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Akt (Protein kinase B) is a key signaling protein in eukaryotic cells that controls many cellular processes such as glucose metabolism and cell proliferation for survival. As obligate intracellular pathogens, viruses modulate host cellular processes, including Akt signaling, for optimal replication. The mechanisms by which viruses modulate Akt and the resulting effects on the infectious cycle differ widely depending on the virus. In this study, we explored the effect of Akt serine 473 phosphorylation (p-Akt) during murine norovirus (MNV) infection. p-Akt increased during infection of murine macrophages with acute MNV-1 and persistent CR3 and CR6 strains. Inhibition of Akt with MK2206, an inhibitor of all three isoforms of Akt (Akt1/2/3), reduced infectious virus progeny of all three virus strains. This reduction was due to decreased viral genome replication (CR3), defective virus assembly (MNV-1), or diminished cellular egress (CR3 and CR6) in a virus strain-dependent manner. Collectively, our data demonstrate that Akt activation increases in macrophages during the later stages of the MNV infectious cycle, which may enhance viral infection in unique ways for different virus strains. The data, for the first time, indicate a role for Akt signaling in viral assembly and highlight additional phenotypic differences between closely related MNV strains. Importance Human noroviruses (HNoV) are a leading cause of viral gastroenteritis, resulting in high annual economic burden and morbidity; yet there are no small animal models supporting productive HNoV infection, or robust culture systems producing cell culture-derived virus stocks. As a result, research on drug discovery and vaccine development against norovirus infection has been challenging, and no targeted antivirals or vaccines against HNoV are approved. On the other hand, murine norovirus (MNV) replicates to high titers in cell culture and is a convenient and widespread model in norovirus research. Our data demonstrate the importance of Akt signaling during the late stage of the MNV life cycle. Notably, the effect of Akt signaling on genome replication, virus assembly and cellular egress is virus strain specific, highlighting the diversity of biological phenotypes despite small genetic variability among norovirus strains. This study is the first to demonstrate a role for Akt in viral assembly.

Natural transformation is the process by which bacteria actively take up and integrate extracellular DNA into their genomes. In cyanobacteria, natural transformation has only been experimentally demonstrated in a handful of species. Although, cyanobacteria are important model systems for studying photosynthesis and circadian cycling, natural transformation in cyanobacteria has not been characterized to the degree that the process has been studied in other gram-negative bacteria. Two cyanobacterial species that are 99.8% genetically identical provide a unique opportunity to better understand the nuances of natural transformation in cyanobacteria: Synechococcus elongatus PCC 7942 and Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 7942 and Synechococcus 2973 respectively). Synechococcus 7942 is a naturally transformable model system, while Synechococcus 2973 is a recently discovered species that is not naturally competent. Taking only 1.5 hours to replicate, Synechococcus 2973 is the fastest growing cyanobacterial species known, and thus is a strong candidate for serving as a model organism. However, the organism's inability to undergo natural transformation has prevented it from becoming a widely used model system. By substituting polymorphic alleles from Synechococcus 7942 for native Synechococcus 2973 alleles, natural transformation was introduced into Synechococcus 2973. Two genetic loci were found to be involved in differential natural competence between the two organisms: transformation pilus component pilN and circadian transcriptional master regulator rpaA. By using targeting genome editing and enrichment outgrowth, a strain that was both naturally transformable and fast-growing was created. This new Synechococcus 2973-T strain will serve as a valuable resource to the cyanobacterial research community. Importance Certain bacterial species have the ability to take up naked extracellular DNA and integrate it into their genomes. This process is known as natural transformation and is widely considered to play a major role in bacterial evolution. Because of the ease of introducing new genes into naturally transformable organisms, this capacity is also highly valued in the laboratory. Cyanobacteria are photosynthetic and can therefore serve as model systems for some important aspects of plant physiology. Here, we describe the creation of a modified cyanobacterial strain (Synechococcus 2973-T) that is capable of undergoing natural transformation and has a replication time that is on par with the fastest-growing cyanobacterium that has been discovered to date. This new cyanobacterium has the potential to serve as a new model organism for the cyanobacterial research community and will allow experiments to be completed in a fraction of the time that it took to complete previous assays.

Streptomyces coelicolor is a model organism for the study of Streptomyces, a genus of Gram-positive bacteria that undergoes a complex life cycle and produces a broad repertoire of bioactive metabolites and extracellular enzymes. This study investigated the production and characterization of membrane vesicles (MVs) in liquid cultures of S. coelicolor M145 from a structural and biochemical point of view; this was achieved by combining microscopic, physical and -omics analyses. Two main populations of MVs, with different sizes and cargos, were isolated and purified. S. coelicolor MV cargo was determined to be complex, containing different kinds of proteins and metabolites. In particular, a total of 166 proteins involved in cell metabolism/differentiation, molecular processing/transport, and stress response were identified in MVs, the latter functional class also being important for bacterial morpho-physiological differentiation. A subset of these proteins was protected from degradation following treatment of MVs with proteinase K, indicating their localization inside the vesicles. Moreover, S. coelicolor MVs contained an array of metabolites, such as antibiotics, vitamins, amino acids, and components of carbon metabolism. In conclusion, this analysis provides detailed information on S. coelicolor MVs under basal conditions and on their corresponding content, which may be useful in the near future to elucidate vesicle biogenesis and functions. IMPORTANCE Streptomycetes are widely distributed in nature and characterized by a complex life cycle that involves morphological differentiation. They are very relevant in industry because they produce about half of all clinically used antibiotics, as well as other important pharmaceutical products of natural origin. Streptomyces coelicolor is a model organism for the study of bacterial differentiation and bioactive molecule production. S. coelicolor produces extracellular vesicles that carry many molecules, such as proteins and metabolites, including antibiotics. The elucidation of S. coelicolor extracellular vesicle cargo will help us to understand different aspects of streptomycete physiology, such as cell communication during differentiation and response to environmental stimuli. Moreover, the capability of these vesicles for carrying different kinds of biomolecules opens up new biotechnological possibilities related to drug delivery. Indeed, decoding the molecular mechanisms involved in cargo selection may lead to the customization of extracellular vesicle content.

Staphylococcus aureus is a common cause of both superficial and invasive infections of humans and animals. Despite a potent host response and apparently appropriate antibiotic therapy, staphylococcal infections frequently become chronic or recurrent, demonstrating a remarkable ability of S. aureus to withstand the hostile host environment. There is growing evidence that staphylococcal DNA repair makes important contributions to the survival of the pathogen in host tissues, as well as promoting the emergence of mutants that resist host defenses and antibiotics. While much of what we know about DNA repair in S. aureus is inferred from studies with model organisms, the roles of specific repair mechanisms in infection are becoming clear and differences with Bacillus subtilis and Escherichia coli have been identified. Furthermore, there is growing interest in staphylococcal DNA repair as a target for novel therapeutics that sensitize the pathogen to host defenses and antibiotics. In this review, we discuss what is known about staphylococcal DNA repair and its role in infection, examine how repair in S. aureus is similar to, or differs from, repair in well-characterized model organisms, and assess the potential of staphylococcal DNA repair as a novel therapeutic target.

The adhesion of noroviruses to strawberry, turkey slices, ham, and cheddar cheese was studied using murine norovirus 1 (MNV-1) as a surrogate for human norovirus (NoV). Based on plaque assay, the recovery and adhesion of MNV-1 depended on the food type (turkey versus strawberry), pH of the initial suspension buffer (pH 4 versus pH 7), and food fat composition (C(8) versus C(18)). Recovery of infectious particles from turkey was 68% compared to 9.4% from strawberry. On turkey, adhesion of MNV-1 was lower at pH 7 (pH of fecal matter), and virus particles adhered to this pH were recovered more easily (33,875 PFU) than at pH 4 (pH of vomitus). The presence of fat and the composition of fatty acids seemed to increase MNV-1 recovery and adherent viral particles recovered but did not affect adhesion (68% on fat-free turkey and regular turkey). Adherent MNV-1 particles recovered from stainless steel coated with saturated fatty acid (C(8), C(14), C(18)) increased significantly with chain length (P

Horizontal gene transfer (HGT) is a driving force of microbial evolution. The gut of animals acts as a potent reservoir for the lateral transfer of virulence, fitness, and antimicrobial resistance genes through plasmids. Reduced-complexity models for the examination of host-microbe interactions involved in plasmid transfer are greatly desired. Thus, this study identifies the use of Drosophila melanogaster as a model organism for the conjugation of plasmids of various incompatibility groups in the gut. Enterobacteriaceae conjugation pairs were identified in vitro and used for oral inoculation of the Drosophila gut. Flies were enumerated for the donor, recipient, and transconjugant populations. Each donor-recipient pair was observed to persist in fly guts for the duration of the experiment. Gut concentrations of the donors and recipients were significantly different between male and female flies, with females generally demonstrating increased concentrations. Furthermore, host genetics significantly altered the concentrations of donors and recipients. However, transconjugant concentrations were not affected by host sex or genetics and were detected only in the IncPε and IncI1 plasmid groups. This study demonstrates Drosophila melanogaster as a model for gut-mediated plasmid transfer. IMPORTANCE Microbial evolution in the gut of animals due to horizontal gene transfer (HGT) is of significant interest for microbial evolution as well as within the context of human and animal health. Microbial populations evolve within the host, and factors from the bacteria and host interact to regulate this evolution. However, little is currently known about how host and bacterial factors regulate plasmid-mediated HGT in the gut. This study demonstrates the use of Drosophila and the roles of sexual dimorphism as well as plasmid incompatibility groups in HGT in the gut.

