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Cell growth rate is regulated in response to the abundance and molecular form of essential nutrients. In Saccharomyces cerevisiae (budding yeast), the molecular form of environmental nitrogen is a major determinant of cell growth rate, supporting growth rates that vary at least threefold. Transcriptional control of nitrogen use is mediated in large part by nitrogen catabolite repression (NCR), which results in the repression of specific transcripts in the presence of a preferred nitrogen source that supports a fast growth rate, such as glutamine, that are otherwise expressed in the presence of a nonpreferred nitrogen source, such as proline, which supports a slower growth rate. Differential expression of the NCR regulon and additional nitrogen-responsive genes results in >500 transcripts that are differentially expressed in cells growing in the presence of different nitrogen sources in batch cultures. Here we find that in growth rate–controlled cultures using nitrogen-limited chemostats, gene expression programs are strikingly similar regardless of nitrogen source. NCR expression is derepressed in all nitrogen-limiting chemostat conditions regardless of nitrogen source, and in these conditions, only 34 transcripts exhibit nitrogen source–specific differential gene expression. Addition of either the preferred nitrogen source, glutamine, or the nonpreferred nitrogen source, proline, to cells growing in nitrogen-limited chemostats results in rapid, dose-dependent repression of the NCR regulon. Using a novel means of computational normalization to compare global gene expression programs in steady-state and dynamic conditions, we find evidence that the addition of nitrogen to nitrogen-limited cells results in the transient overproduction of transcripts required for protein translation. Simultaneously, we find that that accelerated mRNA degradation underlies the rapid clearing of a subset of transcripts, which is most pronounced for the highly expressed NCR-regulated permease genes GAP1, MEP2, DAL5, PUT4, and DIP5. Our results reveal novel aspects of nitrogen-regulated gene expression and highlight the need for a quantitative approach to study how the cell coordinates protein translation and nitrogen assimilation to optimize cell growth in different environments.

Copy number variants (CNVs)-gains and losses of genomic sequences-are an important source of genetic variation underlying rapid adaptation and genome evolution. However, despite their central role in evolution little is known about the factors that contribute to the structure, size, formation rate, and fitness effects of adaptive CNVs. Local genomic sequences are likely to be an important determinant of these properties. Whereas it is known that point mutation rates vary with genomic location and local DNA sequence features, the role of genome architecture in the formation, selection, and the resulting evolutionary dynamics of CNVs is poorly understood. Previously, we have found that the GAP1 gene in Saccharomyces cerevisiae undergoes frequent and repeated amplification and selection under long-term experimental evolution in glutamine-limiting conditions. The GAP1 gene has a unique genomic architecture consisting of two flanking long terminal repeats (LTRs) and a proximate origin of DNA replication (autonomously replicating sequence, ARS), which are likely to promote rapid GAP1 CNV formation. To test the role of these genomic elements on CNV-mediated adaptive evolution, we performed experimental evolution in glutamine-limited chemostats using engineered strains lacking either the adjacent LTRs, ARS, or all elements. Using a CNV reporter system and neural network simulation-based inference (nnSBI) we quantified the formation rate and fitness effect of CNVs for each strain. We find that although GAP1 CNVs repeatedly form and sweep to high frequency in strains with modified genome architecture, removal of local DNA elements significantly impacts the rate and fitness effect of CNVs and the rate of adaptation. We performed genome sequence analysis to define the molecular mechanisms of CNV formation for 177 CNV lineages. We find that across all four strain backgrounds, between 26% and 80% of all GAP1 CNVs are mediated by Origin Dependent Inverted Repeat Amplification (ODIRA) which results from template switching between the leading and lagging strand during DNA synthesis. In the absence of the local ARS, a distal ARS can mediate CNV formation via ODIRA. In the absence of local LTRs, homologous recombination mechanisms still mediate gene amplification following de novo insertion of retrotransposon elements at the locus. Our study demonstrates the remarkable plasticity of the genome and reveals that template switching during DNA replication is a frequent source of adaptive CNVs.

