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

Recent advances in understanding transcription termination by RNA polymerase II [version 1; referees: 2 approved]

Transcription termination is a fundamental process in which RNA polymerase ceases RNA chain extension and dissociates from the chromatin template, thereby defining the end of the transcription unit. Our understanding of the biological role and functional importance of termination by RNA polymerase II and the range of processes in which it is involved has grown significantly in recent years. A large set of nucleic acid-binding proteins and enzymes have been identified as part of the termination machinery. A greater appreciation for the coupling of termination to RNA processing and metabolism has been recognized. In addition to serving as an essential step at the end of the transcription cycle, termination is involved in the regulation of a broad range of cellular processes. More recently, a role for termination in pervasive transcription, non-coding RNA regulation, genetic stability, chromatin remodeling, the immune response, and disease has come to the fore. Interesting mechanistic questions remain, but the last several years have resulted in significant insights into termination and an increasing recognition of its biological importance

The hnRNP-like Nab3 termination factor can employ heterologous prion-like domains in place of its own essential low complexity domain

<div><p>Many RNA-binding proteins possess domains with a biased amino acid content. A common property of these low complexity domains (LCDs) is that they assemble into an ordered amyloid form, juxtaposing RNA recognition motifs in a subcellular compartment in which RNA metabolism is focused. Yeast Nab3 is one such protein that contains RNA-binding domains and a low complexity, glutamine/proline-rich, prion-like domain that can self-assemble. Nab3 also contains a region of structural homology to human hnRNP-C that resembles a leucine zipper which can oligomerize. Here we show that the LCD and the human hnRNP-C homology domains of Nab3 were experimentally separable, as cells were viable with either segment, but not when both were missing. In exploiting the lethality of deleting these regions of Nab3, we were able to test if heterologous prion-like domains known to assemble into amyloid, could substitute for the native sequence. Those from the hnRNP-like protein Hrp1, the canonical prion Sup35, or the epsin-related protein Ent2, could rescue viability and enable the new Nab3 chimeric protein to support transcription termination. Other low complexity domains from RNA-binding, termination-related proteins or a yeast prion, could not. As well, an unbiased genetic selection revealed a new protein sequence that could rescue the loss of Nab3’s essential domain via multimerization. This new sequence and Sup35’s prion domain could also rescue the lethal loss of Hrp1’s prion-like domain when substituted for it. This suggests there are different cross-functional classes of amyloid-forming LCDs and that appending merely any assembly-competent LCD to Nab3 does not restore function or rescue viability. The analysis has revealed the functional complexity of LCDs and provides a means by which the differing classes of LCD can be dissected and understood.</p></div

Bipartite nature of the Nab3 C-terminal essential region.

<p>A) Yeast strains containing a plasmid bearing wildtype <i>NAB3</i> on a URA3-marked plasmid and either the <i>nab3</i>Δ<i>134</i>α allele (strain DY353) or <i>nab3</i>Δ<i>134</i>α<i>L800A</i> allele (strain DY377) on <i>LEU2</i>-marked plasmids, were grown on complete medium (SC) or medium with FOA to test for viability after the loss of the former plasmid. Cells were diluted, spotted, and grown on a single plate of each indicated solid media at 30°C, along with a positive control (DY351; with wildtype <i>NAB3</i> on a <i>LEU2</i>-marked plasmid) and negative control strain with a non-viable allele (<i>nab3</i>Δ<i>191</i>) of <i>NAB3</i> (DY3183). B) The resulting shuffled FOA-resistant strains (DY359 [gray line] and DY379 [black line]) were tested for their termination competence as described in Materials and Methods. In lieu of reproducing the control strain peaks seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186187#pone.0186187.g003" target="_blank">Fig 3</a>, only the position of the fluorescence maxima (arrows) for the termination-competent (+) (DY3217) and termination-defective (-) (DY3218) control strains are shown for reference.</p

Frequency in proteins studied here of the residues commonly over-represented in amyloid-forming LCDs.

<p>Frequency in proteins studied here of the residues commonly over-represented in amyloid-forming LCDs.</p

Viability of cells with Hrp1-chimeric proteins.