β-Hydroxy-α-amino acids are useful compounds for pharmaceutical development. Enzymatic synthesis of β-hydroxy-α-amino acids has attracted considerable interest as a selective, sustainable, and environmentally benign process. In this study, we identified a novel amino acid hydroxylase, AEP14369, from Sulfobacillus thermotolerans Y0017, which is included in a previously constructed CAS-like superfamily protein library, to widen the variety of amino acid hydroxylases. The detailed structures determined by nuclear magnetic resonance and X-ray crystallography analysis of the enzymatically produced compounds revealed that AEP14369 catalyzed threo-β-selective hydroxylation of l-His and l-Gln in a 2-oxoglutarate-dependent manner. Furthermore, the production of l-threo-β-hydroxy-His and l-threo-β-hydroxy-Gln was achieved using Escherichia coli expressing the gene encoding AEP14369 as a whole-cell biocatalyst. Under optimized reaction conditions, 137 mM (23.4 g liter-1) l-threo-β-hydroxy-His and 150 mM l-threo-β-hydroxy-Gln (24.3 g liter-1) were obtained, indicating that the enzyme is applicable for preparative-scale production. AEP14369, an l-His/l-Gln threo-β-hydroxylase, increases the availability of 2-oxoglutarate-dependent hydroxylase and opens the way for the practical production of β-hydroxy-α-amino acids in the future. The amino acids produced in this study would also contribute to the structural diversification of pharmaceuticals that affect important bioactivities. IMPORTANCE Owing to an increasing concern for sustainability, enzymatic approaches for producing industrially useful compounds have attracted considerable attention as a powerful complement to chemical synthesis for environment-friendly synthesis. In this study, we developed a bioproduction method for β-hydroxy-α-amino acid synthesis using a newly discovered enzyme. AEP14369 from the moderate thermophilic bacterium Sulfobacillus thermotolerans Y0017 catalyzed the hydroxylation of l-His and l-Gln in a regioselective and stereoselective fashion. Furthermore, we biotechnologically synthesized both l-threo-β-hydroxy-His and l-threo-β-hydroxy-Gln with a titer of over 20 g liter-1 through whole-cell bioconversion using recombinant Escherichia coli cells. As β-hydroxy-α-amino acids are important compounds for pharmaceutical development, this achievement would facilitate future sustainable and economical industrial applications.

Murine norovirus (MNV) infection results in a late translation shutoff that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the α subunit of eukaryotic initiation factor 2 (eIF2α) kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signaling during metabolic stress. While viral infection is usually associated with activation of double-stranded RNA (dsRNA) binding pattern recognition receptor PKR, we hypothesized that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterize cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signaling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation. IMPORTANCE During viral infection, host defenses are typically characterized by the secretion of proinflammatory autocrine and paracrine cytokines, potentiation of the interferon (IFN) response, and induction of the antiviral response via activation of JAK and Stat signaling. To avoid these and propagate, viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signaling. This highlights novel tools in the viral countermeasures arsenal and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.

Linezolid is an important last-resort antibiotic for the treatment of multidrug-resistant enterococci. The aim of this study was to further characterize the genetic context of optrA and poxtA in 10 florfenicol-resistant enterococci isolated from flowing surface water. In most genomes, optrA and poxtA were embedded in transposition units integrated into plasmids or into the chromosomal radC . For the first time, a chromosomally integrated optrA in an Enterococcus raffinosus isolate is described.

The lipid production potentials of 8 microalgal species were investigated. Among these 8 species, the best strain was a dominant bloom-causing dinoflagellate, Prorocentrum donghaiense; this species had a lipid content of 49.32% ± 1.99% and exhibited a lipid productivity of 95.47 ± 0.99 mg liter−1 day−1, which was 2-fold higher than the corresponding values obtained for the oleaginous microalgae Nannochloropsis gaditana and Phaeodactylum tricornutum. P. donghaiense, which is enriched in C16:0 and C22:6, is appropriate for commercial docosahexaenoic acid (DHA) production. Nitrogen or phosphorus stress markedly induced lipid accumulation to levels surpassing 75% of the dry weight, increased the C18:0 and C17:1 contents, and decreased the C18:5 and C22:6 contents, and these effects resulted in decreases in the unsaturated fatty acid levels and changes in the lipid properties of P. donghaiense such that the species met the biodiesel specification standards. Compared with the results obtained under N-deficient conditions, the enhancement in the activity of alkaline phosphatase of P. donghaiense observed under P-deficient conditions partly alleviated the adverse effects on the photosynthetic system exerted by P deficiency to induce the production of more carbohydrates for lipogenesis. The supernatant of the algicidal bacterium Paracoccus sp. strain Y42 culture lysed P. donghaiense without decreasing its lipid content, which resulted in facilitation of the downstream oil extraction process and energy savings through the lysis of algal cells. The Y42 supernatant treatment improved the lipid profiles of algal cells by increasing their C16:0, C18:0, and C18:1 contents and decreasing their C18:5 and C22:6 contents, which is favorable for biodiesel production. IMPORTANCE This study demonstrates the high potential of Prorocentrum donghaiense, a dominant bloom-causing dinoflagellate, for lipid production. Compared with previously studied oleaginous microalgae, P. donghaiense exhibit greater potential for practical application due to its higher biomass and lipid contents. Nutrient deficiency and the algicidal bacterium Paracoccus sp. strain Y42 improved the suitability of the lipid profile of P. donghaiense for biodiesel production. Furthermore, Paracoccus sp. Y42 effectively lysed algal cells, which facilitates the downstream oil extraction process for biodiesel production and results in energy savings through the lysing of algal cells. This study provides a more promising candidate for the production of docosahexaenoic acid (DHA) for human nutritional products and of microalgal biofuel as well as a more cost-effective method for breaking algal cells. The high lipid productivity of P. donghaiense and algal cell lysis by algicidal bacteria contribute to reductions in the production cost of microalgal oil.

Host-bacterial interactions over the course of aging are understudied due to complexities of the human microbiome and challenges of collecting samples that span a lifetime. To investigate the role of host-microbial interactions in aging, we performed transcriptomics using wild-type Caenorhabditis elegans (N2) and three long-lived mutants (daf-2, eat-2, and asm-3) fed Escherichia coli OP50 and sampled at days 5, 7.5, and 10 of adulthood. We found host age is a better predictor of the E. coli expression profiles than host genotype. Specifically, host age was associated with clustering (permutational multivariate analysis of variance [PERMANOVA], P = 0.001) and variation (Adonis, P = 0.001, R2 = 11.5%) among E. coli expression profiles, whereas host genotype was not (PERMANOVA, P > 0.05; Adonis, P > 0.05, R2 = 5.9%). Differential analysis of the E. coli transcriptome yielded 22 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 100 KEGG genes enriched when samples were grouped by time point [LDA, linear discriminant analysis; log(LDA), ≥2; P ≤ 0.05], including several involved in biofilm formation. Coexpression analysis of host and bacterial genes yielded six modules of C. elegans genes that were coexpressed with one bacterial regulator gene over time. The three most significant bacterial regulators included genes relating to biofilm formation, lipopolysaccharide production, and thiamine biosynthesis. Age was significantly associated with clustering and variation among transcriptomic samples, supporting the idea that microbes are active and plastic within C. elegans throughout life. Coexpression analysis further revealed interactions between E. coli and C. elegans that occurred over time, building on a growing literature of host-microbial interactions. IMPORTANCE Previous research has reported effects of the microbiome on health span and life span of Caenorhabditis elegans, including interactions with evolutionarily conserved pathways in humans. We build on this literature by reporting the gene expression of Escherichia coli OP50 in wild-type (N2) and three long-lived mutants of C. elegans. The manuscript represents the first study, to our knowledge, to perform temporal host-microbial transcriptomics in the model organism C. elegans. Understanding changes to the microbial transcriptome over time is an important step toward elucidating host-microbial interactions and their potential relationship to aging. We found that age was significantly associated with clustering and variation among transcriptomic samples, supporting the idea that microbes are active and plastic within C. elegans throughout life. Coexpression analysis further revealed interactions between E. coli and C. elegans that occurred over time, which contributes to our growing knowledge about host-microbial interactions.

Accumulating evidence links the gut microbiome to neuronal functions in the brain. Given the increasing prevalence of brain disorders, there is a critical need to understand how gut microbes impact neuronal functions so that targeted therapeutic interventions can be developed. In this commentary, we discuss what makes the nematode Caenorhabditis elegans a valuable model for dissecting the molecular basis of gut microbiome-brain interactions. With a fully mapped neuronal circuitry, C. elegans is an effective model for studying signaling of the nervous system in a context that bears translational relevance to human disease. We highlight C. elegans as a potent but underexploited tool to interrogate the influence of the bacterial variable on the complex equation of the nervous system. We envision that routine use of gnotobiotic C. elegans to examine the gut–brain axis will be an enabling technology for the development of novel therapeutic interventions for brain diseases.

