Abstract
Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic in vivo and in vitro studies. During the last years, a few plasmid vectors have been developed aimed at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species Lactococcus lactis, we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter PX or PM from the pneumococcal mal gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying PM also harbor the malR gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (∼115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (∼8–45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.
Highlights
Since the cloning and expression of the gfp cDNA encoding the green fluorescent protein (GFP) from the Aequorea victoria jellyfish in Escherichia coli (Prasher et al, 1992; Chalfie et al, 1994), the number of different applications of a battery of cnidarian-derived GFP-like fluorescent proteins (FPs) and their variants, as well as the range of bacterial species in which they can be used, have greatly increased
We show the utility of the labeling vector carrying the maltose-inducible egfp expression system for direct and easy quantification of the plasmid loss
A similar increase of the specific fluorescence over time was observed irrespective of the presence or absence of the antibiotic, demonstrating that it was due to the maturation of pre-existing mCherry. These results suggested that, fluorescence emission of both EGFP and mCherry requires post-translational maturation of the protein chromophore (Zimmer, 2002; Miyawaki et al, 2012), that of EGFP would occur faster, so that it would have been virtually completed by the time the fluorescence was measured after collecting, washing and resuspension of the bacterial cells in PBS
Summary
Since the cloning and expression of the gfp cDNA encoding the green fluorescent protein (GFP) from the Aequorea victoria jellyfish in Escherichia coli (Prasher et al, 1992; Chalfie et al, 1994), the number of different applications of a battery of cnidarian-derived GFP-like fluorescent proteins (FPs) and their variants, as well as the range of bacterial species in which they can be used, have greatly increased. These FPs have been used in biosensors systems to monitor intracellular physiological parameters, like the pH, the oxygen level or the antioxidant activity (Potzkei et al, 2012; Crone et al, 2013; Germond et al, 2016) They are useful as reporters of gene expression and transcription regulation (Chalfie et al, 1994; Webb et al, 1995; Ruiz-Cruz et al, 2010; Uliczka et al, 2011; Mohedano et al, 2014), or as biomarkers for imaging the subcellular location of fusion proteins and their targets (Webb et al, 1995; Phillips, 2001; Belardinelli and Jackson, 2017; Kapanidis et al, 2018), for direct detection and quantification of plasmid conjugation and plasmid loss (Nancharaiah et al, 2003; Sørensen et al, 2003; Bahl et al, 2004; Karimi et al, 2016), and for tracking the bacterial cells in in vivo and in vitro competition and interactomic studies (Singer et al, 2010; Campbell-Valois and Sansonetti, 2014; Jarchum, 2015; Russo et al, 2015). Due to their spectral properties, these GFP variants can be properly combined with red FPs, like DsRed from Indo-Pacific reef coral Discosoma sp. and its improved monomeric variant mCherry (Shaner et al, 2004; Singer et al, 2010), for simultaneous dual fluorescent tagging of bacterial cells (Nancharaiah et al, 2003; Doherty et al, 2010)
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