Improvement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering.

In phenylketonuria (PKU) patients, a genetic defect in the enzyme phenylalanine hydroxylase (PAH) leads to elevated systemic phenylalanine (Phe), which can result in severe neurological impairment. As a treatment for PKU, Escherichia coli Nissle (EcN) strain SYNB1618 was developed under Synlogic's Synthetic Biotic™ platform to degrade Phe from within the gastrointestinal (GI) tract. This clinical-stage engineered strain expresses the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL), catalyzing the deamination of Phe to the non-toxic product trans-cinnamate (TCA). In the present work, we generate a more potent EcN-based PKU strain through optimization of whole cell PAL activity, using biosensor-based high-throughput screening of mutant PAL libraries. A lead enzyme candidate from this screen is used in the construction of SYNB1934, a chromosomally integrated strain containing the additional Phe-metabolizing and biosafety features found in SYNB1618. Head-to-head, SYNB1934 demonstrates an approximate two-fold increase in in vivo PAL activity compared to SYNB1618.

ArrayIDer: automated structural re-annotation pipeline for DNA microarrays.

BACKGROUND: Systems biology modeling from microarray data requires the most contemporary structural and functional array annotation. However, microarray annotations, especially for non-commercial, non-traditional biomedical model organisms, are often dated. In addition, most microarray analysis tools do not readily accept EST clone names, which are abundantly represented on arrays. Manual re-annotation of microarrays is impracticable and so we developed a computational re-annotation tool (ArrayIDer) to retrieve the most recent accession mapping files from public databases based on EST clone names or accessions and rapidly generate database accessions for entire microarrays. RESULTS: We utilized the Fred Hutchinson Cancer Research Centre 13K chicken cDNA array - a widely-used non-commercial chicken microarray - to demonstrate the principle that ArrayIDer could markedly improve annotation. We structurally re-annotated 55% of the entire array. Moreover, we decreased non-chicken functional annotations by 2 fold. One beneficial consequence of our re-annotation was to identify 290 pseudogenes, of which 66 were previously incorrectly annotated. CONCLUSION: ArrayIDer allows rapid automated structural re-annotation of entire arrays and provides multiple accession types for use in subsequent functional analysis. This information is especially valuable for systems biology modeling in the non-traditional biomedical model organisms.

BioNetBuilder2.0: bringing systems biology to chicken and other model organisms.

BACKGROUND: Systems Biology research tools, such as Cytoscape, have greatly extended the reach of genomic research. By providing platforms to integrate data with molecular interaction networks, researchers can more rapidly begin interpretation of large data sets collected for a system of interest. BioNetBuilder is an open-source client-server Cytoscape plugin that automatically integrates molecular interactions from all major public interaction databases and serves them directly to the user's Cytoscape environment. Until recently however, chicken and other eukaryotic model systems had little interaction data available. RESULTS: Version 2.0 of BioNetBuilder includes a redesigned synonyms resolution engine that enables transfer and integration of interactions across species; this engine translates between alternate gene names as well as between orthologs in multiple species. Additionally, BioNetBuilder is now implemented to be part of the Gaggle, thereby allowing seamless communication of interaction data to any software implementing the widely used Gaggle software. Using BioNetBuilder, we constructed a chicken interactome possessing 72,000 interactions among 8,140 genes directly in the Cytoscape environment. In this paper, we present a tutorial on how to do so and analysis of a specific use case. CONCLUSION: BioNetBuilder 2.0 provides numerous user-friendly systems biology tools that were otherwise inaccessible to researchers in chicken genomics, as well as other model systems. We provide a detailed tutorial spanning all required steps in the analysis. BioNetBuilder 2.0, the tools for maintaining its data bases, standard operating procedures for creating local copies of its back-end data bases, as well as all of the Gaggle and Cytoscape codes required, are open-source and freely available at http://err.bio.nyu.edu/cytoscape/bionetbuilder/.

Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites.

As a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computational and experimental strategies, based on the tyrosine recombinase Flp and its target site FRT, to overcome this impediment. We document the systematic evolution of Flp variants that can utilize, in a bacterial assay, two sites from the human interleukin 10 gene, IL10, as recombination substrates. Recombination competence on an end target site is acquired via chimeric sites containing mixed sequences from FRT and the genomic locus. This is the first time that a tyrosine site-specific recombinase has been coaxed successfully to perform DNA exchange within naturally occurring sequences derived from a foreign genomic context. We demonstrate the ability of an Flp variant to mediate integration of a reporter cassette in Escherichia coli via recombination at one of the IL10-derived sites.

Secondary structure switching in Cro protein evolution.

We report the solution structure of the Cro protein from bacteriophage P22. Comparisons of its sequence and structure to those of lambda Cro strongly suggest an alpha-to-beta secondary structure switching event during Cro evolution. The folds of P22 Cro and lambda Cro share a three alpha helix fragment comprising the N-terminal half of the domain. However, P22 Cro's C terminus folds as two helices, while lambda Cro's folds as a beta hairpin. The all-alpha fold found for P22 Cro appears to be ancestral, since it also occurs in cI proteins, which are anciently duplicated paralogues of Cro. PSI-BLAST and transitive homology analyses strongly suggest that the sequences of P22 Cro and lambda Cro are globally homologous despite encoding different folds. The alpha+beta fold of lambda Cro therefore likely evolved from its all-alpha ancestor by homologous secondary structure switching, rather than by nonhomologous replacement of both sequence and structure.

