Visualizing Bdellovibrio bacteriovorus by Using the tdTomato Fluorescent Protein

Mukherjee, Somdatta; Shanks, Robert M. Q.; Kadouri, Daniel E.

doi:10.1128/aem.03611-15

Cited by 42 publications

(32 citation statements)

References 41 publications

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“…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 .…”

Section: Discussionsupporting

confidence: 90%

Effect of Predatory Bacteria on Human Cell Lines

et al. 2016

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Predatory bacteria are Gram-negative bacteria that prey on other Gram-negative bacteria and have been considered as potential therapeutic agents against multi-drug resistant pathogens. In vivo animal models have demonstrated that predatory bacteria are non-toxic and non-immunogenic in rodents. In order to consider the use of predatory bacteria as live antibiotics, it is important to investigate their effect on human cells. The aim of this study was to determine the effect of Bdellovibrio bacteriovorus strains 109J and HD100, and Micavibrio aeruginosavorus strain ARL-13 on cell viability and inflammatory responses of five human cell lines, representative of clinically relevant tissues. We found that the predators were not cytotoxic to any of the human cell lines tested. Microscopic imaging showed no signs of cell detachment, as compared to predator-free cells. In comparison to an E. coli control, exposure to higher concentrations of the predators did not trigger a significant elevation of pro-inflammatory cytokines in four of the five human cell lines tested. Our work underlines the non-pathogenic attributes of predatory bacteria on human cells and highlights their potential use as live antibiotics against human pathogens.

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Section: Discussionsupporting

confidence: 90%

Effect of Predatory Bacteria on Human Cell Lines

et al. 2016

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“…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.…”

Section: Genetic Tools For Modification Of B Bacteriovorusmentioning

confidence: 88%

“…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.…”

Section: Balos Regulation Of Prey and Non-prey Biofilmsmentioning

confidence: 99%

Biotechnological Potential of Bdellovibrio and Like Organisms and Their Secreted Enzymes

et al. 2020

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Bdellovibrio and like organisms (BALOs) are obligate predatory bacteria that selectively prey on a broad range of Gram-negative bacteria, including multidrug-resistant human pathogens. Due to their unique lifestyle, they have been long recognized as a potential therapeutic and biocontrol agent. Research on BALOs has rapidly grown over the recent decade, resulting in many publications concerning molecular details of bacterial predation as well as applications thereof in medicine and biotechnology. This review summarizes the current knowledge on biotechnological potential of obligate predatory bacteria and their secreted enzymes.

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“…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).…”

Section: Methodsmentioning

confidence: 99%

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

Callaghan

Stella

Lehner

et al. 2020

Preprint

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ABSTRACTTunable control of gene expression is an invaluable tool for biological experiments. In this study, we describe a new xylose-inducible promoter system and evaluate it in both Pseudomonas aeruginosa and P. fluorescens. The Pxut promoter derived from the P. flurorescens xut operon was incorporated into a broad host-range pBBR1-based plasmid and compared to the Escherichia coli-derived PBAD promoter using gfp as a reporter. GFP-fluorescence from the Pxut promoter was inducible in both Pseudomonas species, but not in E. coli, which may facilitate cloning of toxic genes using E. coli to generate plasmids. The Pxut promoter was expressed at a lower inducer concentration than PBAD in P. fluorescens and higher gfp levels were achieved using Pxut. Flow cytometry analysis indicated that Pxut was more leaky than PBAD in the tested Pseudomonas species, but was expressed in a higher proportion of cells when induced. D-xylose did not support growth of P. aeruginosa or P. fluorescens as a sole carbon source and is less expensive than many other commonly used inducers which could facilitate large scale applications. The efficacy of this system aided in demonstrating a role for the P. aeruginosa type II secretion system gene from xcpQ in bacterial inhibition of corneal epithelial cell wound closure. This study introduces a new inducible promoter system for gene expression for use in Pseudomonas species.ImportancePseudomonas species are enormously important in human infections, biotechnology, and as a model system for interrogating basic science questions. In this study we have developed a xylose-inducible promoter system and evaluated it in P. aeruginosa and P. fluorescens and found it to be suitable for the strong induction of gene expression. Furthermore, we have demonstrated its efficacy in controlled gene expression to show that a type 2 secretion system protein from P. aeruginosa, XcpQ, is important for host-pathogen interactions in a corneal wound closure model.

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Visualizing Bdellovibrio bacteriovorus by Using the tdTomato Fluorescent Protein

Cited by 42 publications

References 41 publications

Effect of Predatory Bacteria on Human Cell Lines

Effect of Predatory Bacteria on Human Cell Lines

Biotechnological Potential of Bdellovibrio and Like Organisms and Their Secreted Enzymes

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

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