Cell wall under attack - bacterial response to antibiotics

I took a quick break away from synthetic biology and DNA synthesis research the other day, to dive back into my happy little world of antibiotic research, in preparation for my new project in October. I'll be working with Streptomyces bacteria again, which after a whole summer of E. coli I'm quite looking forward to. What I'll be doing with them is examining the response of the cell wall to antibiotics.

The bacterial cell wall is made up of glycopeptide molecules (sugars and proteins joined together) and surrounds the whole cell. Without it, bacteria swiftly loose their integrity and salt-balance across the membrane, which is why many antibiotics target the cell wall in order to kill bacteria. Both for antibiotic resistance, and for surviving conditions that could damage the cell wall, bacteria have a system of monitoring the state of the cell membrane and responding quickly to any changes.

The system that was discovered in Streptomyces coelicolor (which I'll be working on) was named the sigE system, and consisted of an operon (string of genes) encoding four genes:

SigE encodes for a sigma-factor, a protein used in bacteria to switch on certain sets of genes. The cseA codes for a cell membrane lipoprotein, possibly used in a sensor system, while cseB and C are a two-component signalling system (very common in bacteria). CseC is a sensor (a histadine protein-kinase sensor for those who are interested) while cseB is the response regulator, acting out a response when it receives a signal from cseC.

And now...the science :)

In order to test that this operon was involved in cell membrane responses to antibiotics the lab carried out a variety of experiments, all producing evidence that lead towards this conclusion. The main experiments were as follows:

1. Removing the sigE operon and placing it on a separate plasmid, that activated resistance to Kanamycin. The bacteria were then plated on agar containing antibiotics and challenged with a kanamycin disk. Cell wall attacking antibiotics induced kanamycin, whereas antibiotics that attacked (say) the ribosome didn't.
2. Keeping the sigE in its original chromosomal context, the group then challenged it with different concentrations of vancomycin (an antibiotic which attacks bacterial cell walls). They then measured the level of the sigE operon proteins being produced in the cell. Higher concentrations of vancomycin, lead to more proteins.
3. Going back to the sigE-kanamycin resistant protein, they tried knocking out the sigE promoter, effectively switching all these genes off. The effect seen previously disappeared.
4. Leaving the lab, they then did some computational work, scanning the database to see what genes the sigE sigma-factor actually switched on. They found a group of 12 genes, all of which coded for cell-wall synthesis enzymes.

All of this leads up to some pretty conclusive evidence - in case of cell wall damage, the sigE operon is switched on. The interesting thing is, is that this isn't just a response to antibiotics either. It is highly unlikely that the system is able to respond to every different cell-wall destroying antibiotic, instead, the response is triggered by cell-wall intermediates, or degradation products that signal "Help - cell wall is being destroyed!" and switch on the sigE response, which produces proteins to mend it again.

But there are still a lot of unanswered questions. What is the cseC actually sensing? What is the exact purpose of the cseA? Why produce both a sigma-factor and a heafty response pathway? Which intermediates are used for activating? And, most importantly, can we hijack this somehow to kill bacteria?

I can't wait to get to work with it :D

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Hutchings, M., Hong, H., Leibovitz, E., Sutcliffe, I., & Buttner, M. (2006). The E Cell Envelope Stress Response of Streptomyces coelicolor Is Influenced by a Novel Lipoprotein, CseA Journal of Bacteriology, 188 (20), 7222-7229 DOI: 10.1128/JB.00818-06

Hong, H., Paget, M., & Buttner, M. (2002). A signal transduction system in Streptomyces coelicolor that activates the expression of a putative cell wall glycan operon in response to vancomycin and other cell wall-specific antibiotics Molecular Microbiology, 44 (5), 1199-1211 DOI: 10.1046/j.1365-2958.2002.02960.x

Jacobs C, Frère JM, & Normark S (1997). Cytosolic intermediates for cell wall biosynthesis and degradation control inducible beta-lactam resistance in gram-negative bacteria. Cell, 88 (6), 823-32 PMID: 9118225