Coincubation Assay for Quantifying Competitive Interactions between <em>Vibrio fischeri</em> Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...

University of North Carolina, Chapel Hill

University of North Carolina

Intraspecific competition describes the negative interaction that occurs when different populations of the same species attempt to fill the same niche. Such competition is predicted to occur among host-associated bacteria but has been challenging to study in natural biological systems. Although many bioluminescent  strains exist in seawater, only a few strains are found in the light-organ crypts of an individual wild-caught  squid, suggesting a possible role for intraspecific competition during early colonization. Using a culture-based assay to investigate the interactions of different  strains, we found "lethal" and "nonlethal" isolates that could kill or not kill the well-studied light-organ isolate ES114, respectively. The killing phenotype of these lethal strains required a type VI secretion system (T6SS) encoded in a 50-kb genomic island. Multiple lethal and nonlethal strains could be cultured from the light organs of individual wild-caught adult squid. Although lethal strains eliminate nonlethal strains in vitro, two lethal strains could coexist in interspersed microcolonies that formed in a T6SS-dependent manner. This coexistence was destabilized upon physical mixing, resulting in one lethal strain consistently eliminating the other. When juvenile squid were coinoculated with lethal and nonlethal strains, they occupied different crypts, yet they were observed to coexist within crypts when T6SS function was disrupted. These findings, using a combination of natural isolates and experimental approaches in vitro and in the animal host, reveal the importance of T6SS in spatially separating strains during the establishment of host colonization in a natural symbiosis.

Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host.

The type VI secretion system (T6SS) facilitates lethal competition between bacteria through direct contact. Comparative genomics has facilitated the study of these systems in , which colonizes the squid host  Here, we report the draft genome sequences of two lethal  strains that encode the T6SS, FQ-A001 and ES401.

Draft Genome Sequences of Type VI Secretion System-Encoding Vibrio fischeri Strains FQ-A001 and ES401.

The symbiosis between  squid and its bioluminescent bacterial symbiont, , is a valuable model system to study a natural, coevolved host-microbe association. Over the past 30 years, researchers have developed and optimized many experimental methods to study both partners in isolation and during symbiosis. These powerful tools, along with a strong foundational knowledge about the system, position the -squid symbiosis at the forefront of host-microbe interactions because this system is uniquely suited to investigation of symbiosis from both host and bacterial perspectives. Moreover, the ability to isolate and characterize different strains of  has revealed exciting new insights about how different genotypes evolve to compete for a host niche, including deploying interbacterial weapons early during host colonization. This Perspective explores how interbacterial warfare influences the diversity and spatial structure of the symbiotic population, as well as the possible effects that intraspecific competition might have on the host.

The -Squid Symbiosis as a Model for Studying Interbacterial Competition.

Symbiotic bacteria use diverse strategies to compete for host colonization sites. However, little is known about the environmental cues that modulate interbacterial competition as they transition between free-living and host-associated lifestyles. We used the mutualistic relationship between  squid and  bacteria to investigate how intraspecific competition is regulated as symbionts move from the seawater to a host-like environment. We recently reported that  uses a type VI secretion system (T6SS) for intraspecific competition during host colonization. Here, we investigated how environmental viscosity impacts T6SS-mediated competition by using a liquid hydrogel medium that mimics the viscous host environment. Our data demonstrate that although the T6SS is functionally inactive when cells are grown under low-viscosity liquid conditions similar to those found in seawater, exposure to a host-like high-viscosity hydrogel enhances T6SS expression and sheath formation, activates T6SS-mediated killing in as little as 30 min, and promotes the coaggregation of competing genotypes. Finally, the use of mass spectrometry-based proteomics revealed insights into how cells may prepare for T6SS competition during this habitat transition. These findings, which establish the use of a new hydrogel culture condition for studying T6SS interactions, indicate that  rapidly responds to the physical environment to activate the competitive mechanisms used during host colonization. Bacteria often engage in interference competition to gain access to an ecological niche, such as a host. However, little is known about how the physical environment experienced by free-living or host-associated bacteria influences such competition. We used the bioluminescent squid symbiont  to study how environmental viscosity impacts bacterial competition. Our results suggest that upon transition from a planktonic environment to a host-like environment,  cells activate their type VI secretion system, a contact-dependent interbacterial nanoweapon, to eliminate natural competitors. This work shows that competitor cells form aggregates under host-like conditions, thereby facilitating the contact required for killing, and reveals how  regulates a key competitive mechanism in response to the physical environment.

Environmental Viscosity Modulates Interbacterial Killing during Habitat Transition.

 species of the Harveyi clade are commonly found in free-living and host-associated marine habitats. Here, we report the draft genome sequence for a Harveyi clade bacterium,  sp. strain LB10LO1, which was isolated from the Atlantic brief squid .

Draft Genome Sequence of a Harveyi Clade Bacterium Isolated from Lolliguncula brevis Squid.

