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

The purpose of this qualitative study was to identify perspectives of female spouses/intimate partners regarding posttraumatic stress disorder (PTSD) in returning Iraq and Afghanistan combat Veterans. Through the use of a self-administered questionnaire based on Flanagan's critical incident technique, reports were obtained from a purposive sample of 34 spouses/intimate partners of Veterans recruited through a social group for military spouses and a university in southeastern North Carolina. Two-thirds of the participants reported not having received formal education about PTSD. The main perceived barriers to PTSD treatment seeking were denial of symptoms, fear, and stigma about disclosing PTSD symptoms. Spouses/intimate partners observed Veterans for changes in behavior and routines, disturbed sleep patterns, and nightmares. In the event of PTSD treatment resistance, spouses/intimate partners reported they would suggest the need for treatment, issue an ultimatum, take action, or offer patience and support without taking any action.

Awareness of posttraumatic stress disorder in veterans: a female spouse/intimate partner perspective.

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.

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 Association of Community Health Nursing Educators (ACHNE) Research Committee Subcommittee on Evidence-Based Practice (EBP) presents a paper on the state of translation of science into public health nursing education practice. The paper builds from the integrative research review completed by the ACHNE Research Priorities Subcommittee (McElroy et al., Public Health Nursing, 2020) offering updated research priorities and a Research in Action model. Four EBP Project Priorities were established using the Johns Hopkins EBP Model guided by Dang and Dearholt's (Johns Hopkins nursing evidence-based practice: Model and guidelines, 2018) 19-step process. The EBP Project Priorities emphasize the need for EBP projects to align ACHNE strategic plans and research priorities. The authors recommend that the Research in Action model guide deliberate unification of both the Research and EBP Project Priorities. Standards and criteria for essential effort unifying collaborative effort between PhD and Doctorate in Nursing Practice scholars, coordination of research efforts, and innovative practice partnerships is provided.

Association of Community Health Nursing Educators', 2020 evidence-based project priorities: A national blueprint for unifying research and evidence-based practice priorities.

The Association of Community/Public Health Nurse Educators (ACHNE) Research Priorities Subcommittee presents a report on the state of the science of public health nursing education.

Association of Community Health Nursing Educators 2020 Research Priorities and Research in Action Model.

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.

Stephanie Smith

Department of Earth,
Marine,
and Environmental Sciences,
Department of Earth, Marine, and Environmental Sciences

University of North Carolina

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Stephanie Smith

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

University of North Carolina

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Department of Earth,
Marine,
and Environmental Sciences,
Department of Earth, Marine, and Environmental Sciences