Omnivorous animals, including humans, harbor diverse, species-rich gut communities that impact their growth, development, and homeostasis. Model invertebrates are broadly accessible experimental platforms that enable linking specific species or species groups to host phenotypes, yet often their specialized diets and distinct gut microbiota make them less comparable to human and other mammalian and gut communities. The omnivorous cockroach Periplaneta americana harbors ∼4 × 102 bacterial genera within its digestive tract and is enriched with taxa commonly found in omnivorous mammals (i.e., Proteobacteria, Bacteroidetes, and Firmicutes). These features make P. americana a valuable platform for identifying microbe-mediated host phenotypes with potential translations to mammals. Rearing P. americana insects under germfree conditions resulted in prolonging development time by ∼30% and an up to ∼8% reduction in body size along three dimensions. Germfree rearing resulted in downregulation of gene networks involved in growth, energy homeostasis, and nutrient availability. Reintroduction of a defined microbiota comprised of a subset of P. americana commensals to germfree insects did not recover normal growth and developmental phenotypes or transcriptional profiles observed in conventionally reared insects. These results are in contrast with specialist-feeding model insects (e.g., Drosophila), where introduction of a single endemic bacterial species to germfree condition-reared specimens recovered normal host phenotypes. These data suggest that understanding microbe-mediated host outcomes in animals with species-rich communities should include models that typically maintain similarly diverse microbiomes. The dramatic transcriptional, developmental, and morphological phenotypes linked to gut microbiome status in this study illustrates how microbes are key players in animal growth and evolution. IMPORTANCE Broadly accessible model organisms are essential for illustrating how microbes are engaged in the growth, development, and evolution of animals. We report that germfree rearing of omnivorous Periplaneta americana cockroaches resulted in growth defects and severely disrupted gene networks that regulate development, which highlights the importance of gut microbiota in these host processes. Absence of gut microbiota elicited a starvation-like transcriptional response in which growth and development were inhibited while nutrient scavenging was enhanced. Additionally, reintroduction of a subset of cockroach gut bacterial commensals did not broadly recover normal expression patterns, illustrating that a particular microbiome composition may be necessary for normal host development. Invertebrate microbiota model systems that enable disentangling complex, species-rich communities are essential for linking microbial taxa to specific host phenotypes.

Microorganisms live in dense and diverse communities, with interactions between cells guiding community development and phenotype. The ability to perturb specific intercellular interactions in space and time provides a powerful route to determining the critical interactions and design rules for microbial communities. Approaches using optogenetic tools to modulate these interactions offer promise, as light can be exquisitely controlled in space and time. We report new plasmids for rapid integration of an optogenetic system into Saccharomyces cerevisiae to engineer light control of expression of a gene of interest. In a proof-of-principle study, we demonstrate the ability to control a model cooperative interaction, namely, the expression of the enzyme invertase (SUC2) which allows S. cerevisiae to hydrolyze sucrose and utilize it as a carbon source. We demonstrate that the strength of this cooperative interaction can be tuned in space and time by modulating light intensity and through spatial control of illumination. Spatial control of light allows cooperators and cheaters to be spatially segregated, and we show that the interplay between cooperative and inhibitory interactions in space can lead to pattern formation. Our strategy can be applied to achieve spatiotemporal control of expression of a gene of interest in S. cerevisiae to perturb both intercellular and interspecies interactions. IMPORTANCE Recent advances in microbial ecology have highlighted the importance of intercellular interactions in controlling the development, composition, and resilience of microbial communities. In order to better understand the role of these interactions in governing community development, it is critical to be able to alter them in a controlled manner. Optogenetically controlled interactions offer advantages over static perturbations or chemically controlled interactions, as light can be manipulated in space and time and does not require the addition of nutrients or antibiotics. Here, we report a system for rapidly achieving light control of a gene of interest in the important model organism Saccharomyces cerevisiae and demonstrate that by controlling expression of the enzyme invertase, we can control cooperative interactions. This approach will be useful for understanding intercellular and interspecies interactions in natural and synthetic microbial consortia containing S. cerevisiae and serves as a proof of principle for implementing this approach in other consortia.

Chronic wasting disease (CWD) is a progressive and fatal spongiform encephalopathy of deer and elk species, caused by a misfolded variant of the normal prion protein. Horizontal transmission of the misfolded CWD prion between animals is thought to occur through shedding in saliva and other forms of excreta. The role of blood in CWD transmission is less clear, though infectivity has been demonstrated in various blood fractions. Blood-feeding insects, including ticks, are known vectors for a range of bacterial and viral infections in animals and humans, though to date, there has been no evidence for their involvement in prion disease transmission. In the present study, we evaluated winter ticks (Dermacentor albipictus) collected from 136 North American elk (Cervus canadensis) in an area where CWD is endemic for evidence of CWD prion amplification using the real-time quaking-induced conversion assay (RT-QuIC). Although 30 elk were found to be CWD positive (22%) postmortem, amplifiable prions were found in just a single tick collected from an elk in advanced stages of CWD infection, with some evidence for prions in ticks collected from elk in mid-stage infection. These findings suggest that further investigation of ticks as reservoirs for prion disease may be warranted. IMPORTANCE This study reports the first finding of detectable levels of prions linked to chronic wasting disease in a tick collected from a clinically infected elk. Using the real-time quaking-induced conversion assay (RT-QuIC), "suspect" samples were also identified; these suspect ticks were more likely to have been collected from CWD-positive elk, though suspect amplification was also observed in ticks collected from CWD-negative elk. Observed levels were at the lower end of our detection limits, though our findings suggest that additional research evaluating ticks collected from animals in late-stage disease may be warranted to further evaluate the role of ticks as potential vectors of chronic wasting disease.

Zebrafish (Danio rerio) is an attractive model organism to use for an array of scientific studies, including host-microbe interactions. Zebrafish contain a core (i.e., consistently detected) intestinal microbiome consisting primarily of Proteobacteria. Furthermore, this core intestinal microbiome is plastic and can be significantly altered due to external factors. Zebrafish are particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae. As an intestinal pathogen, V. cholerae must colonize the intestine of an exposed host for pathogenicity to occur. Members of the resident intestinal microbial community likely must be reduced or eliminated by V. cholerae for colonization, and subsequent disease, to occur. Many studies have explored a variety of aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms but few have researched how a V. cholerae infection changes the resident intestinal microbiome. In this study, 16S rRNA gene sequencing was used to examine how five genetically diverse V. cholerae strains alter the intestinal microbiome following an infection. We found that V. cholerae colonization induced significant changes in the zebrafish intestinal microbiome. Notably, changes in the microbial profile were significantly different from each other, based on the particular strain of V. cholerae used to infect zebrafish hosts. We conclude that V. cholerae significantly modulates the zebrafish intestinal microbiota to enable colonization and that specific microbes that are targeted depend on the V. cholerae genotype.

Zebrafish (Danio rerio) are an attractive model organism for a variety of scientific studies, including host-microbe interactions. The organism is particularly useful for the study of aquatic microbes that can colonize vertebrate hosts, including Vibrio cholerae, an intestinal pathogen. V. cholerae must colonize the intestine of an exposed host for pathogenicity to occur. While numerous studies have explored various aspects of the pathogenic effects of V. cholerae on zebrafish and other model organisms, few, if any, have examined how a V. cholerae infection alters the resident intestinal microbiome and the role of the type six secretion system (T6SS) in that process. In this study, 16S rRNA gene sequencing was utilized to investigate how strains of V. cholerae both with and without the T6SS alter the aforementioned microbial profiles following an infection. V. cholerae infection induced significant changes in the zebrafish intestinal microbiome, and while not necessary for colonization, the T6SS was important for inducing mucin secretion, a marker for diarrhea. Additional salient differences to the microbiome were observed based on the presence or absence of the T6SS in the V. cholerae utilized for challenging the zebrafish hosts. We conclude that V. cholerae significantly modulates the zebrafish intestinal microbiome to enable colonization and that the T6SS is important for pathogenesis induced by the examined V. cholerae strains. Furthermore, the presence or absence of T6SS differentially and significantly affected the composition and structure of the intestinal microbiome, with an increased abundance of other Vibrio bacteria observed in the absence of V. cholerae T6SS.

Fungi are responsible for diseases that result in the deaths of over a million individuals each year and devastating crop infestations that threaten global food supplies. However, outside of a select few model organisms, the majority of fungal genes are uncharacterized. The roles of these genes in the biology of the organism, pathogenesis, and mediating interactions with the environment and other microbes are unknown. Historically, fungal gene characterization has primarily relied on classical genetic screens. However, advances in sequencing technology have enabled more rapid methods of gene functional characterization. Large-scale transcriptional profiling projects are one solution to generating hypotheses about fungal gene function. Together with other ‘omics techniques and newer tools that enable massively parallel mutant screens, knowledge of fungal gene function will be substantially improved. Understanding the function of fungal genes will be instrumental in increasing global food security, protecting ecosystems, and improving health outcomes.

