Adaptive mutations that prevent crosstalk enable the expansion of paralogous signaling protein families.

Orthologous proteins often harbor numerous substitutions, but whether these differences result from neutral or adaptive processes is usually unclear. To tackle this challenge, we examined the divergent evolution of a model bacterial signaling pathway comprising the kinase PhoR and its cognate substrate PhoB. We show that the specificity-determining residues of these proteins are typically under purifying selection but have, in α-proteobacteria, undergone a burst of diversification followed by extended stasis. By reversing mutations that accumulated in an α-proteobacterial PhoR, we demonstrate that these substitutions were adaptive, enabling PhoR to avoid crosstalk with a paralogous pathway that arose specifically in α-proteobacteria. Our findings demonstrate that duplication and the subsequent need to avoid crosstalk strongly influence signaling protein evolution. These results provide a concrete example of how system-wide insulation can be achieved postduplication through a surprisingly limited number of mutations. Our work may help explain the apparent ease with which paralogous protein families expanded in all organisms.

Microevolution in the pansecondary metabolome of Aspergillus flavus and its potential macroevolutionary implications for filamentous fungi.

Fungi produce a wealth of pharmacologically bioactive secondary metabolites (SMs) from biosynthetic gene clusters (BGCs). It is common practice for drug discovery efforts to treat species' secondary metabolomes as being well represented by a single or a small number of representative genomes. However, this approach misses the possibility that intraspecific population dynamics, such as adaptation to environmental conditions or local microbiomes, may harbor novel BGCs that contribute to the overall niche breadth of species. Using 94 isolates of Aspergillus flavus, a cosmopolitan model fungus, sampled from seven states in the United States, we dereplicate 7,821 BGCs into 92 unique BGCs. We find that more than 25% of pangenomic BGCs show population-specific patterns of presence/absence or protein divergence. Population-specific BGCs make up most of the accessory-genome BGCs, suggesting that different ecological forces that maintain accessory genomes may be partially mediated by population-specific differences in secondary metabolism. We use ultra-high-performance high-resolution mass spectrometry to confirm that these genetic differences in BGCs also result in chemotypic differences in SM production in different populations, which could mediate ecological interactions and be acted on by selection. Thus, our results suggest a paradigm shift that previously unrealized population-level reservoirs of SM diversity may be of significant evolutionary, ecological, and pharmacological importance. Last, we find that several population-specific BGCs from A. flavus are present in Aspergillus parasiticus and Aspergillus minisclerotigenes and discuss how the microevolutionary patterns we uncover inform macroevolutionary inferences and help to align fungal secondary metabolism with existing evolutionary theory.

Rewiring the specificity of two-component signal transduction systems.

Two-component signal transduction systems are the predominant means by which bacteria sense and respond to environmental stimuli. Bacteria often employ tens or hundreds of these paralogous signaling systems, comprised of histidine kinases (HKs) and their cognate response regulators (RRs). Faithful transmission of information through these signaling pathways and avoidance of detrimental crosstalk demand exquisite specificity of HK-RR interactions. To identify the determinants of two-component signaling specificity, we examined patterns of amino acid coevolution in large, multiple sequence alignments of cognate kinase-regulator pairs. Guided by these results, we demonstrate that a subset of the coevolving residues is sufficient, when mutated, to completely switch the substrate specificity of the kinase EnvZ. Our results shed light on the basis of molecular discrimination in two-component signaling pathways, provide a general approach for the rational rewiring of these pathways, and suggest that analyses of coevolution may facilitate the reprogramming of other signaling systems and protein-protein interactions.

Transposon insertional mutagenesis in Saccharomyces uvarum reveals trans-acting effects influencing species-dependent essential genes.

To understand how complex genetic networks perform and regulate diverse cellular processes, the function of each individual component must be defined. Comprehensive phenotypic studies of mutant alleles have been successful in model organisms in determining what processes depend on the normal function of a gene. These results are often ported to newly sequenced genomes by using sequence homology. However, sequence similarity does not always mean identical function or phenotype, suggesting that new methods are required to functionally annotate newly sequenced species. We have implemented comparative analysis by high-throughput experimental testing of gene dispensability in Saccharomyces uvarum, a sister species of Saccharomyces cerevisiae. We created haploid and heterozygous diploid Tn7 insertional mutagenesis libraries in S. uvarum to identify species-dependent essential genes, with the goal of detecting genes with divergent functions and/or different genetic interactions. Comprehensive gene dispensability comparisons with S. cerevisiae predicted diverged dispensability at 12% of conserved orthologs, and validation experiments confirmed 22 differentially essential genes. Despite their differences in essentiality, these genes were capable of cross-species complementation, demonstrating that trans-acting factors that are background-dependent contribute to differential gene essentiality. This study shows that direct experimental testing of gene disruption phenotypes across species can inform comparative genomic analyses and improve gene annotations. Our method can be widely applied in microorganisms to further our understanding of genome evolution.

