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1. Dynamic Control of Gene Expression with Riboregulated Switchable Feedback Promoters

   https://doi.org/10.1021/acssynbio.1c00015  

   Glasscock, Cameron J. ; Biggs, Bradley W. ; Lazar, John T. ; Arnold, Jack H. ; Burdette, Lisa A. ; Valdes, Aliki ; Kang, Min-Kyoung ; Tullman-Ercek, Danielle ; Tyo, Keith E. ; Lucks, Julius B. ( April 2021 , ACS Synthetic Biology)

   null (Ed.)

   One major challenge in synthetic biology is the deleterious impacts of cellular stress caused by expression of heterologous pathways, sensors, and circuits. Feedback control and dynamic regulation are broadly proposed strategies to mitigate this cellular stress by optimizing gene expression levels temporally and in response to biological cues. While a variety of approaches for feedback implementation exist, they are often complex and cannot be easily manipulated. Here, we report a strategy that uses RNA transcriptional regulators to integrate additional layers of control over the output of natural and engineered feedback responsive circuits. Called riboregulated switchable feedback promoters (rSFPs), these gene expression cassettes can be modularly activated using multiple mechanisms, from manual induction to autonomous quorum sensing, allowing control over the timing, magnitude, and autonomy of expression. We develop rSFPs in Escherichia coli to regulate multiple feedback networks and apply them to control the output of two metabolic pathways. We envision that rSFPs will become a valuable tool for flexible and dynamic control of gene expression in metabolic engineering, biological therapeutic production, and many other applications. 

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2. Regulatory small RNA, Qrr2 is expressed independently of sigma factor-54 and can function as the sole Qrr sRNA to control quorum sensing in Vibrio parahaemolyticus

   https://doi.org/10.1128/JB.00350-21  

   Tague, J.G. ; Hong, J. ; Kalburge, S.S. ; Boyd, E.F. ( October 2021 , Journal of Bacteriology)

   null (Ed.)

   Bacterial cells alter gene expression in response to changes in population density in a process called quorum sensing (QS). In Vibrio harveyi, LuxO, a low cell density activator of sigma factor-54 (RpoN), is required for transcription of five non-coding regulatory sRNAs, Qrr1-Qrr5, which each repress translation of the master QS regulator LuxR. Vibrio parahaemolyticus, the leading cause of bacterial seafood-borne gastroenteritis, also contains five Qrr sRNAs that control OpaR (the LuxR homolog), controlling capsule polysaccharide (CPS), motility, and metabolism. We show that in a Δ luxO deletion mutant, opaR was de-repressed and CPS and biofilm were produced. However, in a Δ rpoN mutant, opaR was repressed, no CPS was produced, and less biofilm production was observed compared to wild type. To determine why opaR was repressed, expression analysis in Δ luxO showed all five qrr genes were repressed, while in Δ rpoN the qrr2 gene was significantly de-repressed. Reporter assays and mutant analysis showed Qrr2 sRNA can act alone to control OpaR. Bioinformatics analysis identified a sigma-70 (RpoD) -35 -10 promoter overlapping the canonical sigma-54 (RpoN) -24 -12 promoter in the qrr2 regulatory region. The qrr2 sigma-70 promoter element was also present in additional Vibrio species indicating it is widespread. Mutagenesis of the sigma-70 -10 promoter site in the Δ rpoN mutant background, resulted in repression of qrr2. Analysis of qrr quadruple deletion mutants, in which only a single qrr gene is present, showed that only Qrr2 sRNA can act independently to regulate opaR . Mutant and expression data also demonstrated that RpoN and the global regulator, Fis, act additively to repress qrr2 . Our data has uncovered a new mechanism of qrr expression and shows that Qrr2 sRNA is sufficient for OpaR regulation. Importance The quorum sensing non-coding sRNAs are present in all Vibrio species but vary in number and regulatory roles among species. In the Harveyi clade, all species contain five qrr genes, and in V. harveyi these are transcribed by sigma-54 and are additive in function. In the Cholerae clade, four qrr genes are present, and in V. cholerae the qrr genes are redundant in function. In V. parahaemolyticus , qrr2 is controlled by two overlapping promoters. In an rpoN mutant, qrr2 is transcribed from a sigma-70 promoter that is present in all V. parahaemolyticus strains and in other species of the Harveyi clade suggesting a conserved mechanism of regulation. Qrr2 sRNA can function as the sole Qrr sRNA to control OpaR. 

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3. Beyond the Biosynthetic Gene Cluster Paradigm: Genome-Wide Coexpression Networks Connect Clustered and Unclustered Transcription Factors to Secondary Metabolic Pathways

   https://doi.org/10.1128/Spectrum.00898-21  

   Kwon, Min Jin ; Steiniger, Charlotte ; Cairns, Timothy C. ; Wisecaver, Jennifer H. ; Lind, Abigail L. ; Pohl, Carsten ; Regner, Carmen ; Rokas, Antonis ; Meyer, Vera ( October 2021 , Microbiology Spectrum)

   Goldman, Gustavo H. (Ed.)

