A Surface Exposed, Two-Domain Lipoprotein Cargo of a Type XI Secretion System Promotes Colonization of Host Intestinal Epithelia Expressing Glycans.

The only known required component of the newly described Type XI secretion system (TXISS) is an outer membrane protein (OMP) of the DUF560 family. TXISSOMPs are broadly distributed across proteobacteria, but properties of the cargo proteins they secrete are largely unexplored. We report biophysical, histochemical, and phenotypic evidence that Xenorhabdus nematophila NilC is surface exposed. Biophysical data and structure predictions indicate that NilC is a two-domain protein with a C-terminal, 8-stranded ß-barrel. This structure has been noted as a common feature of TXISS effectors and may be important for interactions with the TXISSOMP. The NilC N-terminal domain is more enigmatic, but our results indicate it is ordered and forms a ß-sheet structure, and bioinformatics suggest structural similarities to carbohydrate-binding proteins. X. nematophila NilC and its presumptive TXISSOMP partner NilB are required for colonizing the anterior intestine of Steinernema carpocapsae nematodes: the receptacle of free-living, infective juveniles and the anterior intestinal cecum (AIC) in juveniles and adults. We show that, in adult nematodes, the AIC expresses a Wheat Germ Agglutinin (WGA)-reactive material, indicating the presence of N-acetylglucosamine or N-acetylneuraminic acid sugars on the AIC surface. A role for this material in colonization is supported by the fact that exogenous addition of WGA can inhibit AIC colonization by X. nematophila. Conversely, the addition of exogenous purified NilC increases the frequency with which X. nematophila is observed at the AIC, demonstrating that abundant extracellular NilC can enhance colonization. NilC may facilitate X. nematophila adherence to the nematode intestinal surface by binding to host glycans, it might support X. nematophila nutrition by cleaving sugars from the host surface, or it might help protect X. nematophila from nematode host immunity. Proteomic and metabolomic analyses of wild type X. nematophila compared to those lacking nilB and nilC revealed differences in cell wall and secreted polysaccharide metabolic pathways. Additionally, purified NilC is capable of binding peptidoglycan, suggesting that periplasmic NilC may interact with the bacterial cell wall. Overall, these findings support a model that NilB-regulated surface exposure of NilC mediates interactions between X. nematophila and host surface glycans during colonization. This is a previously unknown function for a TXISS.

Apex Predator Nematodes and Meso-Predator Bacteria Consume Their Basal Insect Prey through Discrete Stages of Chemical Transformations.

Microbial symbiosis drives physiological processes of higher-order systems, including the acquisition and consumption of nutrients that support symbiotic partner reproduction. Metabolic analytics provide new avenues to examine how chemical ecology, or the conversion of existing biomass to new forms, changes over a symbiotic life cycle. We applied these approaches to the nematode Steinernema carpocapsae, its mutualist bacterium, Xenorhabdus nematophila, and the insects they infect. The nematode-bacterium pair infects, kills, and reproduces in an insect until nutrients are depleted. To understand the conversion of insect biomass over time into either nematode or bacterium biomass, we integrated information from trophic, metabolomic, and gene regulation analyses. Trophic analysis established bacteria as meso-predators and primary insect consumers. Nematodes hold a trophic position of 4.6, indicative of an apex predator, consuming bacteria and likely other nematodes. Metabolic changes associated with Galleria mellonella insect bioconversion were assessed using multivariate statistical analyses of metabolomics data sets derived from sampling over an infection time course. Statistically significant, discrete phases were detected, indicating the insect chemical environment changes reproducibly during bioconversion. A novel hierarchical clustering method was designed to probe molecular abundance fluctuation patterns over time, revealing distinct metabolite clusters that exhibit similar abundance shifts across the time course. Composite data suggest bacterial tryptophan and nematode kynurenine pathways are coordinated for reciprocal exchange of tryptophan and NAD+ and for synthesis of intermediates that can have complex effects on bacterial phenotypes and nematode behaviors. Our analysis of pathways and metabolites reveals the chemistry underlying the recycling of organic material during carnivory. IMPORTANCE The processes by which organic life is consumed and reborn in a complex ecosystem were investigated through a multiomics approach applied to the tripartite Xenorhabdus bacterium-Steinernema nematode-Galleria insect symbiosis. Trophic analyses demonstrate the primary consumers of the insect are the bacteria, and the nematode in turn consumes the bacteria. This suggests the Steinernema-Xenorhabdus mutualism is a form of agriculture in which the nematode cultivates the bacterial food sources by inoculating them into insect hosts. Metabolomics analysis revealed a shift in biological material throughout progression of the life cycle: active infection, insect death, and conversion of cadaver tissues into bacterial biomass and nematode tissue. We show that each phase of the life cycle is metabolically distinct, with significant differences including those in the tricarboxylic acid cycle and amino acid pathways. Our findings demonstrate that symbiotic life cycles can be defined by reproducible stage-specific chemical signatures, enhancing our broad understanding of metabolic processes that underpin a three-way symbiosis.

Cylindrospermopsis raciborskii Virus and host: genomic characterization and ecological relevance.

Cylindrospermopsis (Raphidiopsis) raciborskii is an invasive, filamentous, nitrogen-fixing cyanobacterium that forms frequent blooms in freshwater habitats. While viruses play key roles in regulating the abundance, production and diversity of their hosts in aquatic ecosystems, the role(s) of viruses in the ecology of C. raciborskii is almost unexplored. Progress in this field has been hindered by the absence of a characterized virus-host system in C. raciborskii. To bridge this gap, we sequenced the genome of CrV-01T, a previously isolated cyanosiphovirus, and its host, C. raciborskii strain Cr2010. Analyses suggest that CrV-01T represents a distinct clade of siphoviruses infecting, and perhaps lysogenizing, filamentous cyanobacteria. Its genome contains unique features that include an intact CRISPR array and a 12 kb inverted duplication. Evidence suggests CrV-01T recently gained the ability to infect Cr2010 and recently lost the ability to form lysogens. The cyanobacterial host contains a CRISPR-Cas system with CRISPR spacers matching protospacers within the inverted duplication of the CrV-01T genome. Examination of metagenomes demonstrates that viruses with high genetic identity to CrV-01T, but lacking the inverted duplication, are present in C. raciborskii blooms in Australia. The unique genomic features of the CrV/Cr2010 system offers opportunities to investigate in more detail virus-host interactions in an ecologically important bloom-forming cyanobacterium.