Exploiting nonionic surfactants to enhance fatty alcohol production in Rhodosporidium toruloides.

Fatty alcohols (FOHs) are important feedstocks in the chemical industry to produce detergents, cosmetics, and lubricants. Microbial production of FOHs has become an attractive alternative to production in plants and animals due to growing energy demands and environmental concerns. However, inhibition of cell growth caused by intracellular FOH accumulation is one major issue that limits FOH titers in microbial hosts. In addition, identification of FOH-specific exporters remains a challenge and previous studies towards this end are limited. To alleviate the toxicity issue, we exploited nonionic surfactants to promote the export of FOHs in Rhodosporidium toruloides, an oleaginous yeast that is considered an attractive next-generation host for the production of fatty acid-derived chemicals. Our results showed FOH export efficiency was dramatically improved and the growth inhibition was alleviated in the presence of small amounts of tergitol and other surfactants. As a result, FOH titers increase by 4.3-fold at bench scale to 352.6 mg/L. With further process optimization in a 2-L bioreactor, the titer was further increased to 1.6 g/L. The method we show here can potentially be applied to other microbial hosts and may facilitate the commercialization of microbial FOH production.

Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides.

BACKGROUND: Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. RESULTS: The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L. CONCLUSION: This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism.

Monoterpene production by the carotenogenic yeast Rhodosporidium toruloides.

BACKGROUND: Due to their high energy density and compatible physical properties, several monoterpenes have been investigated as potential renewable transportation fuels, either as blendstocks with petroleum or as drop-in replacements for use in vehicles (both heavy and light-weight) or in aviation. Sustainable microbial production of these biofuels requires the ability to utilize cheap and readily available feedstocks such as lignocellulosic biomass, which can be depolymerized into fermentable carbon sources such as glucose and xylose. However, common microbial production platforms such as the yeast Saccharomyces cerevisiae are not naturally capable of utilizing xylose, hence requiring extensive strain engineering and optimization to efficiently utilize lignocellulosic feedstocks. In contrast, the oleaginous red yeast Rhodosporidium toruloides is capable of efficiently metabolizing both xylose and glucose, suggesting that it may be a suitable host for the production of lignocellulosic bioproducts. In addition, R. toruloides naturally produces several carotenoids (C40 terpenoids), indicating that it may have a naturally high carbon flux through its mevalonate (MVA) pathway, providing pools of intermediates for the production of a wide range of heterologous terpene-based biofuels and bioproducts from lignocellulose. RESULTS: Sixteen terpene synthases (TS) originating from plants, bacteria and fungi were evaluated for their ability to produce a total of nine different monoterpenes in R. toruloides. Eight of these TS were functional and produced several different monoterpenes, either as individual compounds or as mixtures, with 1,8-cineole, sabinene, ocimene, pinene, limonene, and carene being produced at the highest levels. The 1,8-cineole synthase HYP3 from Hypoxylon sp. E74060B produced the highest titer of 14.94 ± 1.84 mg/L 1,8-cineole in YPD medium and was selected for further optimization and fuel properties study. Production of 1,8-cineole from lignocellulose was also demonstrated in a 2L batch fermentation, and cineole production titers reached 34.6 mg/L in DMR-EH (Deacetylated, Mechanically Refined, Enzymatically Hydorlized) hydrolysate. Finally, the fuel properties of 1,8-cineole were examined, and indicate that it may be a suitable petroleum blend stock or drop-in replacement fuel for spark ignition engines. CONCLUSION: Our results demonstrate that Rhodosporidium toruloides is a suitable microbial platform for the production of non-native monoterpenes with biofuel applications from lignocellulosic biomass.

A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides.

