Biology subjects

Mandel, M. J.

Publications and source records attributed to Mandel, M. J..

15 recordsLinked to original sources

bioRxiv2025

RtmR is a membrane-embedded RRM-family RNA-binding protein that regulates biofilm formation

The animal symbiont Vibrio fischeri has served as a model organism for molecular processes underlying bacterial group behaviors, including quorum sensing and biofilm development. Here, using a genetic approach to identify negative regulators of biofilm formation in V. fischeri, we identified a membrane-bound RNA-binding protein, RtmR (VF_2432), that acts as an inhibitor of the symbiosis polysaccharide (SYP) biofilm. Membrane localization of the protein seems to be required for protein stability or expression, as truncation of the transmembrane helices led to an inability to detect the protein. The conserved RNP1 and RNP2 motifs in RtmRs cytoplasmic RNA recognition motif (RRM) domain are required for function, and we demonstrate binding to RNA substrates. Identification of RtmR RNA ligands was conducted with a iiCLIP-seq approach that revealed a large interactome. One transcript identified was that of the biofilm regulatory histidine kinase RscS. We found that RtmR biofilm inhibition depends on RscS activity and that RtmR negatively regulates levels of RscS. Overall, this work characterizes a novel type of bacterial RNA-binding protein. IMPORTANCEBacterial RNA-binding proteins (RBPs) perform key functions to regulate stress responses and development. Bacterial RBPs including the RNA chaperones Hfq and ProQ, the global regulator CsrA, and the cold shock proteins (Csps) have been extensively studied, although additional classes of RBPs have been predicted by bioinformatic methods including those carrying an RRM domain. This work expands on recent studies of RRM domain proteins in bacteria to characterize a membrane-bound RRM protein that regulates bacterial biofilm development. Given our rapidly-expanding knowledge regarding the role for RNA-binding proteins in bacterial molecular biology, this work contributes a new class of membrane-bound regulators with homologs in human pathogens and marine symbionts.

microbiology↗

bioRxiv2025

Sedative choice alters Klebsiella pneumoniae lung pathogenesis and dissemination

Klebsiella pneumoniae make up 85% of carbapenem-resistant Enterobacteriaceae (CRE), bacteria that have become an urgent threat to public health. K. pneumoniae is largely transmitted in healthcare settings, where inpatients and outpatients are often anesthetized with the widely-used anesthetic induction agent propofol. Recent evidence obtained from rodent infection models indicates that propofol exposure can dramatically increase host susceptibility to microbial infections. Given that intensive care patients who are at a greater risk for K. pneumoniae lung infections are often given propofol during their hospitalization, we investigated the outcome of K. pneumoniae infections in mice briefly sedated with either propofol or ketamine/xylazine as control. Propofol-sedated mice experienced more rapid dissemination from the lungs to secondary sites of infection and their lungs exhibited more severe pathology. Based on these observations, we investigated bacterial factors involved in infection and dissemination in mice with propofol or ketamine/xylazine sedation using a high throughput insertion sequencing (INSeq) approach. We identified numerous novel potential virulence factors together with previously identified gene products, confirming the validity of our screen. We further characterized a mutant lacking the phospholipid retrograde trafficking chaperone MlaC and found that the degree of mutant attenuation was dependent upon sedation method. These results highlight the importance of sedative choice when studying hospital-acquired microbial infections and suggest that sedation can influence outcome of K. pneumoniae infection and dissemination in animal models. IMPORTANCEHost sedation by either propofol or ketamine exposure differentially impacted the severity of K. pneumoniae lung infection following intranasal inoculation of mice. While propofol-sedated mice exhibited increased lung pathology, some bacterial mutants, such as those lacking the MlaC gene product associated with the maintenance of inner and outer membrane lipid asymmetry, exhibited more severe attenuation following propofol sedation versus ketamine/xylazine. Given the dominating use of propofol in health care settings for the induction and maintenance of anesthesia, procedural sedation, and sedation for intensive care patients, this work provides important perspective as to how the choice of an anesthetic agent may impact the outcome of healthcare-associated infections.