   ABSTRACT Fungal secondary metabolites are widely used as therapeutics and are vital components of drug discovery programs. A major challenge hindering discovery of novel secondary metabolites is that the underlying pathways involved in their biosynthesis are transcriptionally silent under typical laboratory growth conditions, making it difficult to identify the transcriptional networks that they are embedded in. Furthermore, while the genes participating in secondary metabolic pathways are typically found in contiguous clusters on the genome, known as biosynthetic gene clusters (BGCs), this is not always the case, especially for global and pathway-specific regulators of pathways’ activities. To address these challenges, we used 283 genome-wide gene expression data sets of the ascomycete cell factory Aspergillus niger generated during growth under 155 different conditions to construct two gene coexpression networks based on Spearman’s correlation coefficients (SCCs) and on mutual rank-transformed Pearson’s correlation coefficients (MR-PCCs). By mining these networks, we predicted six transcription factors, named MjkA to MjkF, to regulate secondary metabolism in A. niger . Overexpression of each transcription factor using the Tet-On cassette modulated the production of multiple secondary metabolites. We found that the SCC and MR-PCC approaches complemented each other, enabling the delineation of putative global (SCC) and pathway-specific (MR-PCC) transcription factors. These results highlight the potential of coexpression network approaches to identify and activate fungal secondary metabolic pathways and their products. More broadly, we argue that drug discovery programs in fungi should move beyond the BGC paradigm and focus on understanding the global regulatory networks in which secondary metabolic pathways are embedded. IMPORTANCE There is an urgent need for novel bioactive molecules in both agriculture and medicine. The genomes of fungi are thought to contain vast numbers of metabolic pathways involved in the biosynthesis of secondary metabolites with diverse bioactivities. Because these metabolites are biosynthesized only under specific conditions, the vast majority of the fungal pharmacopeia awaits discovery. To discover the genetic networks that regulate the activity of secondary metabolites, we examined the genome-wide profiles of gene activity of the cell factory Aspergillus niger across hundreds of conditions. By constructing global networks that link genes with similar activities across conditions, we identified six putative global and pathway-specific regulators of secondary metabolite biosynthesis. Our study shows that elucidating the behavior of the genetic networks of fungi under diverse conditions harbors enormous promise for understanding fungal secondary metabolism, which ultimately may lead to novel drug candidates. 

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4. Coordination of host and symbiont gene expression reveals a metabolic tug-of-war between aphids and Buchnera

   https://doi.org/10.1073/pnas.1916748117  

   Smith, Thomas E. ; Moran, Nancy A. ( January 2020 , Proceedings of the National Academy of Sciences)

   Symbioses between animals and microbes are often described as mutualistic, but are subject to tradeoffs that may manifest as shifts in host and symbiont metabolism, cellular processes, or symbiont density. In pea aphids, the bacterial symbiontBuchnerais confined to specialized aphid cells called bacteriocytes, where it produces essential amino acids needed by hosts. This relationship is dynamic;Buchneratiter varies within individual aphids and among different clonal aphid lineages, and is affected by environmental and host genetic factors. We examined how host genotypic variation relates to host and symbiont function among seven aphid clones differing inBuchneratiter. We found that bacteriocyte gene expression varies among individual aphids and among aphid clones, and thatBuchneragene expression changes in response. By comparing hosts with low and highBuchneratiter, we found that aphids andBuchneraoppositely regulate genes underlying amino acid biosynthesis and cell growth. In high-titer hosts, both bacteriocytes and symbionts show elevated expression of genes underlying energy metabolism. Several eukaryotic cell signaling pathways are differentially expressed in bacteriocytes of low- versus high-titer hosts: Cell-growth pathways are up-regulated in low-titer genotypes, while membrane trafficking, lysosomal processes, and mechanistic target of rapamycin (mTOR) and cytokine pathways are up-regulated in high-titer genotypes. SpecificBuchnerafunctions are up-regulated within different bacteriocyte environments, with genes underlying flagellar body secretion and flagellar assembly overexpressed in low- and high-titer hosts, respectively. Overall, our results reveal allowances and demands made by both host and symbiont engaged in a metabolic “tug-of-war.”

    

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5. Model metabolic strategy for heterotrophic bacteria in the cold ocean based on Colwellia psychrerythraea 34H

   https://doi.org/10.1073/pnas.1807804115  

   Czajka, Jeffrey J. ; Abernathy, Mary H. ; Benites, Veronica T. ; Baidoo, Edward E. K. ; Deming, Jody W. ; Tang, Yinjie J. ( November 2018 , Proceedings of the National Academy of Sciences)

   Colwellia psychrerythraea34H is a model psychrophilic bacterium found in the cold ocean—polar sediments, sea ice, and the deep sea. Although the genomes of such psychrophiles have been sequenced, their metabolic strategies at low temperature have not been quantified. We measured the metabolic fluxes and gene expression of 34H at 4 °C (the mean global-ocean temperature and a normal-growth temperature for 34H), making comparative analyses at room temperature (above its upper-growth temperature of 18 °C) and with mesophilicEscherichia coli. When grown at 4 °C, 34H utilized multiple carbon substrates without catabolite repression or overflow byproducts; its anaplerotic pathways increased flux network flexibility and enabled CO₂fixation. In glucose-only medium, the Entner–Doudoroff (ED) pathway was the primary glycolytic route; in lactate-only medium, gluconeogenesis and the glyoxylate shunt became active. In comparison,E. coli, cold stressed at 4 °C, had rapid glycolytic fluxes but no biomass synthesis. At their respective normal-growth temperatures, intracellular concentrations of TCA cycle metabolites (α-ketoglutarate, succinate, malate) were 4–17 times higher in 34H than inE. coli, while levels of energy molecules (ATP, NADH, NADPH) were 10- to 100-fold lower. Experiments withE. colimutants supported the thermodynamic advantage of the ED pathway at cold temperature. Heat-stressed 34H at room temperature (2 hours) revealed significant down-regulation of genes associated with glycolytic enzymes and flagella, while 24 hours at room temperature caused irreversible cellular damage. We suggest that marine heterotrophic bacteria in general may rely upon simplified metabolic strategies to overcome thermodynamic constraints and thrive in the cold ocean.

    

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