BACKGROUND: Rhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides. RESULTS: This data describes a set of native R. toruloides promoters, characterized over time in four different media commonly used for cultivation of this yeast. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. Promoter expression strength was determined by measurement of EGFP and mRuby2 reporters by flow cytometry. A total of 20 constitutive promoters (12 monodirectional and 8 bidirectional) were found, and are expected to be of potential value for genetic engineering of R. toruloides. CONCLUSIONS: A set of robust and constitutive promoters to facilitate genetic engineering of R. toruloides is presented here, ranging from a promoter previously used for this purpose (P7, glyceraldehyde 3-phosphate dehydrogenase, GAPDH) to stronger monodirectional (e.g., P15, mitochondrial adenine nucleotide translocator, ANT) and bidirectional (e.g., P9 and P9R, histones H3 and H4, respectively) promoters. We also identified promoters that may be useful for specific applications such as late-stage expression (e.g., P3, voltage-dependent anion channel protein 2, VDAC2). This set of characterized promoters significantly expands the range of engineering tools available for this yeast and can be applied in future metabolic engineering studies.

Metabolic engineering of a haploid strain derived from a triploid industrial yeast for producing cellulosic ethanol.

Many desired phenotypes for producing cellulosic biofuels are often observed in industrial Saccharomyces cerevisiae strains. However, many industrial yeast strains are polyploid and have low spore viability, making it difficult to use these strains for metabolic engineering applications. We selected the polyploid industrial strain S. cerevisiae ATCC 4124 exhibiting rapid glucose fermentation capability, high ethanol productivity, strong heat and inhibitor tolerance in order to construct an optimal yeast strain for producing cellulosic ethanol. Here, we focused on developing a general approach and high-throughput screening method to isolate stable haploid segregants derived from a polyploid parent, such as triploid ATCC 4124 with a poor spore viability. Specifically, we deleted the HO genes, performed random sporulation, and screened the resulting segregants based on growth rate, mating type, and ploidy. Only one stable haploid derivative (4124-S60) was isolated, while 14 other segregants with a stable mating type were aneuploid. The 4124-S60 strain inherited only a subset of desirable traits present in the parent strain, same as other aneuploids, suggesting that glucose fermentation and specific ethanol productivity are likely to be genetically complex traits and/or they might depend on ploidy. Nonetheless, the 4124-60 strain did inherit the ability to tolerate fermentation inhibitors. When additional genetic perturbations known to improve xylose fermentation were introduced into the 4124-60 strain, the resulting engineered strain (IIK1) was able to ferment a Miscanthus hydrolysate better than a previously engineered laboratory strain (SR8), built by making the same genetic changes. However, the IIK1 strain showed higher glycerol and xylitol yields than the SR8 strain. In order to decrease glycerol and xylitol production, an NADH-dependent acetate reduction pathway was introduced into the IIK1 strain. By consuming 2.4g/L of acetate, the resulting strain (IIK1A) exhibited a 14% higher ethanol yield and 46% lower byproduct yield than the IIK1 strain from anaerobic fermentation of the Miscanthus hydrolysate. Our results demonstrate that industrial yeast strains can be engineered via haploid isolation. The isolated haploid strain (4124-S60) can be used for metabolic engineering to produce fuels and chemicals.

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae.

UNLABELLED: Efficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular ß-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1 We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an efficient strategy for yeast metabolic engineering. IMPORTANCE: In order to enable rapid and efficient fermentation of cellulosic hydrolysates by engineered yeast, we delve into the limiting factors of cellobiose fermentation by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular ß-glucosidase (encoded by gh1-1). Through laboratory evolution, we isolated mutant strains capable of fermenting cellobiose much faster than a parental strain. Genome sequencing of the fast cellobiose-fermenting mutant reveals that there are massive amplifications of cdt-1 and gh1-1 in the yeast genome. We also found positive and quantitative relationships between the rates of cellobiose consumption and the copy numbers of cdt-1 and gh1-1 in the evolved strains. Our results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step for efficient cellobiose fermentation. We demonstrate the feasibility of optimizing not only heterologous metabolic pathways in yeast through laboratory evolution but also on-demand gene amplification in yeast, which can be broadly applicable for metabolic engineering.

Engineering Rhodosporidium toruloides for increased lipid production.

Oleaginous yeast are promising organisms for the production of lipid-based chemicals and fuels from simple sugars. In this work, we explored Rhodosporidium toruloides for the production of lipid-based products. This oleaginous yeast natively produces lipids at high titers and can grow on glucose and xylose. As a first step, we sequenced the genomes of two strains, IFO0880, and IFO0559, and generated draft assemblies and annotations. We then used this information to engineer two R. toruloides strains for increased lipid production by over-expressing the native acetyl-CoA carboxylase and diacylglycerol acyltransferase genes using Agrobacterium tumefaciens mediated transformation. Our best strain, derived from IFO0880, was able to produce 16.4 ± 1.1 g/L lipid from 70 g/L glucose and 9.5 ± 1.3 g/L lipid from 70 g/L xylose in shake-flask experiments. This work represents one of the first examples of metabolic engineering in R. toruloides and establishes this yeast as a new platform for production of fatty-acid derived products.

