20 research outputs found
Robust estimation of bacterial cell count from optical density
Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data
Isolation and Analysis of the Yeast TEA1 Gene, Which Encodes a Zinc Cluster Ty Enhancer-Binding Protein
A genetic screen for mutants that affect the activity of internal regulatory sequences of Ty retrotransposons led to the identification of a new gene encoding a DNA-binding protein that interacts with the downstream enhancer-like region of Ty1 elements. The TEA1 (Ty enhancer activator) gene sequence predicts a protein of 86.9 kDa whose N terminus contains a zinc cluster and dimerization motif typical of the Gal4-type family of DNA-binding proteins. The C terminus encodes an acidic domain with a net negative charge of �10 and the ability to mediate transcriptional activation. Like other zinc cluster proteins, purified Tea1 was found to bind to a partially palindromic CGGN xCCG repeat motif located in the Ty1 enhancer region. The Ty1 Tea1 binding site has a spacing of 10 and is located near binding sites for the DNA-binding proteins Rap1 and Mcm1. Analysis of the phenotype of tea1 deletion mutants confirmed that the TEA1 gene is required for activation from the internal Ty1 enhancer characterized in this study and makes a modest contribution to normal Ty1 levels in the cell. Hence, Tea1, like Rap1, is a member of a small family of downstream activators in Saccharomyces cerevisiae. Further analysis of the Tea1 protein and its interactions may provide insight into the mechanism of downstream activation in yeast cells. In contrast to the majority of yeast RNA polymerase IItranscribed genes whose activities depend on upstream activatio
Phylogenetic analysis confirming absence of <i>LYS4</i> and <i>LYS20</i> in animals.
<p>(<b>A</b>) Phylogenetic inference using Bayesian MCMC methods shows that the closest sequence matches in animal genome sequences to the <i>LYS4</i> genes are the <i>ACO1</i> genes, which encodes the aconitase enzyme of the tricarboxylic acid (TCA) cycle. Choanoflagellates (triangles) have <i>LYS4</i> and <i>ACO1</i> but animals (squares) have only <i>ACO1</i>. The tree is rooted between the two gene clades for Lys4 and Aco1. (<b>B</b>) A representative neighbor-joining (NJ) tree shows the presence of <i>LYS4</i> (blue) and <i>ACO2</i> (pink) in choanoflagellates (triangles), but not in animals (squares), for which sequences could not be found. One related gene <i>ACO1</i> (maroon), which encodes an enzymatic activity of the TCA cycle, is still present in animals, while <i>LEU1</i> (mustard yellow), a second related gene, involved in the leucine biosynthesis as part of the branched chain amino acid pathway, is lost in both choanoflagellates and animals. <i>ACO2</i> could not be found in the colonial choanoflagellate, <i>Salpingoeca rosetta</i>. Numbers indicate bootstrap support from 500 replicates. The tree is rooted between the Leu1 outgroup clade and theLys4/Aco1/Aco2 superclade. (<b>C</b>) A representative neighbor-joining tree shows the presence of <i>LYS20</i>/21 gene in choanoflagellates (triangles) and other organisms. <i>LYS20</i> and <i>LYS21</i> correspond to yeast specific duplications of this genetic function. No related homologs are found in animals (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117192#pone.0117192.g001" target="_blank">Fig. 1</a>). Distantly-related bacterial outgroups do however possess these genes. The tree is rooted between the eukaryotic Lys20/21 clade and the bacterial (Deinococcus-Thermus) gene clade.</p
Twenty-five genetic functions undetectable in animals.
<p>(<b>A</b>) Heat map of twenty-seven genes (first twenty-seven rows with gene names) from the budding yeast <i>S</i>. <i>cerevisiae</i> (<i>S</i>. <i>cer</i>.) and their orthologs in related organisms with whole genome sequence assemblies and their closest matches in animals. Six of the twenty-seven yeast genes are the result of lineage-specific duplications in yeast (indicated by dark gray brackets, left of table), leaving twenty-four genetic functions either lost or not measurably conserved in metazoans. The heat map is based on the BLASTP expect values of the best match to the indicated yeast gene (see key, lower right of table). Species abbreviations: <i>S</i>. <i>cer</i>., <i>Saccharomyces cerevisiae</i>; <i>S</i>. <i>pom</i>., <i>Schizosaccharomyces pombe</i>; <i>C</i>. <i>neo</i>., <i>Cryptococcus neoformans</i>; <i>P</i>. <i>gra</i>., <i>Puccinia