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

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 in vitro and in situ. 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. Although ESI is an efficient, widely adopted ionization method, its widespread use may contribute to the re-identification of known metabolites. Here, we present the use of a dielectric barrier discharge ionization (DBDI) for microbial metabolomics applications through the use of soft ionization chemical reaction in-transfer (SICRIT). Additionally, we compared SICRIT to ESI using two different Vibrio species-Vibrio fischeri, a symbiotic marine bacterium, and Vibrio cholerae, a pathogenic bacterium. Overall, we found that the SICRIT source ionizes a different set of metabolites than ESI, and it has the ability to ionize lipids more efficiently than ESI in positive mode. This work highlights the value of using more than one ionization source for the detection of metabolites.

biochemistry↗

bioRxiv2024

Mobile-CRISPRi as a powerful tool for modulating Vibrio 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 dcas9 and sgRNA genes into the genome using Tn7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio campbellii. We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments.

molecular biology↗

bioRxiv2023

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. We have developed a label-free method for the direct detection of c-di-GMP in microbial colony biofilms using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). We applied this method to the enteric pathogen Vibrio cholerae, the marine symbiont V. fischeri, and the opportunistic pathogen Pseudomonas aeruginosa PA14 and detected spatial and temporal changes in c-di-GMP signal that accompanied genetic alterations in factors that synthesize and degrade the compound. We further demonstrated how this method can be simultaneously applied to detect additional metabolites of interest in a single experiment.

microbiology↗

bioRxiv2023

Transcriptional pathways across colony biofilm models in the symbiont Vibrio fischeri

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 Vibrio fischeri, 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 V. fischeri biofilm formation, yet much remains unknown regarding the breadth of target genes induced by these factors. Here, we probed two widely-used in vitro models of biofilm formation to identify novel regulatory pathways in the squid symbiont V. fischeri ES114. We discovered a shared set of 232 genes that demonstrated similar patterns in expression in both models. These genes comprise multiple exopolysaccharide loci that are upregulated and flagellar motility genes that are downregulated, with a consistent decrease in measured swimming motility. Furthermore, we identified genes regulated downstream of the key sensor kinase RscS that are induced independent of the response regulator SypG. Our data suggest that putative response regulator VpsR plays a strong role in expression of at least a subset of these genes. Overall, this study adds to our understanding of the genes involved in V. fischeri biofilm regulation, while revealing new regulatory pathways branching from previously characterized signaling networks. IMPORTANCEThe V. fischeri-squid system provides an opportunity to study biofilm development both in the animal host and in culture-based biofilm models that capture key aspects of in vivo signaling. In this work, we report the results of the transcriptomic profiling of two V. fischeri biofilm models followed by phenotypic validation and examination of novel signaling pathway architecture. Remarkable consistency between the models provides a strong basis for future studies using either-or both-approaches. A subset of the factors identified by the approaches were validated in the work, and the body of transcriptomic data provides a number of leads for future studies in culture and during animal colonization.

