Predicted bacterial interactions affect in vivo microbial colonization dynamics in Nematostella

The maintenance and resilience of host-associated microbiota during development is a fundamental process influencing the fitness of many organisms. Several host properties were identified as influencing factors on bacterial colonization, including the innate immune system, mucus composition and diet. In contrast, the importance of bacteria-bacteria interactions on host colonization is less understood. Here, we use bacterial abundance data of the marine model organism Nematostella vectensis to reconstruct potential bacteria-bacteria interactions through co-occurrence networks. The analysis indicates that bacteria-bacteria interactions are dynamic during host colonization and change according to the host’s developmental stage. To assess the predictive power of inferred interactions, we tested bacterial isolates with predicted cooperative or competitive behavior for their ability to influence bacterial recolonization dynamics. Within three days of recolonization all tested bacterial isolates affected bacterial community structure, while only competitive bacteria increased bacterial diversity. Already one week after recolonization almost no differences in bacterial community structure could be observed between control and treatments. These results show that predicted competitive bacteria can influence community structure for a short period of time, verifying the in silico predictions. However, within one week, the effects of the bacterial isolates are neutralized, indicating a high degree of resilience of the bacterial community. Hanna Domin1, Yazmín H. Zurita-Gutiérrez2, Marco Scotti3, Jann Buttlar1, Ute Hentschel Humeida2 and Sebastian Fraune1

Analysis of the Microprocessor in Dictyostelium: The Role of RbdB, a dsRNA Binding Protein

We identified the dsRNA binding protein RbdB as an essential component in miRNA processing in Dictyostelium discoideum. RbdB is a nuclear protein that accumulates, together with Dicer B, in nucleolar foci reminiscent of plant dicing bodies. Disruption of rbdB results in loss of miRNAs and accumulation of primary miRNAs. The phenotype can be rescued by ectopic expression of RbdB thus allowing for a detailed analysis of domain function. The lack of cytoplasmic dsRBD proteins involved in miRNA processing, suggests that both processing steps take place in the nucleus thus resembling the plant pathway. However, we also find features e.g. in the domain structure of Dicer which suggest similarities to animals. Reduction of miRNAs in the rbdB- strain and their increase in the Argonaute A knock out allowed the definition of new miRNAs one of which appears to belong to a new non-canonical class

Image_1_Predicted Bacterial Interactions Affect in Vivo Microbial Colonization Dynamics in Nematostella.pdf

<p>The maintenance and resilience of host-associated microbiota during development is a fundamental process influencing the fitness of many organisms. Several host properties were identified as influencing factors on bacterial colonization, including the innate immune system, mucus composition, and diet. In contrast, the importance of bacteria–bacteria interactions on host colonization is less understood. Here, we use bacterial abundance data of the marine model organism Nematostella vectensis to reconstruct potential bacteria–bacteria interactions through co-occurrence networks. The analysis indicates that bacteria–bacteria interactions are dynamic during host colonization and change according to the host’s developmental stage. To assess the predictive power of inferred interactions, we tested bacterial isolates with predicted cooperative or competitive behavior for their ability to influence bacterial recolonization dynamics. Within 3 days of recolonization, all tested bacterial isolates affected bacterial community structure, while only competitive bacteria increased bacterial diversity. Only 1 week after recolonization, almost no differences in bacterial community structure could be observed between control and treatments. These results show that predicted competitive bacteria can influence community structure for a short period of time, verifying the in silico predictions. However, within 1 week, the effects of the bacterial isolates are neutralized, indicating a high degree of resilience of the bacterial community.</p

Table_1_Predicted Bacterial Interactions Affect in Vivo Microbial Colonization Dynamics in Nematostella.XLSX

<p>The maintenance and resilience of host-associated microbiota during development is a fundamental process influencing the fitness of many organisms. Several host properties were identified as influencing factors on bacterial colonization, including the innate immune system, mucus composition, and diet. In contrast, the importance of bacteria–bacteria interactions on host colonization is less understood. Here, we use bacterial abundance data of the marine model organism Nematostella vectensis to reconstruct potential bacteria–bacteria interactions through co-occurrence networks. The analysis indicates that bacteria–bacteria interactions are dynamic during host colonization and change according to the host’s developmental stage. To assess the predictive power of inferred interactions, we tested bacterial isolates with predicted cooperative or competitive behavior for their ability to influence bacterial recolonization dynamics. Within 3 days of recolonization, all tested bacterial isolates affected bacterial community structure, while only competitive bacteria increased bacterial diversity. Only 1 week after recolonization, almost no differences in bacterial community structure could be observed between control and treatments. These results show that predicted competitive bacteria can influence community structure for a short period of time, verifying the in silico predictions. However, within 1 week, the effects of the bacterial isolates are neutralized, indicating a high degree of resilience of the bacterial community.</p

Image_3_Predicted Bacterial Interactions Affect in Vivo Microbial Colonization Dynamics in Nematostella.PDF