graminis</i>; <i>B</i>. <i>den</i>., <i>Batrachochytrium dendrobatidis; C</i>. <i>owc</i>., <i>Capsaspora owczarzaki; M</i>. <i>bre</i>., <i>Monosiga brevicollis</i>; <i>S</i>. <i>ros</i>., <i>Salpingoeca rosetta; A</i>. <i>que</i>., <i>Amphimedon queenslandica</i>; <i>T</i>. <i>adh</i>., <i>Trichoplax adhaerens; M</i>. <i>lei</i>., <i>Mnemiopsis leidyi; N</i>. <i>vec</i>., <i>Nematostella vectensis; L</i>. <i>gig</i>., <i>Lottia gigantea; C</i>. <i>tel</i>., <i>Capitella teleta</i>; <i>H</i>. <i>rob</i>., <i>Helobdella robusta</i>; <i>D</i>. <i>mel</i>., <i>Drosophila melanogaster</i>; and <i>H</i>. <i>sap</i>., <i>Homo sapiens</i>. Other abbreviations: Chytridio., Chytridiomycota; Placo., Placozoa; Ctenoph., Ctenophora; Ecdyso., Ecdysozoa; and Chor., Chordata. The last row is from an alternative screen for lost animal genes (see text) in which Amoebozoa PFAM domains that are absent in animals were identified (five genes already identified plus one new gene, <i>PURB</i>). (<b>B</b>) Close-up of the lost animal gene columns from panel A. This table shows either the taxonomic origin of a weak animal match, or the name of the gene if it is not directly related (<i>i</i>.<i>e</i>., not orthologous) to the named yeast gene (first column) for all matches with BLASTP expectation values < 1e-20. Most of the animal matches to the candidate lost genes correspond to lineage-specific horizontal gene transfers and/or environmental contaminants from unrelated (non-opisthokont) clades. Some matches correspond to non-orthologous genes such as <i>ANKCLP</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117192#pone.0117192.s002" target="_blank">S2 Fig.</a>) in the case of the eukaryotic clpB genes <i>HSP78</i> and <i>HSP104</i>, or <i>ACO1</i> in the case of <i>LYS4</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117192#pone.0117192.g003" target="_blank">Fig. 3</a>). Only <i>HIS2</i> might have been lost soon after early animal radiation based on the presence of a weak match in <i>Trichoplax adhaerens</i>. This gene is most similar to the version from <i>Capsaspora</i> and not to either of the two choanoflagellates, so it is of uncertain origin.</p
Phylogenetic analyses confirming absence of intact <i>clpB</i> genes in animals.
<p>(<b>A</b>) Phylogenetic inference using Bayesian MCMC methods shows that the closest matching sequence in known animal genome sequences is a partial ClpB fragment in the sponge <i>Amphimedon queenslandica</i>. This fragment does not contain both nucleotide binding domains (NBDs) seen in bacterial or eukaryotic (Hsp78 and Hsp104) ClpB sequences, which are at least 800 amino acids long. The top three sequence hits in GenBank databases using the <i>Amphimedon</i> ClpB fragment as query are included. Two of these are intact ClpB protein sequences from bacterial (γ-proteobacteria) endosymbionts of marine tubeworms. Topological convergence was achieved after 300,000 generations with a relative burn-in phase of 25% (MrBayes) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117192#pone.0117192.ref080" target="_blank">80</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117192#pone.0117192.ref082" target="_blank">82</a>]. Mixed models were tested, but WAG [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117192#pone.0117192.ref083" target="_blank">83</a>] was the sole model favored upon completion. Average standard deviation of split frequencies was < 0.002. The tree was rooted between the bacterial clpB clade, including mitochondrial Hsp78, and the eukaryotic Hsp104 clade. (<b>B</b>) Maximum likelihood phylogenetic analysis of the ClpB family with choanoflagellates and chytridiomycetes (<i>Batrachochytrium dendrobatidis</i> and <i>Gonapodya prolifera</i>) representing fungal lineages shows that Hsp78 is the mitochondrially inherited <i>clpB</i> gene based on its sister grouping with α-proteobacteria. Dikarya sequences were omitted to simplify the tree. Numbers indicate bootstrap support from 500 replicates using MEGA5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117192#pone.0117192.ref085" target="_blank">85</a>]. (<b>C</b>) A simple, unrooted, neighbor-joining tree of the highly-conserved <i>clpB</i> genes across all of life shows that the ClpB functions are maintained in all major domains of life and in all endosymbiotic eukaryotic compartments. The ubiquity of the <i>clpB</i> genes makes the loss in animals more striking.</p
Yeast <i>HSP78</i> and <i>HSP104</i> share a unique regulatory architecture.