Meiosis is required for the formation of gametes in all sexually reproducing species and the process is well conserved across the tree of life. However, meiosis is sensitive to a variety of external factors, which can impact chromosome pairing, recombination, and fertility. For example, the optimal temperature for successful meiosis varies between species of plants and animals. This suggests that meiosis is temperature sensitive, and that natural selection may act on variation in meiotic success as organisms adapt to different environmental conditions. To understand how temperature alters the successful completion of meiosis, we utilized two species of the budding yeast Saccharomyces with different temperature preferences: thermotolerant Saccharomyces cerevisiae and cold tolerant Saccharomyces uvarum . We surveyed three metrics of meiosis: sporulation efficiency, spore viability, and recombination rate in multiple strains of each species. As per our predictions, the proportion of cells that complete meiosis and form spores is temperature sensitive, with thermotolerant S. cerevisiae having a higher temperature threshold for successful meiosis than cold tolerant S. uvarum . We confirmed previous observations that S. cerevisiae recombination rate varies between strains and across genomic regions, and add new results that S. uvarum has higher recombination rates than S. cerevisiae . We find that temperature significantly influences recombination rate plasticity in S. cerevisiae and S. uvarum , in agreement with studies in animals and plants. Overall, these results suggest that meiotic thermal sensitivity is associated with organismal thermal tolerance, and may even result in temporal reproductive isolation as populations diverge in thermal profiles.

Meiotic recombination is an evolutionary force that acts by breaking up genomic linkage, increasing the efficacy of selection. Recombination is initiated with a double-strand break which is resolved via a crossover, which involves the reciprocal exchange of genetic material between homologous chromosomes, or a non-crossover, which results in small tracts of non-reciprocal exchange of genetic material. Crossover and non-crossover rates vary between species, populations, individuals, and across the genome. In recent years, recombination rate has been associated with the distribution of ancestry derived from past interspecific hybridization (introgression) in a variety of species. We explore this interaction of recombination and introgression by sequencing spores and detecting crossovers and non-crossovers from two crosses of the yeast Saccharomyces uvarum. One cross is between strains which each contain introgression from their sister species, S . eubayanus, while the other cross has no introgression present. We find that the recombination landscape is significantly different between S . uvarum crosses, and that some of these differences can be explained by the presence of introgression in one cross. Crossovers are reduced and non-crossovers are increased in heterozygous introgression compared to syntenic regions in the cross without introgression. This translates to reduced allele shuffling within introgressed regions, and an overall reduction of shuffling on most chromosomes with introgression compared to the syntenic regions and chromosomes without introgression. Our results suggest that hybridization can significantly influence the recombination landscape, and that the reduction in allele shuffling contributes to the initial purging of introgression in the generations following a hybridization event.

FlhDC is a heterohexameric complex that acts as a master regulator of flagellar biosynthesis genes in numerous bacteria. Previous studies have identified a single flhDC operon encoding this complex. However, we found that two flhDC loci are present throughout Paraburkholderia , and two additional flhC copies are also present in Paraburkholderia unamae. Systematic deletion analysis in P. unamae of the different flhDC copies showed that one of the operons, flhDC1 , plays the predominant role, with deletion of its genes resulting in a severe inhibition of motility and biofilm formation. Expression analysis using promoter- lacZ fusions and real-time quantitative PCR support the primary role of flhDC1 in flagellar gene regulation, with flhDC2 a secondary contributor. Phylogenetic analysis shows the presence of the flhDC1 and flhDC2 operons throughout Paraburkholderia . In contrast, Burkholderia and other bacteria only carry the copy syntenous with flhDC2 . The variations in impact each copy of flhDC has on downstream processes indicate that regulation of FlhDC in P. unamae , and likely other Paraburkholderia species, is regulated at least in part by the presence of multiple copies of these genes. IMPORTANCE Motility is important in the colonization of plant roots by beneficial and pathogenic bacteria, with flagella playing essential roles in host cell adhesion, entrance, and biofilm formation. Flagellar biosynthesis is energetically expensive. Its complex regulation by the FlhDC master regulator is well studied in peritrichous flagella expressing enterics. We report the unique presence throughout Paraburkholderia of multiple copies of flhDC . In P. unamae , the flhDC1 copy showed higher expression and a greater effect on swim motility, flagellar development, and regulation of downstream genes, than the flhDC2 copy that is syntenous to flhDC in Escherichia coli and pathogenic Burkholderia spp. The flhDC genes have evolved differently in these plant-growth-promoting bacteria, giving an additional layer of complexity in gene regulation by FlhDC.