Biology subjects

Septer, A. N.

Publications and source records attributed to Septer, A. N..

10 recordsLinked to original sources

bioRxiv2025

Differential metaproteomics of bacteria grown in vitro and in planta reveals functions used during growth on maize roots

Microbes are ubiquitous in the rhizosphere and play crucial roles in plant health, yet the metabolisms and physiologies of individual species in planta remain poorly understood. In this study, we examined microbial gene expression in response to the maize root environment for seven bacterial species originally isolated from maize roots. We grew each species individually, both in vitro in a minimal medium and in planta, and used differential proteomics to identify functions upregulated specifically when bacteria are grown on maize roots. We identified between 1,500 and 2,100 proteins from each species, with 20-60% of these proteins being differentially abundant between the two conditions. While we found that transporter proteins were upregulated in all species in planta, all other differentially abundant functions varied greatly between species, suggesting niche specialization in root-associated microbes. Indeed, in vitro assays confirmed that Curtobacterium pusillum likely degrades plant hemicellulose, Enterobacter ludwigii may benefit the plant by phosphate solubilization, and Herbaspirillum robiniae colonizes maize roots more effectively when both of its Type VI Secretion Systems are functional. Together, our findings highlight both conserved and species-specific bacterial strategies for growth in the root environment and lay a foundation for future work investigating the mechanisms underlying plant-microbiota interactions. ImportanceBacteria that live on and around plant roots are important for plant growth and health, yet we still know relatively little about how individual bacterial species behave in this environment. In this study, we looked at seven bacterial species originally isolated from maize roots to understand how they change their metabolism and physiology when grown on the plant versus when grown under laboratory conditions. By doing this, we identified key strategies bacteria use to survive and thrive in the root environment, including changes in nutrient uptake, metabolism, and secretion systems. We also substantiated some of these behaviors using lab experiments and bacterial mutants. Understanding these species-specific functions helps us learn how bacteria establish themselves on roots and interact with the plant. This knowledge is critical for future efforts to design effective microbial communities that improve crop performance and resilience.

microbiology↗

bioRxiv2025

H-NS is a conserved repressor of the type VI secretion system in Vibrio fischeri

The type VI secretion system (T6SS) is a broadly distributed interbacterial weapon found in both beneficial and pathogenic bacteria and can enhance a microbes ability to colonize a host. Vibrio fischeri is a beneficial symbiont of fish and squid and a model organism for T6SS function, which is activated in high-viscosity conditions. Previously, we isolated an hns mutant in a transposon screen to identify regulators of the T6SS in the fish symbiont V. fischeri MJ11. The hns gene encodes the DNA-binding protein, H-NS, a conserved global regulator of gene expression that aids in adaptation to changing environments. Quantitative transcriptomes of the hns mutant and parent strains grown in liquid or hydrogel media revealed hns is required for the global transcriptional changes that occur during transition from lower to higher viscosity conditions. Furthermore, T6SS gene transcripts are more abundant in the hns mutant in both conditions, suggesting H-NS represses T6SS in the parent. Single-cell fluorescence microscopy confirmed hns mutant cells make more T6SS weapons in both liquid and hydrogel medium, where the hns mutant is more proficient at killing a competitor strain, compared to the wild-type parent. Finally, disrupting the hns gene in additional light organ isolates resulted in a similar derepression of T6SS, indicating H-NS is a conserved repressor of this interbacterial weapon. This work furthers our understanding of the role of H-NS as a global regulator during environmental shifts in a host-associated bacterial symbiont and expands the list of species where H-NS represses T6SS to include V. fischeri.

microbiology↗

bioRxiv2025

Euprymna berryi as a comparative model host for Vibrio fischeri light organ symbiosis

