Visualizing Bdellovibrio bacteriovorus by Using the tdTomato Fluorescent Protein

Abstract

bBdellovibrio bacteriovorus is a Gram-negative bacterium that belongs to the delta subgroup of proteobacteria and is characterized by a predatory life cycle. In recent years, work has highlighted the potential use of this predator to control bacteria and biofilms. Traditionally, the reduction in prey cells was used to monitor predation dynamics. In this study, we introduced pMQ414, a plasmid that expresses the tdTomato fluorescent reporter protein, into a host-independent strain and a host-dependent strain of B. bacteriovorus 109J. The new construct was used to conveniently monitor predator proliferation in real time, in different growth conditions, in the presence of lytic enzymes, and on several prey bacteria, replicating previous studies that used plaque analysis to quantify B. bacteriovorus. The new fluorescent plasmid also enabled us to visualize the predator in liquid cultures, in the context of a biofilm, and in association with human epithelial cells.

Effect of Predatory Bacteria on Human Cell Lines

“…aeruginosa CFU numbers at time 24 hour might have been higher than that of the initial inoculation. A slight, but non-significant increase in cell viability after exposure to the predators seen in HaCaT, HepG2 and THP-1 cells, could also be as a result of individual predator cells remaining in the wells, as it was previously demonstrated that the predatory bacteria were able to attach to human corneal epithelial cells (HCLE) [35]. Our data supports earlier findings in which HCLE cells were exposed to high concentrations of B .…”

Biotechnological Potential of Bdellovibrio and Like Organisms and Their Secreted Enzymes

“…Heterologous promoters such as P nptII and P lac from E. coli, were used to express gfp (encoding green fluorescent protein) in B. bacteriovorus. Constitutive nptII promoter was proved to be functional in both, HD and HI growth phase (Mukherjee et al, 2015). Expression of gfp under inducible lac promoter was also observed in HD and HI strains (Flannagan et al, 2004;, but as noted by Flannagan et al (2004) it was independent of IPTG and could not be regulated.…”

“…Rather than its ability to form self-biofilm, it is the ability of BALOs to inhibit formation, as well as reduce preformed biofilms of other bacteria, that has raised general interest. Several methods have been developed to specifically study the role of predatory bacteria in biofilms; including fluorescently labeled B. bacteriovorus (Mukherjee et al, 2015), chip calorimetry assays measuring metabolic heat during biofilm removal (Buchholz et al, 2012), as well as atomic force microscopy for more mechanistic insight in biofilm formation and degradation (Núñez et al, 2005); the latter being limited to single-layered structures.…”

Generation of Xylose-inducible promoter tools forPseudomonasspecies and their use in implicating a role for the Type II secretion system protein XcpQ in inhibition of corneal epithelial wound closure

“…The resulting plasmid, pMQ578, and all plasmids made in this study are listed in Table 1. One variant of pMQ578 was made in which gfp was replaced with tdtomato from pMQ414 (15) using primers 4127 and 4218 and named pMQ643. For another, pMQ650, an artificial DNA sequence containing six restriction enzyme sites (EcoRI, SalI, BamH1, SpeI, SphI, and HindIII) and a sequence with stop codons in three frames was introduced using a 499 bp long double stranded DNA sequence listed as primer number 4068 (gBlock, IDT, Inc).…”