In this project, we designed a predator, SRBioQuencher, for sulfate-reducing bacteria(SRB) in sewers to efficiently and safely solve the problem of high hydrogen sulfide concentration in sewers.
Our design component consists of two parts: a cocktail of sewer SRB elimination and SRBioQuencher chassis optimization.
Fig.1 Project Overview
In urban sewers, SRB produce biofilms to protect themselves, so we designed a cocktail therapy for removing SRB from sewers from three aspects: biofilm removal, biofilm inhibition, and removal of film-producing strains in the hope of establishing a new program for eliminating harmful bacteria by biofilm control.
SRB is a membrane-producing bacteria that produces biofilm that provide an anaerobic environment for its growth and helps it resist external environmental stresses. It has been shown that the structural framework of the SRB biofilm is a glycosidic bonds, so we would like to endow SRBioQuencher with the ability to express glycosidases that degrade the protective barrier of SRB.
Fig.2 Protective effect of SRB biofilm on SRB
It has been shown that disperin b(DspB) is a commonly used glycosidase for degrading biological periplasm, and dispersal hexosaminidase (DisH) is an SRB-derived glycosidase for degrading its own biological periplasm. Two different sources of glycosidases expressed in E. coli differed in their ability to degrade glycosidic bonds, so we wanted to compare the properties of the two glycosidases and screen for glycosidases more suitable for SRBioQuencher.
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Fig.3 Reaction of glycosidic bond degradation by DspB and DisH
The formation of the biofilm cannot be separated from the Quorum sensing (QS) of bacteria. When the number of bacteria multiplies to a certain threshold, it will stimulate QS, then promoting the formation of the biofilm. It has been shown that SRB produces N-acyl homoserine lactones (AHLs: C6-HSL, C8-HSL, C10-HSL, C12HSL) as the signaling molecules for QS, and quenching the quorum sensing molecules can disrupt the QS of SRB. Therefore, we expressed N-acyl homoserine lactonase, which is capable of degrading four AHLs at the same time, in our engineered bacteria to inhibit the formation of SRB biofilm.
Fig.4 The process of biofilm formation promoted by AHLs
Aiia is the most commonly used enzyme for degrading AHLs, but it is a zinc ion-dependent metalloenzyme, whose activity is susceptible to impairment in the sewer environment. We found two more efficient N-acyl homoserine lactonases: AidH enzymes in prokaryotic and Moml in eukaryotes. Besides, in order to obtain enzymes that bind better to AHLs and are more stable, we designed four mutants of Aiia and AidH by protein structure modeling. We verified the efficiency of these enzymes s and their mutants by experiements to screen for more suitable N-acyl homoserine lactonases for SRBioQuencher.
Fig.5 Reaction of AHL degrading enzymes
It is well known that free-bacterium exist in the biofilm, and them have the potential to regenerate the biofilm. Therefore, we linked signaling peptide (OmpA) to broad-spectrum antimicrobial peptide (AMP) to construct secreted-AMP to kill free SRB under the SRB biofilm and other bacterium in the sewer that contribute to SRB biofilm formation. Two AMPs from bovine, indolicidin(Ind) and bactenecin(Bac), have been demonstrated to have the ability to kill SRB. We tested the inhibitory capacity of the two AMPs by the inhibition zone method method and turbidimetric method to screen for antimicrobial peptides that are more suitable for the SRBioQuencher.
Fig.6 Mechanism of antimicrobial peptide inhibition
In many cases, direct expression of antimicrobial peptides (AMP) in host bacteria may be toxic to itself . In order to minimize cytotoxicity, we will tailor our antimicrobial peptide release strategy to the specific microenvironment of SRB. In prokaryotic expression systems, a common approach is to produce antimicrobial peptides as fusion proteins. We expressed DspB by fusion with antimicrobial peptides through disulfide bonds, designed TEV enzyme cleavage strategies in association with the fusion protein, and predicted the efficiency of this fusion protein expression by modeling.
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Fig.7 Mechanism of fusion protein targeting
Since E. coli itself produces small amounts of hydrogen sulfide, and SRBioQuencher's use of E. coli as a chassis organism needs to be optimized to reduce environmental hazards and provide a safer biocontrol solution.
It is well known that E. coli produces hydrogen sulfide gas, contrary to our modification of E. coli to remove hydrogen sulfide from sewers. Reviewing the literature, we found that high concentrations of hydrogen sulfide gas are converted by three pathways when it enters the organism. One of them is the thioredoxin reductase(SQR), a membrane protein that converts extramembranous hydrogen sulfide into polysulfides that can be utilized by bacteria, but E. coli itself cannot produce the SQR. we expressed SQR in SRBioQuencher, in order to rapidly convert hydrogen sulfide generated by E. coli and improve the efficiency of SRBioQuencher. In addition, the properties of SQR membrane proteins show that SQR can convert the excessive hydrogen sulfide in the sewers, which helps the SRBioQuencher treat sewer hydrogen sulfide gas faster.
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Fig.8 Reaction of Hydrogen sulfide oxidation
Biological control is a kind of green control focusing on safety and non-pollution. Our project proposes a strategy to improve the biosafety of our SRBioQuencher, i.e., designing a novel suicide system in the SRBioQuencher targeting the microenvironment of sewer SRB survival. We utilized SRB-produced quorum-sensing signaling molecules as inducers and coupled the luxR regulatory system with the LacI regulatory system to achieve a suicide system that turns off expression in the presence of AHLs and turns on expression in the absence of AHLs to kill the SRBioQuencher. This suicide system allowed the E. coli population to be regulated by the SRB population. The suicide system and the cocktail therapy resulted in a "predator-prey" model between E. coli and SRB, in which the numbers of both were affected by the dynamics of the AHLs in the sewers, preventing the SRBioQuencher from remaining in the sewers and causing secondary pollution.
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Fig.9 Mechanism of quorum-sensing induced suicide system(low AHLs)
In this section, we verified the preference and sensitivity of our system to AHLs, the impact of the expression of ccdB toxic proteins, and finally varified the logic circuit of our regulatory system.
Learn MoreStillger, L., Viau, L., Kamm, L., Holtmann, D. & Müller, D. Optimization of antimicrobial peptides for the application against biocorrosive bacteria. Appl Microbiol Biotechnol 107, 4041–4049 (2023).
Jayaraman, A., Mansfeld, F. B. & Wood, T. K. Inhibiting sulfate-reducing bacteria in biofilms by expressing the antimicrobial peptides indolicidin and bactenecin. Journal of Industrial Microbiology and Biotechnology 22, 167–175 (1999).
Pechsrichuang, P. et al. OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system. SpringerPlus 5, 1200 (2016).
Liu H, Fan K, Li H, Wang Q, Yang Y, Li K, Xia Y, Xun L. Synthetic Gene Circuits Enable Escherichia coli To Use Endogenous H2S as a Signaling Molecule for Quorum Sensing. ACS Synth Biol. 2019 Sep 20;8(9):2113-2120.