The species specificity of papillomaviruses has been a significant roadblock for performing in vivo pathogenesis studies in common model organisms. The Mus musculus papillomavirus type 1 (MmuPV1) causes cutaneous papillomas that can progress to squamous cell carcinomas in laboratory mice. The papillomavirus E6 and E7 genes encode proteins that establish and maintain a cellular milieu that allows for viral genome synthesis and viral progeny synthesis in growth-arrested, terminally differentiated keratinocytes. The E6 and E7 proteins provide this activity by binding to and functionally reprogramming key cellular regulatory proteins. The MmuPV1 E7 protein lacks the canonical LXCXE motif that mediates the binding of multiple viral oncoproteins to the cellular retinoblastoma tumor suppressor protein, RB1. Our proteomic experiments, however, revealed that MmuPV1 E7 still interacts specifically with RB1. We show that MmuPV1 E7 interacts through its C-terminus with the C-terminal domain of RB1. Binding of MmuPV1 E7 to RB1 did not cause significant activation of E2F-regulated cellular genes. MmuPV1 E7 expression was shown to be essential for papilloma formation. Experimental infection of mice with MmuPV1 virus expressing an E7 mutant that is defective for binding to RB1 caused delayed onset, lower incidence, and smaller sizes of papillomas. Our results demonstrate that the MmuPV1 E7 gene is essential and that targeting non-canonical activities of RB1, which are independent of RB1’s ability to modulate the expression of E2F-regulated genes, contribute to papillomavirus-mediated pathogenesis.ImportancePapillomavirus infections cause a variety of epithelial hyperplastic lesions, warts. While most warts are benign, some papillomaviruses cause lesions that can progress to squamous cell carcinomas and approximately 5% of all human cancers are caused by human papillomavirus (HPV) infections. The papillomavirus E6 and E7 proteins are thought to function to reprogram host epithelial cells to enable viral genome replication in terminally differentiated, normally growth-arrested cells. E6 and E7 lack enzymatic activities and function by interacting and functionally altering host cell regulatory proteins. Many cellular proteins that can interact with E6 and E7 have been identified, but the biological relevance of these interactions for viral pathogenesis has not been determined. This is because papillomaviruses are species-specific and do not infect heterologous hosts. Here we use a recently established mouse papillomavirus (MmuPV1) model to investigate the role of the E7 protein in viral pathogenesis. We show that MmuPV1 E7 is necessary for papilloma formation. The retinoblastoma tumor suppressor protein (RB1) is targeted by many papillomaviral E7 proteins, including cancer-associated HPVs. We show that MmuPV1 E7 can bind RB1 and that infection with a mutant MmuPV1 virus that expresses an RB1 binding defective E7 mutant caused smaller and fewer papillomas that arise with delayed kinetics.

Here, we report the genome sequence of bacteriophage Elbi that infects the cyanobacterium Anabaena sp. strain PCC 7120, a model organism for prokaryotic multicellular development. The 68,626 bp encode 108 proteins, of which 31 can be assigned a function. Elbi is similar to two Anabaena myophages, namely, A-1 and N-1, isolated in the 1970s.

Rediscovered as a potential epigenetic mark, N6-methyladenine DNA modification (6mA) was recently reported to be sensitive to environmental stressors in several multicellular eukaryotes. As 6mA distribution and function differ significantly in multicellular and unicellular organisms, whether and how 6mA in unicellular eukaryotes responds to environmental stress remains elusive. Here, we characterized the dynamic changes of 6mA under starvation in the unicellular model organism Tetrahymena thermophila. Single-molecule, real-time (SMRT) sequencing reveals that DNA 6mA levels in starved cells are significantly reduced, especially symmetric 6mA, compared to those in vegetatively growing cells. Despite a global 6mA reduction, the fraction of asymmetric 6mA with a high methylation level was increased, which might be the driving force for stronger nucleosome positioning in starved cells. Starvation affects expression of many metabolism-related genes, the expression level change of which is associated with the amount of 6mA change, thereby linking 6mA with global transcription and starvation adaptation. The reduction of symmetric 6mA and the increase of asymmetric 6mA coincide with the downregulation of AMT1 and upregulation of AMT2 and AMT5, which are supposedly the MT-A70 methyltransferases required for symmetric and asymmetric 6mA, respectively. These results demonstrated that a regulated 6mA response to environmental cues is evolutionarily conserved in eukaryotes. IMPORTANCE Increasing evidence indicated that 6mA could respond to environmental stressors in multicellular eukaryotes. As 6mA distribution and function differ significantly in multicellular and unicellular organisms, whether and how 6mA in unicellular eukaryotes responds to environmental stress remains elusive. In the present work, we characterized the dynamic changes of 6mA under starvation in the unicellular model organism Tetrahymena thermophila. Our results provide insights into how Tetrahymena fine-tunes its 6mA level and composition upon starvation, suggesting that a regulated 6mA response to environmental cues is evolutionarily conserved in eukaryotes.

Research and marketing of probiotics demand holistic strain improvement considering both the biotic and abiotic gut environment. Here, we aim to establish the continuous in vitro colonic fermentation model PolyFermS as a tool for adaptive evolutionary engineering. Immobilized fecal microbiota from adult donors were steadily cultivated up to 72 days in PolyFermS reactors, providing a long-term compositional and functional stable ecosystem akin to the donor’s gut. Inoculation of the gut microbiota with immobilized or planktonic Lactiplantibacillus plantarum NZ3400, a derivative of the probiotic model strain WCFS1, led to successful colonization. Whole-genome sequencing of 45 recovered strains revealed mutations in 16 genes involved in signaling, metabolism, transport, and cell surface. Remarkably, mutations in LP_RS14990, LP_RS15205, and intergenic region LP_RS05100, mSystems, 6 (2), ISSN:2379-5077

Amoxicillin-clavulanic acid (AMC) is one of the most frequently prescribed antibiotic formulations in the Western world. Extensive oral use of this antimicrobial combination influences the gut microbiota. One of the most abundant early colonizers of the human gut microbiota is represented by different taxa of the Bifidobacterium genus, which include many members that are considered to bestow beneficial effects upon their host. In the current study, we investigated the impact of AMC administration on the gut microbiota composition, comparing the gut microbiota of 23 children that had undergone AMC antibiotic therapy to that of 19 children that had not been treated with antibiotics during the preceding 6 months. Moreover, we evaluated AMC sensitivity by MIC test of 261 bifidobacterial strains, including reference strains for the currently recognized 64 bifidobacterial (sub)species, as well as 197 bifidobacterial isolates of human origin. These assessments allowed the identification of four bifidobacterial strains that exhibit a high level of AMC insensitivity, which were subjected to genomic and transcriptomic analyses to identify the putative genetic determinants responsible for this AMC insensitivity. Furthermore, we investigated the ecological role of AMC-resistant bifidobacterial strains by in vitro batch cultures. IMPORTANCE Based on our results, we observed a drastic reduction in gut microbiota diversity of children treated with antibiotics, which also affected the abundance of Bifidobacterium, a bacterial genus commonly found in the infant gut. MIC experiments revealed that more than 98% of bifidobacterial strains tested were shown to be inhibited by the AMC antibiotic. Isolation of four insensitive strains and sequencing of their genomes revealed the identity of possible genes involved in AMC resistance mechanisms. Moreover, gut-simulating in vitro experiments revealed that one strain, i.e., Bifidobacterium breve PRL2020, is able to persist in the presence of a complex microbiota combined with AMC antibiotic.

Diseases caused by mycobacterial species result in millions of deaths per year globally, and present a substantial health and economic burden, especially in immunocompromised patients. Difficulties inherent in working with mycobacterial pathogens have hampered the development and application of high-throughput genetics that can inform genome annotations and subsequent functional assays., Functional characterization of bacterial proteins lags far behind the identification of new protein families. This is especially true for bacterial species that are more difficult to grow and genetically manipulate than model systems such as Escherichia coli and Bacillus subtilis. To facilitate functional characterization of mycobacterial proteins, we have established a Mycobacterial Systems Resource (MSR) using the model organism Mycobacterium smegmatis. This resource focuses specifically on 1,153 highly conserved core genes that are common to many mycobacterial species, including Mycobacterium tuberculosis, in order to provide the most relevant information and resources for the mycobacterial research community. The MSR includes both biological and bioinformatic resources. The biological resource includes (i) an expression plasmid library of 1,116 genes fused to a fluorescent protein for determining protein localization; (ii) a library of 569 precise deletions of nonessential genes; and (iii) a set of 843 CRISPR-interference (CRISPRi) plasmids specifically targeted to silence expression of essential core genes and genes for which a precise deletion was not obtained. The bioinformatic resource includes information about individual genes and a detailed assessment of protein localization. We anticipate that integration of these initial functional analyses and the availability of the biological resource will facilitate studies of these core proteins in many Mycobacterium species, including the less experimentally tractable pathogens M. abscessus, M. avium, M. kansasii, M. leprae, M. marinum, M. tuberculosis, and M. ulcerans.

