FPCountR: Absolute quantification of fluorescent proteins for synthetic biology

This paper presents a generalisable method for the calibration of fluorescence readings on microplate readers, in order to convert arbitrary fluorescence units into absolute units. FPCountR relies on the generation of bespoke fluorescent protein (FP) calibrants, assays to determine protein concentration and activity, and a corresponding analytical workflow. We systematically characterise the assay protocols for accuracy, sensitivity and simplicity, and describe a novel ‘ECmax’ assay that outperforms the others and even enables accurate calibration without requiring the purification of FPs. To obtain cellular protein concentrations, we consider methods for the conversion of optical density to either cell counts or alternatively to cell volumes, as well as examining how cells can interfere with protein counting via fluorescence quenching, which we quantify and correct for the first time. Calibration across different instruments, disparate filter sets and mismatched gains is demonstrated to yield equivalent results. It can also reveal that mCherry absorption at 600nm does not confound cell density measurements unless expressed to over 100,000 proteins per cell. FPCountR is presented as pair of open access tools (protocol and R package) to enable the community to use this method, and ultimately to facilitate the quantitative characterisation of synthetic microbial circuits.

The role of mitochondrial energetics in the origin and diversification of eukaryotes

The origin of eukaryotic cell size and complexity is thought by some to have required an energy excess provided by mitochondria, whereas others claim that mitochondria provide no energetic boost to eukaryotes. Recent observations show that energy demand scales continuously and linearly with cell volume across both prokaryotes and eukaryotes, and thus suggest that eukaryotes do not have an increased energetic capacity over prokaryotes. However, amounts of respiratory membranes and ATP synthases scale super-linearly with cell surface area. Furthermore, the energetic consequences of the contrasting genomic designs between prokaryotes and eukaryotes have yet to be precisely quantified. Here, we investigated (1) potential factors that affect the cell volumes at which prokaryotes become surface area-constrained, and (2) the amount of energy that is divested to increasing amounts of DNA due to the contrasting genomic designs of prokaryotes and eukaryotes. Our analyses suggest that prokaryotes are not necessarily constrained by their cell surfaces at cell volumes of 100-103 µm3, and that the genomic design of eukaryotes is only slightly advantageous at genomes sizes of 106-107 bp. This suggests that eukaryotes may have first evolved without the need for mitochondria as these ranges hypothetically encompass the Last Eukaryote Common Ancestor and its proto-eukaryotic ancestors. However, our analyses also show that increasingly larger and fast-dividing prokaryotes would have a shortage of surface area devoted to respiration and would disproportionally divest more energy to DNA synthesis at larger genome sizes. We thus argue that, even though mitochondria may not have been required by the first eukaryotes, the successful diversification of eukaryotes into larger and more active cells was ultimately contingent upon the origin of mitochondria.

Virio- and bacterioplankton of the Barents Sea in the autumn

In the autumn period 2011, 2015 in the waters of the Barents Sea, the communities of viruses and bacteria were studied, their quantitative composition was determined, and the nature of their distribution was studied. It was shown that the distribution of both virio- and bacterioplankton had pronounced zoning presumably due to increased concentrations of organic matter in more productive coastal and Atlantic waters compared to the Arctic. In September 2011, the number of viruses varied from 0.6 to 46.7 million particles/ml, exceeding the abundance of bacteria by 5 times an average. The quantity of bacterioplankton varied within 0.3–2.9 million cells/ml, biomass – 4.1–35.1 mg C/m3, with a range of mean cell volumes of 0.030–0.115 μm3. In November–December 2015, the abundance of viruses was 0.3–6.4 million particles/ml and quantitatively exceeded their bacterial hosts by 18 times an average. The quantity and biomass of bacteria varied within 0.02–0.3 million cells/ml and 0.3–2.7 mg C/m3, with a range of mean cell volumes of 0.013–0.068 μm3. It was found that the level of development of virio- and bacterioplankton significantly decreased by the late autumn period.

