Project description:Using RNA-Seq analysis we compared the transcriptome of Methylosinus trichosporium OB3b grown in the presence of varying amounts of copper and cerium. When copper was added in the absence of cerium, expression of genes encoding for both soluble and particulate methane monooxygenases varied as expected. Genes encoding for copper uptake, storage, and efflux also increased, indicating that methanotrophs must carefully control copper homeostasis. When cerium was added in the absence of copper, expression of genes encoding for alternative methanol dehydrogenases varied as expected, but few other genes were found have differential expression. When cerium concentrations were varied in the presence of copper, few genes were found to be either up or downregulated, indicating that copper over rules any regulation by cerium. When copper was added in the presence of cerium, however, many genes were upregulated, most notably multiple steps of the central methane oxidation pathway, the serine cycle, and the ethylmalonyl-CoA pathway. Many genes were also downregulated, including those encoding for nitrogenase and hydrogenase.
Project description:Methane oxidation by aerobic methanotrophs is well-known to be strongly regulated by the availability of copper, i.e., the “copper-switch”. That is, there are two forms of the methane monooxygenase: a cytoplasmic or soluble methane monooxygenase (sMMO) and a membrane-bound or particulate methane monooxygenase (pMMO). sMMO is only expressed and active in the absence of copper, while pMMO requires copper. Previous work has also shown that one gene in the operon of the soluble methane monooxygenase – mmoD – also plays a critical role, but its function is still vague. Herein we show that MmoD is not needed for expression of genes in the sMMO gene cluster but is critical for formation of sMMO polypeptides and sMMO activity in Methylosinus trichosporium OB3b, indicating that MmoD plays a key post-transcriptional role in maturation of sMMO. Further, data also show that MmoD controls expression of methanobactin, a unique copper-binding compound used by some methanotrophs for copper collection. Collectively these results provide greater insights into the components of the “copper-switch” and thus provide new strategies to manipulate methanotrophic activity.
Project description:W614A-3S peptide is a modified 3S motif of the HIV-gp41 (mutation W614A). We previously detected the presence of natural neutralizing antibodies directed against W614A-3S peptide (NAbs) in long-term non-progressor HIV+ patients. Here, we compared the efficacy of W614A-3S peptide formulated in either squalene emulsion (SQE) or in aluminum hydroxide (Alum) in inducing broadly-NAbs (bNAbs). Rabbit and mouse models were used to screen the induction of bNAbs following 4 immunizations. SQE was more efficient than Alum formulation in inducing W614A-3S-specific bNAbs with up to 67-93% of HIV strains neutralized. We then analyzed the quality of peptide-specific murine B cells by single-cell gene expression by quantitative Reverse Transcription-PCR and single-cell V(D)J sequencing. We found more frequent germinal center B cells in SQE than in Alum, albeit with a different gene expression profile. The V(D)J sequencing of W614A-3S-specific BCR showed significant differences in BCR sequences and validates the dichotomy between adjuvant formulations. All sixteen BCR sequences which were cloned were specific of peptide. Adjuvant formulations of W614A-3S-peptide-conjugated immunogen impact the quantity and quality of B cell immune responses at both the gene expression level and BCR sequence. We used a single-cell quantitative Reverse Transcription-PCR (qRT-PCR) approach to compare between the two formulations the quality of W614A-3S-specific B cell populations isolated from draining lymph nodes, one week after 2nd and 3rd immunizations (W3 and W5, respectively).
Project description:Aerobic methanotrophic bacteria use methane as their sole source of carbon and energy and serve as a major sink for the potent greenhouse gas methane in freshwater ecosystems. Despite this important environmental role, little is known about the molecular details of how these organisms interact in the environment. Many bacterial species use quorum sensing systems to regulate gene expression in a density-dependent manner. We have identified a quorum sensing system in the genome of Methylobacter tundripaludum, a dominant methane-oxidizer in methane enrichments of sediment from Lake Washington (Seattle, WA, USA). We determined that M. tundripaludum primarily produces N-3-hydroxydecanoyl-L-homoserine lactone (3-OH-C10-HSL) and that production is governed by a positive feedback loop. We then further characterized this system by determining which genes are regulated by quorum sensing in this methane-oxidizer using RNA-seq, and discovered this system regulates the expression of a novel nonribosomal peptide synthetase biosynthetic gene cluster. These results identify and characterize a mode of cellular communication in an aerobic methane-oxidizing bacterium.
Project description:Aerobic methanotrophic bacteria use methane as their sole source of carbon and energy and serve as a major sink for the potent greenhouse gas methane in freshwater ecosystems. Despite this important environmental role, little is known about the molecular details of how these organisms interact in the environment. Many bacterial species use quorum sensing systems to regulate gene expression in a density-dependent manner. We have identified a quorum sensing system in the genome of Methylobacter tundripaludum, a dominant methane-oxidizer in methane enrichments of sediment from Lake Washington (Seattle, WA, USA). We determined that M. tundripaludum primarily produces N-3-hydroxydecanoyl-L-homoserine lactone (3-OH-CÂ10-HSL) and that production is governed by a positive feedback loop. We then further characterized this system by determining which genes are regulated by quorum sensing in this methane-oxidizer using RNA-seq, and discovered this system regulates the expression of a novel nonribosomal peptide synthetase biosynthetic gene cluster. These results identify and characterize a mode of cellular communication in an aerobic methane-oxidizing bacterium. Samples are 2 sets of biological replicates of a Methylobacter tundripaludum strain 21/22 mutant where the acyl-homoserine lactone (AHL) synthase gene mbaI (T451DRAFT_0796) has been deleted. The mutant strain was grown to log (48 hours) or stationary (68 hours) phase in the absence or presence of the AHL 3-OH-C10-HSL.