Isoprene is a valuable petrochemical used for a wide variety of consumer goods, such as adhesives and synthetic rubber. We were able to achieve a high yield of renewable isoprene by taking advantage of the naturally high-flux mevalonate lipid synthesis pathway in anaerobic methane-producing archaea (methanogens). Our study illustrates that by genetically manipulating Methanosarcina species methanogens, it is possible to create organisms that grow by producing the hemiterpene isoprene. Mass balance measurements show that engineered methanogens direct up to 4% of total carbon flux to isoprene, demonstrating that methanogens produce higher isoprene yields than engineered yeast, bacteria, or cyanobacteria, and from inexpensive feedstocks. Expression of isoprene synthase resulted in increased biomass and changes in gene expression that indicate that isoprene synthesis depletes membrane precursors and redirects electron flux, enabling isoprene to be a major metabolic product. Our results demonstrate that methanogens are a promising engineering chassis for renewable isoprene synthesis. IMPORTANCE A significant barrier to implementing renewable chemical technologies is high production costs relative to those for petroleum-derived products. Existing technologies using engineered organisms have difficulty competing with petroleum-derived chemicals due to the cost of feedstocks (such as glucose), product extraction, and purification. The hemiterpene monomer isoprene is one such chemical that cannot currently be produced using cost-competitive renewable biotechnologies. To reduce the cost of renewable isoprene, we have engineered methanogens to synthesize it from inexpensive feedstocks such as methane, methanol, acetate, and carbon dioxide. The “isoprenogen” strains we developed have potential to be used for industrial production of inexpensive renewable isoprene.

There is an urgent need for sustainable protein supply routes with low environmental footprint. Recently, the use of hydrogen-oxidizing bacteria (HOB) as a platform for high-quality microbial protein (MP) production has regained interest. This study aims to investigate the added value of using conditions such as salt and temperature to steer HOB communities to lower diversities, while maintaining a high protein content and a high-quality amino acid profile. Pressure drop and hydrogen consumption were measured for 56 days to evaluate autotrophy of a total of six communities in serum flasks. Of the six communities, four were enriched under saline (0.0, 0.25, 0.5, and 1.0 mol NaCl liter−1) and two under thermophilic conditions (65°C). Five communities enriched for HOB were subsequently cultivated in continuously stirred reactors under the same conditions to evaluate their potential as microbial protein producers. The protein percentages ranged from 41 to 80%. The highest protein content was obtained for the thermophilic enrichments. Amino acid profiles were comparable to protein sources commonly used for feed purposes. Members of the genus Achromobacter were found to dominate the saline enrichments, while members of the genus Hydrogenibacillus were found to dominate the thermophilic enrichments. Here, we show that enriching for HOB while steering the community toward low diversity and maintaining a high-quality protein content can be successfully achieved, in both saline and thermophilic conditions. IMPORTANCE Alternative feed and food supply chains are required to decrease water and land use. HOB offer a promising substitute for traditional agricultural practice to produce microbial protein (MP) from residual materials and renewable energy. To safeguard product stability, the composition of the HOB community should be controlled. Defining strategies to maintain the stability of the communities is therefore key for optimization purposes. In this study, we use salt and temperature as independent conditions to stabilize the composition of the HOB communities. Based on the results presented, we conclude that HOB communities can be steered to have low diversity using the presented conditions while producing a desirable protein content with a valuable amino acid profile.

We report the complete genome sequence of the virulent Aeromonas salmonicida subsp. masoucida strain BR19001YR, isolated from diseased black rockfish ( Sebastes schlegelii ). Sequencing of the circular chromosome and three plasmids using the PacBio and Illumina platforms yielded 4,982,192 bp with a 58.24% G+C content.

Bacteria have evolved a variety of signal transduction mechanisms that generate different outputs in response to external stimuli. Chemosensory pathways are widespread in bacteria and are among the most complex signaling mechanisms, requiring the participation of at least six proteins. These pathways mediate flagellar chemotaxis, in addition to controlling alternative functions such as second messenger levels or twitching motility. The human pathogen Pseudomonas aeruginosa has four different chemosensory pathways that carry out different functions and are stimulated by signal binding to 26 chemoreceptors. Recent research employing a diverse range of experimental approaches has advanced enormously our knowledge on these four pathways, establishing P. aeruginosa as a primary model organism in this field. In the first part of this article, we review data on the function and physiological relevance of chemosensory pathways as well as their involvement in virulence, whereas the different transcriptional and posttranscriptional regulatory mechanisms that govern pathway function are summarized in the second part. The information presented will be of help to advance the understanding of pathway function in other organisms., This study was supported by FEDER funds and the Fondo Social Europeo through grants from the Spanish Ministry for Science and Innovation to M.A.M. (PID2019-103972GA-I00) and the Spanish Ministry of Economy and Competitiveness to J.A.G. (BIO2016-74875-P) and T.K. (BIO2016-76779-P).

Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a Cd300lf conditional knockout (CD300lf(F/F)) mouse to elucidate the cell tropism of persistent and nonpersistent strains of murine norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoV(CR6)) in vivo. In contrast, the nonpersistent MNoV strain CW3 (MNoV(CW3)) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (LysM Cre+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (CD19 Cre), neutrophils (Mrp8 Cre), and dendritic cells (CD11c Cre) did not affect MNoV(CW3) viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoV(CW3) to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoV(CR6); that myelomonocytic cells are a major, but not exclusive, target cell of MNoV(CW3); and that STAT1 signaling restricts the cellular tropism of MNoV(CW3). This study provides the first genetic system for studying the cell type-specific role of CD300lf in norovirus pathogenesis. IMPORTANCE Human noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a Cd300lf conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoV(CR6) requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoV(CW3) infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoV(CW3) infection. Mortality associated with the MNoV(CW3) strain in Stat1(−/−) mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism in vivo.

Departamento de Tecnología de Alimentos​​ (INIA), The draft genome sequence of Bifidobacterium breve INIA P734, a strain shared by mother and child, is reported. It consists of 50 contigs, with 2,391,925 bp, 2,099 genes, and a G+C content of 58.8%. The genome analysis revealed the absence of antibiotic resistance and pathogenicity-related genes., This work was supported by project no. RTA2017-00002-00-00 from the Spanish Ministry of Science and Innovation and RMP2015-00001-00-00 from the National Institute for Agricultural and Food Research and Technology (INIA) and the Spanish Agency for Research (AEI-MCIU), cofinanced by the European Regional Development Fund., 2 Pág.

Arbuscular mycorrhizal (AM) fungi form tight symbiotic relationships with the majority of terrestrial plants and play critical roles in plant P acquisition, adding a further dimension of complexity. The plant-AM fungus-bacterium system is considered a continuum, with the bacteria colonizing not only the plant roots, but also the associated mycorrhizal hyphal network, known as the hyphosphere microbiome. Plant roots are usually colonized by different AM fungal species which form an independent phosphorus uptake pathway from the root pathway, i.e., the mycorrhizal pathway., Plant roots are usually colonized by various arbuscular mycorrhizal (AM) fungal species, which vary in morphological, physiological, and genetic traits. This colonization constitutes the mycorrhizal nutrient uptake pathway (MP) and supplements the pathway through roots. Simultaneously, the extraradical hyphae of each AM fungus is associated with a community of bacteria. However, whether the community structure and function of the microbiome on the extraradical hyphae differ between AM fungal species remains unknown. In order to understand the community structure and the predicted functions of the microbiome associated with different AM fungal species, a split-root compartmented rhizobox cultivation system, which allowed us to inoculate two AM fungal species separately in two root compartments, was used. We inoculated two separate AM fungal species combinations, (i) Funneliformis mosseae and Gigaspora margarita and (ii) Rhizophagus intraradices and G. margarita, on a single root system of cotton. The hyphal exudate-fed, active microbiome was measured by combining 13C-DNA stable isotope probing with MiSeq sequencing. We found that different AM fungal species, which were simultaneously colonizing a single root system, hosted active microbiomes that were distinct from one another. Moreover, the predicted potential functions of the different microbiomes were distinct. We conclude that the arbuscular mycorrhizal fungal component of the system is responsible for the recruitment of distinct microbiomes in the hyphosphere. The potential significance of the predicted functions of the microbial ecosystem services is discussed. IMPORTANCE Arbuscular mycorrhizal (AM) fungi form tight symbiotic relationships with the majority of terrestrial plants and play critical roles in plant P acquisition, adding a further dimension of complexity. The plant-AM fungus-bacterium system is considered a continuum, with the bacteria colonizing not only the plant roots, but also the associated mycorrhizal hyphal network, known as the hyphosphere microbiome. Plant roots are usually colonized by different AM fungal species which form an independent phosphorus uptake pathway from the root pathway, i.e., the mycorrhizal pathway. The community structure and function of the hyphosphere microbiome of different AM species are completely unknown. In this novel study, we found that arbuscular mycorrhizal fungi cocolonizing on single plant roots recruit their own specific microbiomes, which should be considered in evaluating plant microbiome form and function. Our findings demonstrate the importance of understanding trophic interactions in order to gain insight into the plant-AM fungus-bacterium symbiosis.

Rickettsiales -like organisms are important for the survival and functioning of corals, prompting an investigation of their complete genomes. Earlier reports of the genomes of these organisms remain incomplete. Here, we report a novel draft genome of Rickettsiales bacterial strain SESOKO1, found in Acropora tenuis coral, using single-cell genome technology.