Stepwise manipulation of DNA specificity in Flp recombinase: progressively adapting Flp to individual and combinatorial mutations in its target site.

The Flp protein from Saccharomyces cerevisiae is one of the site-specific tyrosine family recombinases that are used widely in genomic engineering. As a first step towards mediating directed DNA rearrangements at non-native Flp recombination targets (mFRTs), we have evolved three separate groups of Flp variants that preferentially act on mFRTs containing substitutions at the first, seventh or both positions of the Flp-binding elements. The variants that recombine the double-mutant mFRT contain a subset of the mutations present in those that are active on the single-mutant mFRTs, plus additional mutations. Specificity for and discrimination between target sites, effected primarily by amino acid residues that contact DNA, can be modulated by those that do not interact with DNA or with a DNA-contacting residue. The degree of modulation can range from relaxed DNA specificity to almost completely altered specificity. Our results suggest that combined DNA shuffling and mutagenesis of libraries of Flp variants active on distinct mFRTs can yield variants that can recombine mFRTs containing combinations of the individual mutations.

Recombination of hybrid target sites by binary combinations of Flp variants: mutations that foster interprotomer collaboration and enlarge substrate tolerance.

Strategies of directed evolution and combinatorial mutagenesis applied to the Flp site-specific recombinase have yielded recombination systems that utilize bi-specific hybrid target sites. A hybrid site is assembled from two half-sites, each harboring a distinct binding specificity. Satisfying the two specificities by a binary combination of Flp variants, while necessary, may not be sufficient to elicit recombination. We have identified amino acid substitutions that foster interprotomer collaboration between partner Flp variants to potentiate strand exchange in hybrid sites. One such substitution, A35T, acts specifically in cis with one of the two partners of a variant pair, Flp(K82M) and Flp(A35T, R281V). The same A35T mutation is also present within a group of mutations that rescue a Flp variant, Flp(Y60S), that is defective in establishing monomer-monomer interactions on the native Flp target site. Strikingly, these mutations are localized to peptide regions involved in interdomain and interprotomer interactions within the recombination complex. The same group of mutations, when transferred to the context of wild-type Flp, can relax its specificity to include non-native target sites. The hybrid Flp systems described here mimic the naturally occurring XerC/XerD recombination system that utilizes two recombinases with distinct DNA binding specificities. The ability to overcome the constraints of binding site symmetry in Flp recombination has important implications in the targeted manipulations of genomes.

Evolutionary principles of modular gene regulation in yeasts.

Divergence in gene regulation can play a major role in evolution. Here, we used a phylogenetic framework to measure mRNA profiles in 15 yeast species from the phylum Ascomycota and reconstruct the evolution of their modular regulatory programs along a time course of growth on glucose over 300 million years [corrected]. We found that modules have diverged proportionally to phylogenetic distance, with prominent changes in gene regulation accompanying changes in lifestyle and ploidy, especially in carbon metabolism. Paralogs have significantly contributed to regulatory divergence, typically within a very short window from their duplication. Paralogs from a whole genome duplication (WGD) event have a uniquely substantial contribution that extends over a longer span. Similar patterns occur when considering the evolution of the heat shock regulatory program measured in eight of the species, suggesting that these are general evolutionary principles. DOI:http://dx.doi.org/10.7554/eLife.00603.001.

Correction: Evolutionary principles of modular gene regulation in yeasts.

[This corrects the article DOI: 10.7554/eLife.00603.].

Convergent Evolution of Calcineurin Pathway Roles in Thermotolerance and Virulence in Candida glabrata.

Candida glabrata is an emerging human fungal pathogen that is frequently drug tolerant, resulting in difficulties in treatment and a higher mortality in immunocompromised patients. The calcium-activated protein phosphatase calcineurin plays critical roles in controlling drug tolerance, hyphal growth, and virulence in diverse fungal pathogens via distinct mechanisms involving survival in serum or growth at host temperature (37° and higher). Here, we comprehensively studied the calcineurin signaling cascade in C. glabrata and found novel and uncharacterized functions of calcineurin and its downstream target Crz1 in governing thermotolerance, intracellular architecture, and pathogenesis in murine ocular, urinary tract, and systemic infections. This represents a second independent origin of a role for calcineurin in thermotolerant growth of a major human fungal pathogen, distinct from that which arose independently in Cryptococcus neoformans. Calcineurin also promotes survival of C. glabrata in serum via mechanisms distinct from C. albicans and thereby enables establishment of tissue colonization in a murine systemic infection model. To understand calcineurin signaling in detail, we performed global transcript profiling analysis and identified calcineurin- and Crz1-dependent genes in C. glabrata involved in cell wall biosynthesis, heat shock responses, and calcineurin function. Regulators of calcineurin (RCN) are a novel family of calcineurin modifiers, and two members of this family were identified in C. glabrata: Rcn1 and Rcn2. Our studies demonstrate that Rcn2 expression is controlled by calcineurin and Crz1 to function as a feedback inhibitor of calcineurin in a circuit required for calcium tolerance in C. glabrata. In contrast, the calcineurin regulator Rcn1 activates calcineurin signaling. Interestingly, neither Rcn1 nor Rcn2 is required for virulence in a murine systemic infection model. Taken together, our findings show that calcineurin signaling plays critical roles in thermotolerance and virulence, and that Rcn1 and Rcn2 have opposing functions in controlling calcineurin signaling in C. glabrata.