<p>(<b>A</b>) A dot plot between 320 bp sequence blocks located -10 bp and -65 bp upstream of the <i>S</i>. <i>cerevisiae HSP78</i> (vertical) and <i>HSP104</i> (horizontal) open reading frames, respectively. These coordinates were chosen because it anchors their HSE4 elements at the same position (starting 80 bp from the left) for ease of comparison. This comparison reveals a serial ordered alignment corresponding to an HSE4 element (blue box), a short alignment partially overlapping a triple array of STRE elements (pink boxes), and a TATA-containing core promoter element (green box). (<b>B</b>) Graph of the relevant details of the <i>HSP78</i> (top) and <i>HSP104</i> (bottom) upstream regulatory sequences. Each track (numbered lines 1–6) provides locations of the dot plot motifs (DP-I and DP-II) and known binding motifs (colored boxes in lines 1–5). Labels above certain binding sites indicate binding of a known transcription factor at this position under the specific conditions listed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117192#pone.0117192.ref039" target="_blank">39</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117192#pone.0117192.ref049" target="_blank">49</a>]. Line 6 shows conserved non-coding sequences (CNS) as determined from comparisons of corresponding orthologs from other species of <i>Saccharomyces</i>. Dots and lines in the ruler mark off 10 bp and 100 bp intervals, respectively. The transcriptional arrows represent only approximate positions of the transcriptional +1 based on distance from the TATA-containing promoter motif. (<b>C</b>) Motif key for panel B. (<b>D</b>) Results using the Serial Pattern of Expression Levels Locator (SPELL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117192#pone.0117192.ref031" target="_blank">31</a>]) algorithm/query engine to identify the closest regulated genes to <i>HSP78</i> (left graph), <i>HSP104</i> (center graph), or both genes combined (right graph). Plotted are the transcriptional correlation scores (SPELL, Adjusted Correlation Scores). The SPELL scores are based on pair-wise Pearson correlation co-efficients with Fisher z-transforms across 9,190 gene expression studies in yeast [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117192#pone.0117192.ref031" target="_blank">31</a>]. These graphs show that the two <i>clpB</i> genes are co-regulated with one another (red arrows) and to Hsp70/DnaK (K) and J-domain/DnaJ co-chaperones (J), which bind and recruit unfolded proteins to chaperone machinery in the cytoplasm (blue bars) and in the mitochondria (purple bars).</p
The <i>clpB</i> genes are co-regulated by HSE4s in Fungi and Holozoa.
<p>* Each locus matching the indicated motif in each genome is either listed as a named gene or enumerated. For example, “+2” means “plus two more loci” with matches in upstream regulatory region and “+2” in ORF” means “plus two more in ORFs”.</p><p>The <i>clpB</i> genes are co-regulated by HSE4s in Fungi and Holozoa.</p
Yeast interactome for the lost genes shows specific associations between lysine biosynthetic enzymes and the ClpB chaperones.
<p>(<b>A</b>) Yeast interactome graph showing high-throughput physical interactions (pink links), medium throughput TAP-tag physical interaction data for diverse chaperones (gray links), and genetic interactions (green links) amongst the yeast counterparts to the twenty-four lost genetic functions. Genetic interactions are based on 108 separate data sets, while physical interactions, not including the separate TAP-tag data set, are based on 103 data sets. The gene nodes are also colored by functional category. The largest statistically significant category of enrichment is for “biosynthetic process” with an expect value in random sampling of 2.3E-24 (17/27 lost genes). The two lost eukaryotic ClpB chaperones encoded by HSP104 and HSP78 interact physically with only two of the lost genes, LYS4 and LYS20, which are mitochondrial components of the lysine biosynthesis pathway. (<b>B</b>) Gene association analysis shows that Lys4 and Lys20 make contact with ribosome-associated chaperones (RAC) but no other Hsp70 and J-domain co-chaperones. Proteins localized to the mitochondria are indicated with purple labels. Genes with known roles in protein targeting and import to the mitochondria or refolding after import are indicated by asterisks (see key). Genes with HSE4 elements in their promoters (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117192#pone.0117192.t001" target="_blank">Table 1</a>) are indicated by yellow halos. The edge and node color combination is as in (A).</p
A perfect HSE4 element is specific to a <i>clpB</i> refolding regulon in <i>S</i>. <i>cerevisiae</i>.
<p>Abbreviations: DP-I, dot plot motif I; IUPAC, International Union of Pure and Applied Chemistry; PWM, Position-Weighted Matrix; Ψ, pseudo-count correction.</p><p>* Models 1–3 describe the genomic distributions of the <i>clpB</i>-specific HSE4 element using different descriptions. IUPAC DNA codes are used as needed except ‘N’ is represented by a dot (‘.’). Y = pYrimidine (C or T), and R = puRine (A or G).</p><p>** Percentages refer to the fraction of all genomic loci matching the model signature that are known to be involved in prion homeostasis. All such loci are listed in bold in the penultimate column. Hits in CDS regions are not listed but are counted for the precision metric.</p><p>A perfect HSE4 element is specific to a <i>clpB</i> refolding regulon in <i>S</i>. <i>cerevisiae</i>.</p
Hsp78 and Hsp104 are required for thermotolerance.
<p>Viability of <i>clpB</i> mutants cultured in rich media (YPD). Cultures were grown to log phase at 30°C, pre-treated at 37°C and diluted 1:1 into 50°C media (20 min) or diluted directly into cold water. Values are average colony forming unit (cfu) counts from YPD plates incubated at 30°C from three strains of each genotype. Error bars correspond to the standard deviation. Strains are: wild type: BY4730, BY4700, BY4738; <i>hsp78</i>: JF2478, JF2479, JF2480; <i>hsp104</i>: JF2473, JF2474, JF2498; <i>hsp78 hsp104</i>: JF2494, JF2495, JF2516. Double asterisks correspond to a p-value of < 0.001 in an unpaired Student’s t-test.</p