<p>The maintenance and resilience of host-associated microbiota during development is a fundamental process influencing the fitness of many organisms. Several host properties were identified as influencing factors on bacterial colonization, including the innate immune system, mucus composition, and diet. In contrast, the importance of bacteria–bacteria interactions on host colonization is less understood. Here, we use bacterial abundance data of the marine model organism Nematostella vectensis to reconstruct potential bacteria–bacteria interactions through co-occurrence networks. The analysis indicates that bacteria–bacteria interactions are dynamic during host colonization and change according to the host’s developmental stage. To assess the predictive power of inferred interactions, we tested bacterial isolates with predicted cooperative or competitive behavior for their ability to influence bacterial recolonization dynamics. Within 3 days of recolonization, all tested bacterial isolates affected bacterial community structure, while only competitive bacteria increased bacterial diversity. Only 1 week after recolonization, almost no differences in bacterial community structure could be observed between control and treatments. These results show that predicted competitive bacteria can influence community structure for a short period of time, verifying the in silico predictions. However, within 1 week, the effects of the bacterial isolates are neutralized, indicating a high degree of resilience of the bacterial community.</p

Image_2_Predicted Bacterial Interactions Affect in Vivo Microbial Colonization Dynamics in Nematostella.PDF

<p>The maintenance and resilience of host-associated microbiota during development is a fundamental process influencing the fitness of many organisms. Several host properties were identified as influencing factors on bacterial colonization, including the innate immune system, mucus composition, and diet. In contrast, the importance of bacteria–bacteria interactions on host colonization is less understood. Here, we use bacterial abundance data of the marine model organism Nematostella vectensis to reconstruct potential bacteria–bacteria interactions through co-occurrence networks. The analysis indicates that bacteria–bacteria interactions are dynamic during host colonization and change according to the host’s developmental stage. To assess the predictive power of inferred interactions, we tested bacterial isolates with predicted cooperative or competitive behavior for their ability to influence bacterial recolonization dynamics. Within 3 days of recolonization, all tested bacterial isolates affected bacterial community structure, while only competitive bacteria increased bacterial diversity. Only 1 week after recolonization, almost no differences in bacterial community structure could be observed between control and treatments. These results show that predicted competitive bacteria can influence community structure for a short period of time, verifying the in silico predictions. However, within 1 week, the effects of the bacterial isolates are neutralized, indicating a high degree of resilience of the bacterial community.</p

Image_4_Predicted Bacterial Interactions Affect in Vivo Microbial Colonization Dynamics in Nematostella.PDF

<p>The maintenance and resilience of host-associated microbiota during development is a fundamental process influencing the fitness of many organisms. Several host properties were identified as influencing factors on bacterial colonization, including the innate immune system, mucus composition, and diet. In contrast, the importance of bacteria–bacteria interactions on host colonization is less understood. Here, we use bacterial abundance data of the marine model organism Nematostella vectensis to reconstruct potential bacteria–bacteria interactions through co-occurrence networks. The analysis indicates that bacteria–bacteria interactions are dynamic during host colonization and change according to the host’s developmental stage. To assess the predictive power of inferred interactions, we tested bacterial isolates with predicted cooperative or competitive behavior for their ability to influence bacterial recolonization dynamics. Within 3 days of recolonization, all tested bacterial isolates affected bacterial community structure, while only competitive bacteria increased bacterial diversity. Only 1 week after recolonization, almost no differences in bacterial community structure could be observed between control and treatments. These results show that predicted competitive bacteria can influence community structure for a short period of time, verifying the in silico predictions. However, within 1 week, the effects of the bacterial isolates are neutralized, indicating a high degree of resilience of the bacterial community.</p

Subcellular localization of RbdB GFP and co-localization with DrnB.

<p>AX2 cells were transformed with the integrating plasmid pDneo2a RbdB GFP and subcellular localization was analyzed by fluorescence microscopy. A: Living cells were analyzed in low fluorescence axenic medium showing a diffuse distribution of the fusion proteins in the nucleoplasm and distinct foci at the periphery of the nuclei. Scale bar represents 5 μm. B: To better localize the subnuclear foci, cells were fixed with methanol and analyzed by an OptiGrid microscope (Leica DM 5500). Genomic DNA was stained by DAPI (red). The nucleoli showed no or only a very weak staining. Merging GFP (green) and DAPI (red) signals indicated that RbdB-GFP foci were enriched adjacent to areas with weak or no DAPI staining. Scale bar represents 2.5 μm. C: Co-localization of GFP DrnB and RbdB mRFP in nucleoli associated foci was monitored by fluorescence microscopy using methanol fixed cells. Shown is a single nucleus. Fusion proteins were expressed from extrachromosomally replicating plasmids. Scale bar represents 2.5 μm.</p

primary miRNAs accumulated in rbdB- and drnB- strains.

<p>A: Schematic representation of ddi-miR-1176 and ddi-miR-1177 transcripts and predicted pre-miRNA structures. Arrows indicate primers that were used to amplify primary miRNA transcripts. As a control, RT-PCR on <i>corA</i> mRNA was performed. B: Gene specific (reverse) primers were used to generate cDNA molecules: #1828 (<i>corA</i>), DM059 (pri-ddi-miR-1176), DM083 (pri-ddi-miR-1177). The primer sets P1/P2 (DM058/DM059), P2/P3 (DM082, DM083) and #1828/#1829 were used in the following PCR reaction to amplify pri-ddi-miR-1176 (390 bp), pri-ddi-miR-1177 (283 bp) and <i>corA</i> (200 bp) fragments respectively. The number in brackets indicates number of PCR-cycles. For unknown reasons, the minus RT control for pri-ddi-miR-1176 in the AX2 wild type always produced a weak signal.</p