Functional studies of host-microbe interactions benefit from natural model systems that enable exploration of molecular mechanisms at the host-microbe interface. Bioluminescent Vibrio fischeri colonize the light organ of the Hawaiian bobtail squid, Euprymna scolopes, and this binary model has enabled advances in understanding host-microbe communication, colonization specificity, in vivo biofilms, intraspecific competition, and quorum sensing. The hummingbird bobtail squid, Euprymna berryi, can be generationally bred and maintained in lab settings and has had multiple genes deleted by CRISPR approaches. The prospect of expanding the utility of the light organ model system by producing multigenerational host lines led us to determine the extent to which the E. berryi light organ symbiosis parallels known processes in E. scolopes. However, the nature of the E. berryi light organ, including its microbial constituency and specificity for microbial partners, have not been examined. In this report, we isolate bacteria from E. berryi animals and tank water. Assays of bacterial behaviors required in the host, as well as host responses to bacterial colonization, illustrate largely parallel phenotypes in E. berryi and E. scolopes hatchlings. This study reveals E. berryi to be a valuable comparative model to complement studies in E. scolopes. IMPORTANCEMicrobiome studies have been substantially advanced by model systems that enable functional interrogation of the roles of the partners and the molecular communication between those partners. The Euprymna scolopes-Vibrio fischeri system has contributed foundational knowledge, revealing key roles for bacterial quorum sensing broadly and in animal hosts, for bacteria in stimulating animal development, for bacterial motility in accessing host sites, and for in vivo biofilm formation in development and specificity of an animals microbiome. Euprymna berryi is a second bobtail squid host, and one that has recently been shown to be robust to laboratory husbandry and amenable to gene knockout. This study identifies E. berryi as a strong symbiosis model host due to features that are conserved with those of E. scolopes, which will enable extension of functional studies in bobtail squid symbioses.

microbiology↗

bioRxiv2024

The conserved global regulator H-NS has a strain-specific impact on biofilm formation in Vibrio fischeri symbionts

Strain-level variation among host-associated bacteria often determines host range and the extent to which colonization is beneficial, benign, or pathogenic. Vibrio fischeri is a beneficial symbiont of the light organs of fish and squid with known strain-specific differences that impact host specificity, colonization efficiency, and interbacterial competition. Here, we describe how the conserved global regulator, H-NS, has a strain-specific impact on a critical colonization behavior: biofilm formation. We isolated a mutant of the fish symbiont V. fischeri MJ11 with a transposon insertion in the hns gene. This mutant formed sticky, moderately wrinkled colonies on LBS plates, a condition not known to induce biofilm in this species. A reconstructed hns mutant displayed the same wrinkled colony, which became smooth when hns was complemented in trans, indicating the hns disruption is causal for biofilm formation in MJ11. Transcriptomes revealed differential expression for the syp biofilm locus in the hns mutant, relative to the parent, suggesting biofilm may in part involve SYP polysaccharide. However, enhanced biofilm in the MJ11 hns mutant was not sufficient to allow colonization of a non-native squid host. Finally, moving the hns mutation into other V. fischeri strains, including the squid symbionts ES114 and ES401, and seawater isolate PP3, revealed strain-specific biofilm phenotypes: ES114 and ES401 hns mutants displayed minimal biofilm phenotypes while PP3 hns mutant colonies were more wrinkled than the MJ11 hns mutant. These findings together define H-NS as a novel regulator of V. fischeri symbiotic biofilm and demonstrate key strain specificity in that role. ImportanceThis work, which shows how H-NS has strain-specific impacts on biofilm in Vibrio fischeri, underscores the importance of studying multiple strains, even when examining highly conserved genes and functions. Our observation that knocking out a conserved regulator can result in a wide range of biofilm phenotypes, depending on the isolate, serves as a powerful reminder that strain-level variation is common and worthy of exploration. Indeed, uncovering the mechanisms of strain-specific phenotypic differences is essential to understand drivers of niche differentiation and bacterial evolution. Thus, it is important to carefully match the number and type of strains used in a study with the research question to accurately interpret and extrapolate the results beyond a single genotype. The additional work required for multi-strain studies is often worth the investment of time and resources, as it provides a broader view of the complexity of within-species diversity in microbial systems.

microbiology↗

bioRxiv2024

A mutualistic model bacterium is lethal to non-symbiotic hosts via the type VI secretion system

