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

Unlabelled: Strain-level variation among host-associated bacteria often determines host range and the extent to which colonization is beneficial, benign, or pathogenic. 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.

Generation of Barcode-Tagged Vibrio fischeri Deletion Strains and Barcode Sequencing (BarSeq) for Multiplex Strain Competitions.

Vibrio fischeri is a model mutualist for studying molecular processes affecting microbial colonization of animal hosts. We present a detailed protocol for a barcode sequencing (BarSeq) approach that combines targeted gene deletion with short-read sequencing technology to enable studies of mixed bacterial populations. This protocol includes wet lab steps to plan and produce the deletions, approaches to scale up mutant generation, protocols to prepare and conduct the strain competition, library preparation for sequencing on an Illumina iSeq 100 instrument, and data analysis with the barseq python package.

Functional analysis of cyclic diguanylate-modulating proteins in .

As bacterial symbionts transition from a motile free-living state to a sessile biofilm state, they must coordinate behavior changes suitable to each lifestyle. Cyclic diguanylate (c-di-GMP) is an intracellular signaling molecule that can regulate this transition, and it is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. Generally, c-di-GMP inhibits motility and promotes biofilm formation.

Microbial Metabolomics' Latest SICRIT: Soft Ionization by Chemical Reaction In-Transfer Mass Spectrometry.

Microbial metabolomics studies are a common approach for identifying microbial strains that have a capacity to produce new chemistries both and . A limitation to applying microbial metabolomics to the discovery of new chemical entities is the rediscovery of known compounds, or "known unknowns." One factor contributing to this rediscovery is that the majority of laboratories use one ionization source─electrospray ionization (ESI)─to conduct metabolomics studies.

Microbial metabolomics' latest SICRIT: Soft ionization by Chemical Reaction in-Transfer mass spectrometry.

Microbial metabolomics studies are a common approach to identifying microbial strains that have a capacity to produce new chemistries both and . A limitation to applying microbial metabolomics to the discovery of new chemical entities is the rediscovery of known compounds, or "known unknowns." One contributing factor to this rediscovery is the majority of laboratories use one ionization source-electrospray ionization (ESI)-to conduct metabolomics studies.

Mobile-CRISPRi protocol optimization for .

Mobile clustered regularly interspaced palindromic repeats interference (Mobile-CRISPRi) is an established method for bacterial gene expression knockdown. The deactivated Cas9 protein and guide RNA are isopropyl β-D-1-thiogalactopyranoside inducible, and all components are integrated into the chromosome via Tn transposition. Here, we optimized methods specific for applying Mobile-CRISPRi in multiple species.

Mobile-CRISPRi as a powerful tool for modulating gene expression.

CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible and genes into the genome using Tn transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple species: , , , , and .

A Label-Free Approach for Relative Spatial Quantitation of c-di-GMP in Microbial Biofilms.

Microbial biofilms represent an important lifestyle for bacteria and are dynamic three-dimensional structures. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous signaling molecule that is known to be tightly regulated with biofilm processes. While measurements of global levels of c-di-GMP have proven valuable toward understanding the genetic control of c-di-GMP production, there is a need for tools to observe the local changes of c-di-GMP production in biofilm processes.

Mobile-CRISPRi as a powerful tool for modulating gene expression.

CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible and genes into the genome using Tn transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple species: , and .

Transcriptional pathways across colony biofilm models in the symbiont .

Beneficial microbial symbionts that are horizontally acquired by their animal hosts undergo a lifestyle transition from free-living in the environment to associating with host tissues. In the model symbiosis between the Hawaiian bobtail squid and its microbial symbiont , one mechanism used to make this transition during host colonization is the formation of biofilm-like aggregates in host mucosa. Previous work identified factors that are sufficient to induce biofilm formation, yet much remains unknown regarding the breadth of target genes induced by these factors.

A label-free approach for relative spatial quantitation of c-di-GMP in microbial biofilms.

Microbial biofilms represent an important lifestyle for bacteria and are dynamic three dimensional structures. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous signaling molecule that is known to be tightly regulated with biofilm processes. While measurements of global levels of c-di-GMP have proven valuable towards understanding the genetic control of c-di-GMP production, there is a need for tools to observe the local changes of c-di-GMP production in biofilm processes.

Transcriptional pathways across colony biofilm models in the symbiont .

