algD promoter

The algD promoter is an inducible promoter that activates under the presence of a range of RDX. This sequence is derived from the genome of P. aeruginosa spp and was codon-optimized for E. coli DH5 alpha.

Usage and Biology

The RUM-UPRM iGEM 2022 team successfully characterized the algD promoter. This inducible promoter is activated by the presence of RDX from 0.2 to 0.5mM in Pseudomonas HK-6 (Lee et al., 2013). Pseudomonas species contain the algD operon which is in charge of alginate biosynthesis and is composed of a 12 gene cluster (Chitnis and Ohman, 1993). The regulation of this operon includes 15 genes in which 4 of them directly bond to the algD promoter being AlgR, AlgB, AmrZ and AlgU which is the recognition site (σ²²)shown in Figure 1.(Hay et al., 2013). For RDX detection one of the most important genes involved in the regulation is algA, it was shown that expression of this gene is directly related to stress induced by RDX (Lee et al., 2013). If a future iGEM team decides to use this part, they must use a Pseudomonas or Azotobacter as final chassis or integrate all the regulators needed to induce transcription in the desired chassis.

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Figure 1. Regulation of alginate biosynthesis

Team Aix-Marseille 2023 : Bibliographic research of the regulation of algD promoter in P. aeruginosa

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 AlgU [1][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 aeruginosa[4]

Figure 2. Simplified structure of PalgD (adapted from references [1] to [4])

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]

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