At a Glance
Worldwide, multidrug-resistant bacteria are on the rise and provoke the intensive search for novel effective compounds. To simplify the search for new antibiotics and to track the antibiotic pollution in water samples, whole-cell biosensors constitute a helpful investigative tool. In this part of EncaBcillus, we developed a functional and independent heterologous β-lactam biosensor in Bacillus subtilis. These specialised cells are capable of sensing a compound of the β-lactam family and will respond by the production of an easily measurable luminescence signal. We analysed the detection range and sensitivity of the biosensor in response to six different β-lactam antibiotics from various subclasses. The evaluated biosensor was then encapsulated into Peptidosomes to prove the concept of our project EncaBcillus. The encapsulation of engineered bacteria allows a simplified handling and increased biosafety, potentially raising the chances for their application e.g. sewage treatment plants.
To achieve our goal of encapsulating bacteria into Peptidosomes that can sense antibiotics of the β-lactam family, we first needed to develop a reliable biosensor strain. In Staphylococcus aureus the bla-operon encodes a one-component system, which is responsible for sensing and mediating resistance against β-lactam antibiotics. The idea was to transfer the regulatory elements of this operon to Bacillus subtilis and replace the native output – being the β-lactamase BlaZ – by an easy detectable signal. Thus, making Bacillus subtilis a β-lactam sensing biosensor. (see Figure 2).
For the creation of our biosensor in B. subtilis, the bla-operon from S. aureus was split into three genetic constructs: (A) The Receptor gene blaR1 under control of a strong constitutive promotor (Pveg), (B) the repressor gene blaI under control moderate strong constitutive promoter (PlepA) and (C) the target promoter region of the bla-operon (PblaZ and PblaR1I) in front of the lux-operon (luxABCDE) (see Figure 3). In addition, an inducible version of the blaR1 construct was made by inserting the PxylA promoter upstream of the blaR1 gene (A).
All genetic constructs and plasmids have been created using the RFC10 and/or RFC25 cloning standard. Enzymes used were obtained from New England BioLabs©. Cloning procedures were carried out according to the manufacturer`s protocols.
For submission of our parts to the registry, all Biobricks were cloned into the pSB1C3 backbone. The created genetic constructs were verified by sequencing (Eurofins or GATC sequencing services). All designed plasmids were stored in Escherichia coli DH10β (see Experiments and Protocols for details). In this project, we used integrative single-copy B. subtilis specific vectors that stably integrate into the genome at designated loci.
|Part in pSB1C3 backbone||BioBrick Number|
First, we investigated the detection range towards different β-lactam families as well as the sensitivity of the created biosensor. Therefore, we conducted plate reader experiments and disk diffusion assays to test our biosensor in liquid as well as on solid conditions. We recorded the luminescence signal and growth behavior (see Experiments and Protocols for details) of our biosensor strains in the presence of six different β-lactam antibiotics. We also included physiological controls that lack one or two of the genetic constructs of the complete biosensor machinery. Furthermore, we analyzed the impact of deleting the Bacillus subtilis gene penP - encoding a β-lactamase (which has not been studied intensively yet) - on the luminescence output. The strain W168 penP::kanR was created via Long-Flanking Homology PCR (see Experiments and Protocols for details). We also investigated, if the different β-lactam antibiotics induce the promoter driving penP.
Encapsulation into Peptidosomes
1. Determination of Inhibitory Antibiotic Concentrations
Before starting the actual tests regarding the functionality of the β-lactam biosensor, several pretests have been conducted to determine the optimal antibiotic concentration for the subsequent experiments. Therefore, we analyzed the concentration dependent effect of six different β-lactam antibiotics on the growth of Bacillus subtilis W168 and a strain lacking the B. subtilis native β-lactamase PenP (W168 penP::kanR). We decided to test the following β-lactams in our assays: ampicillin, carbenicillin, cefoperazone, cefoxitin, cefalexin and penicillin G. As controls we chose water (dH2O) and the peptide antibiotic bacitracin, which does not belong to the group of β-lactams.
We expected a higher growth inhibition with rising antibiotic concentrations. In the penP mutant we expected an increased sensitivity towards the β-lactam antibiotics. Addition of water to the culture should not show any effect on the growth and serves as a control. We included a non β-lactam (the peptide antibiotic bacitracin) in all our assays to demonstrate the specificity of the biosensor.
2. Analysis of Detection Range and Sensitivity
2.1 Assessing the Detection Range via Plate Reader Assays
During this project, we generated several strains to investigate the functionality of the heterologous constructs constituting the biosensor in Bacillus subtilis. This result section though will focus on the evaluation of the strains shown in Table 5 as these represent the most interesting ones.
In our first experiment, we performed plate reader assays in a 96 well plate format and measured growth (OD600) and luminescence output for 18 hours every 5 minutes. Induction with the β-lactam antibiotics occurred after 1 hour. All strains have been tested in triplicates under the same conditions. Strains with the genotype penP::kanR have been induced with lower concentrations compared to the wild type strain W168 (see Table 4 above).
After induction, we anticipate a strong increase in luminescence signal for strains containing the full set of constructs (PblaZ_lux or PblaR1I_lux, Pveg_blaR1 or Pxyl_blaR1, PlepA_blaI), thus representing functional biosensors. Besides the biosensor constructs, we also tested all physiological controls missing one essential composite of the biosensors' heterologous one-component system (data not shown). The control strain W168 (wild type) and control 1, will presumably not show any luminescence output, while the positive control 2 is expected to show a steady luminescence signal regardless of the presence of any antibiotic compound.
We could not observe a substantial activation of the PblaR1I promoter by the β-lactam compounds, which is why we are not taking it into account in the evaluation below. The bar charts in Figure 5 illustrate the best biosensor constructs identified in the plate reader experiments and compare the RLU/OD600 values of the strains 2 hours post induction with the antibiotics.
2.2 Analyzing the biosensors' behavior on solid medium conducting Disk Diffusion assays
|||C. Lee Ventola, MS (2015) The antibiotic resistance crisis: part 2: management strategies and new agents. Pharmacy and Therapeutics 40(5), 344–352|
|||www.aerzteblatt.de, visited 08/23/17 (5:34pm)|
|||www.who.int, visited 09/04/17 (3:21pm)|
|||https://en.wikipedia.org/wiki/Β-lactam_antibiotic, visited 10/27/17 (4:42pm)|
|||Leticia I. Llarrull, Mary Prorok, and Shahriar Mobashery (2010) Binding of the Gene Repressor BlaI to the bla Operon in Methicillin-Resistant Staphylococcus aureus. Biochemistry 49(37), 7975–7977|
|||Radeck, J., Kraft, K., Bartels, J., Cikovic, T., Dürr, F., Emenegger, J., Kelterborn, S., Sauer, C., Fritz, G., Gebhard, S., and Mascher, T. (2013) The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J Biol Eng 7(29),|
|||Toth, M., Antunes, N.T., Stewart, N.K., Frase, H., Bhattacharya, M., Smith, C. and Vakulenko, S. (2016) Class D β-lactamases do exist in Gram-positive bacteria. Nature Chemical Biology 12(1),9-14|