Bacterial colonization of Hydra hatchlings follows a robust temporal pattern

Animals are colonized by complex bacterial communities. The processes controlling community membership and influencing the establishment of the microbial ecosystem during development are poorly understood. Here we aimed to explore the assembly of bacterial communities in Hydra with the broader goal of elucidating the general rules that determine the temporal progression of bacterial colonization of animal epithelia. We profiled the microbial communities in polyps at various time points after hatching in four replicates. The composition and temporal patterns of the bacterial communities were strikingly similar in all replicates. Distinct features included high diversity of community profiles in the first week, a remarkable but transient adult-like profile 2 weeks after hatching, followed by progressive emergence of a stable adult-like pattern characterized by low species diversity and the preponderance of the Betaproteobacterium Curvibacter. Intriguingly, this process displayed important parallels to the assembly of human fecal communities after birth. In addition, a mathematical modeling approach was used to uncover the organizational principles of this colonization process, suggesting that both, local environmental or host-derived factor(s) modulating the colonization rate, as well as frequency-dependent interactions of individual bacterial community members are important aspects in the emergence of a stable bacterial community at the end of development.     

Cdk2 knockout mice are viable

BACKGROUND: Cyclin-dependent kinases (Cdks) and their cyclin regulatory subunits control cell growth and division. Cdk2/cyclin E complexes are thought to be required because they phosphorylate the retinoblastoma protein and drive cells through the G1/S transition into the S phase of the cell cycle. In addition, Cdk2 associates with cyclin A, which itself is essential for cell proliferation during early embryonic development. RESULTS: In order to study the functions of Cdk2 in vivo, we generated Cdk2 knockout mice. Surprisingly, these mice are viable, and therefore Cdk2 is not an essential gene in the mouse. However, Cdk2 is required for germ cell development; both male and female Cdk2(-/-) mice are sterile. Immunoprecipitates of cyclin E1 complexes from Cdk2(-/-) spleen extracts displayed no activity toward histone H1. Cyclin A2 complexes were active in primary mouse embryonic fibroblasts (MEFs), embryo extracts and in spleen extracts from young animals. In contrast, there was little cyclin A2 kinase activity in immortalized MEFs and spleen extracts from adult animals. Cdk2(-/-) MEFs proliferate but enter delayed into S phase. Ectopic expression of Cdk2 in Cdk2(-/-) MEFs rescued the delayed entry into S phase. CONCLUSIONS: Although Cdk2 is not an essential gene in the mouse, it is required for germ cell development and meiosis. Loss of Cdk2 affects the timing of S phase, suggesting that Cdk2 is involved in regulating progression through the mitotic cell cycle.     

Endosomal translocation of vertebrate DNA activates dendritic cells via TLR9-dependent and -independent pathways

TLRs discriminate foreign from self via their specificity for pathogen-derived invariant ligands, an example being TLR9 recognizing bacterial unmethylated CpG motifs. In this study we report that endosomal translocation of CpG DNA via the natural endocytotic pathway is inefficient and highly saturable, whereas endosomal translocation of DNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) is not. Interestingly, DOTAP-mediated enhanced endosomal translocation of otherwise nonstimulatory vertebrate DNA or of certain noncanonical CpG motifs triggers robust dendritic cell activation in terms of both up-regulation of CD40/CD69 and cytokine production, such as type I IFN and IL-6. We report that the stimulatory activity of phosphorothioated noncanonical CpG oligodeoxynucleotides is TLR9 dependent, whereas phosphodiester DNA, such as vertebrate DNA, in addition trigger TLR9-independent pathways. We propose that the inefficiency of the natural route for DNA internalization hinders low affinity TLR9 ligands in endosomes to reach threshold concentrations required for TLR9 activation. Endosomal compartmentalization of TLR9 may thus reflect an evolutionary strategy to avoid TLR9 activation by self-DNA.     

Gut Symbionts from Distinct Hosts Exhibit Genotoxic Activity via Divergent Colibactin Biosynthesis Pathways

