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

Enaminone formation drives the coupling of biosynthetic pathways to generate cyclic lipopeptides

A family of novel cyclic lipopeptides named tasikamides A H (Tsk A H) were discovered recently in Streptomyces tasikensis P46. Aside from the unique cyclic pentapeptide scaffold shared by the tasikamides, Tsk A C contain a hydrazone bridge that connects the cyclic pentapeptide to the lipophilic alkyl 5- hydroxylanthranilate (AHA) moiety. Here we report the production of tasikamides I K (Tsk I K) by a mutant strain of S. tasikensis P46 that overexpresses two pathway-specific transcription regulators. Unlike Tsk A C, Tsk I K feature a rare enaminone-bridge that links the cyclic peptide scaffold to the AHA moiety. Our experimental data suggest that Tsk I K are generated by the coupling of two biosynthetic pathways via a nonenzymatic condensation reaction between an arylamine and a β-keto aldehyde-containing precursor. The results underscore the nucleophilic and electrophilic reactivity of the β-keto aldehyde moiety and its ability toMinistry of Education (MOE)National Research Foundation (NRF)Submitted/Accepted versionWe acknowledge the generous funding support from the Ministry of Education of Singapore (ARC Tier 2 grant (T2EP30221-0029) awarded to Z.-X.L.) and National Research Foundation of Singapore (SBP01 grant awarded to Z.-X.L.)