114
et al. 2018), which is a fusion of a slower maturing red fluorescent protein (Tdimer2)
(Vrzheshch et al. 2000) and a faster maturing super folder green fluorescent protein
(sfGFP) (Pedelacq et al. 2006). This fluorescent timer is expressed using a constitutive promoter, which can mark the slow growing and growth arrested P. aeruginosa
cells at single cell level (Fig. 5.1c).
The systems discussed above are based on the constitutive expressed fluorescent
proteins. Next, we will focus on reporter systems that rely on the inducer and determine the replication dynamics based on fluorescence dilution. In this system, a fluorescent protein is expressed under the control of tightly regulated inducible promoter.
After induction, the division of individual cells in inducer free condition can be
monitored by measuring the concentration of GFP (Roostalu et al. 2008). In the
exponentially growing cultures, vast majority of E. coli cells were divided uniformly. However, dividing and non growing subpopulation were observed when
stationary phase culture was diluted into fresh medium (Roostalu et al. 2008).
Helaine and coworkers modified this fluorescence dilution system by putting an
inducible red fluorescent protein (DsRed or mCherry) and another constitutive
enhanced green fluorescent protein (EGFP) on one plasmid (Fig. 5.1e). Using this
approach, they demonstrated that many Salmonella cells do not replicate but appear
to enter a dormant like state and therefore survive in vivo antibiotic treatment in the
macrophage infection model (Figueira et al. 2013; Helaine et al. 2010, 2014; Saliba
et al. 2016; Stapels et al. 2018).
One common feature of persister cells is low intracellular ATP level (Conlon
et al. 2016; Dorr et al. 2010; Pu et al. 2019; Shan et al. 2017). Genetically encoded
fluorescent sensors for bacteria intracellular ATP measurement have been developed, including QUEEN (quantitative evaluator of cellular energy) (Yaginuma et al.
2014) and ATeam (Adenosine 5′-Triphosphate indicator based on Epsilon subunit
for Analytical Measurements) (Maglica et al. 2015). QUEEN is ratiometric ATP
indicator consisting of a single GFP and a bacterial ATP binding protein (Fig. 5.1f).
ATeam is a fluorescence resonance energy transfer (FRET) sensor, which is composed of a cyan fluorescent protein (CFP) and YFP that were connected by the ε
subunit of Bacillus subtilis FoF1-ATP synthase (Fig. 5.1g) (Imamura et al. 2009).
However, there is no study that applies ATP sensors to study persister cells at single
cell level.
Fluorescent reporters are very powerful tool to detect heterogeneity within bacterial populations. Combining these reporters with advanced microscope, flow cytometry and microfluidics platform can help to isolate the persister cells within the
whole population.
X. Duan et al.
et al. 2018), which is a fusion of a slower maturing red fluorescent protein (Tdimer2)
(Vrzheshch et al. 2000) and a faster maturing super folder green fluorescent protein
(sfGFP) (Pedelacq et al. 2006). This fluorescent timer is expressed using a constitutive promoter, which can mark the slow growing and growth arrested P. aeruginosa
cells at single cell level (Fig. 5.1c).
The systems discussed above are based on the constitutive expressed fluorescent
proteins. Next, we will focus on reporter systems that rely on the inducer and determine the replication dynamics based on fluorescence dilution. In this system, a fluorescent protein is expressed under the control of tightly regulated inducible promoter.
After induction, the division of individual cells in inducer free condition can be
monitored by measuring the concentration of GFP (Roostalu et al. 2008). In the
exponentially growing cultures, vast majority of E. coli cells were divided uniformly. However, dividing and non growing subpopulation were observed when
stationary phase culture was diluted into fresh medium (Roostalu et al. 2008).
Helaine and coworkers modified this fluorescence dilution system by putting an
inducible red fluorescent protein (DsRed or mCherry) and another constitutive
enhanced green fluorescent protein (EGFP) on one plasmid (Fig. 5.1e). Using this
approach, they demonstrated that many Salmonella cells do not replicate but appear
to enter a dormant like state and therefore survive in vivo antibiotic treatment in the
macrophage infection model (Figueira et al. 2013; Helaine et al. 2010, 2014; Saliba
et al. 2016; Stapels et al. 2018).
One common feature of persister cells is low intracellular ATP level (Conlon
et al. 2016; Dorr et al. 2010; Pu et al. 2019; Shan et al. 2017). Genetically encoded
fluorescent sensors for bacteria intracellular ATP measurement have been developed, including QUEEN (quantitative evaluator of cellular energy) (Yaginuma et al.
2014) and ATeam (Adenosine 5′-Triphosphate indicator based on Epsilon subunit
for Analytical Measurements) (Maglica et al. 2015). QUEEN is ratiometric ATP
indicator consisting of a single GFP and a bacterial ATP binding protein (Fig. 5.1f).
ATeam is a fluorescence resonance energy transfer (FRET) sensor, which is composed of a cyan fluorescent protein (CFP) and YFP that were connected by the ε
subunit of Bacillus subtilis FoF1-ATP synthase (Fig. 5.1g) (Imamura et al. 2009).
However, there is no study that applies ATP sensors to study persister cells at single
cell level.
Fluorescent reporters are very powerful tool to detect heterogeneity within bacterial populations. Combining these reporters with advanced microscope, flow cytometry and microfluidics platform can help to isolate the persister cells within the
whole population.
X. Duan et al.