A genome-wide analysis of FRT-like sequences in the human genome.

Efficient and precise genome manipulations can be achieved by the Flp/FRT system of site-specific DNA recombination. Applications of this system are limited, however, to cases when target sites for Flp recombinase, FRT sites, are pre-introduced into a genome locale of interest. To expand use of the Flp/FRT system in genome engineering, variants of Flp recombinase can be evolved to recognize pre-existing genomic sequences that resemble FRT and thus can serve as recombination sites. To understand the distribution and sequence properties of genomic FRT-like sites, we performed a genome-wide analysis of FRT-like sites in the human genome using the experimentally-derived parameters. Out of 642,151 identified FRT-like sequences, 581,157 sequences were unique and 12,452 sequences had at least one exact duplicate. Duplicated FRT-like sequences are located mostly within LINE1, but also within LTRs of endogenous retroviruses, Alu repeats and other repetitive DNA sequences. The unique FRT-like sequences were classified based on the number of matches to FRT within the first four proximal bases pairs of the Flp binding elements of FRT and the nature of mismatched base pairs in the same region. The data obtained will be useful for the emerging field of genome engineering.

Arsenic exposure perturbs epithelial-mesenchymal cell transition and gene expression in a collagen gel assay.

Arsenic is a naturally occurring metalloid and environmental contaminant. Arsenic exposure in drinking water is reported to cause cancer of the liver, kidneys, lung, bladder, and skin as well as birth defects, including neural tube, facial, and vasculogenic defects. The early embryonic period most sensitive to arsenic includes a variety of cellular processes. One key cellular process is epithelial-mesenchymal transition (EMT) where epithelial sheets develop into three-dimensional structures. An embryonic prototype of EMT is found in the atrioventricular (AV) canal of the developing heart, where endothelia differentiate to form heart valves. Effects of arsenic on this cellular process were examined by collagen gel invasion assay (EMT assay) using explanted AV canals from chicken embryo hearts. AV canals treated with 12.5-500 ppb arsenic showed a loss of mesenchyme at 12.5 ppb, and mesenchyme formation was completely inhibited at 500 ppb. Altered gene expression in arsenic-treated explants was investigated by microarray analysis. Genes whose expression was altered consistently at exposure levels of 10, 25, and 100 ppb were identified, and results showed that 25 ppb in vitro was particularly effective. Three hundred and eighty two genes were significantly altered at this exposure level. Cytoscape analysis of the microarray data using the chicken interactome identified four clusters of altered genes based on published relationships and pathways. This analysis identified cytoskeleton and cell adhesion-related genes whose disruption is consistent with an altered ability to undergo EMT. These studies show that EMT is sensitive to arsenic and that an interactome-based approach can be useful in identifying targets.

MicroRNA expression during chick embryo development.

MicroRNAs (miRNAs) are small, abundant, noncoding RNAs that modulate protein abundance by interfering with target mRNA translation or stability. miRNAs are detected in organisms from all domains and may regulate 30% of transcripts in vertebrates. Understanding miRNA function requires a detailed determination of expression, yet this has not been reported in an amniote species. High-throughput whole mount in situ hybridization was performed on chicken embryos to map expression of 135 miRNA genes including five miRNAs that had not been previously reported in chicken. Eighty-four miRNAs were detected before day 5 of embryogenesis, and 75 miRNAs showed differential expression. Whereas few miRNAs were expressed during formation of the primary germ layers, the number of miRNAs detected increased rapidly during organogenesis. Patterns highlighted cell-type, organ or structure-specific expression, localization within germ layers and their derivatives, and expression in multiple cell and tissue types and within sub-regions of structures and tissues. A novel group of miRNAs was highly expressed in most tissues but much reduced in one or a few organs, including the heart. This study presents the first comprehensive overview of miRNA expression in an amniote organism and provides an important foundation for investigations of miRNA gene regulation and function.

Genomic resources for chicken.

The recent sequencing and draft assembly of a chicken genome has provided biologists with an invaluable research tool that complements a growing list of additional avian genomic resources. For many researchers, finding and using these resources is challenging, because information is presented through an increasing number of Web sites and browser navigation frequently requires specific knowledge and expertise. This primer provides an overview of online genomic resources for the chicken, including the Ensembl, UCSC, and NCBI annotated chicken genome browsers; expressed sequence tag and in situ hybridization databases; and sources for microarrays, cDNAs, and bacterial artificial chromosomes (BACs). Several short tutorials oriented toward the biologist with limited bioinformatics skills outline how to retrieve several types of commonly needed information and reagents.