microbiology↗
bioRxiv2018

Multiple Flagellin Proteins Have Distinct and Synergistic Roles in Agrobacterium tumefaciens Motility

Rotary flagella propel bacteria through liquid and across semi-solid environments. Flagella are composed of the basal body that constitutes the motor for rotation, the curved hook that connects to the basal body, and the flagellar filament that propels the cell. Flagellar filaments can be comprised of a single flagellin protein such as in Escherichia coli or with multiple flagellins such is in Agrobacterium tumefaciens. The four distinct flagellins FlaA, FlaB, FlaC and FlaD produced by wild type A. tumefaciens, are not redundant in function, but have specific properties. FlaA and FlaB are much more abundant than FlaC and FlaD and are readily observable in mature flagellar filaments, when either FlaA or FlaB is fluorescently labeled. Cells having FlaA with any one of the other three flagellins can generate functional filaments and thus are motile, but FlaA alone cannot constitute a functional filament. In flaA mutants that manifest swimming deficiencies, there are multiple ways by which these mutations can be phenotypically suppressed. These suppressor mutations primarily occur within or upstream of the flaB flagellin gene or in the transcriptional factor sciP regulating flagellar expression. The helical conformation of the flagellar filament appears to require a key asparagine residue present in FlaA and absent in other flagellins. However, FlaB can be spontaneously mutated to render helical flagella in absence of FlaA, reflecting their overall similarity and perhaps the subtle differences in the specific functions they have evolved to fulfill.\n\nImportanceFlagellins are abundant bacterial proteins comprising the flagellar filaments that propel bacterial movement. Several members of the Alphaproteobacterial group express multiple flagellins, in contrast to model systems such as Escherichia coli that has only one flagellin protein. The plant pathogen Agrobacterium tumefaciens has four flagellins, the abundant and readily detected FlaA and FlaB, and lower levels of FlaC and FlaD. Mutational analysis reveals that FlaA requires at least one of the other flagellins to function - flaA mutants produce non-helical flagella and cannot swim efficiently. Suppressor mutations can rescue this swimming defect through mutations in the remaining flagellins, including structural changes imparting flagellar helical shape, and putative regulators. Our findings shed light on how multiple flagellins contribute to motility.

microbiology↗
bioRxiv2017

Reciprocal control of motility and biofilm formation by the PdhS2 two-component sensor kinase of Agrobacterium tumefaciens

A core regulatory pathway that directs developmental transitions and cellular asymmetries in Agrobacterium tumefaciens involves two overlapping, integrated phosphorelays. One of these phosphorelays putatively includes four histidine sensor kinase homologues, DivJ, PleC, PdhS1, and PdhS2, and two response regulators, DivK and PleD. In several different alphaproteobacteria, this pathway influences a conserved downstream phosphorelay that ultimately controls the phosphorylation state of the CtrA master response regulator. The PdhS2 sensor kinase reciprocally regulates biofilm formation and swimming motility. In the current study the mechanisms by which the A. tumefaciens sensor kinase PdhS2 directs this regulation are delineated. PdhS2 lacking a key residue for phosphatase activity is markedly deficient in proper control of attachment and motility phenotypes, whereas a kinase-deficient PdhS2 mutant is only modestly affected. A genetic interaction between DivK and PdhS2 is revealed, unmasking one of several connections between PdhS2-dependent phenotypes and transcriptional control by CtrA. Epistasis experiments suggest that PdhS2 can function independently of the CckA sensor kinase, the cognate sensor kinase for CtrA which is inhibited by DivK. PdhS2 dynamically localizes to the daughter cell pole in dividing cells. Global expression analysis of the pdhS2 mutant reveals a restricted regulon, functioning through CtrA to separately control motility and regulate levels of the intracellular signal cyclic diguanylate monophosphate (cdGMP), thereby affecting production of adhesive polysaccharides and attachment. We hypothesize that in A. tumefaciens the CtrA regulatory circuit has expanded to include additional inputs through addition of PdhS-type sensor kinases, likely fine-tuning the response of this organism to the soil microenvironment.\n\nIMPORTANCEBacterial developmental processes, such as morphological transformations and behavioral transitions, are tightly regulated. In many alphaproteobacteria cell division and development are coordinated by a specific suite of conserved histidine kinases and their partnered regulatory proteins. Here we describe how the histidine kinase PdhS2 of Agrobacterium tumefaciens regulates complex phenotypes including biofilm formation and motility. PdhS2 genetically interacts with a single-domain response regulator, DivK, and the intracellular signal cyclic diguanylate monophosphate. PdhS2 dynamically localizes to the new pole of recently divided cells, contributing to the regulatory processes that dictate whether these cells remain motile or initiate biofilm formation. These findings expand our understanding of the complex network that integrates cell division and developmental control in A. tumefaciens and related alphaproteobacteria.

microbiology↗