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

Molecular characterization of chicken glutamate receptor, metabotropic1 (GRM 1)

Glutamate is the most abundant excitatory neurotransmitter in the mammalian nervous system. Ionotropic glutamate receptors used to be the only type of glutamate receptors, bringing about essential functions including synaptic transmissions. Since 1991, eight metabotropic glutamate receptors have been discovered. Belonging to the subfamily C of G protein-coupled receptor (GPCR) superfamily, these receptors have unique structural features. They couple to their own specific G proteins and transduce signals via pathways not recognized in other subfamilies. To date, little information on these receptors have been revealed in mammals, and even less is known about them in non-mammalian species including chicken. In the present study, various cDNAs of the chicken glutamate receptor, metabotropic 1 (GRM1) as well as its splice variants were cloned from adult brain tissue. At least 11 exons were identified in the chicken (c-) GRM1 gene, in which the alternative usage of exons and splice acceptor sites results in at least three variants, namely cGRM1a, cGRM1b and cGRM1f. The predicted coding regions of cGRM1a, cGRM1b and cGRM1f are 3459 base pairs (bp), 2736 bp and 2697 bp in length, which were deduced to encode receptor peptides of 1152 amino acids (aa), 911 aa and 898 aa, respectively. The predicted cGRM1a peptide shows high amino acid sequence identities (87.5% to 88%) to its counterparts in humans, rats, mice, chimpanzees and cattle. cGRM1b transcript differs from cGRM1a transcript by inclusion of two additional exons (7b and 7c), which contains a premature stop codon and results in its shorter C-terminal tail. cGRM1f is a novel splice variant that lacks exon 7b and is 13 aa shorter than cGRM1b. Reverse transcription-polymerase chain reaction (RT-PCR) assays showed that the transcripts of cGRM1a, cGRM1b and cGRM1f were preferentially expressed in adult chicken brains, in which cGRM1f mRNA was additionally identified in pituitary, lungs and gonads. Functional assay demonstrated that cGRM1a and cGRM1b receptors, expressed in Chinese hamster ovary cells, were induced by glutamate in dose-dependent manners via the Fura-2 dye calcium assays. In addition, dual luciferase reporter assays suggested that cGRM1a and cGRM1b receptors have no significant effects on the activation of cAMP/PKA and MAPK/ERK signaling pathways upon glutamate treatment. Taken together, the present study has provided the first step in understanding the possible roles of GRM1 in chickens.published_or_final_versionBiological SciencesMasterMaster of Philosoph

Genomic alignment of <i>Enterobacter cloacae</i>.

<p>MAUVE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074487#B35" target="_blank">35</a>] alignment of the genome sequences of <i>E. cloacae</i> subsp. <i>cloacae</i> ATCC13047, <i>E. cloacae</i> subsp. <i>dissolvens</i> SDM, <i>E. cloacae</i> EcWSU1 and <i>E. cloacae</i> subsp. <i>cloacae</i> ENHKU01. Same color boxes represent homologous regions of sequence, without rearrangement (locally collinear blocks or LCB), shared between <i>E. cloacae</i> genomes. Black arrows show the genomic position of the T4SS located in ATCC13047.</p

Antagonistic activity of <i>E. cloacae</i> subsp. <i>cloacae</i> ENHKU01 against fungi.

<p>(A) Visualization of fungal growth with and without ENHKU01: (from left to right, upper row) <i>Alternaria</i> sp., <i>Colletotrichum</i><i>capsici</i>, <i>Didymellabryoniae</i>, (from left to right, lower row) <i>Fusarium oxysporum</i> and <i>Sclerotinia sclerotiorum</i>. Photos were taken 7 days after incubation; (B) Growth of <i>Colletotrichum</i><i>capsici</i> (Col) and <i>Sclerotiniascleotiorum</i> (Scl) were closely monitored with and without ENHKU01. Challenging fungi were grown on PDA plates as described in Methods and Materials, the radius of growth of hyphae (in cm) was measured. Numbers show an average of 10 plates, and error bars represent the S.D. from the mean.</p

Phylogenetic analysis of ClpV.