The  clade is a group of alphaproteobacteria 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  DSS-3 by coculturing it with a group of phylogenetically diverse bacteria. The killing mechanism is diffusible and occurs when cells are grown both on surfaces and in suspension and is dependent on cell density. A screen of random transposon mutants revealed that the killing phenotype, as well as resistance to killing, 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, including antimicrobials. Transcriptomics revealed the gene cluster is highly upregulated in wild-type DSS-3 compared to a nonkiller mutant when grown in liquid coculture with a roseobacter target. Our findings show that  has the capability to eliminate closely and distantly related competitors, providing a mechanism to alter the community structure and function in its native habitats. Bacteria carry out critical ecological and biogeochemical processes and form the foundations of ecosystems. Identifying the factors that influence microbial community composition and the functional capabilities encoded within them is key to predicting how microbes impact an ecosystem. Because microorganisms must compete for limited space and nutrients to promote their own propagation, they have evolved diverse mechanisms to outcompete or kill competitors. However, the genes and regulatory strategies that promote such competitive abilities are largely underexplored, particularly in free-living marine bacteria. Here, genetics and omics techniques are used to investigate how a model marine bacterium is capable of quickly eliminating natural competitors in coculture. We determined that a previously uncharacterized horizontally acquired gene cluster is required for this bacterium to kill diverse competitors. This work represents an important step toward understanding the mechanisms bacterial populations can use to become dominant members in marine microbial communities.

A Model , Ruegeria pomeroyi DSS-3, Employs a Diffusible Killing Mechanism To Eliminate Competitors.

Bacteria employ diverse competitive strategies to enhance fitness and promote their own propagation. However, little is known about how symbiotic bacteria modulate competitive mechanisms as they compete for a host niche. The bacterium Vibrio fischeri forms a symbiotic relationship with marine animals and encodes a type VI secretion system (T6SS), which is a contact-dependent killing mechanism used to eliminate competitors during colonization of the Euprymna scolopes squid light organ. Like other horizontally acquired symbionts, V. fischeri experiences changes in its physical and chemical environment during symbiosis establishment. Therefore, we probed both environmental and host-like conditions to identify ecologically relevant cues that control T6SS-dependent competition during habitat transition. Although the T6SS did not confer a competitive advantage for V. fischeri strain ES401 under planktonic conditions, a combination of both host-like pH and viscosity was necessary for T6SS competition. For ES401, high viscosity activates T6SS expression and neutral/acidic pH promotes cell-cell contact for killing, and this pH-dependent phenotype was conserved in the majority of T6SS-encoding strains examined. We also identified a subset of V. fischeri isolates that engaged in T6SS-mediated competition at high viscosity under both planktonic and host-like pH conditions. T6SS phylogeny revealed that strains with pH-dependent phenotypes cluster together to form a subclade within the pH-independent strains, suggesting that V. fischeri may have recently evolved to limit competition to the host niche.  Bacteria have evolved diverse strategies to compete for limited space and resources. Because these mechanisms can be costly to use, their expression and function are often restricted to specific environments where the benefits outweigh the costs. However, little is known about the specific cues that modulate competitive mechanisms as bacterial symbionts transition between free-living and host habitats. Here, we used the bioluminescent squid and fish symbiont Vibrio fischeri to probe for host and environmental conditions that control interbacterial competition via the type VI secretion system. Our findings identify a new host-specific cue that promotes competition among many but not all V. fischeri isolates, underscoring the utility of studying multiple strains to reveal how competitive mechanisms may be differentially regulated among closely related populations as they evolve to fill distinct niches.

Host-Like Conditions Are Required for T6SS-Mediated Competition among Vibrio fischeri Light Organ Symbionts.

Bacteria have evolved diverse strategies to compete for a niche, including the type VI secretion system (T6SS), a contact-dependent killing mechanism. T6SSs are common in bacterial pathogens, commensals, and beneficial symbionts, where they affect the diversity and spatial structure of host-associated microbial communities. Although T6SS gene clusters are often located on genomic islands (GIs), which may be transferred as a unit, the regulatory strategies that promote gene expression once the T6SS genes are transferred into a new cell are not known. We used the squid symbiont, , to identify essential regulatory factors that control expression of a strain-specific T6SS encoded on a GI. We found that a transcriptional reporter for this T6SS is active only in strains that contain the T6SS-encoding GI, suggesting the GI encodes at least one essential regulator. A transposon screen identified seven mutants that could not activate the reporter. These mutations mapped exclusively to three genes on the T6SS-containing GI that encode two essential structural proteins (a TssA-like protein and TssM) and a transcriptional regulator (TasR). Using T6SS reporters, RT-PCR, competition assays, and differential proteomics, we found that all three genes are required for expression of many T6SS components, except for the TssA-like protein and TssM, which are constitutively expressed. Based on these findings, we propose a model whereby T6SS expression requires conserved structural proteins, in addition to the essential regulator TasR, and this ability to self-regulate may be a strategy to activate T6SS expression upon transfer of T6SS-encoding elements into a new bacterial host.  Interbacterial weapons like the T6SS are often located on mobile genetic elements and their expression is highly regulated. We found that two conserved structural proteins are required for T6SS expression in . These structural proteins also contain predicted GTPase and GTP binding domains, suggesting their role in promoting T6SS expression may involve sensing the energetic state of the cell. Such a mechanism would provide a direct link between T6SS activation and cellular energy levels, providing a "checkpoint" to ensure the cell has sufficient energy to build such a costly weapon. Because these regulatory factors are encoded within the T6SS gene cluster, they are predicted to move with the genetic element to activate T6SS expression in a new host cell.

Activation of the type VI secretion system in the squid symbiont  requires the transcriptional regulator TasR and the structural proteins TssM and TssA.

Alecia N. Septer

Department of Marine Sciences

University of North Carolina, Chapel Hill

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Alecia N. Septer

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Marine Sciences

University of North Carolina, Chapel Hill

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Department of Marine Sciences