<p>A schematic depiction of the wildtype Hrp1 and the indicated LCD derivatives is shown at the top. Strains (DY388, DY387, DY389, DY1638, DY3242, and DY4500, from top to bottom) carrying the indicated plasmid-encoded proteins, as well as a <i>URA3</i>-marked plasmid with <i>HRP1</i>, were grown in SC leu^-, diluted to OD₆₀₀ of 0.05 and serially diluted ten-fold four times. Ten microliters of each dilution were spotted onto a single plate of the indicated solid media and cells were grown at 30°C.</p

GFP expression in strains with chimeric Nab3 proteins.

<p>(A) Flow cytometry profiles for control and experimental yeast strains. A positive control strain [DY3217 (+) in green] with strong termination due to the insertion of the <i>IMD2</i> intergenic terminator between the <i>GAL1</i> promoter and GFP is shown. A negative control strain [DY3218 (-) in red] with no terminator between the <i>GAL1</i> promoter and GFP is also shown. Results for experimental strains expressing Nab3Hrp1 (DY3197), Nab3Sup35 (DY3187), Nab3Ent2 (DY3246) or Nab3CT25 (DY4014) expressed from a plasmid as the sole Nab3 protein, are shown. (B) GFP fluorescence for DY3187 was monitored for colonies grown on SC leu^- solid medium supplemented with galactose and illuminated with the Dark Reader optical system (Clare Chemical Research, Inc.). (C) Flow cytometry of a Nab3-Sup35-expressing strain before (DY3187, blue) and after (DY3204, orange) conversion to the reporter-positive state (see text). A control strain was analyzed that lacks terminator function [DY3218 (-)]. (D) GFP fluorescence for DY3204 was monitored for colonies grown on SC leu^- containing galactose after illumination with the Dark Reader optical system (Clare Chemical Research, Inc.).</p

Growth and protein expression in yeast strains with Nab3-chimeras.

<p>Yeast cultures were diluted to 0.05 OD₆₀₀ and serially ten-fold therefrom. Ten μl were spotted onto a single plate of the indicated solid media, and incubated at 30°C. From the top down, the strains expressed wildtype Nab3 and a second Nab3 protein, either wildtype Nab3 (DY351), the Nab3Δ191 ‘stem’ (DY3183), the Nab3Hrp1 chimera (DY3193), the Nab3Sup35 chimera (DY4002), the Nab3Hrp1scrambled chimera (DY3213), the Nab3Ent2 chimera (DY3244), the Nab3CT25 chimera (DY4001), the Nab3Rnq1 chimera (DY3186), the Nab3Rat1 chimera (DY3184), or the Nab3Pcf11 chimera (DY3185). The right side of the figure is a western blot of whole cell lysates from logarithmically growing strains containing the indicated Nab3 protein (left to right: DY3033, DY3196, DY3182, DY4006, DY3245, DY4004, DY3183) or wildtype Nab3 protein with an additional Nab3 derivative that could not support growth on its own (DY3184, DY3185, DY3186) lysed in sample buffer and subjected to SDS-PAGE and western blotting with an antibody against Nab3, as described in Methods. Each of the two western blot panels were run on separate gels and processed separately.</p

Nab3's localization to a nuclear granule in response to nutrient deprivation is determined by its essential prion-like domain.

Ribonucleoprotein (RNP) granules are higher order assemblies of RNA, RNA-binding proteins, and other proteins, that regulate the transcriptome and protect RNAs from environmental challenge. There is a diverse range of RNP granules, many cytoplasmic, which provide various levels of regulation of RNA metabolism. Here we present evidence that the yeast transcription termination factor, Nab3, is targeted to intranuclear granules in response to glucose starvation by Nab3's proline/glutamine-rich, prion-like domain (PrLD) which can assemble into amyloid in vitro. Localization to the granule is reversible and sensitive to the chemical probe 1,6 hexanediol suggesting condensation is driven by phase separation. Nab3's RNA recognition motif is also required for localization as seen for other PrLD-containing RNA-binding proteins that phase separate. Although the PrLD is necessary, it is not sufficient to localize to the granule. A heterologous PrLD that functionally replaces Nab3's essential PrLD, directed localization to the nuclear granule, however a chimeric Nab3 molecule with a heterologous PrLD that cannot restore termination function or viability, does not form granules. The Nab3 nuclear granule shows properties similar to well characterized cytoplasmic compartments formed by phase separation, suggesting that, as seen for other elements of the transcription machinery, termination factor condensation is functionally important