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1. Goal

     Since complex biological pathways are used in an industrial way in order to produce molecules of interest, it has become crucial to understand and improve these pathways. However, biological systems are so complex that it is sometimes impossible to have a complete understanding of the reactions and mechanisms of different pathways.

     The goal of our project is to solve this problem using a natural tool: the integron. With this technique, it could be possible to alter the order of genes along their operon. So, an optimization of a pathway is thinkable if the natural order of genes is not the most productive. The project is based on an article of Bikard et al. (2010), in which the production of tryptophan is improved thanks to the integron. We are going to use the production of microcin as a proof of concept.

2. Introduction

2.1. Integron

     Integrons are natural cloning and expression systems composed of a tyrosine recombinase (the integrase, IntI), a primary recombination site (attI), a promoter and an array of gene cassettes. Those gene cassettes are small mobile elements that contain a promoterless ORF flanked by attC specific recombination site. Most gene cassettes currently known are antibiotic-resistance genes (Cambray et al. 1995).

     Usually, gene cassettes are found integrated in the integron. They can move from one site in the integron to another by site-specific recombination catalyzed by the integrase. The integrase gene is expressed upon stress and Int allows recombination at attC sites. This leads to the excision of circular cassettes that can be reintegrated at the attI site. In the end, the gene cassettes movements can lead to deletions, rearrangements and new genes captures by lateral gene transfer. A single promoter drives the expression of the gene cassettes (Cambray et al. 1995).

2.2. Microcins

     Microcins are antibacterial peptides produced by Enterobacteriaceae. They are synthesized by ribosomes and are active against closely related bacterial species. Some microcins must be heavily modified by maturation enzymes to be active while others are known as unmodified peptides (Severinov et al. 2007).

     In our project, we focused on 2 microcins: microcin B17 (MccB17) and microcins C7 (MccC7). Both of them are produced by Escherichia coli and share many characteristics (Severinov et al. 2007).

      Firstly, they have low molecular weights (respectively 3,093 Da and 1,178 Da) (Severinov et al. 2007).

     Secondly, both microcins are subjected to extensive post-translational modifications. The operons responsible for the production of microcins are carried on plasmids. Each operon contains a structural gene coding for a microcin precursor as well as genes whose products are essential enzymes for microcin modifications and maturation. To provide resistance to the producing bacteria, the operons usually contain a gene encoding a immunity protein that provide resistance to the producer cell. In addition, the operons contain gene(s) coding for a pump exporting microcins outside of the cell. Export is thought to contribute to resistance. This export protein is highly specific to the microcin that is produced which means that the producing cell remains sensitive to other microcins (Severinov et al. 2007).

     Thirdly, both McB17 and mcC7 target essential processes in the cytoplasm of sensitive cells (see below) (Severinov et al. 2007).

      Microcin synthesis is activated when cells reach stationary phase. However, it can also be activated by phosphate, carbon or nitrogen starvation. It has been proposed that the expression of microcin genes allows the producing cells to kill sensitive cells in order to make more resources available. (Severinov et al. 2007).

2.2.1. Microcin B17

     Seven genes are found in the mccB17 operon. mcBA is the first gene and encodes a 69 amino acid precursor. This precursor is then modified by the mcbBCD gene products (Severinov et al. 2007).

     The export of microcin B17 is carried out by the mcbEF gene products which are predicted to be an integral membrane protein and an ATP-binding protein respectively. Together with McbG, McbEF confer immunity to Microcin B17 producing cells (Destoumieux-Garzón et al. 2002).

     The target of microcin B17 is the DNA-gyrase, a type II DNA topoisomerase that regulates DNA supercoiling. The activity of Microcin B17 on DNA-gyrase rapidly causes DNA replication arrest and SOS response induction, and eventually cell death (Severinov et al. 2007).

2.2.2. Microcin C7

     The mccC7 operon consists of 6 genes: mccABCDEF. Microcin C7 consists of a heptapeptide encoded by the first gene of the MccC7 cluster, mccA, and modified with an adenosine monophosphate attached to its C terminal end (Severinov et al. 2007, Severinov & Nair 2012).

     mccBDE are genes required for microcin C7 maturation. The MccB enzyme begins the maturation process by attaching adenosine monophosphate to the precursor peptide. Then, MccD and MccE gene products further modify the heptapeptide and attach an aminopropyl moiety to the phosphate (Severinov et al. 2007, Severinov & Nair 2012).

It appears that MccCEF provide immunity to producing cells towards microcin C7. MccC gene encodes for an efflux pump from the major facilitator superfamily that exports Microcin C7. MccE modifies the peptide and renders it harmless. Finally, MccF cleaves the amide bond connecting the peptidyl and nucleotidyl part of Microcin C7 and inactivates it (Severinov et al. 2007, Severinov & Nair 2012).

     Mature microcin C7 enters sensitive cells via a membrane transporter. At that point, microcin C7 is subjected to further processing by the enzymes of the sensitive cell. The resulting product has a structure analogue to aspartyl-adenylate an intermediate of the reaction catalyzed by aspartyl-tRNA synthase. It prevents the loading of the aspartyll-tRNA with aspartate and the result is the inhibition of translation (Severinov et al. 2007, Severinov & Nair 2012).

References

Bikard D., Julié-Galau S., Cambray G., Mazel D., 2010, The synthetic integrin: an in vivo genetic shuffling device, Nucleic Acids Res., 38 (15), e153

Cambray G., Guerout A-M, Mazel D., Integrons, 2010, Annual Review of Genetics, Vol. 44: 141-166

Destoumieux-Garzón D., Peduzzi J., Rebuffat S., 2002, Focus on modified microcins: structural features and mechanisms of action, Biochimie 84, 511-519

Severinov K. & Nair S., 2012, Microcin C: Biosynthesis and mechanisms of bacterial resistance, Future Microbiology, 7 (2), 1-xxx

Severinov K., Semenova E., Kazakov A. & Gelfand M., 2007, Low-molecular-weight post-translationally modified microcins, Molecular Microbiology 65 (6), 1380-1394

Mazel D., 2006, Integrons: agents of bacterial evolution, Nature Reviews Microbiology, 610 (4): 608-620