Difference between revisions of "Part:BBa K3447003"

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priR

The priR gene, also called ptr, is a 1506 bp gene encodes a 501 amino acids’ 50 kDa protein, which is resistant to pristinamycin I and II antibiotics and other antibiotics.

Introduction

Aminoglycoside antibiotics

The antibacterial mechanism of aminoglycoside antibiotics works by interfering with the accuracy of translation and the subsequent accumulation of error proteins. The misfolded protein that appears in the bacterial cell membrane increases the permeability of the drug, resulting in an increase in the concentration of intracellular aminoglycoside antibiotics, which plays a key role in sterilization and post-antibiotic effects. The ingestion of aminoglycoside antibiotics by bacteria is essential for their biological activity. The penetration of aminoglycoside antibiotics into bacterial cells requires three steps. The first is an energy-independent step, followed by two energy-dependent steps. Ribosomes and the respiratory chain that bind to the cell membrane are two essential substances for the accumulation of aminoglycoside antibiotic molecules in cells. That’s why anaerobic bacteria lacking an electron transport system are resistant to aminoglycoside antibiotics, such as enterococcus. Can resist low concentrations of amino sugar antibiotics. When aminoglycoside molecules come into contact with bacterial cells, polycationic antibiotic molecules bind to the anionic compounds of Gram-negative bacteria on the cell surface, such as lipopolysaccharides, phospholipids, and outer membrane proteins. The result of binding to the anionic site on the outer membrane is that the bridging divalent cations adjacent to the lipopolysaccharide molecule are displaced, resulting in increased permeability, forming "self-promoting absorption", and penetrating aminoglycoside molecules into the cytoplasmic space. The next process is called energy-dependent process I. In state I, only a small number of antibiotic molecules cross the cytoplasmic membrane, and a small number of molecules reaching the cytoplasm cause protein synthesis errors. The insertion of the incorrectly translated membrane protein destroys the integrity of the cytoplasmic membrane and initiates the next "energy-dependent process" Ⅱ". In this state, a large amount of amino sugars are transported across the injured cell plasma membrane. The higher the concentration of amino sugar, the faster the II state will be turned on. The abnormal protein in the injured lipid membrane is conducive to the transport of more antibiotic molecules, which enhances the interference with normal protein synthesis, causing greater damage to the cell membrane, forming an "autocatalytic type" to accelerate the absorption of antibiotics, and ultimately leading to cell death.

The molecular mechanism

The fidelity of the bacterial protein synthesis process is maintained by "monitoring" the base pairing between the mRNA codon and the tRNA anticodon at the ribosome coding site. The binding of ribosomes to the recognizable tRNA causes a conformational change in the coding site of ribosomal RNA, thus forming a signal to accelerate translation. The A 1492 and A 1493 adenylate residues contained in the inner loop of the 30S ribosomal subunit 16S rRNA constitute a major part of the bacterial coding site. The conformational flexible residues A 1492 and A1493 are in contact with the minor groove of the codon-inverse codon hybrid. It is speculated that this is a geometric structure that "senses" the base pairing between mRNA and tRNA. Aminoglycoside antibiotics specifically bind to bacteria The coding site RNA increases the probability of translation errors, and locks the residues A 1492 and A1493 to interact with the mRNA-tRNA hybrid in a misplaced conformation. This leads to a decrease in the discrimination of unrecognizable tRNAs, thereby reducing translation accuracy Therefore, amino sugars can interfere with the coding process and eventually cause cell death, and the clinical effect of aminoglycoside antibiotics is reduced due to the modification of aminoglycoside antibiotics by bacterial resistance enzymes.

Pristinamycin is a typical aminoglycoside antibiotic.

Usage and Biology

Pristinamycin, also known as protomycin, is a member of the Streptomyces antibiotic family and consists of two different ring structures. It has strong activity against methicillin, penicillin and vancomycin resistant bacteria. So far, there has been no report that gram-positive cocci are resistant to pristinamycin. Pristinamycin mainly acts on bacterial ribosomes, inhibits protein synthesis, and is produced by Streptomyces pristinaespiralis. During the fermentation process, Pristinamycin itself inhibits the growth of mycelium and the biosynthesis of pristinamycin, while the multi-drug resistance gene ptr is scattered in a single gene cluster, which gives it a variety of compounds. Resistance. It has been pointed out that the introduction of drug resistance gene overexpression can increase the resistance of bacteria to antibiotics and enhance the production of secondary metabolites in Streptomyces. At the same time, it shows that this actinomycete can be engineered.

The ptr gene is interesting not only because of its potential biochemical or genetic connection with the biosynthesis of protomycin, but also because of its abnormal multidrug resistance phenotype and regulatory mechanism. Putting ptr into a growing family of homologous integral membrane proteins has "easy" substrate specificity for a variety of toxic compounds with different structures.

Design

Design Notes

There is a repetitive sequence between cvi and cvaC gene, and other than that there is no need to pay attention.

Source

This gene is derived from Escherichia coli (strain: A2363), we get the genomic sequence by NCBI.

• https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP033091.2?report=genbank&from=43597&to=43908&strand=true
  
• https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP033091.2?report=genbank&from=43886&to=44122&strand=true
  

We ordered the part DNA from a synthesis company.

Sequence and Features

References

1. Coproduction of colicin V and lactic acid bacteria bacteriocins in lactococci and enterococci strains of biotechnological interest., Journal of Applied Microbiology, 122, 1159-1167.
2. Colicin V can be produced by lactic acid bacteria.(1999), Letters in Applied Microbiology, 29, 37-41.
3. Mutational Analysis of Cvab, an ABC Transporter Involved in the Secretion of Active Colicin V., Kai-Hui Wu., Ying-Hsin Hsieh., Phang C. Tai, Plos One, 7(4), e35382.
4. Purification and Characterization of Colicin V from Escherichia coli Culture Supernatants.(1994), Biochemistry, 33, 6911-6917.