What makes a bacterium pathogenic? Since the early days of germ theory, researchers have categorized bacteria as pathogens or non-pathogens, those that cause harm and those that do not, but this binary view is not always accurate. Vibrio fischeri is an exclusive mutualistic symbiont found within the light organs of Hawaiian bobtail squid. This symbiotic interaction requires V. fischeri to utilize a range of behaviors and produce molecules that are often associated with pathogenicity. This juxtaposition of employing "pathogenic" behaviors for a symbiotic relationship led the field to focus on how V. fischeri establishes a beneficial association with its host. In this study, we observe that V. fischeri induces mortality in zebrafish embryos and Artemia nauplii. Non-lethal doses of V. fischeri leads to zebrafish growth delays and phenotypes indicative of disease. Our data also provide evidence that the conserved type VI secretion system on chromosome I (T6SS1) plays a role in the V. fischeri-induced mortality of zebrafish embryos and Artemia nauplii. These results support the hypothesis that the V. fischeri T6SS1 is involved in eukaryotic cell interactions. Despite its traditional view as a beneficial symbiont, we provide evidence that V. fischeri is capable of harming aquatic organisms, indicating its potential to be pathogenic toward non-symbiotic hosts.

microbiology↗

bioRxiv2024

Flagella are required to coordinately activate competition and host colonization factors in response to a mechanical signal

Bacteria employ antagonistic strategies to eliminate competitors of an ecological niche. Contact-dependent mechanisms, such as the type VI secretion system (T6SS), are prevalent in host-associated bacteria, yet we know relatively little about how T6SS+ strains make contact with competitors in highly viscous environments, such as host mucus. To better understand how cells respond to and contact one another in such environments, we performed a genome-wide transposon mutant screen of the T6SS-wielding beneficial bacterial symbiont, Vibrio fischeri, and identified two sets of genes that are conditionally required for killing. LPS/capsule and flagellar-associated genes do not affect T6SS directly and are therefore not required for interbacterial killing when cell contact is forced yet are necessary for killing in high-viscosity liquid (hydrogel) where cell-cell contact must be biologically mediated. Quantitative transcriptomics revealed that V. fischeri significantly increases expression of both T6SS genes and cell surface modification factors upon transition from low-to high-viscosity media. Consistent with coincubation and fluorescence microscopy data, flagella are not required for T6SS expression in hydrogel. However, flagella play a key role in responding to the physical environment by promoting expression of the surface modification genes identified in our screen, as well as additional functional pathways important for host colonization including uptake of host-relevant iron and carbon sources, and nitric oxide detoxification enzymes. Our findings suggest that flagella may act as a mechanosensor for V. fischeri to coordinately activate competitive strategies and host colonization factors, underscoring the significance of the physical environment in directing complex bacterial behaviors. SignificanceThe physical environment can have dramatic effects on bacterial behavior, but little is known about how mechanical signals impact antagonistic interactions. Symbiotic bacteria use molecular weapons to eliminate competitors for limited space within highly viscous host tissue and mucus. To better understand how the physical environment affects competition and adhesion within eukaryotic hosts, we used quantitative transcriptomics to reveal the flagella-dependent transcriptional response to bacterial transition from lower to a higher viscosity environment. This work revealed the T6SS interbacterial weapon is coordinately activated with host colonization factors, emphasizing the importance of integrating activation of interbacterial weapons into host colonization pathways to enhance a symbionts ability to successfully colonize the host while efficiently eliminating potential competitors from the host niche.

microbiology↗

bioRxiv2023

The Vibrio fischeri type VI secretion system incurs a fitness cost under host-like conditions

The type VI secretion system (T6SS) is an interbacterial weapon composed of thousands of protein subunits and predicted to require significant cellular energy to deploy, yet a fitness cost from T6SS use is rarely observed. Here, we identify host-like conditions where the T6SS incurs a fitness cost using the beneficial symbiont, Vibrio fischeri, which uses its T6SS to eliminate competitors in the natural squid host. We hypothesized that a fitness cost for the T6SS could be dependent on the cellular energetic state and used theoretical ATP cost estimates to predict when a T6SS-dependent fitness cost may be apparent. Theoretical energetic cost estimates predicted a minor relative cost for T6SS use in fast-growing populations (0.4-0.45% of total ATP used cell-1), and a higher relative cost (3.1-13.6%) for stationary phase cells. Consistent with these predictions, we observed no significant T6SS-dependent fitness cost for fast-growing populations typically used for competition assays. However, the stationary phase cell density was significantly lower in the wild-type strain, compared to a regulator mutant that does not express the T6SS, and this T6SS-dependent fitness cost was between 11 and 23%. Such a fitness cost could influence the prevalence and biogeography of T6SSs in animal-associated bacteria. While the T6SS may be required in kill or be killed scenarios, once the competitor is eliminated there is no longer selective pressure to maintain the weapon. Our findings indicate an evolved genotype lacking the T6SS would have a growth advantage over its parent, resulting in the eventual dominance of the unarmed population.