Toward Minimalistic Model of Cellular Volume Dynamics in Neurovascular Unit

The neurovascular unit (NVU) concept denotes cells and their communication mechanisms that autoregulate blood supply in the brain parenchyma. Over the past two decades, it has become clear that besides its primary function, NVU is involved in many important processes associated with maintaining brain health and that altering the proportion of the extracellular space plays a vital role in this. While biologists have studied the process of cells swelling or shrinking, the consequences of the NVU’s operation are not well understood. In addition to direct quantitative modeling of cellular processes in the NVU, there is room for developing a minimalistic mathematical description, similar to how computational neuroscience operates with very simple models of neurons, which, however, capture the main features of dynamics. In this work, we have developed a minimalistic model of cell volumes regulation in the NVU. We based our model on the FitzHugh–Nagumo model with noise excitation and supplemented it with a variable extracellular space volume. We show that such a model acquires new dynamic properties in comparison with the traditional neuron model. To validate our approach, we adjusted the parameters of the minimalistic model so that its behavior fits the dynamics computed using the high-dimensional quantitative and biophysically relevant model. The results show that our model correctly describes the change in cell volume and intercellular space in the NVU.

A hydraulic instability drives the cell death decision in the nematode germline

Oocytes are large cells that develop into an embryo upon fertilization1. As interconnected germ cells mature into oocytes, some of them grow—typically at the expense of others that undergo cell death2–4. We present evidence that in the nematode Caenorhabditis elegans, this cell-fate decision is mechanical and related to tissue hydraulics. An analysis of germ cell volumes and material fluxes identifies a hydraulic instability that amplifies volume differences and causes some germ cells to grow and others to shrink, a phenomenon that is related to the two-balloon instability5. Shrinking germ cells are extruded and they die, as we demonstrate by artificially reducing germ cell volumes via thermoviscous pumping6. Our work reveals a hydraulic symmetry-breaking transition central to the decision between life and death in the nematode germline.

Taniajacoite and Strontioruizite, Two New Minerals Isostructural with Ruizite from the N'Chwaning III Mine, Kalahari Manganese Field, South Africa

ABSTRACT Two new mineral species, taniajacoite and strontioruizite, ideally SrCaMn3+2Si4O11(OH)4·2H2O and Sr2Mn3+2Si4O11(OH)4·2H2O, respectively, have been identified from the N'Chwaning III mine, Kalahari manganese field, South Africa. Both minerals occur as brown radiating groups or aggregates of acicular or prismatic crystals, with individual crystals up to 0.15 × 0.04 × 0.02 mm for taniajacoite and 1.3 × 0.2 × 0.2 mm for strontioruizite. Minerals associated with taniajacoite include sugilite, aegirine, pectolite, richterite, potassic-ferri-leakeite, and lipuite, whereas those associated with strontioruizite include sugilite, potassic-magnesio-arfvedsonite, and lipuite. Both taniajacoite and strontioruizite are brown in transmitted light, transparent with very light brown streak and vitreous luster. They are brittle and have a Mohs hardness of 5–5.5; cleavage is good on {010} and no parting or twinning is observed macroscopically. The measured and calculated densities are 3.05(2) and 3.09 g/cm3, respectively, for taniajacoite and 3.20(2) and 3.16 g/cm3 for strontioruizite. Optically, both taniajacoite and strontioruizite are biaxial (–), with α = 1.686(2), β = 1.729(2), γ = 1.746(2) (white light), 2V (meas.) = 63.7(5)°, 2V (calc.) = 62.5° for the former and α = 1.692(2), β = 1.734(2), γ = 1.747(2) (white light), 2V (meas.) = 59.1(5)°, 2V (calc.) = 56.6° for the latter. The calculated compatibility index based on the empirical formula is 0.008 for taniajacoite and 0.015 for strontioruizite. An electron microprobe analysis yielded an empirical formula (based on 17 O apfu) of Sr(Ca0.81Sr0.19)Σ1.00(Mn3+1.90Fe3+0.15Al0.01)Σ2.06Si3.96O11(OH)4·2H2O for taniajacoite and (Sr1.61Ca0.42)Σ2.03(Mn3+1.95Fe3+0.05)Σ2.00Si3.98O11(OH)4·2H2O for strontioruizite. Taniajacoite and strontioruizite are isostructural with ruizite. Strontioruizite, like ruizite, is monoclinic with space group C2 and unit-cell parameters a = 9.1575(4), b = 6.2857(4), c = 12.0431(6) Å, β = 91.744(4)°, and V = 692.90(6) Å3, whereas taniajacoite is triclinic, with space group C1 and a = 9.1386(5), b = 6.2566(3), c = 12.0043(6) Å, α = 90.019(4), β = 91.643(4), γ = 89.900(4)°, and V = 686.08(6) Å3. Their structures are characterized by chains of edge-sharing MnO6 octahedra extended along [010], which are linked together by corner-shared SiO4 tetrahedra in four-membered [Si4O11(OH)2] linear clusters, giving rise to a so-called “hetero-polyhedral framework”. The large cations Sr2+ and Ca2+ occupy the seven-coordinated interstices. Unlike monoclinic ruizite and strontioruizite, taniajacoite with Sr:Ca ≈ 1:1 is triclinic, owing to the ordering of Sr2+ and Ca2+ into two crystallographically distinct sites, indicating an incomplete solid solution between Ca and Sr endmembers. The unit-cell volumes for ruizite, taniajacoite, and strontioruizite appear to vary linearly with the Sr/(Ca + Sr) ratio.

