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

Phosphorylation from Inorganic Phosphate and ATP Synthesis of Sarcoplasmic Membranes

The incorporation of inorganic phosphate in the fragmented sarcoplasmic membranes induced by the removal of calcium ions bound to high affinity binding sites at the cytoplasmic surface of the membranes gives rise to the formation of two species of phosphoenzyme. The properties of the phosphoproteins formed depend on the absence or the presence of a gradient of calcium ions across the membranes. The phosphoenzymes differ by the affinity of the protein for phosphate, the enthalpy of formation, the kinetics of phosphate incorporation, and by the sensitivity to ionophores and ADP. In the absence of a calcium gradient less than 0.5 nmol phosphoenzyme per mg protein are formed in media containing < 5 mM phosphate at pH 7 and 10°C. Under the same conditions ∼ 2 nmol of phosphoenzyme per mg protein are formed with an initial rate of 0.5 nmol mg−1· s−1 when a calcium gradient exists. When the gradient is abolished by the addition of the ionophore X537A, the level of phosphoprotein drops to the same value as observed in the absence of a gradient. On addition of ADP at concentrations increasing from 0.3 to 10 μM continuous ATP formation is activated to its maximum rate, and simultaneously, the level of phosphoprotein declines. These concentrations of ADP scarcely affect phosphoprotein formed in the absence of a gradient, the phosphoryl residue of which is displaced when the concentration of ADP exceeds 10 μM without the formation of an equivalent amount of ATP. Minimum mechanisms for the formation of gradient‐independent and gradient‐dependent phosphoprotein are discussed

Phosphorylation by inorganic phosphate of sarcoplasmic membranes

Phosphorylation, Dephosphorylation, Inorganic Phosphate, Sarcoplasmic Membranes The calcium transport protein of the sarcoplasmic reticulum accepts inorganic phosphate rapidly when phosphorylation is initiated either by the addition of phosphate or m agnesium ions to the calcium free protein. Phosphorylation proceeds much more slowly when it is initiated by the addition of the calcium chelator ethyleneglycol-bis (β-aminoethyl ether) -N ,N′-tetraacetic acid (EGTA) to the phosphate and magnesium containing assay. The time course of phosphorylation following immediately calcium removal is monophasic at all temperatures between 20° and 37 0|C. In contrast, phosphorylation of the calcium free enzyme becomes biphasic at temperatures above 25 °C. The biphasic time course does not only apply to net formation of phosphoprotein but also to its exchange with medium phosphate. On addition of calcium, the phosphoprotein decays in a biphasic process the time constants of which are much longer than those observed for phosphoprotein formation. The temperature dependence of the rate as well as of the extent of phosphoprotein formation indicate a discontinuity in the reactivity of the protein

Variable Ca2+ Transport: Phosphoprotein Ratios in the Early Part of the GTP‐Driven Calcium‐Transport Reaction of the Sarcoplasmic Reticulum

Initial Ca2+ transport and phosphoprotein formation of the sarcoplasmic reticulum membrane with GTP were investigated in a comparative study. While saturation of the high-affinity sites for Ca2+ binding and transporting as well as for GTP binding on the external surface of the membrane resulted in Ca2+ transport and phosphoprotein formation in a molar ratio of 2, the variation of the concentrations of the two reactants yielded ratios between 1.7 and 5.7. The ratios varied with a similar dependence on the concentrations of Ca2+ and GTP, except at 500 microM Ca2+, if the reaction was started by Ca2+ instead of GTP but the overall rates decreased. 1 mM DL-propranolol in the preincubation medium selectively inhibited Ca2+ transport but had no effect on initial phosphoprotein formation. These observations indicate that:L (a) phosphorylation of one enzyme molecule induces Ca2+ transport by a variable but limited number of neighbouring molecules, (b) not all Ca2+ bound is essential for phosphorylation but can be transported in parallel, (c) Ca2+ bound to low-affinity sites occupied at 500 microM Ca2+ in the reaction medium is also transported initially, (d) the accessibility of the high-affinity Ca2+ binding sites for DL-propranolol differs, (e) DL-propranolol interacts with Ca2+ binding and transporting sites only in that conformation of the enzyme that can be phosphorylated by the nucleotide

Competition between oxalate and phosphate during active calcium accumulation by sarcoplasmic vesicles

Sarcoplasmic Calcium Uptake, Oxalate and Phosphate Uptake, Oxalate-Phosphate Competition, Calcium Turnover 1. During ATP supported active calcium uptake oxalate as well as phosphate are accumulated with calcium. The uptake of calcium exceeds that of both anions by a small quantity - accounting for calcium binding to vesicular proteins and lipids. 2. From assay media containing phosphate and oxalate - nearly exclusively either oxalate or phosphate are taken up together with calcium by the sarcoplasmic reticulum vesicles. The mutual exclusion occurs in a very narrow concentration range of the anions. 3. In solutions containing phosphate and oxalate, calcium phosphate or calcium oxalate pre­ cipitates are formed according to their solubility properties. 4. When phosphate prevents oxalate from being taken up, calcium transport is inhibited. In­ hibition occurs, because the concentration of ionized calcium inside the vesicles rises approximately 100-fold when oxalate is replaced by phosphate. The activity of the calcium dependent ATPase parallels the calcium uptake activity. 5. It is excluded that the inhibition of calcium uptake produced by phosphate is caused by an enhanced permeability of the sarcoplasmic reticulum membranes for calcium in the presence of phosphate