An antibiotic is a substance or compound that that kills or inhibits the growth of bacteria. They are broadly classified based on lab behavior as bactericidal (kill bacteria directly) or bacteriostatic (prevent cell division).Also categorized by target-specificity: Narrow-spectrum antibiotics target specific bacteria such as Gram-positive or Gram-negative bacteria, while broad-spectrum antibiotics affect a wider range of bacteria.
This diagram shows the various targets of antibiotics on the structure and functions carried out by bacteria. The beta-lactams group of antibiotics, which I will talk more about shortly, has an effect on the construction of cell walls by many different types of bacteria.
Ampicillin is a type of beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961. It is considered part of the aminopenicillin family and belongs to the B-lactam group of antibiotics because of its characteristic B-lactam ring that consists of 3 carbon atoms and 1 nitrogren atom. (Show Slide 4)Ampicillin is a broad-range antibiotic so it can penetrate both Gram-positive and Gram-negative bacteria. The penicillin derivative acts as a competitive inhibitor of the enzyme transpeptidase, found on the inner surface of the cell surface membrane and which is essential for bacterial cell wall synthesis. It inhibits the third and final stage of cell wall synthesis, which ultimately leads to cell lysis.Ampicillin can also act as a bactericidal in the presence of E. Coli bacteria.However, as I mentioned before, beta-lactam drugs such as ampicillin have been popular for decades for the treatment of bacterial infection, hence certain bacteria have been able to develop counter-measures to nullify the antibiotics effectiveness. This evolutionary process is known as a buildup of drug resistance. There are several different underlying molecular mechanisms to antibiotic resistance, and I will explain the mechanism specific to ampicillin.
Ampicillin resistance is achieved by the cleavage of the beta-lactam ring by the beta-lactamase enzyme.
The enzyme is coded for by the plasmid-linked bla gene which hydrolyzes ampicillin by cleaving the beta-lactam ring. This can be witnessed when a plasmid that has been inserted into a bacterium is first coupled with the bla gene and then the bacterium is placed in a culture medium containing ampicillin – causing ampicillin levels to be continually depleted.
So now the most important question, is how the ampicillin-beta lactamase mechanism can be useful to us as a synthetic biology technique?It is often used as a selective agent to confirm the uptake of genes by bacteria (eg. plasmids). As we heard in a previous presentation, bacterial transformation results in the integration of the same of foreign DNA from the media surrounding the bacteria to produce chemically competent cells.If the exogenous DNA is tagged with an antibiotic resistance gene eg beta-lactamase and then grown in a medium containing ampicillin, only the bacteria that had successfully taken up the desired DNA become ampicillin resistant and do not become lysed by the ampicillin. This is then quite an accurate way to confirm whether or not successful bacterial transformation has occurred.
Kanamycin is a chemical compound which targets the 30s ribosomal subunit in prokaryotes, binding in such a way as to cause a frameshift in every translation. This has a "bacteriostatic" effect: the bacterium is unable to produce any proteins correctly, leading to a halt in growth and eventually cell death.
Over-use of kanamycin has led to many wild bacteria possessing resistance, which is encoded in plasmids. As a result of this (as well as a lot of side effects in humans), kanamycin is widely used for genetic purposes rather than medicinal purposes, especially in transgenic plants.Resistance is often to a family of related antibiotics, and comes in three variaties: antibiotic-degrading enzymes, reduced membrane permeability, or proteins protecting the 30s subunit.
Chloroamphenicol is also bacterio static; it halts bacterial growth by inhibiting the enzyme peptidyl transferase, a protein that assists in the binding of tRNA to the 50s ribosomal subunit in prokaryotes.Three methods of resistance exist: reduced membrane permeability, mutation of the 50s subunit, and an enzyme called chloramphenicol acetyltransferase, which inactivates chloramphenicol by covaltly linking groups.Indeed, there is much similarity in function, use and resistance between kanamycin and Chloroamphenicol.
APPLICATIONS OFENZYMES INANTIBIOTICS….
ANTIBIOTICS A chemical compound acting against life is known as antibiotics. It can obtain either from natural sources ,microbes or by synthetic methods.
THE BASICS• Used to kill or inhibit the growth of bacteria• Classified as bactericidal or bacteriostatic Kill bacteria directly Prevent cell division• Classified by target specificity:Narrow-spectrum vs Broad range• Most modified chemically from original compounds found in nature, some isolated and produced from living organisms
MOST COMMONLY USEDANTIBIOTICSPenicillinStreptomycinAmpicillinKanamycinchloramphenicol
PENICILLIN most widely used antibiotic. It is drug of choice when infection is caused by organisms susceptible to it . It is effective drug against gram + bacteriaand also against rickettsia . Mostly penicillin are produced by species of penicillium .
PENICILLIN BIOSYNTHESIS Overall, there are three main and important steps to the biosynthesis of penicillin G (benzylpenicillin).
