86 research outputs found
The Early Interferon Alpha Subtype Response in Infant Macaques Infected Orally With SIV
Type I interferons play an important role in the early defense against viral and other pathogens. These innate responses are also critically important in shaping the subsequent adaptive response. Thus, a more thorough knowledge of innate response types and mechanisms will improve our understanding of pathogenesis and guide the development of new therapeutics
ChIP-exo interrogation of Crp, DNA, and RNAP holoenzyme interactions
Numerous in vitro studies have yielded a refined picture of the structural and molecular associations between Cyclic-AMP receptor protein (Crp), the DNA motif, and RNA polymerase (RNAP) holoenzyme. In this study, high-resolution ChIP-exonuclease (ChIP-exo) was applied to study Crp binding in vivo and at genome-scale. Surprisingly, Crp was found to provide little to no protection of the DNA motif under activating conditions. Instead, Crp demonstrated binding patterns that closely resembled those generated by σ70. The binding patterns of both Crp and σ70 are indicative of RNAP holoenzyme DNA footprinting profiles associated with stages during transcription initiation that occur post-recruitment. This is marked by a pronounced advancement of the template strand footprint profile to the +20 position relative to the transcription start site and a multimodal distribution on the nontemplate strand. This trend was also observed in the familial transcription factor, Fnr, but full protection of the motif was seen in the repressor ArcA. Given the time-scale of ChIP studies and that the rate-limiting step in transcription initiation is typically post recruitment, we propose a hypothesis where Crp is absent from the DNA motif but remains associated with RNAP holoenzyme post-recruitment during transcription initiation. The release of Crp from the DNA motif may be a result of energetic changes that occur as RNAP holoenzyme traverses the various stable intermediates towards elongation complex formation
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Multiple Optimal Phenotypes Overcome Redox and Glycolytic Intermediate Metabolite Imbalances in <i>Escherichia coli pgi</i> Knockout Evolutions
A mechanistic understanding of how new phenotypes develop to overcome the loss of a gene product provides valuable insight on both the metabolic and regulatory functions of the lost gene. The pgi gene, whose product catalyzes the second step in glycolysis, was deleted in a growth-optimized Escherichia coli K-12 MG1655 strain. The initial knockout (KO) strain exhibited an 80% drop in growth rate that was largely recovered in eight replicate, but phenotypically distinct, cultures after undergoing adaptive laboratory evolution (ALE). Multi-omic data sets showed that the loss of pgi substantially shifted pathway usage, leading to a redox and sugar phosphate stress response. These stress responses were overcome by unique combinations of innovative mutations selected for by ALE. Thus, the coordinated mechanisms from genome to metabolome that lead to multiple optimal phenotypes after the loss of a major gene product were revealed.IMPORTANCE A mechanistic understanding of how microbes are able to overcome the loss of a gene through regulatory and metabolic changes is not well understood. Eight independent adaptive laboratory evolution (ALE) experiments with pgi knockout strains resulted in eight phenotypically distinct endpoints that were able to overcome the gene loss. Utilizing multi-omics analysis, the coordinated mechanisms from genome to metabolome that lead to multiple optimal phenotypes after the loss of a major gene product were revealed
Evolution of gene knockout strains of <i>E-coli</i> reveal regulatory architectures governed by metabolism
The function of metabolic genes in the context of regulatory networks is not well understood. Here, the authors investigate the adaptive responses of E. coli after knockout of metabolic genes and highlight the influence of metabolite levels in the evolution of regulatory function
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