microbiology↗

bioRxiv2023

Functional analysis of cyclic diguanylate-modulating proteins in Vibrio fischeri

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. While c-di-GMP and the enzymes that contribute to its metabolism have been well-studied in pathogens, considerably less focus has been placed on c-di-GMP regulation in beneficial symbionts. Vibrio fischeri is the sole beneficial symbiont of the Hawaiian bobtail squid (Euprymna scolopes) light organ, and the bacterium requires both motility and biofilm formation to efficiently colonize. C-di-GMP regulates swimming motility and cellulose exopolysaccharide production in V. fischeri. The genome encodes 50 DGCs and PDEs, and while a few of these proteins have been characterized, the majority have not undergone comprehensive characterization. In this study, we use protein overexpression to systematically characterize the functional potential of all 50 V. fischeri proteins. All 28 predicted DGCs and 14 predicted PDEs displayed at least one phenotype consistent with their predicted function, and a majority of each displayed multiple phenotypes. Finally, active site mutant analysis of proteins with the potential for both DGC and PDE activities revealed potential activities for these proteins. This work presents a systems-level functional analysis of a family of signaling proteins in a tractable animal symbiont and will inform future efforts to characterize the roles of individual proteins during lifestyle transitions. IMPORTANCEC-di-GMP is a critical second messenger that mediates bacterial behaviors, and V. fischeri colonization of its Hawaiian bobtail squid host presents a tractable model in which to interrogate the role of c-di-GMP during animal colonization. This work provides systems-level characterization of the 50 proteins predicted to modulate c-di-GMP levels. By combining multiple assays, we generated a rich understanding of which proteins have the capacity to influence c-di-GMP levels and behaviors. Our functional approach yielded insights into how proteins with domains to both synthesize and degrade c-di-GMP may impact bacterial behaviors. Finally, we integrated published data to provide a broader picture of each of the 50 proteins analyzed. This study will inform future work to define specific pathways by which c-di-GMP regulates symbiotic behaviors and transitions.

microbiology↗

bioRxiv2022

High levels of cyclic diguanylate interfere with beneficial bacterial colonization

During colonization of the Hawaiian bobtail squid (Euprymna scolopes), 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. fischeri encodes 50 proteins predicted to synthesize and/or degrade c-di-GMP, but a role for c-di-GMP regulation during host colonization has not been investigated. We examined strains exhibiting either low or high levels of c-di-GMP during squid colonization and found that while a low c-di-GMP strain had no colonization defect, a high c-di-GMP strain was severely impaired. Expression of a heterologous c-di-GMP phosphodiesterase restored colonization, demonstrating that the effect is due to high c-di-GMP levels. In the constitutive high c-di-GMP state, colonizing V. fischeri exhibited reduced motility, altered biofilm aggregate morphology, and a regulatory interaction where transcription of one polysaccharide locus is inhibited by the presence of the other polysaccharide. Our results highlight the importance of proper c-di-GMP regulation during beneficial animal colonization, illustrate multiple pathways regulated by c-di-GMP in the host, and uncover an interplay of multiple exopolysaccharide systems in host-associated aggregates. IMPORTANCEThere is substantial interest in studying cyclic diguanylate (c-di-GMP) in pathogenic and environmental bacteria, which has led to an accepted paradigm in which high c-di-GMP levels promote biofilm formation and reduce motility. However, considerably less focus has been placed on understanding how this compound contributes to beneficial colonization. Using the Vibrio fischeri-Hawaiian bobtail squid study system, we took advantage of recent genetic advances in the bacterium to modulate c-di-GMP levels and measure colonization and track c-di-GMP phenotypes in a symbiotic interaction. Studies in the animal host revealed a c-di-GMP-dependent genetic interaction between two distinct biofilm polysaccharides, Syp and cellulose, that was not evident in culture-based studies: elevated c-di-GMP altered the composition and abundance of the in vivo biofilm by decreasing syp transcription due to increased cellulose synthesis. This study reveals important parallels between pathogenic and beneficial colonization and additionally identifies c-di-GMP-dependent regulation that occurs specifically in the squid host.