Metabolic engineering of the oleaginous yeast Rhodosporidium toruloides IFO0880 for lipid overproduction during high-density fermentation.

Natural lipids can be used to make biodiesel and many other value-added compounds. In this work, we explored a number of different metabolic engineering strategies for increasing lipid production in the oleaginous yeast Rhodosporidium toruloides IFO0880. These included increasing the expression of enzymes involved in different aspects of lipid biosynthesis-malic enzyme (ME), pyruvate carboxylase (PYC1), glycerol-3-P dehydrogenase (GPD), and stearoyl-CoA desaturase (SCD)-and deleting the gene PEX10, required for peroxisome biogenesis. Only malic enzyme and stearoyl-CoA desaturase, when overexpressed, were found to significantly increase lipid production. Only stearoyl-CoA desaturase, when overexpressed, further increased lipid production in a strain previously engineered to overexpress acetyl-CoA carboxylase (ACC1) and diacylglycerol acyltransferase (DGA1). Our best strain produced 27.4 g/L lipid with an average productivity of 0.31 g/L/h during batch growth on glucose and 89.4 g/L lipid with an average productivity of 0.61 g/L/h during fed-batch growth on glucose. These results further establish R. toruloides as a platform organism for the production of lipids and potentially other lipid-derived compounds from sugars.

Towards an informative mutant phenotype for every bacterial gene.

Mutant phenotypes provide strong clues to the functions of the underlying genes and could allow annotation of the millions of sequenced yet uncharacterized bacterial genes. However, it is not known how many genes have a phenotype under laboratory conditions, how many phenotypes are biologically interpretable for predicting gene function, and what experimental conditions are optimal to maximize the number of genes with a phenotype. To address these issues, we measured the mutant fitness of 1,586 genes of the ethanol-producing bacterium Zymomonas mobilis ZM4 across 492 diverse experiments and found statistically significant phenotypes for 89% of all assayed genes. Thus, in Z. mobilis, most genes have a functional consequence under laboratory conditions. We demonstrate that 41% of Z. mobilis genes have both a strong phenotype and a similar fitness pattern (cofitness) to another gene, and are therefore good candidates for functional annotation using mutant fitness. Among 502 poorly characterized Z. mobilis genes, we identified a significant cofitness relationship for 174. For 57 of these genes without a specific functional annotation, we found additional evidence to support the biological significance of these gene-gene associations, and in 33 instances, we were able to predict specific physiological or biochemical roles for the poorly characterized genes. Last, we identified a set of 79 diverse mutant fitness experiments in Z. mobilis that are nearly as biologically informative as the entire set of 492 experiments. Therefore, our work provides a blueprint for the functional annotation of diverse bacteria using mutant fitness.

Indirect and suboptimal control of gene expression is widespread in bacteria.

Gene regulation in bacteria is usually described as an adaptive response to an environmental change so that genes are expressed when they are required. We instead propose that most genes are under indirect control: their expression responds to signal(s) that are not directly related to the genes' function. Indirect control should perform poorly in artificial conditions, and we show that gene regulation is often maladaptive in the laboratory. In Shewanella oneidensis MR-1, 24% of genes are detrimental to fitness in some conditions, and detrimental genes tend to be highly expressed instead of being repressed when not needed. In diverse bacteria, there is little correlation between when genes are important for optimal growth or fitness and when those genes are upregulated. Two common types of indirect control are constitutive expression and regulation by growth rate; these occur for genes with diverse functions and often seem to be suboptimal. Because genes that have closely related functions can have dissimilar expression patterns, regulation may be suboptimal in the wild as well as in the laboratory.

Dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates.

