ResearchBlogging.orgAs a community here @sciamblogs we decided to each cover something chemistry related on each of our individual blogs to coincide with the World Chemistry Congress taking place in Puerto Rico. This scared the bejeezus out of me as I’m a biologist, not a chemist, and I’ve never been brilliant at the textbook chemistry stuff from my undergraduate classes. Also, a wise biology teacher once told me that all chemistry is boring until it starts moving, then its biology.

Working in the molecular side of biology however, requires some interest and ability in chemistry which is fine, as long as I don’t make anything accidentally explode.

So in fitting with this chemistry theme I went looking for a more bio-chemistry type topic to write about and found a cool paper entitled “Antibiotics as probes of biological complexity” in the appropriately named Nature Chemical Biology. Note the 'Chemical' in Nature Chemical Biology, it still counts :)

The observation that penicillin killed bacterial cultures was once of the most profoundly important advances in biomedical science, and science generally, as it opened up an avenue to combat bacterial infection. But at the same time it provided researchers with a new tool to investigate bacterial physiology. In fact penicillin and the family of antibiotics it comes from, the β-lactams, have provided huge amounts of information about what makes up bacteria and how all those bits do, well, stuff.

Following the observation of the penicillin's activity the first question that had to be answered by the biochemists was 'how’s it do that?'. The initial experiments showed that following the addition of penicillin the quantity of compounds referred to as uridine diphosphates (UDP) increased.

Peptidoglycan synthesis

I knew memorising peptidoglycan synthesis would come in handy one day... (1) The pentapeptide side group is assembled on the cytoplasmic side of the bacterial membrane while attached to a UDP intermediate. (2) The pentapeptide group, attached to the sugar NAM is joined the the bactoprenol carrier. (3) The UDP carrier is removed from the sugar NAG, allowing NAG to be attached to the NAM-Pentapeptide. (4) The bactoprenol-sugar-pentapeptide compound was flipped to the exterior surface of the bacterial membrane. (5) The bactoprenol carrier is freed of the sugar-pentapeptide compound ad it is incorporated into peptidoglycan structure. (6) the pentapeptide side groups attach to adjacent sugars to form cross-bridges (also spot the mistake I made...). (7) The bactoprenol group is recycled back to the cytoplasmic side of the bacterial membrane. Credit: Me

This observation was interpreted alongside the knowledge that penicillin treated cells tended to swell then burst. This generated the (later proved) hypothesis that uridine diphosphate sugar peptides were the precursors for bacterial cell wall peptidoglycan synthesis and so by virtue of its activity the first important steps in bacterial cell wall synthesis began to unravel. By interfering with peptiodoglycan synthesis the bacterial cell walls fell apart resulting in the cell becoming susceptible to osmotic changes. Water moves into the cell and cell goes pop. The whole process is pretty well covered in this video.

Developed and produced by http://www.MechanismsinMedicine.com

50 years later penicillin was still teaching us about bacterial physiology and was involved in the development of our understanding of the bacterial SOS response.Like any good SOS signal it should be turned 'off' most of the time and be activated only when needed. To achieve this a protein called LexA blocks access to certain SOS genes turning them 'off' but in response to DNA damage another protein, RecA, is produced. RecA cleaves LexA forcing these SOS elements 'on' and this prevents cell division and increases the production of proteins to scan for and repair DNA damage. The weird thing is this pathway is also activated when penicillin starts breaking down the cell wall.

It turns out that pencillin's activity interferes with cell division (as you would expect, the cell's wall is falling apart) and this interference activates a reporter system called the dpiAB operon. Once activated the products of the dpiAB operon could also activate the SOS response.

Other antibiotics such as puromycin have illuminated other cellular activity like protein synthesis. Puromycin is an extremely effective antibiotic because it looks like aminoacyl-tRNA, the molecules used to bring amino acids into the ribosome facilitating protein synthesis. When puromycin is incorporated into the ribosome in the place of aminoacyl-tRNA the ribosome stalls and falls apart, the protein manufacture is aborted and a cell without proteins is a dead cell.

Puromycin activity

Translation with and without puromycin. Credit: Me

This antibiotic continues to be used to investigate ribosomal activity but also at sub-lethal concentrations to investigate protein synthesis abortion and escape mechanisms of ribosome stress.

A final example is the investigation of the activity of gyrase which was probed and understood using antibiotics. Continued research in DNA replication and synthesis relies heavily on compounds such as nalidixic acid, oxolinic acid, novobiocin and coumermycin.

The job of gyrase is very simple to explain. If you take a helix and open it at one end (which would let the DNA replication machinery in) the helix tightens or supercoils and the job of gyrase is to spin the helix back the other way such that you don’t break the DNA. Gyrase is comprised of 4 subunits, two subunits of GyrA and 2 subunits GyrB, each with its own role to play.

Helix (Gyrase)

Opening the helix just supercoils it, unless gyrase can do its work. Credit: Me

The compounds novobiocin and coumermycin killed cells very effectively and analysis showed that following treatment DNA wouldn’t supercoil. As it could also be shown that no DNA replication was taking place either it indicated that DNA supercoiling was involved in DNA synthesis. These observations put together suggested that novobiocin and coumermycin somehow prevented DNA supercoiling and that resulted in cell death.

In addition to anti-coiling the DNA to prevent supercoiling gyrase also relaxes the coil of DNA to facilitate its easier separation. It does this by making cuts in the double helix and allowing the coil to resolve itself. Another antibiotic, nalidixic acid and its analog oxolinic acid were observed to kill cells and prevent the relaxation of DNA. Again these observations suggested without the ability to relax the DNA helix the cell would die.

Much later these four compounds were fitted against the subunits of gyrase and found to block its activity specifically. These four compounds described the function of a protein before it had even been discovered.

It’s somewhat ironic that the utility and usefulness of antibiotics as biological probes has remained strong since their initial discovery despite the decrease in clinical efficacy over the same time period and its scary to wonder if, with the decline in antibiotic development, we are also handcuffing our ability to further probe bacterial physiology.

References

Falconer, S., Czarny, T., & Brown, E. (2011). Antibiotics as probes of biological complexity Nature Chemical Biology, 415-423 DOI: 10.1038/nchembio.590