<p>(A) The neighbor-joining tree of T6SSs using ClpV orthologs from 348 T6SS clusters in 231 species. The 231 species are grouped according to their class, which is indicated using color lines: α for the subdivision of Proteobacteria (Pink), β subdivision (Green), γ subdivision (Blue), δ/α subdivision (Black) and other bacteria unrelated to Proteobacteria (Gray). ClpV orthologs are distributed in five clades and named I-V. Naming of clades is according to Boyer et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074487#B86" target="_blank">86</a>]. Each clade is contributed by different bacterial families of Proteobacteria. Our result is consistent with a previous phylogenetic analysis of T6SS using 13 T6SS conserved component genes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074487#B86" target="_blank">86</a>]. ClpV of <i>E. cloacae</i> are distributed in clades II and III of the phylogenetic tree and are clustered together with other strains and species in the family of Enterobacteriaceae possessing T6SS, thus forming sub-trees in each clade (indicated by gray circles). A simplified and enlarged version of the neighbor-joining tree with bootstrap values showing (B) the sub-tree of clade III formed by ClpV orthologs associated with ECENHK_13140 and (C) the sub-tree of clade II associated with ECENHK_15865. Color squares indicate the habitats of the corresponding bacterial species: plant/ soil-associated (light green), insect-associated (green) and Human/ animal associated (orange).</p

Phylogenetic analysis of chitinase genes.

<p>Neighbor-joining tree with bootstrap values of chitinase genes associated with (A) ECENHK_07430 and (B) ECENHK_08915 constructed by twenty-five representing orthologs from each species using MEGA5. The blue box indicates the corresponding chitinase gene of ENHKU01. Chitinases that have been functionally characterized for their antifungal activities are highlighted in green boxes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074487#B87" target="_blank">87</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074487#B88" target="_blank">88</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074487#B89" target="_blank">89</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074487#B90" target="_blank">90</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074487#B91" target="_blank">91</a>].</p

Genetic organization of T6SS in <i>Enterobacter</i>.

<p>(A) and (B) show genetic organization of the two T6SS clusters commonly found in <i>Enterobacter</i>. ENHKU01 and ATCC13047 contain both T6SS, and EcWSU1, SDM and LF7a have one of the two, and other <i>Enterobacter</i> have none. The two clusters of T6SS in <i>Enterobacter</i> have different genetic organization but are aligned across the <i>Enterobacter</i> genomes at the corresponding loci. Each T6SS cluster is composed of conserved regions formed by conserved T6SS core component genes, which are indicated in solid blue/ green color boxes, and variable regions that are indicated by arrows. The variable regions contain a variable number of conserved genes (solid gray color) and unique genes (white color boxes). Most genes located in the variable regions are described as hypothetical proteins. Genes possibly involved in bacteriocin activity are shown in red. Details of the genetic organization of T6SSs in <i>Enterobacter</i> are listed in Data S4.</p

Antagonistic activity of <i>E. cloacae</i> subsp. <i>cloacae</i> ENHKU01 against <i>R. solanacearum</i>.

<p>Figures show results of ENHKU01 – <i>R. solanacearum</i> competition assays: (A) the planktonic culture with the quantities of <i>E. cloacae</i> subsp. <i>cloacae</i> ENHKU01 (ENHK) and <i>R. solanacearum</i> (RSHK) in log C.F.U. per ml recorded at 0, 2, 4, 6 and 24 hours after incubation. Numbers show an average of 4 replications, and error bars represent the S.D. from the mean; (B) Biofilm culture, relative percentage of <i>E. cloacae</i> subsp. <i>cloacae</i> ENHKU01 (ENHK): <i>R. solanacearum</i> (RSHK) at day 0 and day 1 of incubation.</p

Comparison of subsystem features between <i>E. cloacae</i>.

<p>Genome sequences of ATCC13047, SDM, EcWSU1 and ENHKU01 were uploaded to the SEED Viewer server (<a href="http://rast.nmpdr.org/seedviewer.cgi" target="_blank"><u>http://rast.nmpdr.org/seedviewer.cgi</u></a>) independently. Functional roles of RAST annotated genes were assigned and grouped in subsystem feature categories as shown in the figure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074487#B26" target="_blank">26</a>], and colored bars indicate the number of genes assigned to each category. Details for subsystem and functional role assignment for the genes of each strain are listed in Data S1.</p