Secondary metabolites produced by nonribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways are chemical mediators of microbial interactions in diverse environments. However, little is known about their distribution, evolution, and functional roles in bacterial symbionts associated with animals. A prominent example is colibactin, a largely unknown family of secondary metabolites produced by Escherichia coli via a hybrid NRPS-PKS biosynthetic pathway that inflicts DNA damage upon eukaryotic cells and contributes to colorectal cancer and tumor formation in the mammalian gut. Thus far, homologs of this pathway have only been found in closely related Enterobacteriaceae, while a divergent variant of this gene cluster was recently discovered in a marine alphaproteobacterial Pseudovibrio strain. Herein, we sequenced the genome of Frischella perrara PEB0191, a bacterial gut symbiont of honey bees and identified a homologous colibactin biosynthetic pathway related to those found in Enterobacteriaceae. We show that the colibactin genomic island (GI) has conserved gene synteny and biosynthetic module architecture across F. perrara, Enterobacteriaceae, and the Pseudovibrio strain. Comparative metabolomics analyses of F. perrara and E. coli further reveal that these two bacteria produce related colibactin pathway-dependent metabolites. Finally, we demonstrate that F. perrara, like E. coli, causes DNA damage in eukaryotic cells in vitro in a colibactin pathway-dependent manner. Together, these results support that divergent variants of the colibactin biosynthetic pathway are widely distributed among bacterial symbionts, producing related secondary metabolites and likely endowing its producer with functional capabilities important for diverse symbiotic associations.     

Note on using nuclear 28S rDNA for sequencing ancient and strongly degraded insect DNA

A protocol using insect specimens or parts thereof allows for sequencing of sections of nuclear 28S rDNA. In the present note it is demonstrated that this protocol can readily be applied to strongly degraded DNA (ancient, fixed or contaminated). Primers that are specifically designed to discriminate against human DNA but also other non‐arthropod species are tested on a range of species covering all insect groups (59 insect species from all 33 orders). Additionally, the samples represent a selection of various, mostly DNA‐degrading, preservation methods, including the most common fixatives used for morphological investigations and for long‐term storage in collections. Successful amplification was possible for all tested samples including ca. 200 year‐old dried museum specimens as well as for over 4000 year‐old fossil insects embedded in copal. When the NCBI database contained information on the tested species an unambiguous taxonomic discrimination was possible. This approach is based on a standardized protocol that guarantees easy application. This note presents primer pairs for 28S rDNA that can be a useful tool for ancient DNA (aDNA) research.     

Chemical composition, intake by sheep, and in situ disappearance in cannulated cows of bermudagrass hayed at two moisture concentrations and treated with a non-viable Lactobacillus-lactic acid preservative

Bermudagrass [Cynodon dactylon (L.) Pers.] is commonly used for grazing and haying in the southern USA, but hay curing can be challenging due to frequent rainfall events during spring and early summer. An existing stand of ‘Greenfield’ bermudagrass was divided into 12 plots using a randomized complete block design with a 2×2 factorial treatment arrangement to evaluate the influence of a non-viable Lactobacillus-lactic acid preservative and moisture concentration at baling on chemical composition, intake by sheep, and in situ disappearance in cattle. At time of mowing, half of the plots in each block were either spray-treated (T) or not treated (U) with 81 mL/t forage dry matter (DM) of the preservative solution. Hay was then baled at target moisture concentrations of either 174 g/kg DM (L) or 267 g/kg DM (H). Maximum temperature and heating degree days were greater (P<0.05) from H compared with L during the 42-d storage period. An interaction between spray and moisture treatments tended (P<0.10) to affect recovery of DM; recoveries for LT (0.992) differed (P<0.10) from HT (0.913), but LU and HU were intermediate between the spray-treated hays, and did not differ from either (P>0.10). Post-storage nutritive value was largely influenced by moisture treatments only. Intake and digestibility, and in situ DM disappearance of these same hays were determined using 16 wether lambs (43 ± 3.7 kg initial BW), or six ruminally cannulated cows (617 ± 3.5 kg initial BW), respectively. Dry matter intake by sheep was not affected by either treatment factor (P>0.05), but DM digestibility and digestible DM intake were greater (P<0.05) from U compared with T. The in situ immediately soluble DM portion was greater from (P<0.05) L compared with H, but the reverse was true for the potentially degradable DM fraction. The lag time tended (P<0.10) to be greater from H compared with L. Treating bermudagrass with a non-viable Lactobacillus acidophilus-lactic acid spray product at time of baling may not offset the negative effects on forage quality and digestibility of baling bermudagrass hay at excessive moisture concentrations.     

RNA and β-Hemolysin of Group B Streptococcus Induce Interleukin-1β (IL-1β) by Activating NLRP3 Inflammasomes in Mouse Macrophages

The inflammatory cytokine IL-1β is critical for host responses against many human pathogens. Here, we define Group B Streptococcus (GBS)-mediated activation of the Nod-like receptor-P3 (NLRP3) inflammasome in macrophages. NLRP3 activation requires GBS expression of the cytolytic toxin, β-hemolysin, lysosomal acidification, and leakage. These processes allow the interaction of GBS RNA with cytosolic NLRP3. The present study supports a model in which GBS RNA, along with lysosomal components including cathepsins, leaks out of lysosomes and interacts with NLRP3 to induce IL-1β production.