Pseudomonas aeruginosa is an opportunistic pathogen that is frequently associated with both acute and chronic infections. P. aeruginosa possesses a complex regulatory network that modulates nutrient acquisition and virulence, but our knowledge of these networks is largely based on studies with shaking cultures, which are not likely representative of conditions during infection. Here, we provide proteomic, metabolic, and genetic evidence that regulation by iron, a critical metallonutrient, is altered in static P. aeruginosa cultures. Specifically, we observed a loss of iron-induced expression of proteins for oxidative phosphorylation, tricarboxylic acid (TCA) cycle metabolism under static conditions. Moreover, we identified type VI secretion as a target of iron regulation in P. aeruginosa cells under static but not shaking conditions, and we present evidence that this regulation occurs via PrrF small regulatory RNA (sRNA)-dependent production of 2-alkyl-4(1H)-quinolone metabolites. These results yield new iron regulation paradigms in an important opportunistic pathogen and highlight the need to redefine iron homeostasis in static microbial communities. IMPORTANCE Host-mediated iron starvation is a broadly conserved signal for microbial pathogens to upregulate expression of virulence traits required for successful infection. Historically, global iron regulatory studies in microorganisms have been conducted in shaking cultures to ensure culture homogeneity, yet these conditions are likely not reflective of growth during infection. Pseudomonas aeruginosa is a well-studied opportunistic pathogen and model organism for iron regulatory studies. Iron homeostasis is maintained through the Fur protein and PrrF small regulatory sRNAs, the functions of which are highly conserved in many other bacterial species. In the current study, we examined how static growth affects the known iron and PrrF regulons of P. aeruginosa, leading to the discovery of novel PrrF-regulated virulence processes. This study demonstrates how the utilization of distinct growth models can enhance our understanding of basic physiological processes that may also affect pathogenesis.

Plasmodium parasites, which cause malaria, and related apicomplexans are important human and veterinary pathogens. These parasites represent a highly divergent and understudied branch of eukaryotes, and as such often defy the expectations set by model organisms. One striking example of unique apicomplexan biology is the apicoplast, an essential but nonphotosynthetic plastid derived from an unusual secondary (eukaryote-eukaryote) endosymbiosis. Endosymbioses are a major driver of cellular innovation, and apicoplast biogenesis pathways represent a hot spot for molecular evolution. We previously conducted an unbiased screen for apicoplast biogenesis genes in P. falciparum to uncover these essential and innovative pathways. Here, we validate a novel gene candidate from our screen and show that its role in apicoplast biogenesis does not match its functional annotation predicted by model eukaryotes. Our findings suggest that an uncharacterized chloroplast maintenance pathway has been reused for complex plastid biogenesis in this divergent branch of pathogens., Plasmodium parasites and related apicomplexans contain an essential “complex plastid” organelle of secondary endosymbiotic origin, the apicoplast. Biogenesis of this complex plastid poses a unique challenge requiring evolution of new cellular machinery. We previously conducted a mutagenesis screen for essential apicoplast biogenesis genes to discover organellar pathways with evolutionary and biomedical significance. Here we validate and characterize a gene candidate from our screen, Pf3D7_0913500. Using a conditional knockdown strain, we show that Pf3D7_0913500 depletion causes growth inhibition that is rescued by the sole essential product of the apicoplast, isopentenyl pyrophosphate (IPP), and results in apicoplast loss. Because Pf3D7_0913500 had no previous functional annotation, we name it apicoplast-minus IPP-rescued 4 (AMR4). AMR4 has an annotated CaaX protease and bacteriocin processing (CPBP) domain, which in eukaryotes typically indicates a role in CaaX postprenylation processing. Indeed, AMR4 is the only putative CaaX-like protease in Plasmodium parasites which are known to require protein prenylation, and we confirm that the conserved catalytic residue of AMR4 (E352) is required for its apicoplast function. However, we unexpectedly find that AMR4 does not act in a CaaX postprenylation processing pathway in Plasmodium falciparum. Instead, we find that AMR4 is imported into the apicoplast and is derived from a cyanobacterial CPBP gene which was retained through both primary and secondary endosymbiosis. Our findings suggest that AMR4 is not a true CaaX protease, but instead it performs a conserved, uncharacterized chloroplast function that has been retained for complex plastid biogenesis.

Colorless plastids incapable of photosynthesis evolved in many plant and algal groups, but what functions they perform is still unknown in many cases. Here, we study the elusive plastid of Euglena longa, a nonphotosynthetic cousin of the familiar green flagellate Euglena gracilis. We document an unprecedented combination of metabolic functions that the E. longa plastid exhibits in comparison with previously characterized nonphotosynthetic plastids. For example, and truly surprisingly, it has retained the synthesis of tocopherols (vitamin E) and a phylloquinone (vitamin K) derivative. In addition, we offer a possible solution of the long-standing conundrum of the presence of the CO2-fixing enzyme RuBisCO in E. longa. Our work provides a detailed account on a unique variant of relic plastids, the first among nonphotosynthetic plastids that evolved by secondary endosymbiosis from a green algal ancestor, and suggests that it has persisted for reasons not previously considered in relation to nonphotosynthetic plastids., Most secondary nonphotosynthetic eukaryotes have retained residual plastids whose physiological role is often still unknown. One such example is Euglena longa, a close nonphotosynthetic relative of Euglena gracilis harboring a plastid organelle of enigmatic function. By mining transcriptome data from E. longa, we finally provide an overview of metabolic processes localized to its elusive plastid. The organelle plays no role in the biosynthesis of isoprenoid precursors and fatty acids and has a very limited repertoire of pathways concerning nitrogen-containing metabolites. In contrast, the synthesis of phospholipids and glycolipids has been preserved, curiously with the last step of sulfoquinovosyldiacylglycerol synthesis being catalyzed by the SqdX form of an enzyme so far known only from bacteria. Notably, we show that the E. longa plastid synthesizes tocopherols and a phylloquinone derivative, the first such report for nonphotosynthetic plastids studied so far. The most striking attribute of the organelle could be the presence of a linearized Calvin-Benson (CB) pathway, including RuBisCO yet lacking the gluconeogenetic part of the standard cycle, together with ferredoxin-NADP+ reductase (FNR) and the ferredoxin/thioredoxin system. We hypothesize that the ferredoxin/thioredoxin system activates the linear CB pathway in response to the redox status of the E. longa cell and speculate on the role of the pathway in keeping the redox balance of the cell. Altogether, the E. longa plastid defines a new class of relic plastids that is drastically different from the best-studied organelle of this category, the apicoplast. IMPORTANCE Colorless plastids incapable of photosynthesis evolved in many plant and algal groups, but what functions they perform is still unknown in many cases. Here, we study the elusive plastid of Euglena longa, a nonphotosynthetic cousin of the familiar green flagellate Euglena gracilis. We document an unprecedented combination of metabolic functions that the E. longa plastid exhibits in comparison with previously characterized nonphotosynthetic plastids. For example, and truly surprisingly, it has retained the synthesis of tocopherols (vitamin E) and a phylloquinone (vitamin K) derivative. In addition, we offer a possible solution of the long-standing conundrum of the presence of the CO2-fixing enzyme RuBisCO in E. longa. Our work provides a detailed account on a unique variant of relic plastids, the first among nonphotosynthetic plastids that evolved by secondary endosymbiosis from a green algal ancestor, and suggests that it has persisted for reasons not previously considered in relation to nonphotosynthetic plastids.

Partitiviruses are segmented, multipartite dsRNA viruses that until recently were only known to infect fungi, plants, and protozoans. Metagenomic surveys have revealed that partitivirus-like sequences are also commonly associated with arthropods. One arthropod-associated partitivirus, galbut virus, is extraordinarily common in wild populations of Drosophila melanogaster fruit flies. To begin to understand the processes that underlie this virus’s high global prevalence, we established colonies of wild-caught infected flies. Infection remained at stably high levels over three years, with between 63-100% of individual flies infected. Galbut virus infects fly cells and replicates in tissues throughout infected adults, including reproductive tissues and the gut epithelium. We detected no evidence of horizontal transmission via ingestion but vertical transmission from either infected females or infected males was ~100% efficient. Vertical transmission of a related partitivirus, verdadero virus, that we discovered in a laboratory colony of Aedes aegypti mosquitoes was similarly efficient. This suggests that efficient biparental vertical transmission may be a feature of at least a subset of insect-infecting partitiviruses. To study the impact of galbut virus infection free from the confounding effect of other viruses, we generated an inbred line of flies with galbut virus as the only detectable virus infection. We were able to transmit infection experimentally via microinjection of homogenate from these galbut-only flies. This sets the stage for experiments to understand the biological impact and possible utility of partitiviruses infecting important model organisms and disease vectors.ImportanceGalbut virus is a recently discovered partitivirus that is extraordinarly common in wild populations of the model organism Drosophila melanogaster. Like most viruses discovered through metagenomics, most of the basic biological questions about this virus remain unanswered. We found that galbut virus, along with a closely related partitivirus found in Aedes aegypti mosquitoes, is transmitted from infected females or males to offspring with ~100% efficiency and can be maintained in laboratory colonies over years. This represents one of the most efficient means of virus transmission described, and likely underlies the successful spread of these viruses through insect populations. We created Drosophila lines that contained galbut virus as the only virus infection and showed that these flies can be used as a source for experimental infections. This provides insight into how arthropod-infecting partitiviruses may be maintained in nature and sets the stage for exploration of their biology and potential utility.