microbiology↗

bioRxiv2021

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 Syp symbiotic polysaccharide. TCS systems are broadly important for bacteria to sense environmental cues and then direct changes in behavior. Previously, we identified hybrid histidine kinase BinK as a strong negative regulator of V. fischeri biofilm regulation, and here we further explore the function of BinK. To inhibit biofilm formation, BinK requires the predicted phosphorylation sites in both the histidine kinase (H362) and receiver (D794) domains. Furthermore, we show that RscS is not essential for host colonization when binK is deleted from strain ES114, and imaging of aggregate size revealed no benefit to the presence of RscS in a background lacking BinK. Strains lacking RscS still suffered in competition. Finally, we show that BinK functions to inhibit biofilm gene expression in the light organ crypts, providing evidence for biofilm gene regulation at later stages of host colonization. Overall, this study provides direct evidence for opposing activities of RscS and BinK and yields novel insights into biofilm regulation during the maturation of a beneficial symbiosis. IMPORTANCEBacteria are often in a biofilm state, and transitions between planktonic and biofilm lifestyles are important for pathogenic, beneficial, and environmental microbes. The critical nature of biofilm formation during Vibrio fischeri colonization of the Hawaiian bobtail squid light organ provides an opportunity to study development of this process in vivo using a combination of genetic and imaging approaches. The current work refines the signaling circuitry of the biofilm pathway in V. fischeri, provides evidence that biofilm regulatory changes occur in the host, and identifies BinK as one of the regulators of that process. This study provides information about how bacteria regulate biofilm gene expression in an intact animal host.

microbiology↗

bioRxiv2020

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 Vibrio fischeri and its squid host Euprymna scolopes 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 V. fischeri. 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). Our deletion approach improves on previous techniques based on splicing-by-overlap extension PCR (SOE-PCR) and tfoX-based natural transformation by incorporating a randomized barcode that results in unique DNA sequences within each deletion scar. Amplicon sequencing of the pool of barcoded strains before and after colonization faithfully reports on known colonization factors and provides increased sensitivity over colony counting methods. BarSeq enables rapid and sensitive characterization of the molecular factors involved in establishing the Vibrio-squid symbiosis and provides a valuable tool to interrogate the molecular dialogue at microbe-animal host interfaces. ImportanceBeneficial microbes play essential roles in the health and development of their hosts. However, the complexity of animal microbiomes and general genetic intractability of their symbionts have made it difficult to study the coevolved mechanisms for establishing and maintaining specificity at the microbe-animal host interface. Model symbioses are therefore invaluable for studying the mechanisms of beneficial microbe-host interactions. Here we present a combined barcode-tagged deletion and BarSeq approach to interrogate the molecular dialogue that ensures specific and reproducible colonization of the Hawaiian bobtail squid by Vibrio fischeri. The ability to precisely manipulate the bacterial genome, combined with multiplex colonization assays, will accelerate the use of this valuable model system for mechanistic studies of how environmental microbes--both beneficial and pathogenic--colonize specific animal hosts.

microbiology↗

bioRxiv2020

Discovery of a microbially produced small molecule in a host-specific organ

The lifelong relationship between the Hawaiian bobtail squid, Euprymna scolopes, and its microbial symbiont, Vibrio fischeri, 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 hosts in a process termed counterillumination. The squid hosts hatch without their symbionts, which must be acquired from the ocean amid a diversity of non-beneficial bacteria, so precise molecular communication is required for initiation of the specific relationship. It is therefore likely that there may be specialized metabolites used in the light organ microenvironment to modulate these processes. To identify small molecules that may influence the establishment of this symbiosis, we used imaging mass spectrometry to analyze metabolite production in V. fischeri with altered biofilm production, which correlates directly to colonization capability in its host. Biofilm-Up and Biofilm-Down mutants were compared to a wild-type strain, and masses that were more abundantly produced by the biofilm-up mutant were detected. Using a combination of structure elucidation and synthetic chemistry, one such signal was determined to be a diketopiperazine, cyclo(O_SCPLOWDC_SCPLOW-histidyl-O_SCPLOWLC_SCPLOW-proline). This diketopiperazine modulated luminescence in V. fischeri and, using label-free imaging mass spectrometry, was directly detected in the light organ of the colonized host. This work highlights the continued need for untargeted discovery efforts in host-microbe interactions and showcases the benefits of the squid-Vibrio system for identification and characterization of small molecules that modulate microbiome behaviors. Significance StatementThe complexity of animal microbiomes presents challenges to defining signaling molecules within the microbial consortium and between the microbes and the host. By focusing on the binary symbiosis between Vibrio fischeri and Euprymna scolopes, we have combined genetic analysis with direct imaging to define and study small molecules in the intact symbiosis. We have detected and characterized a diketopiperazine produced by strong biofilm-forming V. fischeri strains that was detectable in the host symbiotic organ, and which influences bacterial luminescence. Biofilm formation and luminescence are critical for initiation and maintenance of the association, respectively, suggesting that the compound may link early and later development stages, providing further evidence that multiple small molecules are important in establishing these beneficial relationships.

