Regulation of algD promoter in P. aeruginosa: documentation of the part BBa_K3857000
Since one of our objectives this year was to overproduce alginate produced by P. putida, we delved into the regulation of the algD operon, particularly in its cousin, P. aeruginosa. The algD operon exhibits complex regulation, but it is crucial to understand its elements. Thus, through this literature review, we aim to contribute to future teams interested in working on the algD operon in P. aeruginosa by summarizing the regulatory elements of the algD promoter. P. aeruginosa alginate operon is composed of twelve genes regulated by three promoters: the main PalgD promoter located upstream of the algD gene and two additional internal promoters upstream of algG and algI. In the PalgD promoter, the initiation codon (start) is 367 bp from the transcription start site (+1). The upstream non-coding region (around 500 base pairs long) contains binding sites for multiple positive regulators and the sigma factor AlgU1, 2.
A recent study revisited the role of the previously described regulators of the alg operon using a transcriptional reporter system 4. The promoter region of algD was transcriptionally fused to the gfp sequence and its activity was monitored in various mutant backgrounds compared to the WT strain. Among the nine tested regulators of the alg operon (AmrZ, AlgP, IHFα, IHFβ, CysB, Vfr, AlgR, AlgB, and AlgQ), the AmrZ, AlgR and AlgB proteins were found to be strictly required for transcription of the alginate operon, while deletion of vfr or cysB led to decreased transcription 4
Finally, the second messenger c-di-GMP was found to positively regulates transcription of the alginate operon in Pseudomonas aeruginosa4
AlgU / MucA
In P. aeruginosa, AlgU and the Muc proteins form a major regulatory switch that controls the synthesis of alginate and the conversion of the bacteria to a mucoid phenotype. AlgU (also referred to as σ22 or as AlgT) is an alternative sigma factor homologous to RpoE in E. coli. AlgU exhibits autoregulation of the PalgU promoter and activates other promoters essential for alginate biosynthesis, including genes encoding AlgR, AlgB, AmrZ and the algD operon 2. The membrane complex composed of MucA and MucB plays a pivotal role in the post-transcriptional control of AlgU. Specifically, MucA functions as an anti-sigma factor that sequestrates AlgU, thus controlling alginate production in P. aeruginosa by inhibiting the DNA polymerase activity and stopping the transcription of the algD operon. This regulatory mechanism maintains alginate production at a controlled level in the bacteria. However, when MucA is mutated, it causes increased expression of algD, leading to increased alginate production and to mucoid phenotype. Thus, regulation of MucA directly influences alginate synthesis.1, 2
AlgR / AlgB
AlgB is two-component response regulators of the NtrC family, a group of receptor-like regulatory proteins usually responding to environmental signals 1, 2. Experimental data from in vivo sandwich ELISA assays, corroborated by EMSA experiments, confirmed a direct interaction between the AlgB protein and the PalgD promoter in mucoid strains of P. aeruginosa. Enrichment methods like SELEX have identified a small 50 bp region responsible for AlgB binding on PalgD, between positions -274 and -224 2. However, its mechanism of action remains unclear, as it seems not to be dependent of its phosphorylation state. Indeed, phosphorylation of AlgB by its related kinase, KinB, is not necessary for the induction of the transcriptional activity of the algD operon.
AlgR is a member of the Lytr family. The AlgR protein functions by activating the expression of the algD operon by direct binding to three distinct segments of the PalgD promoter. It has been proposed that AlgB binding to PalgD induces a bending of the DNA resulting in bringing the regulator AlgR closer to the sigma factor AlgU (σ22).
AmrZ
AmrZ (also called AlgZ) is composed of a flexible N-terminal end (extending from residue 1 to 11), a C-terminal tetramerization domain (CTD) (spanning from residue 67 to residue 108), and a ribbon-helix-helix DNA-binding domain (from residue 12 to residue 66), which is found in proteins of the Arc superfamily. AmrZ binds to four Zinc Binding Sites (ZBS) in the PalgD region (ZBS1, ZBS2, ZBS3, ZBS4), each consisting of 18 base pairs. It is interesting to note that the ZBS4 binding site overlaps with a binding site of the host integration factor, IHF, which is not involved in alginate production 3. Based on the tetramerization of AmrZ observed in solution, it has been hypothesized that AmrZ oligomers interact with each other when binding to the four ZBS, facilitated by a bending of the DNA that brings the two ends of the DNA (ZBS1-ZBS2 and ZBS3-ZBS4) closer together. This hypothesis has been tested and confirmed through fluorescence resonance energy transfer (FRET) experiments.3
Sources
- Insights into the Assembly of the Alginate Biosynthesis Machinery In Pseudomonas aeruginosa, Zahid U.Rehman et al, Appl Environ Microbiol. (2013). doi: 10.1128/AEM.0460-13
- The NtrC Family Regulator AlgB, Which Controls Alginate Biosynthesis in Mucoid Pseudomonas aeruginosa, Binds Directly to the algD Promoter, Andrew J.Leech et al, J. bacteriol (2008). doi: 10.1128/JB.01307-07
- Pseudomonas aeruginosa AmrZ Binds to Four Sites in the algD Promoter, Inducing DNA-AmrZ Complex Formation and Transcriptional Activation, Binjie Xu et al, J. bacteriol (2016). doi: 10.1128/JB.00259-16
- Transcription of the Alginate Operon in Pseudomonas aeruginosa Is Regulated by c-di-GMP Ziwei Liang et al., Microbiol. Spectr. (2022). doi: 10.1128/spectrum.00675-22
Alginate beads standardization
Why ?
One of the major challenges we faced was finding a way to standardize the size of alginate beads.
On one hand, the size and weight of the beads affect water retention, and on the other hand, different tests to optimize the characteristics and use of our beads require standardized bead sizes for meaningful and interpretable results.
Importantly, we engaged in discussions with viticulturists and farmers who emphasized the need to mechanize the bead distribution process in the fields.
Together, we concluded that using a seeder to distribute the beads in the soil would be a good idea, and therefore, the bead size must be compatible and standardized to work seamlessly with the seeder. These discussions made us realize the importance of addressing how to obtain standardized bead sizes. Standardized bead design is a step toward industrial-scale development.
A first version
To address this challenge, we envisioned 3D parts forming a bead mold.
For the mold model design, we based it on several principles and constraints, with the most important being that the alginate solution and the CaCl
Additionally, the contractor, "L'atelier du Prado" by Julien Lucas, with whom we had discussions regarding the design, emphasized the importance of keeping the parts simple, as industrial molds are often rudimentary. Furthermore, practicality, such as the ease of assembling the parts, is a crucial factor to consider.
With all this information, we envisioned an initial version of the mold, consisting of two pieces that could be easily assembled and attached. The resulting mold includes a closed chamber for bead formation connected to two injection wells, allowing the injection of alginate and CaCl
A better version
Subsequently, we designed and printed a version 2 , which is an extension of the first version, enabling the production of multiple beads with a single mold.Mold testing
We had the opportunity to test our molds to optimize their use, and we designed a practical guide to simplify their use for everyone.
The next steps in characterizing the functionality of our molds will involve testing the size and weight of the obtained beads to confirm that the molds enable standardized alginate bead production. As alginate beads have numerous applications and have been used by other iGEM teams in the past, our molds offer a straightforward opportunity to create standardized alginate beads.
If you want to use it, think to read our usage guide .
Finally, you can see images of our molds here:
