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. 2021 Apr;23(4):2070-2085.
doi: 10.1111/1462-2920.15296. Epub 2021 Jan 22.

From macro to micro: a combined bioluminescence-fluorescence approach to monitor bacterial localization

Riccardo Soldan  1 , Nattapong Sanguankiattichai  1 , Marcel Bach-Pages  1 , Indra Bervoets  2 , Wei E Huang  3 , Gail M Preston  1
Affiliations

From macro to micro: a combined bioluminescence-fluorescence approach to monitor bacterial localization

Riccardo Soldan et al. Environ Microbiol. 2021 Apr.

Abstract

Bacterial bioluminescence is widely used to study the spatiotemporal dynamics of bacterial populations and gene expression in vivo at a population level but cannot easily be used to study bacterial activity at the level of individual cells. In this study, we describe the development of a new library of mini-Tn7-lux and lux::eyfp reporter constructs that provide a wide range of lux expression levels, and which combine the advantages of both bacterial bioluminescence and fluorescent proteins to bridge the gap between macro- and micro-scale imaging techniques. We demonstrate that a dual bioluminescence-fluorescence approach using the lux operon and eYFP can be used to monitor bacterial movement in plants both macro- and microscopically and demonstrate that Pseudomonas syringae pv phaseolicola can colonize the leaf vascular system and systemically infect leaves of common bean (Phaseolus vulgaris). We also show that bacterial bioluminescence can be used to study the impact of plant immune responses on bacterial multiplication, viability and spread within plant tissues. The constructs and approach described in this study can be used to study the spatiotemporal dynamics of bacterial colonization and to link population dynamics and cellular interactions in a wide range of biological contexts.

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Figures

Fig. 1
Fig. 1
A library of mini‐Tn7 constructs with different promoters upstream of theluxoperon provides a wide dynamic range of luminescence when introduced intoPseudomonas syringaepv. phaseolicola1302A (Pph1302A). Luminescence values were detected at 4 h. Time series data are presented in the Supporting Information Fig. S6.OXB11:Pph1302A pOXB11::lux‐pA1/04/03::eYFP;OXB13:Pph1302A pOXB13::lux‐pA1/04/03::eYFP;OXB16:Pph1302A pOXB16::lux‐pA1/04/03::eYFP;OXB20:Pph1302A pOXB20::lux‐pA1/04/03::eYFP;nptII:Pph1302A pnptII::lux‐pA1/04/03:::eYFP. KB (King's B medium), M9 (M9 minimal medium). OD = optical density at 600 nm. Error bar ± SE.n = 3. [Color figure can be viewed at wileyonlinelibrary.com]
Fig. 2
Fig. 2
Pseudomonas syringae pv. phaseolicola RJ3 (Pph RJ3) colonizes the leaf vasculature of P. vulgaris cultivar TG. A. Pph RJ3 pnptII::lux‐pA1/04/03::eYFP was syringe‐infiltrated in localized areas on the abaxial surface of Phaseolus vulgaris cv. Tendergreen leaves. Images were taken after 0, 1, 3, 5 DPI with the nightOWL LB 983 at 10‐min exposure. The black line indicates cross sections used for confocal microscopy imaging (B). The white arrows indicate co‐localization of Pph RJ3 pnptII::lux‐pA1/04/03::eYFP with the leaf vasculature. The black arrows indicate regions of the leaves that have cts values higher than the maximum detection limit. B. Maximum projections of the leaf vasculature cross section. Pph RJ3 pnptII::lux‐pA1/04/03::eYFP colonies (yellow). Xylem autofluorescence (bright grey). Collenchyma autofluorescence (dark grey). co; collenchyma. xy; xylem. Red squares indicate areas shown at higher magnification in panels C–E. Scale bar: 40 μm. C. Pph RJ3 pnptII::lux‐pA1/04/03::eYFP colonies (yellow; white arrow). Xylem autofluorescence (grey). D. Pph RJ3 pnptII::lux‐pA1/04/03::eYFP colonies (yellow; white arrow). Collenchyma autofluorescence (dark grey). E. Zoom of panel B. Pph RJ3 pnptII::lux‐pA1/04/03::eYFP colonies (yellow; white arrow). Collenchyma autofluorescence (dark grey). Scale bar: 10 μm. [Color figure can be viewed at wileyonlinelibrary.com]
Fig. 3
Fig. 3
Bacterial bioluminescence can be used to study the effect of plant immune responses on bacterial growth and viability. Phaseolus vulgaris cultivar Tendergreen leaves were syringe infiltrated with 106 CFU ml−1 of Pseudomonas syringae pv. phaseolicola 1302A (Pph 1302A) ΔhrpA, ΔPphB and ΔxerC tagged with pOXB13::lux. A. After infiltration, leaves were kept in the dark for 10 min and then imaged with the nightOWL LB 983 at 2 min exposure. B. After 2 days post inoculation, leaves were detached from the plants, kept in the dark for 10 min and imaged with the nightOWL LB 983 at 5 min exposure. 1302A ΔPphB : Pph 1302A ΔPphB pOXB13::lux; 1302A ΔxerC : Pph1302A ΔxerC pOXB13::lux; 1302A ΔhrpA : Pph 1302A ΔhrpA pOXB13::lux; 1302A ΔxerC no lux: Pph 1302A ΔxerC. Luminescence and area values are reported for the eight biological replicates. Technical replicates (spots within the same leaf) were averaged. Error bar ± SE. n = 8. Significant differences (Student's T‐test, P < 0.05) are indicated by letters. [Color figure can be viewed at wileyonlinelibrary.com]
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