Changes in Cell Size and Shape during 50,000 Generations of Experimental Evolution with Escherichia coli

ABSTRACT Bacteria adopt a wide variety of sizes and shapes, with many species exhibiting stereotypical morphologies. How morphology changes, and over what timescales, is less clear. Previous work examining cell morphology in an experiment with Escherichia coli showed that populations evolved larger cells and, in some cases, cells that were less rod-like. That experiment has now run for over two more decades. Meanwhile, genome sequence data are available for these populations, and new computational methods enable high-throughput microscopic analyses. In this study, we measured stationary-phase cell volumes for the ancestor and 12 populations at 2,000, 10,000, and 50,000 generations, including measurements during exponential growth at the last time point. We measured the distribution of cell volumes for each sample using a Coulter counter and microscopy, the latter of which also provided data on cell shape. Our data confirm the trend toward larger cells while also revealing substantial variation in size and shape across replicate populations. Most populations first evolved wider cells but later reverted to the ancestral length-to-width ratio. All but one population evolved mutations in rod shape maintenance genes. We also observed many ghost-like cells in the only population that evolved the novel ability to grow on citrate, supporting the hypothesis that this lineage struggles with maintaining balanced growth. Lastly, we show that cell size and fitness remain correlated across 50,000 generations. Our results suggest that larger cells are beneficial in the experimental environment, while the reversion toward ancestral length-to-width ratios suggests partial compensation for the less favorable surface area-to-volume ratios of the evolved cells. IMPORTANCE Bacteria exhibit great morphological diversity, yet we have only a limited understanding of how their cell sizes and shapes evolve and of how these features affect organismal fitness. This knowledge gap reflects, in part, the paucity of the fossil record for bacteria. In this study, we revived and analyzed samples extending over 50,000 generations from 12 populations of experimentally evolving Escherichia coli to investigate the relation between cell size, shape, and fitness. Using this “frozen fossil record,” we show that all 12 populations evolved larger cells concomitant with increased fitness, with substantial heterogeneity in cell size and shape across the replicate lines. Our work demonstrates that cell morphology can readily evolve and diversify, even among populations living in identical environments.

Antibiotics modulate attractive interactions in bacterial colonies affecting survivability under combined treatment

Biofilm formation protects bacteria from antibiotics. Very little is known about the response of biofilm-dwelling bacteria to antibiotics at the single cell level. Here, we developed a cell-tracking approach to investigate how antibiotics affect structure and dynamics of colonies formed by the human pathogen Neisseria gonorrhoeae. Antibiotics targeting different cellular functions enlarge the cell volumes and modulate within-colony motility. Focusing on azithromycin and ceftriaxone, we identify changes in type 4 pilus (T4P) mediated cell-to-cell attraction as the molecular mechanism for different effects on motility. By using strongly attractive mutant strains, we reveal that the survivability under ceftriaxone treatment depends on motility. Combining our results, we find that sequential treatment with azithromycin and ceftriaxone is synergistic. Taken together, we demonstrate that antibiotics modulate T4P-mediated attractions and hence cell motility and colony fluidity.