Project description:The identification of processes activated by specific microbes during microbiota colonization of plant roots has been hampered by technical constraints in metatranscriptomics. These include lack of reference genomes, high representation of host or microbial rRNA sequences in datasets, or difficulty to experimentally validate gene functions. Here, we recolonized germ-free Arabidopsis thaliana with a synthetic, yet representative root microbiota comprising 106 genome-sequenced bacterial and fungal isolates. We used multi-kingdom rRNA depletion, deep RNA-sequencing and read mapping against reference microbial genomes to analyse the in-planta metatranscriptome of abundant colonizers. We identified over 3,000 microbial genes that were differentially regulated at the soil-root interface. Translation and energy production processes were consistently activated in planta, and their induction correlated with bacterial strains’ abundance in roots. Finally, we used targeted mutagenesis to show that several genes consistently induced by multiple bacteria are required for root colonization in one of the abundant bacterial strains (a genetically tractable Rhodanobacter). Our results indicate that microbiota members activate strain-specific processes but also common gene sets to colonize plant roots.
Project description:The human gut microbiota harbors methanogens represented by the dominant archaeon, Methanobrevibacter smithii, a polyphyletic group of acetogens, and sulfate-reducing bacteria. Defining their roles in the H2-economy of the gut has potential therapeutic importance for modulating the efficiency of fermentation of dietary components. We quantified methanogens in fecal samples from 40 healthy adult female monozygotic(MZ) and 28 dizygotic(DZ) twin pairs, analyzed bacterial 16S rRNA datasets generated from their fecal samples to identify taxa that co-occur with methanogens, sequenced the genomes of 20 M. smithii strains isolated from families of MZ and DZ twins, and performed RNA-Seq of a subset of strains to identify their responses to varied formate concentrations. The concordance rate for methanogen carriage was significantly higher for MZ versus DZ twin pairs. Co-occurrence analysis revealed 22 bacterial species-level taxa positively correlated with methanogens: all but two were members of the Clostridiales, with several being, or related to, known hydrogen-producing and -consuming bacteria. The M. smithii pan-genome contains 987 genes conserved in all strains, and 1860 variably represented genes. Strains from MZ and DZ twin pairs had a similar degree of shared genes and SNPs, and were significantly more similar than strains isolated from mothers or members of other families. The 101 adhesin-like proteins(ALPs) in the pan-genome (45±6/strain) exhibit strain-specific differences in expression and responsiveness to formate. We hypothesize that M. smithii strains use their different repertoires of ALPs to create diversity in their metabolic niches, by allowing them to establish syntrophic relationships with bacterial partners with differing metabolic capabilities and patterns of co-occurrence These strains were isolated from human feces, but they are in pure culture now. All the information about each species is associated with the genome accession number Fecal samples from 40 healthy adult female monozygotic(MZ) and 28 dizygotic(DZ) twin pairs, analyzed bacterial 16S rRNA datasets generated from their fecal samples to identify taxa that co-occur with methanogens, sequenced the genomes of 20 M. smithii strains isolated from families of MZ and DZ twins, and performed RNA-Seq of a subset of strains to identify their responses to varied formate concentrations. Strains of Methanobrevibacter smithii were grown in vitro (modified MBC media) to mid-log phase, at 37°C in serum bottles pressurized with 80% hydrogen, 20% CO2 gasses at 30psi. Cells were harvested by centrifugation, and DNA was isolated by phenol-chloroform and ethanol precipitation.
Project description:Gene regulation is one of the most ubiquitous processes in biology. And yet, while the catalogue of 15 bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in 16 these genomes are regulated. Characterizing the molecular mechanisms by which regulatory sequences 17 operate still requires focused efforts using low-throughput methods. Here we show how a combination of 18 massively parallel reporter assays, mass spectrometry, and information-theoretic modeling can be used 19 to dissect bacterial promoters in a systematic and scalable way. We demonstrate this method on both 20 well-studied and previously uncharacterized promoters in the enteric bacterium Escherichia coli. In all 21 cases we recover nucleotide-resolution models of promoter mechanism. For some promoters, including 22 previously unannotated ones, we can further extract quantitative biophysical models describing 23 input-output relationships. This method opens up the possibility of exhaustively dissecting the 24 mechanisms of promoter function in E. coli and a wide range of other bacteria.
Project description:Rhizobia are soil bacteria that can associate with some legumes and participate in symbiotic nitrogen fixation. Bacterial CspA family members are small, single stranded nucleic acid binding proteins. Differentiation of rhizobial bacteria from a free-living to symbiotic state within legume root nodules follows a massive re-programming of bacterial gene expression. Here, the role of Sinorhizobium meliloti CspA family members in symbiotic development with Medicago sativa (alfalfa) was investigated. We defined expression patterns of CspA family members, identified CspA interacting RNAs, and investigated phenotypes and transcriptional defects associated with cspA deletion strains. We propose that these proteins affect rhizobial physiology through their global control of the cellular RNA secondary structure strength environment and through specific modulation of small non-coding RNA (sRNA) structures involved in cis-regulation of stress responsive sigma factor expression. This work describes an RNA structure mediated mechanism important for bacterial stress adaptation and symbiotic development within a plant host.