Arrayed in vivo barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries.

Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45 000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.

Arrayed in vivo barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries.

Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45,000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.

Differential Hsp90-dependent gene expression is strain-specific and common among yeast strains.

Enhanced phenotypic diversity increases a population's likelihood of surviving catastrophic conditions. Hsp90, an essential molecular chaperone and a central network hub in eukaryotes, has been observed to suppress or enhance the effects of genetic variation on phenotypic diversity in response to environmental cues. Because many Hsp90-interacting genes are involved in signaling transduction pathways and transcriptional regulation, we tested how common Hsp90-dependent differential gene expression is in natural populations. Many genes exhibited Hsp90-dependent strain-specific differential expression in five diverse yeast strains. We further identified transcription factors (TFs) potentially contributing to variable expression. We found that on Hsp90 inhibition or environmental stress, activities or abundances of Hsp90-dependent TFs varied among strains, resulting in differential strain-specific expression of their target genes, which consequently led to phenotypic diversity. We provide evidence that individual strains can readily display specific Hsp90-dependent gene expression, suggesting that the evolutionary impacts of Hsp90 are widespread in nature.

Plastic Rewiring of Sef1 Transcriptional Networks and the Potential of Nonfunctional Transcription Factor Binding in Facilitating Adaptive Evolution.

Prior and extensive plastic rewiring of a transcriptional network, followed by a functional switch of the conserved transcriptional regulator, can shape the evolution of a new network with diverged functions. The presence of three distinct iron regulatory systems in fungi that use orthologous transcriptional regulators suggests that these systems evolved in that manner. Orthologs of the transcriptional activator Sef1 are believed to be central to how iron regulatory systems developed in fungi, involving gene gain, plastic network rewiring, and switches in regulatory function. We show that, in the protoploid yeast Lachancea kluyveri, plastic rewiring of the L. kluyveri Sef1 (Lk-Sef1) network, together with a functional switch, enabled Lk-Sef1 to regulate TCA cycle genes, unlike Candida albicans Sef1 that mainly regulates iron-uptake genes. Moreover, we observed pervasive nonfunctional binding of Sef1 to its target genes. Enhancing Lk-Sef1 activity resuscitated the corresponding transcriptional network, providing immediate adaptive benefits in changing environments. Our study not only sheds light on the evolution of Sef1-centered transcriptional networks but also shows the adaptive potential of nonfunctional transcription factor binding for evolving phenotypic novelty and diversity.

Hsp90 regulates nongenetic variation in response to environmental stress.

Nongenetic cell-to-cell variability often plays an important role for the survival of a clonal population in the face of fluctuating environments. However, the underlying mechanisms regulating such nongenetic heterogeneity remain elusive in most organisms. We report here that a clonal yeast population exhibits morphological heterogeneity when the level of Hsp90, a molecular chaperone, is reduced. The morphological heterogeneity is driven by the dosage of Cdc28 and Cla4, a key regulator of septin formation. Low Hsp90 levels reduce Cla4 protein stability and cause a subpopulation of cells to switch to a filamentous form that has been previously suggested to be beneficial under certain hostile environments. Moreover, Hsp90-dependent morphological heterogeneity can be induced by environmental stress and is conserved across diverse yeast species. Our results suggest that Hsp90 provides an evolutionarily conserved mechanism that links environmental stress to the induction of morphological diversity.