Beneficial microbial symbionts that are horizontally acquired by their animal hosts undergo a lifestyle transition from free-living in the environment to associated with host tissues. In the model symbiosis between the Hawaiian bobtail squid and its microbial symbiont one mechanism used to make this transition during host colonization is the formation of biofilm-like aggregates in host mucosa. Previous work identified factors that are sufficient to induce biofilm formation, yet much remains unknown regarding the breadth of target genes induced by these factors.

Functional analysis of cyclic diguanylate-modulating proteins in .

As bacterial symbionts transition from a motile free-living state to a sessile biofilm state, they must coordinate behavior changes suitable to each lifestyle. Cyclic diguanylate (c-di-GMP) is an intracellular signaling molecule that can regulate this transition, and it is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. Generally, c-di-GMP inhibits motility and promotes biofilm formation.

Two enhancer binding proteins activate σ-dependent transcription of a quorum regulatory RNA in a bacterial symbiont.

To colonize a host, bacteria depend on an ensemble of signaling systems to convert information about the various environments encountered within the host into specific cellular activities. How these signaling systems coordinate transitions between cellular states in vivo remains poorly understood. To address this knowledge gap, we investigated how the bacterial symbiont initially colonizes the light organ of the Hawaiian bobtail squid .

High Levels of Cyclic Diguanylate Interfere with Beneficial Bacterial Colonization.

During colonization of the Hawaiian bobtail squid (), Vibrio fischeri bacteria undergo a lifestyle transition from a planktonic motile state in the environment to a biofilm state in host mucus. Cyclic diguanylate (c-di-GMP) is a cytoplasmic signaling molecule that is important for regulating motility-biofilm transitions in many bacterial species. V.

: a new causative species of infectious keratitis.

A 25-year-old man presented with a 2-month history of progressively worsening left eye pain and an atypical corneal ring infiltrate. His condition deteriorated despite topical antibiotic therapy. Cultures for bacteria, fungus and acanthamoeba, repeated twice, all demonstrated no growth.

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

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.

Hybrid Histidine Kinase BinK Represses Vibrio fischeri Biofilm Signaling at Multiple Developmental Stages.

The symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and its exclusive light organ symbiont, Vibrio fischeri, provides a natural system in which to study host-microbe specificity and gene regulation during the establishment of a mutually beneficial symbiosis. Colonization of the host relies on bacterial biofilm-like aggregation in the squid mucus field. Symbiotic biofilm formation is controlled by a two-component signaling (TCS) system consisting of regulators RscS-SypF-SypG, which together direct transcription of the symbiosis polysaccharide Syp.

A Small Molecule Coordinates Symbiotic Behaviors in a Host Organ.

The lifelong relationship between the Hawaiian bobtail squid and its microbial symbiont represents a simplified model system for studying microbiome establishment and maintenance. The bacteria colonize a dedicated symbiotic light organ in the squid, from which bacterial luminescence camouflages the host in a process termed counterillumination. The squid host hatches without its symbionts, which must be acquired from the ocean amidst a diversity of nonbeneficial bacteria, such that precise molecular communication is required for initiation of the specific relationship.

Hip Dysplasia and Osteogenesis Imperfecta: A Case Report.

Case: A 1-week-old female patient presented to our clinic with bilateral dislocated hips and was subsequently treated in a Pavlik harness. Harness treatment failed requiring a closed reduction and spica cast application. In the Post-Anesthesia Care Unit (PACU), the patient was found to have a right humerus fracture.

Multiplexed Competition in a Synthetic Squid Light Organ Microbiome Using Barcode-Tagged Gene Deletions.

Beneficial symbioses between microbes and their eukaryotic hosts are ubiquitous and have widespread impacts on host health and development. The binary symbiosis between the bioluminescent bacterium and its squid host serves as a model system to study molecular mechanisms at the microbe-animal interface. To identify colonization factors in this system, our lab previously conducted a global transposon insertion sequencing (INSeq) screen and identified over 300 putative novel squid colonization factors in To pursue mechanistic studies on these candidate genes, we present an approach to quickly generate barcode-tagged gene deletions and perform high-throughput squid competition experiments with detection of the proportion of each strain in the mixture by barcode sequencing (BarSeq).

Improving resident education through unstable chicken hips: a novel way to teach an infant hip examination.

Current clinical screening for infantile hip dysplasia relies on combined Ortolani and Barlow maneuvers, which have a quoted sensitivity and specificity of 74-98 and 84-99%, respectively. Teaching this delicate physical examination maneuver is difficult as it requires a distinct tactile feel. The purpose of this study is to validate a benchtop learning grocery-bought chicken simulator model of newborn hip instability through a pre- and post-test surveys.