microbiology↗

bioRxiv2020

Bacterial gene essentiality under modeled microgravity

To support long-duration spaceflight, identifying genes that enable microbial survival and stability under space stressors is crucial, as these factors can affect both host health and in-space biomanufacturing efforts. Our objective was to determine what bacterial genes are required for growth in culture under modeled, or simulated, microgravity conditions compared to normal gravity controls. We focused on the marine bacterium Vibrio fischeri, which forms a monospecific symbiosis with the Hawaiian bobtail squid, Euprymna scolopes. The symbiosis has been studied during spaceflight and in ground-based modeled microgravity conditions. Using transposon insertion sequencing (INSeq), we identified dozens of genes that exhibited fitness defects under both conditions, yet we identified relatively few genes with differential effects under modeled microgravity or gravity specifically. We additionally compared RNA-seq and INSeq data and determined that expression under microgravity was not predictive of the essentiality of a given gene. In summary, empirical determination of conditional gene essentiality identifies few microgravity-specific genes for environmental growth of V. fischeri, suggesting that the condition of microgravity has a minimal impact on symbiont gene requirement during growth in media. These findings suggest that maintaining beneficial microbial communities during spaceflight, and enabling their use in biomanufacturing applications, may require minimal genetic adaptation, easing the challenges of supporting healthy symbiotic relationships on long-duration missions.

microbiology↗

bioRxiv2018

Natural strain diversity reveals novel biofilm regulation in squid-colonizing Vibrio fischeri

The mutualistic symbiont Vibrio fischeri builds a symbiotic biofilm during colonization of squid hosts. Regulation of the exopolysaccharide component, termed Syp, has been examined in strain ES114, where production is controlled by a phosphorelay that includes the inner membrane hybrid histidine kinase RscS. Most strains that lack RscS or encode divergent RscS proteins cannot colonize a squid host unless RscS from a squid symbiont is heterologously expressed. In this study, we examine V. fischeri isolates worldwide to understand the landscape of biofilm regulation during beneficial colonization. We provide a detailed study of three distinct evolutionary groups of V. fischeri and find that while the RscS-Syp biofilm pathway is required in one of the groups, two other groups of squid symbionts require Syp independent of RscS. Mediterranean squid symbionts, including V. fischeri SR5, colonize without an RscS homolog encoded in their genome. Additionally, Group A V. fischeri strains, which form a tightly-related clade of Hawaii isolates, have a frameshift in rscS and do not require the gene for squid colonization or competitive fitness. These same strains have a frameshift in sypE, and we provide evidence that this Group A sypE allele leads to an upregulation in biofilm activity. This work thus describes the central importance of Syp biofilm in colonization of diverse isolates, and demonstrates that significant evolutionary transitions correspond to regulatory changes in the syp pathway.\n\nIMPORTANCEBiofilms are surface-associated, matrix-encased bacterial aggregates that exhibit enhanced protection to antimicrobial agents. Previous work has established the importance of biofilm formation by a strain of luminous Vibrio fischeri bacteria as the bacteria colonize their host, the Hawaiian bobtail squid. In this study, expansion of this work to many natural isolates revealed that biofilm genes are universally required, yet there has been a shuffling of the regulators of those genes. This work provides evidence that even when bacterial behaviors are conserved, dynamic regulation of those behaviors can underlie evolution of the host colonization phenotype. Furthermore, this work emphasizes the importance of investigating natural diversity as we seek to understand molecular mechanisms in bacteria.

microbiology↗