FIRST STEP is the condensation of three amino acids — L-α- aminoadipic acid, L-cysteine, L-valine into a tripeptide. Before condensing into the tripeptide, the amino acid L-valine must undergo epimerization to become D-valine. The condensed tripeptide is named δ-(L-α-aminoadipyl)-L-cysteine-D-valine (ACV). The condensation reaction and epimerization are both catalyzed by the enzyme δ-(L-α-aminoadipyl)-L- cysteine-D-valine synthetase (ACVS), a nonribosomal peptide synthetase or NRPS.
SECOND STEP The second step in the biosynthesis of penicillin G is the oxidative conversion of linear ACV into the bicyclic intermediate isopenicillin N by isopenicillin N synthase (IPNS), which is encoded by the gene pcbC. Isopenicillin N is a very weak intermediate, because it does not show strong antibiotic activity.
FINAL STEP is an transamidation by isopenicillin N N-acyltransferase, in which the α- aminoadipyl side-chain of isopenicillin N is removed and exchanged for a phenylacetyl side-chain. This reaction is encoded by the gene penDE, which is unique in the process of obtaining penicillins
PRODUCTION Penicillin is a secondary metabolite of certain species of Penicillium and is produced when growth of the fungus is inhibited by stress. It is not produced during active growth. Production is also limited by feedback in the synthesis pathway of penicillin. α-ketoglutarate + AcCoA → homocitrate → L-α-aminoadipic acid → L-lysine + β- lactam The by-product, L-lysine, inhibits the production of homocitrate, so the presence of exogenous lysine should be avoided in penicillin production. The Penicillium cells are grown using a technique called fed-batch culture, in which the cells are constantly subject to stress, which is required for induction of penicillin production. The available carbon sources are also important: Glucose inhibits penicillin production, whereas lactose does not. The pH and the levels of nitrogen, lysine, phosphate, and oxygen of the batches must also be carefully controlled. The biotechnological method of directed evolution has been applied to produce by mutation a large number of Penicillium strains. These techniques includeerror-prone PCR, DNA shuffling, ITCHY, and strand-overlap PCR. Semisynthetic penicillins are prepared starting from the penicillin nucleus 6-APA.
STREPTOMYCIN It is produced using strains of streptomyces griseus . Basic medium for production of sterptomycin containssoybean meal as nitrogen source ,glucose as carbon source and NaCl .Proteolytic enzymatic activity of S.griseus releases ammonia to the medium from the soybean meal causing rise in pH .during this initial fermentation phase there is a littleproduction of streptomycin.
CLASSICFERMENTATIONPROCESS FOR THEPRODUCTION OFSTREPTOMYCIN
A little additional production of mycelia. The glucose added in the medium & the ammonia released from the soybean meal are consumed during this phase. The Ph remains fairly constant (7.6- 8). It is the final phase of the fermentation, after depletion of carbohydrates from the medium, Streptomycin production ceases & the bacterial cells began to lyse. There is a rapid increase in ph because of the release of ammonia from lysed cells. In the end of fermentation, the mycleium is separated from the broth by filtration & the streptomycin is recovered. The purification consists of adsorbing the streptomycin onto activated charcoal & eluting with acid alcohol.
AMPICILLIN Belongs to β-lactam group of antibiotics – contain β-lactam ring Broad-spectrum Penicillin derivative that inhibits bacterial cell wall synthesis (peptidoglycan cross-linking) Inactivates transpeptidases on the inner surface of the bacterial cell membrane Bactericidal only to growing E. Coli
AMPICILLIN RESISTANCE Cleavage of β-lactam ring by β-lactamase enzyme
β-lactamase is encoded by the plasmid-linked bla (TEM-1) geneHydrolyzes ampicillinAmpicillin levels in culture continually depleted
USE IN SYNTHETIC BIOLOGY• To confirm uptake of gene (eg. of plasmids) by bacteria• Bacterial Transformation: DNA integrates into bacteria’s chromosome and made chemically competent• Exogenous DNA tagged with an antibiotic resistance gene eg. β-lactamase• Grown in medium containing ampicillin• Ampicillin resistance indicates successful bacterial transformation
KANAMYCIN Targets 30s ribosomal subunit, causing a frameshift in every translation Bacteriostatic: bacterium is unable to produce any proteins correctly, leading to a halt in growth and eventually cell death
KANAMYCIN USE/RESISTANCE Over-use of kanamycin has led to many wild bacteria possessing resistance plasmids As a result of this (as well as a lot of side effects in humans), kanamycin is widely used for genetic purposes rather than medicinal purposes, especially in transgenic Plants Resistance is often to a family of related antibiotics, and can include antibiotic-degrading enzymes or proteins protecting the 30s subunit.
CHLORAMPHENICOL Bacteriostatic: functions by halting bacterial growth, which is done by inhibiting the enzyme peptidyl transferase, a protein that assists in the binding of tRNA to the 50s ribosomal subunit Three methods of resistance: reduced membrane permeability, mutation of the 50s subunit, and an enzyme called chloramphenicol acetyltransferase, which inactivates chloramphenicol by covaltly linking groups Easy/cheap to manufacture, but unused in western countries because of possible aplastic anemia as a side effect