Fluorescent Protein Expression as a Proxy for Bacterial Fitness in a High-Throughput Assay

Abstract

Bacterial growth is classically assessed by measuring the increase in optical density of pure cultures in shaken liquid media. Measuring growth using optical density has severe limitations when studying multistrain interactions as it is not possible to measure the growth of individual strains within mixed cultures. Here we demonstrated that constitutively expressed fluorescent proteins can be used to track the growth of individual strains in different liquid media. Fluorescence measurements were highly correlated with optical density measurements and cell counts. This allowed us to assess bacterial growth not only in pure cultures, but also in mixed bacterial cultures and determine the impact of competitors on a focal strain, thereby assessing relative fitness. Furthermore, we were able to track the growth of two different strains simultaneously by using fluorescent proteins with differential excitation and emission wavelengths. Bacterial densities measured by fluorescence yielded more consistent data between technical replicates than optical density measurements. Our setup employs fluorescent microplate readers that allow for high throughput and replication. Importance We expand on an important limitation of the concept of measuring bacterial growth which is classically limited to one strain at a time. By adopting this approach, it is possible to measure growth of several bacterial strains simultaneously in high temporal resolution and in a high throughput manner. This is important to investigate bacterial interactions such as competition and facilitation.

Metabolic resource overlap impacts on the competition of phyllosphere bacteria

“…3a). If normalised by the fluorescence signal of a monoculture, constitutive fluorescence expression was shown to serve as a proxy for changes in bacterial biomass of individual strains in pairwise competitions [43].…”

“…Competition assays were performed in MM supplemented with mixed carbon sources (MM 5×C ), composed of a total 0.125% w/v of glucose, fructose, sorbitol, malate, and methanol (0.025% w/v glucose, 0.025% w/v fructose, 0.025% w/v malate, 0.025% w/v sorbitol, and 0.025% v/v methanol). The red fluorescent Pe299R::mSc strain was competed against individual non-fluorescent bacteria by mixing both strains in a 1:1 OD 600 ratio, as described previously [43]. Briefly, flat bottom 96-well microtiter plates (Costar ® , Corning ® , NY, USA) were seeded with 200 µL MM 5×C containing a defined mixed bacterial suspension (final OD 600 = 0.05, three technical replicates).…”

Chromatic Bacteria v.2-A Himar1 Transposon-Based Delivery Vector to Extend the Host Range of a Toolbox to Fluorescently Tag Bacteria

“…As described in Schlechter et al ( 2018), the here-constructed plasmids allow for convenient fluorescent tagging of environmental bacteria and cover a different host range compared to the previously described vectors. Fluorescent protein tags are the prerequisite for many experimental studies that follow different populations simultaneously, identify focal populations in complex environments, or to follow the behaviour of individual cells [4,[29][30][31][32]. Currently, many delivery systems, including the first versions of the Chromatic bacteria, function almost exclusively in Proteobacteria; the Himar transposons have been shown to have a wider range of activity.…”

Experimental heatwaves reduce the effectiveness of ejaculates at occupying female reproductive tracts in a model insect

“…This macro method has been published previously [ 34 , 49 ]. Although efficient, it is currently unable to produce precise sperm quantification, but using fluorescence as a proxy of cell abundance has been highlighted in research on human sperm [ 61 ] and bacterial culture [ 62 ]. Qualitatively, the method produced negligible fluorescence values for reproductive tracts from females which are virgin or have only mated non-GFP males.…”