Project description:We used whole-genome microarrays to identify the global transcriptional changes during biofilm dispersal and also to investigate the molecular mechanism that regulating biofilm dispersal.
Project description:Limitation for amino acids is thought to regulate translation in mammalian cells primarily by signaling through the kinases mTORC1 and GCN2. We find that limitation for the amino acid arginine causes a selective loss of tRNA charging, which regulates translation through ribosome pausing at two of six arginine codons. Interestingly, limitation for leucine, an essential and abundant amino acid in protein, results in little or no ribosome pausing. Chemical and genetic perturbation of mTORC1 and GCN2 signaling revealed that their robust response to leucine limitation prevents ribosome pausing, while an insufficient response to arginine limitation led to loss of arginine tRNA charging and ribosome pausing. Codon-specific ribosome pausing decreased protein production and triggered premature ribosome termination without significantly reducing mRNA levels. Together, our results suggest that amino acids which are not optimally sensed by the mTORC1 and GCN2 pathways still regulate translation through an evolutionarily conserved mechanism based on synonymous codon usage.
Project description:The rate, timing, and mode of species dispersal is recognized as a key driver of the structure and function of communities of macroorganisms, and may be one ecological process that determines the diversity of microbiomes. Many previous studies have quantified the modes and mechanisms of bacterial motility using monocultures of a few model bacterial species. But most microbes live in multispecies microbial communities, where direct interactions between microbes may inhibit or facilitate dispersal through a number of physical (e.g., hydrodynamic) and biological (e.g., chemotaxis) mechanisms, which remain largely unexplored. Using cheese rinds as a model microbiome, we demonstrate that physical networks created by filamentous fungi can impact the extent of small-scale bacterial dispersal and can shape the composition of microbiomes. From the cheese rind of Saint Nectaire, we serendipitously observed the bacterium Serratia proteamaculans actively spreads on networks formed by the fungus Mucor. By experimentally recreating these pairwise interactions in the lab, we show that Serratia spreads on actively growing and previously established fungal networks. The extent of symbiotic dispersal is dependent on the fungal network: diffuse and fast-growing Mucor networks provide the greatest dispersal facilitation of the Serratia species, while dense and slow-growing Penicillium networks provide limited dispersal facilitation. Fungal-mediated dispersal occurs in closely related Serratia species isolated from other environments, suggesting that this bacterial-fungal interaction is widespread in nature. Both RNA-seq and transposon mutagenesis point to specific molecular mechanisms that play key roles in this bacterial-fungal interaction, including chitin utilization and flagellin biosynthesis. By manipulating the presence and type of fungal networks in multispecies communities, we provide the first evidence that fungal networks shape the composition of bacterial communities, with Mucor networks shifting experimental bacterial communities to complete dominance by motile Proteobacteria. Collectively, our work demonstrates that these strong biophysical interactions between bacterial and fungi can have community-level consequences and may be operating in many other microbiomes.
Project description:The global transcriptional regulator Hha of Escherichia coli controls hemolysin activity, biofilm formation, and virulence expressions. Earlier, we have reported that Hha represses initial biofilm formation and disperses biofilms as well as controls prophage excision in E. coli. Since biofilm dispersal is a promising area to control biofilms, here we rewired Hha to control biofilm dispersal and formation. The Hha variant Hha13D6 was obtained to have enhanced biofilm dispersal activity along with increased toxicity compared to wild-type Hha (Hha13D6 induces dispersal 60%, whereas wild-type Hha induces dispersal at early biofilms but not at mature biofilms). Toxic Hha13D6 caused cell death probably by the activation of proteases HslUV, Lon, and PrlC, and deletion of protease gene hslV with overproducing Hh13D6 repressed biofilm dispersal, indicating Hha13D6 induces biofilm dispersal through the activity of protease HslV. Furthermore, another Hha variant Hha24E9 was also obtained to decrease biofilm formation 4-fold compared to wild-type Hha by regulation of gadW, glpT, and phnF. However, the dispersal variant Hha13D6 did not decrease biofilm formation, while the biofilm variant Hha24E9 did