Here, we report the genome sequence and characterization for a Blattabacterium strain isolated from the viviparous cockroach, Diploptera punctata, which provides amino acids critical for intrauterine embryo development. The genome was assembled by sequencing of the cockroach fat body, which is the location of this obligate symbiont.

Experimental evolution studies have elucidated evolutionary processes, but usually in chemically well-defined and/or constant environments. Using complex environments is important to begin to understand how evolution may occur in natural environments, such as soils or within a host. However, characterizing the stresses that cells experience in these complex environments can be challenging. One way to approach this is by determining how cells biochemically acclimate to heterogenous environments. In this study, we began to characterize physiological changes by analyzing the transcriptome of cells in a dynamic complex environment. By characterizing the transcriptional profile of cells in long-term stationary phase, a heterogenous and stressful environment, we can begin to understand how cells physiologically and biochemically react to the laboratory environment, and how this compares to more-natural conditions., Microbes live in complex and constantly changing environments, but it is difficult to replicate this in the laboratory. Escherichia coli has been used as a model organism in experimental evolution studies for years; specifically, we and others have used it to study evolution in complex environments by incubating the cells into long-term stationary phase (LTSP) in rich media. In LTSP, cells experience a variety of stresses and changing conditions. While we have hypothesized that this experimental system is more similar to natural environments than some other lab conditions, we do not yet know how cells respond to this environment biochemically or physiologically. In this study, we began to unravel the cells’ responses to this environment by characterizing the transcriptome of cells during LTSP. We found that cells in LTSP have a unique transcriptional program and that several genes are uniquely upregulated or downregulated in this phase. Further, we identified two genes, cspB and cspI, which are most highly expressed in LTSP, even though these genes are primarily known to respond to cold shock. By competing cells lacking these genes with wild-type cells, we show that these genes are also important for survival during LTSP. These data can help identify gene products that may play a role in survival in this complex environment and lead to identification of novel functions of proteins. IMPORTANCE Experimental evolution studies have elucidated evolutionary processes, but usually in chemically well-defined and/or constant environments. Using complex environments is important to begin to understand how evolution may occur in natural environments, such as soils or within a host. However, characterizing the stresses that cells experience in these complex environments can be challenging. One way to approach this is by determining how cells biochemically acclimate to heterogenous environments. In this study, we began to characterize physiological changes by analyzing the transcriptome of cells in a dynamic complex environment. By characterizing the transcriptional profile of cells in long-term stationary phase, a heterogenous and stressful environment, we can begin to understand how cells physiologically and biochemically react to the laboratory environment, and how this compares to more-natural conditions.

Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated., Although many bacterial cell division factors have been uncovered over the years, evidence from recent studies points to the existence of yet-to-be-discovered factors involved in cell division regulation. Thus, it is important to identify factors and conditions that regulate cell division to obtain a better understanding of this fundamental biological process. We recently reported that in the Gram-positive organisms Bacillus subtilis and Staphylococcus aureus, increased production of YpsA resulted in cell division inhibition. In this study, we isolated spontaneous suppressor mutations to uncover critical residues of YpsA and the pathways through which YpsA may exert its function. Using this technique, we were able to isolate four unique intragenic suppressor mutations in ypsA (E55D, P79L, R111P, and G132E) that rendered the mutated YpsA nontoxic upon overproduction. We also isolated an extragenic suppressor mutation in yfhS, a gene that encodes a protein of unknown function. Subsequent analysis confirmed that cells lacking yfhS were unable to undergo filamentation in response to YpsA overproduction. We also serendipitously discovered that YfhS may play a role in cell size regulation. Finally, we provide evidence showing a mechanistic link between YpsA and YfhS. IMPORTANCE Bacillus subtilis is a rod-shaped Gram-positive model organism. The factors fundamental to the maintenance of cell shape and cell division are of major interest. We show that increased expression of ypsA results in cell division inhibition and impairment of colony formation on solid medium. Colonies that do arise possess compensatory suppressor mutations. We have isolated multiple intragenic (within ypsA) mutants and an extragenic suppressor mutant. Further analysis of the extragenic suppressor mutation led to a protein of unknown function, YfhS, which appears to play a role in regulating cell size. In addition to confirming that the cell division phenotype associated with YpsA is disrupted in a yfhS-null strain, we also discovered that the cell size phenotype of the yfhS knockout mutant is abolished in a strain that also lacks ypsA. This highlights a potential mechanistic link between these two proteins; however, the underlying molecular mechanism remains to be elucidated.

Cholera is a devastating illness that kills tens of thousands of people annually. Vibrio cholerae, the causative agent of cholera, is an important model organism to investigate both bacterial pathogenesis and the impact of horizontal gene transfer on the emergence and dissemination of new virulent strains. Despite the importance of this pathogen, roughly one-third of V. cholerae genes are functionally unannotated, leaving large gaps in our understanding of this microbe. Through coexpression network analysis of existing RNA sequencing data, this work develops an approach to uncover novel gene-gene relationships and contextualize genes with no known function, which will advance our understanding of V. cholerae virulence and evolution., Research into the evolution and pathogenesis of Vibrio cholerae has benefited greatly from the generation of high-throughput sequencing data to drive molecular analyses. The steady accumulation of these data sets now provides a unique opportunity for in silico hypothesis generation via coexpression analysis. Here, we leverage all published V. cholerae RNA sequencing data, in combination with select data from other platforms, to generate a gene coexpression network that validates known gene interactions and identifies novel genetic partners across the entire V. cholerae genome. This network provides direct insights into genes influencing pathogenicity, metabolism, and transcriptional regulation, further clarifies results from previous sequencing experiments in V. cholerae (e.g., transposon insertion sequencing [Tn-seq] and chromatin immunoprecipitation sequencing [ChIP-seq]), and expands upon microarray-based findings in related Gram-negative bacteria. IMPORTANCE Cholera is a devastating illness that kills tens of thousands of people annually. Vibrio cholerae, the causative agent of cholera, is an important model organism to investigate both bacterial pathogenesis and the impact of horizontal gene transfer on the emergence and dissemination of new virulent strains. Despite the importance of this pathogen, roughly one-third of V. cholerae genes are functionally unannotated, leaving large gaps in our understanding of this microbe. Through coexpression network analysis of existing RNA sequencing data, this work develops an approach to uncover novel gene-gene relationships and contextualize genes with no known function, which will advance our understanding of V. cholerae virulence and evolution.

Bifidobacterial species are common inhabitants of the gut of human infants during the period when milk is a major component of the diet. Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum subspecies longum, and B. longum subspecies infantis have been detected frequently in infant feces, but B. longum subsp. infantis may be disadvantaged numerically in the gut of infants in westernized countries. This may be due to the different durations of breast milk feeding in different countries. Supplementation of the infant diet or replacement of breast milk using formula feeds is common in Western countries. Formula milks often contain galacto- and/or fructo-oligosaccharides (GOS and FOS, respectively) as additives to augment the concentration of oligosaccharides in ruminant milks, but the ability of B. longum subsp. infantis to utilize these potential growth substrates when they are in competition with other bifidobacterial species is unknown. We compared the growth and oligosaccharide utilization of GOS and FOS by bifidobacterial species in pure culture and coculture. Short-chain GOS and FOS (degrees of polymerization [DP] 2 and 3) were favored growth substrates for strains of B. bifidum and B. longum subsp. longum, whereas both B. breve and B. longum subsp. infantis had the ability to utilize both short- and longer-chain GOS and FOS (DP 2 to 6). B. breve was nevertheless numerically dominant over B. longum subsp. infantis in cocultures. This was probably related to the slower use of GOS of DP 3 by B. longum subsp. infantis, indicating that the kinetics of substrate utilization is an important ecological factor in the assemblage of gut communities. IMPORTANCE The kinds of bacteria that form the collection of microbes (the microbiota) in the gut of human infants may influence health and well-being. Knowledge of how the composition of the infant diet influences the assemblage of the bacterial collection is therefore important because dietary interventions may offer opportunities to alter the microbiota with the aim of improving health. Bifidobacterium longum subspecies infantis is a well-known bacterial species, but under modern child-rearing conditions it may be disadvantaged in the gut. Modern formula milks often contain particular oligosaccharide additives that are generally considered to support bifidobacterial growth. However, studies of the ability of various bifidobacterial species to grow together in the presence of these oligosaccharides have not been conducted. These kinds of studies are essential for developing concepts of microbial ecology related to the influence of human nutrition on the development of the gut microbiota.

The gammaproteobacterium Marinobacter hydrocarbonoclasticus NCT7M was cultivated from the copepod Acartia tonsa, collected from the coastal western North Atlantic Ocean. The genome was assembled into 45 contigs for a total of 4,128,590 bp, a GC content of 57.3%, and 3,890 protein-coding genes. The genome contains the full gene cluster for denitrification.