The efficient production of biofuels from cellulosic feedstocks will require the efficient fermentation of the sugars in hydrolyzed plant material. Unfortunately, plant hydrolysates also contain many compounds that inhibit microbial growth and fermentation. We used DNA-barcoded mutant libraries to identify genes that are important for hydrolysate tolerance in both Zymomonas mobilis (44 genes) and Saccharomyces cerevisiae (99 genes). Overexpression of a Z. mobilis tolerance gene of unknown function (ZMO1875) improved its specific ethanol productivity 2.4-fold in the presence of miscanthus hydrolysate. However, a mixture of 37 hydrolysate-derived inhibitors was not sufficient to explain the fitness profile of plant hydrolysate. To deconstruct the fitness profile of hydrolysate, we profiled the 37 inhibitors against a library of Z. mobilis mutants and we modeled fitness in hydrolysate as a mixture of fitness in its components. By examining outliers in this model, we identified methylglyoxal as a previously unknown component of hydrolysate. Our work provides a general strategy to dissect how microbes respond to a complex chemical stress and should enable further engineering of hydrolysate tolerance.

Spatial tethering of kinases to their substrates relaxes evolutionary constraints on specificity.

Signal transduction proteins are often multi-domain proteins that arose through the fusion of previously independent proteins. How such a change in the spatial arrangement of proteins impacts their evolution and the selective pressures acting on individual residues is largely unknown. We explored this problem in the context of bacterial two-component signalling pathways, which typically involve a sensor histidine kinase that specifically phosphorylates a single cognate response regulator. Although usually found as separate proteins, these proteins are sometimes fused into a so-called hybrid histidine kinase. Here, we demonstrate that the isolated kinase domains of hybrid kinases exhibit a dramatic reduction in phosphotransfer specificity in vitro relative to canonical histidine kinases. However, hybrid kinases phosphotransfer almost exclusively to their covalently attached response regulator domain, whose effective concentration exceeds that of all soluble response regulators. These findings indicate that the fused response regulator in a hybrid kinase normally prevents detrimental cross-talk between pathways. More generally, our results shed light on how the spatial properties of signalling pathways can significantly affect their evolution, with additional implications for the design of synthetic signalling systems.

Systematic dissection and trajectory-scanning mutagenesis of the molecular interface that ensures specificity of two-component signaling pathways.

Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk.

Draft genome sequence of Pelosinus fermentans JBW45, isolated during in situ stimulation for Cr(VI) reduction.

Pelosinus fermentans JBW45 is an anaerobic, lactate-fermenting bacterium isolated from Cr(VI)-contaminated groundwater at the Hanford Nuclear Reservation 100-H site (Washington) that was collected after stimulation with a polylactate compound. The genome sequence of this organism will provide insight into the metabolic potential of a predominant population during stimulation for metal-reducing conditions.

Regulation of the bacterial cell cycle by an integrated genetic circuit.

How bacteria regulate cell cycle progression at a molecular level is a fundamental but poorly understood problem. In Caulobacter crescentus, two-component signal transduction proteins are crucial for cell cycle regulation, but the connectivity of regulators involved has remained elusive and key factors are unidentified. Here we identify ChpT, an essential histidine phosphotransferase that controls the activity of CtrA, the master cell cycle regulator. We show that the essential histidine kinase CckA initiates two phosphorelays, each requiring ChpT, which lead to the phosphorylation and stabilization of CtrA. Downregulation of CckA activity therefore results in the dephosphorylation and degradation of CtrA, which in turn allow the initiation of DNA replication. Furthermore, we show that CtrA triggers its own destruction by promoting cell division and inducing synthesis of the essential regulator DivK, which feeds back to downregulate CckA immediately before S phase. Our results define a single integrated circuit whose components and connectivity can account for the cell cycle oscillations of CtrA in Caulobacter.

Barcoded reciprocal hemizygosity analysis via sequencing illuminates the complex genetic basis of yeast thermotolerance.

Decades of successes in statistical genetics have revealed the molecular underpinnings of traits as they vary across individuals of a given species. But standard methods in the field cannot be applied to divergences between reproductively isolated taxa. Genome-wide reciprocal hemizygosity mapping (RH-seq), a mutagenesis screen in an interspecies hybrid background, holds promise as a method to accelerate the progress of interspecies genetics research. Here, we describe an improvement to RH-seq in which mutants harbor barcodes for cheap and straightforward sequencing after selection in a condition of interest. As a proof of concept for the new tool, we carried out genetic dissection of the difference in thermotolerance between two reproductively isolated budding yeast species. Experimental screening identified dozens of candidate loci at which variation between the species contributed to the thermotolerance trait. Hits were enriched for mitosis genes and other housekeeping factors, and among them were multiple loci with robust sequence signatures of positive selection. Together, these results shed new light on the mechanisms by which evolution solved the problems of cell survival and division at high temperature in the yeast clade, and they illustrate the power of the barcoded RH-seq approach.