not induce biofilm dispersal. Hence, Hha may have evolved two ways in response to environmental factors to control biofilm dispersal and formation, but both controlling mechanisms come from different regulatory systems. For the whole-transcriptome study of BW25113 hha/pCA24N-hha13D6 versus BW25113 hha/pCA24N-hha biofilm dispersal, cells were grown in 250 mL of LB glucose (0.2%) for 16 h at 125 rpm with 10 g of glass wool (Corning Glass Works, Corning, NY, USA) in 1 L Erlenmeyer flasks to form a robust biofilm (Ren et al., 2004) and incubated an additional 1 h with 1 mM IPTG to induce wild-type Hha and Hha13D6. Similarly, for the whole transcriptome study of BW25113 hha/pCA24N-hha24E9 versus BW25113 hha/pCA24N-hha biofilm formation, cells were grown in 250 mL of LB glucose (0.2%) containing 1 mM IPTG for 7 h at 250 rpm with 10 g of glass wool to form biofilms. Biofilm cells were obtained by rinsing and sonicating the glass wool in sterile 0.85% NaCl solution at 0°C, and RNALater buffer® (Applied Biosystems, Foster City, CA, USA) was added to stabilize RNA during the RNA preparation steps. Total RNA was isolated from biofilm cells using a bead beater (Biospec, Bartlesville, OK, USA). cDNA synthesis, fragmentation, and hybridizations to the E. coli GeneChip Genome 2.0 array (Affymetrix, Santa Clara, CA, USA; P/N 511302). Genes were identified as differentially expressed if the expression ratio was higher than the standard deviation: 2.0-fold (induced and repressed) cutoff for Hha13D6 DNA microarrays (standard deviation 1.3-fold) and 10.0-fold (induced) or 4.0-fold (repressed) for Hha24E9 DNA microarrays (standard deviation 4.0-fold), and if the p-value for comparing two chips was less than 0.05.
Project description:The global transcriptional regulator Hha of Escherichia coli controls hemolysin activity, biofilm formation, and virulence expressions. Earlier, we have reported that Hha represses initial biofilm formation and disperses biofilms as well as controls prophage excision in E. coli. Since biofilm dispersal is a promising area to control biofilms, here we rewired Hha to control biofilm dispersal and formation. The Hha variant Hha13D6 was obtained to have enhanced biofilm dispersal activity along with increased toxicity compared to wild-type Hha (Hha13D6 induces dispersal 60%, whereas wild-type Hha induces dispersal at early biofilms but not at mature biofilms). Toxic Hha13D6 caused cell death probably by the activation of proteases HslUV, Lon, and PrlC, and deletion of protease gene hslV with overproducing Hh13D6 repressed biofilm dispersal, indicating Hha13D6 induces biofilm dispersal through the activity of protease HslV. Furthermore, another Hha variant Hha24E9 was also obtained to decrease biofilm formation 4-fold compared to wild-type Hha by regulation of gadW, glpT, and phnF. However, the dispersal variant Hha13D6 did not decrease biofilm formation, while the biofilm variant Hha24E9 did not induce biofilm dispersal. Hence, Hha may have evolved two ways in response to environmental factors to control biofilm dispersal and formation, but both controlling mechanisms come from different regulatory systems.
Project description:Dendritic cells (DCs) patrol tissues and transport antigens to lymph nodes to initiate adaptive immune responses. Within tissues, DCs constitute a complex cell population composed of distinct subsets that can exhibit different activation states and functions. How tissue-specific cues orchestrate DC diversification remains elusive. Here, we show that the small intestine included two pools of cDC2s originating from common preDC precursors: (1) lamina propria (LP) CD103+CD11b+ cDC2s that were mature-like pro-inflammatory cells and (2) intraepithelial cDC2s that exhibited an immature-like phenotype as well as tolerogenic properties. These phenotypes resulted from the action of food-derived retinoic acid (ATRA), which enhanced actomyosin contractility and promoted LP cDC2 transmigration into the epithelium. There, cDC2s were imprinted by environmental cues including ATRA itself and the mucus component Muc2. Hence, by reaching distinct sub-tissular niches, DCs can exist as immature and mature cells within the same tissue, revealing an additional mechanism of DC functional diversification.
Project description:With a model mimicking GBM tumor cell dispersal, transcriptome changes between core (immotile) and dispersive (motile) cells were analyzed. Many genes are differentially expressed between these populations. This study focused on the genes that are significantly upregulated in dispersive cells. Besides gene sets related with the cell cycle and cell survival, epithelial to mesenchymal transition gene set is upregulated in dispersive cells. In this gene set, this study identified SERPINE1 gene as an important regulator of GBM cell dispersal.