Improving Bioreporter Strain Sensitivity with Lipopolysaccharide Mutants of E.coli

 

The presence of potentially harmful compounds within nature is linked with several human and animal health disorders. These include carcinogenic materials, such as asbestos, and those that lead to the formation of oxidative radicals, such as paraquat, a viologen herbicide that is currently forbidden within many European countries.

In an effort to make bacterial bioreporters more sensitive to their target chemicals, our group has studied the use of membrane permeabilizing conditions and chemicals. The basic premise for this is the selectively permeable nature of the bacterial membrane, which inhibits transport of large, polar molecules both into and out from the cell cytoplasm.2 Since the protein machinery needed to recognize and respond to these compounds is present within the cytoplasm, the membrane effectively inhibits the bacterium from responding fully or the compound from being completely active.3 This was demonstrated in two previous reports where the sensitivities of RecA- and SodA-based reporter strains were improved significantly with serum complement and polyethyleneimines (PEIs), respectively.4,5

In the first study, the bacterial membrane was perforated using human serum complement, a number of different proteins which bind to and create pores within the bacterial membrane.5 By doing so, we increased the sensitivity of the E. coli bioreporter to mitomycin C, a chemotherapeutic drug that covalently binds DNA, by almost 450-fold. Identical experiments with a SodA-based reporter strain, which responds to superoxide radicals, showed a 1.7-fold improved sensitivity. In a subsequent study, we found branched PEIs (BPEIs) increased the sensitivity of an E. coli reporter strain by 2- to 4-fold with several different viologens.4 PEIs, and specifically BPEIs, are thought to interact with and disrupt the bacterial membrane by interacting with the negatively charged moieties within the lipopolysaccharide (LPS),6 leading to a disordered state that increases permeability,7,8 but without loss or release of the LPS.8

Given the role of LPS in maintaining the outer membrane integrity, here we evaluated the effects different LPS mutants have both on the response time and the sensitivity of bacterial reporter strains. In total, 19 different E. coli strains were characterized, each without one of the genes involved in LPS biosynthesis. For several mutations, it is already known that they lead to an increased membrane permeability, as shown in antibiotic sensitivity tests.

However, to date, no study has reported the application of PEIs have on the responses of strain EBS, an E. coli str. BW25113 harbouring a sodA::luxCDABE transcriptional fusion, when exposed to viologens. For this, both linear and branched PEIs of various molecular weights were evaluated to determine which was best suited to improve the responses and sensitivity of this bacterial bioreporter with several different viologens. The results show that PELs increase the responses and hint that they may be used to improve the bioactivity of viologens, such as in microbial fuel cell applications.

Numerous bioreporters have been developed for various applications, including wastewater treatment and environmental toxicity assessments, and these respond to a wide range of chemical effectors, such as genotoxins, oxidative radicals and phenolic compounds. In an effort to  make bacterial bioreporters more sensitive to their target chemicals, our group has studied the use of membrane permeabilizing conditions and chemicals. The basic premise for this is the selectively permeable nature of the bacterial membrane, which inhibits transport of large, polar molecules both into and out from the cell cytoplasm. Since the protein machinery needed to recognize and respond to these compounds is present within the cytoplasm, the membrane effectively inhibits the bacterium from responding fully or the compound from being completely active. This was demonstrated in two previous reports where the sensitivities of Rec A- and SodA-based reporter strains were improved significantly with serum complement and polyethyleneimines (PELs), respectively.

In the serum paper, please look at Figure 3 and Table 1 (Below).

Note the increased sensitivity after treatment with human serum with the RecA and SodA strains, which recognize DNA-damage and superoxide radicals, respectively.

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In the BPEI paper, look at Table 1 (Below) and Figure 4.

Note the increased sensitivity after treatment for most of the chemicals were we trying to detect.

2

The Relative fold induction (RFI) is defined as the RBL from the BPEI-treated culture divided by that of the untreated control at the same time point.

This allows us to compare the responses from the different bacterial samples to the same chemical.

We will use a similar approach in our current study to evaluate the relative responses from the different E. coli mutants.

 

References

1             Revkin, A. C. Paraquat – a Potent Weed Killer Is Killing People. Sci Dig91, 36-& (1983).

2             Nikaido, H. & Vaara, M. Molecular-Basis of Bacterial Outer-Membrane Permeability. Microbiol Rev49, 1-32 (1985).

3             Nikaido, H. Outer-Membrane Barrier as a Mechanism of Antimicrobial Resistance. Antimicrob Agents Ch33, 1831-1836, doi:Doi 10.1128/Aac.33.11.1831 (1989).

4             Mabekou, S. S., Lee, S. C., Dinh, T. H., Won, K. & Mitchell, R. J. Enhanced sensitivity and responses to viologens from a whole-cell bacterial bioreporter treated with branched polyethyleneimine. J Appl Microbiol123, 1478-1487, doi:10.1111/jam.13592 (2017).

5             Lee, S., Amasia, M., Madou, M. & Mitchell, R. J. Serum complement enhances the responses of genotoxin- and oxidative stress-sensitive Escherichia coli bioreporters. Biosens Bioelectron46, 175-182, doi:10.1016/j.bios.2013.02.038 (2013).

6             Khalil, H., Chen, T., Riffon, R., Wang, R. & Wang, Z. Synergy between polyethylenimine and different families of antibiotics against a resistant clinical isolate of Pseudomonas aeruginosa. Antimicrob Agents Ch52, 1635-1641, doi:10.1128/Aac.01071-07 (2008).

7             Helander, I. M., Latva-Kala, K. & Lounatmaa, K. Permeabilizing action of polyethyleneimine on Salmonella typhimurium involves disruption of the outer membrane and interactions with lipopolysaccharide. Microbiology144 ( Pt 2), 385-390, doi:10.1099/00221287-144-2-385 (1998).

8             Helander, I. M., Alakomi, H. L., LatvaKala, K. & Koski, P. Polyethyleneimine is an effective permeabilizer of Gram-negative bacteria. Microbiol-Uk143, 3193-3199, doi:Doi 10.1099/00221287-143-10-3193 (1997).

9             Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol2, 2006 0008, doi:10.1038/msb4100050 (2006).

10           Mitchell, R. J. & Gu, M. B. An Escherichia coli biosensor capable of detecting both genotoxic and oxidative damage. Appl Microbiol Biotechnol64, 46-52, doi:10.1007/s00253-003-1418-0 (2004).

11           Lee, S., Amasia, M., Madou, M. & Mitchell, R. J. Serum complement enhances the responses of genotoxin- and oxidative stress-sensitive Escherichia coli bioreporters. Biosens Bioelectron46, 175-182, doi:10.1016/j.bios.2013.02.038 (2013).

 

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