Spotted fever group rickettsia (SFGR) can cause mild to fatal illness. The early interaction between the host and rickettsia in skin is largely unknown, and the pathogenesis of severe rickettsiosis remains an important topic. A surveillance of SFGR infection by PCR of blood and skin biopsy specimens followed by sequencing and immunohistochemical (IHC) detection was performed on patients with a recent tick bite between 2013 and 2016. Humoral and cutaneous immunoprofiles were evaluated in different SFGR cases by serum cytokine and chemokine detection, skin IHC staining, and transcriptome sequencing (RNA-seq)., Spotted fever group rickettsia (SFGR) can cause mild to fatal illness. The early interaction between the host and rickettsia in skin is largely unknown, and the pathogenesis of severe rickettsiosis remains an important topic. A surveillance of SFGR infection by PCR of blood and skin biopsy specimens followed by sequencing and immunohistochemical (IHC) detection was performed on patients with a recent tick bite between 2013 and 2016. Humoral and cutaneous immunoprofiles were evaluated in different SFGR cases by serum cytokine and chemokine detection, skin IHC staining, and transcriptome sequencing (RNA-seq). A total of 111 SFGR cases were identified, including 79 “Candidatus Rickettsia tarasevichiae,” 22 Rickettsia raoultii, 8 Rickettsia sibirica, and 2 Rickettsia heilongjiangensis cases. The sensitivity to detect SFGR in skin biopsy specimens (9/24, 37.5%) was significantly higher than that in blood samples (105/2,671, 3.9%) (P

We lack fundamental understanding of how phage infection influences bacterial gene expression and, consequently, how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole-genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside their intended host targets., Bacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug-resistant bacterial infections. However, there are major gaps in our understanding of the molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism Enterococcus faecalis, we used two distinct genomic approaches, namely, transposon library screening and RNA sequencing, to investigate the interaction of E. faecalis with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (epa) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for epa mutation during phage infection. Transcriptomic profiling of phage-infected E. faecalis revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intra- and interbacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together, our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities, such as the host microbiota, during phage therapy.

Bacteroides is one of the most prominent genera in the human gut microbiome, and study of this bacterial group provides insights into gut microbial ecology and pathogenesis. In this report, we introduce a negative selection system for rapid and efficient allelic exchange in wild Bacteroides species that does not require any alterations to the genetic background or a nutritionally defined culture medium. In this approach, dual antibacterial effectors normally delivered via type VI secretion are targeted to the bacterial periplasm under the control of tightly regulated anhydrotetracycline (aTC)-inducible promoters. Introduction of aTC selects for recombination events producing the desired genetic modification, and the dual effector design allows for broad applicability across strains that may have immunity to one counterselection effector. We demonstrate the utility of this approach across 21 human gut Bacteroides isolates representing diverse species, including strains isolated directly from human donors. We use this system to establish that antimicrobial peptide resistance in Bacteroides vulgatus is determined by the product of a gene that is not included in the genomes of previously genetically tractable members of the human gut microbiome.IMPORTANCE Human gut Bacteroides species exhibit strain-level differences in their physiology, ecology, and impact on human health and disease. However, existing approaches for genetic manipulation generally require construction of genetically modified parental strains for each microbe of interest or defined medium formulations. In this report, we introduce a robust and efficient strategy for targeted genetic manipulation of diverse wild-type Bacteroides species from the human gut. This system enables genetic investigation of members of human and animal microbiomes beyond existing model organisms.

In many cases, genes for commonly used genetic markers in model organisms have not been identified; therefore, it is of interest to identify the causative genes. Whole-genome sequencing was used to identify potential causative mutations for a col-4 allele of Neurospora crassa.

High-throughput, quantitative approaches have enabled the discovery of fundamental principles describing bacterial physiology. These principles provide a foundation for predicting the behavior of biological systems, a widely held aspiration. However, these approaches are often exclusively applied to the best-known model organism, E. coli. In this report, we investigate to what extent quantitative principles discovered in Gram-negative E. coli are applicable to Gram-positive B. subtilis. We found that these two extremely divergent bacterial species employ deeply similar strategies in order to coordinate growth, cell size, and the cell cycle. These similarities mean that the quantitative physiological principles described here can likely provide a beachhead for others who wish to understand additional, less-studied prokaryotes., Bacillus subtilis and Escherichia coli are evolutionarily divergent model organisms whose analysis has enabled elucidation of fundamental differences between Gram-positive and Gram-negative bacteria, respectively. Despite their differences in cell cycle control at the molecular level, the two organisms follow the same phenomenological principle, known as the adder principle, for cell size homeostasis. We thus asked to what extent B. subtilis and E. coli share common physiological principles in coordinating growth and the cell cycle. We measured physiological parameters of B. subtilis under various steady-state growth conditions with and without translation inhibition at both the population and single-cell levels. These experiments revealed core physiological principles shared between B. subtilis and E. coli. Specifically, both organisms maintain an invariant cell size per replication origin at initiation, under all steady-state conditions, and even during nutrient shifts at the single-cell level. Furthermore, the two organisms also inherit the same “hierarchy” of physiological parameters. On the basis of these findings, we suggest that the basic principles of coordination between growth and the cell cycle in bacteria may have been established early in evolutionary history.

Fusobacterium spp. are Gram-negative, anaerobic, opportunistic pathogens involved in multiple diseases, including a link between the oral pathogen Fusobacterium nucleatum and the progression and severity of colorectal cancer. The identification and characterization of virulence factors in the genus Fusobacterium has been greatly hindered by a lack of properly assembled and annotated genomes. Using newly completed genomes from nine strains and seven species of Fusobacterium, we report the identification and corrected annotation of verified and potential virulence factors from the type 5 secreted autotransporter, FadA, and MORN2 protein families, with a focus on the genetically tractable strain F. nucleatum subsp. nucleatum ATCC 23726 and type strain F. nucleatum subsp. nucleatum ATCC 25586. Within the autotransporters, we used sequence similarity networks to identify protein subsets and show a clear differentiation between the prediction of outer membrane adhesins, serine proteases, and proteins with unknown function. These data have identified unique subsets of type 5a autotransporters, which are key proteins associated with virulence in F. nucleatum. However, we coupled our bioinformatic data with bacterial binding assays to show that a predicted weakly invasive strain of F. necrophorum that lacks a Fap2 autotransporter adhesin strongly binds human colonocytes. These analyses confirm a gap in our understanding of how autotransporters, MORN2 domain proteins, and FadA adhesins contribute to host interactions and invasion. In summary, we identify candidate virulence genes in Fusobacterium, and caution that experimental validation of host-microbe interactions should complement bioinformatic predictions to increase our understanding of virulence protein contributions in Fusobacterium infections and disease. IMPORTANCEFusobacterium spp. are emerging pathogens that contribute to mammalian and human diseases, including colorectal cancer. Despite a validated connection with disease, few proteins have been characterized that define a direct molecular mechanism for Fusobacterium pathogenesis. We report a comprehensive examination of virulence-associated protein families in multiple Fusobacterium species and show that complete genomes facilitate the correction and identification of multiple, large type 5a secreted autotransporter genes in previously misannotated or fragmented genomes. In addition, we use protein sequence similarity networks and human cell interaction experiments to show that previously predicted noninvasive strains can indeed bind to and potentially invade human cells and that this could be due to the expansion of specific virulence proteins that drive Fusobacterium infections and disease.

Type III interferon (IFN), or IFN lambda (IFN-λ), is an essential component of the innate immune response to mucosal viral infections. In both the intestine and the lung, signaling via the IFN-λ receptor (IFNLR) controls clinically important viral pathogens, including influenza virus, norovirus, and rotavirus. While it is thought that IFN-λ cytokines are the exclusive ligands for signaling through IFNLR, it is not known whether genetic ablation of these cytokines phenotypically recapitulates disruption of the receptor. Here, we report the serendipitous establishment of Ifnl2- / - Ifnl3- / - mice, which lack all known functional murine IFN-λ cytokines. We demonstrate that, like Ifnlr1- / - mice lacking IFNLR signaling, these mice display defective control of murine norovirus, reovirus, and influenza virus and therefore genocopy Ifnlr1- / - mice. Thus, for regulation of viral infections at mucosal sites of both the intestine and lung, signaling via IFNLR can be fully explained by the activity of known cytokines IFN-λ2 and IFN-λ3. Our results confirm the current understanding of ligand-receptor interactions for type III IFN signaling and highlight the importance of this pathway in regulation of mucosal viral pathogens.IMPORTANCE Type III interferons are potent antiviral cytokines important for regulation of viruses that infect at mucosal surfaces. Studies using mice lacking the Ifnlr1 gene encoding the type III interferon receptor have demonstrated that signaling through this receptor is critical for protection against influenza virus, norovirus, and reovirus. Using a genetic approach to disrupt murine type III interferon cytokine genes Ifnl2 and Ifnl3, we found that mice lacking these cytokines fully recapitulate the impaired control of viruses observed in mice lacking Ifnlr1 Our results support the idea of an exclusive role for known type III interferon cytokines in signaling via IFNLR to mediate antiviral effects at mucosal surfaces. These findings emphasize the importance of type III interferons in regulation of a variety of viral pathogens and provide important genetic evidence to support our understanding of the ligand-receptor interactions in this pathway.

Here, we report the draft genome sequence of Vibrio cyclitrophicus NCT10V, cultivated from the copepod Acartia tonsa, collected from coastal surface waters of the western North Atlantic Ocean. The assembly is 5,638,575 bp long and has 44 contigs, a GC content of 43.2%, and 5,044 protein coding sequences.