microbiology↗

bioRxiv2021

A subcellular biochemical model for T6SS dynamics reveals winning competitive strategies

The Type VI secretion system (T6SS) is a broadly distributed interbacterial weapon that can be used to eliminate competing bacterial populations. Although unarmed target populations are typically used to study T6SS function, bacteria most likely encounter other T6SS-armed competitors in nature. The outcome of such battles is not well understood, neither is the connection between the outcomes with the subcellular details of the T6SS. Here, we incorporated new biological data derived from natural competitors of Vibrio fischeri light organ symbionts to build a biochemical model for T6SS function at the single cell level. The model accounts for activation of structure formation, structure assembly, and deployment. By developing an integrated agent-based model (IABM) that incorporates strain-specific T6SS parameters, we replicated outcomes of biological competitions, validating our approach. We used the IABM to isolate and manipulate strain-specific physiological differences between competitors, in a way that is not possible using biological samples, to identify winning strategies for T6SS-armed populations. We found that a tipping point exists where the cost of building more T6SS weapons outweighs their protective ability. Furthermore, we found that competitions between a T6SS-armed population and a unarmed target had different outcomes dependent on the geometry of the battlefield: target cells survived at the edges of a range expansion scenario where unlimited territory could be claimed, while competitions within a confined space, much like the light organ crypts where natural V. fischeri compete, resulted in the rapid elimination of the unarmed competitor.

microbiology↗

bioRxiv2021

A large lipoprotein mediates target specificity for T6SS-dependent killing

Interbacterial competition is prevalent in host-associated microbiota, where it can shape community structure and function, impacting host health in both positive and negative ways. However, the factors that permit bacteria to discriminate among their various neighbors for targeted elimination of competitors remain elusive. We identified a specificity factor in Vibrio species that is used to target specific competitors for elimination. Here, we describe this specificity factor, which is associated with the broadly-distributed type VI secretion system (T6SS), by studying symbiotic Vibrio fischeri, which use the T6SS to compete for colonization sites in their squid host. We demonstrate that a large lipoprotein (TasL) allows V. fischeri cells to restrict T6SS-dependent killing to certain genotypes by selectively integrating competitor cells into aggregates while excluding other cell types. TasL is also required for T6SS-dependent competition within juvenile squid, indicating the adhesion factor is active in the host. Because TasL homologs are found in other host-associated bacterial species, this newly-described specificity factor has the potential to impact microbiome structure within diverse hosts.

microbiology↗

bioRxiv2019

A model Roseobacter employs a diffusible killing mechanism to eliminate competitors

The roseobacter clade is a group of -proteobacteria that have diverse metabolic and regulatory capabilities. They are abundant in marine environments and have a substantial role in marine ecology and biogeochemistry. However, interactions between roseobacters and other bacterioplankton have not been extensively explored. In this study, we identify a killing mechanism in the model Roseobacter Ruegeria pomeroyi DSS-3 by competing it against a group of phylogenetically diverse bacteria. The killing mechanism involves an unidentified antimicrobial compound that is produced when cells are grown on both surfaces and in suspension and is dependent on cell density. A screen of random transposon mutants revealed the killing phenotype, as well as resistance to the antimicrobial, require genes within an ~8 kb putative {gamma}-butyrolactone synthesis gene cluster, which resembles similar pheromone-sensing systems in actinomycetes that regulate secondary metabolite production. Transcriptomics revealed the gene cluster is highly upregulated in wild-type DSS-3 compared to a non-killer mutant when grown in liquid coculture with a roseobacter target. Our findings show that R. pomeroyi has the capability to eliminate closely- and distantly-related competitors, providing a mechanism to alter the community structure and function in its native habitats.

microbiology↗