Friday, May 27, 2011

Systems biology approach to stringent response

Bacterial cells constantly need to monitor their environment and act accordingly.

The trouble is, bacteria are very small and when you are so very small, all the effects of being quantized in terms of molecule numbers are becoming very strong: number of mRNA molecules for a certain gene is an integer value, and not a very high at that, events of receptor getting activated or RNA polymerase binding to the promoter are stochastic in nature, and since not too many of the individual events occur at a time, they are not averaged out due to the law of large numbers.

All this results in bacteria gambling all the time: some react to stimulus, some don't, some produce more proteins in response to it, some less. This leads to so called phenotypic heterogeneity, when otherwise (genetically) identical bacteria become very different in terms of their responses.

This could be a good thing and also could be a bad thing. Having a collection of different bugs instead of a clone army will provide certain versatility: some are ready for one conditions, and some are ready for others. For instance, some are ready to grow and divide right away and some are slower and more cautious. Both types of cells can be beneficial in different conditions: the active ones will drive the population growth, but will be sensitive to the antibiotic treatment, and the passive ones will wait until the treatment is over and then they will come to life. Sounds like a good strategy (and it has a name, this strategy - "bed hedging") and I guess it is exactly the reason why clone armies never caught on.

On the other hand, this noise makes it really hard for bacteria to make an educated guess and respond to a stimulus in the best possible way - there is simply too much background to filter through!

One of the widely used cellular response systems used by bacteria is so called stringent response. This one is mediated by a family of proteins called RSH (RelA SpoH Homologue, with RelA and SpoT being the first members to be discovered). These proteins sense different cues (aminoacid starvation, fatty acid starvation, heat shock and so on) and translate these input signals into modulation of the intracellular concentration of ppGpp - a modified G nucleotide which acts as a second messenger modulating numerous cellular processes, such as transcription, translation, replication and lots more.

The RSH-mediated system in M. tuberculosis was shown to be  vital for prolonged life in the host, linking it to the phenotypic heterogeneity. This bug has one RSH protein, Rel, which is capable of both producing and degrading ppGpp. The logical question is then - how is Rel activity and abundance regulated, and what is its distribution in the bacterial population?

In the recent two papers (1 and 2) exactly that was done using M. smegmatis as a non-pathogenic model for M. tuberculosis. Rel promotor was fused with a GFP ORF so that its activity can be monitored on the single cell level using flow cytometry. And indeed, they saw that cells are very strongly heterogeneous in respect to the GFP level, which reflects activity of the rel promoter. This heterogeneity turned out to be brought about by the positive feedback loop feeding Rel expression back to its promoter via activity of the MprAB and SigE proteins.

Positive feed back loops are a common way of creating this sort of bistability in the biological systems and is a very common motif in the gene networks. What would be interesting is to see how common is this positive feedback among different bacteria, especially the ones that have different RSH system, such as the most commonly studied E. coli, which has two RSH proteins instead of one in M. tuberculosis: RelA, which is mostly producing ppGpp, and SpoT, which is mostly degrading it.

Effects of noise in protein expression are also different in E. coli: here synthetic and hydrolytic components are split into two independently produced proteins, thus resulting in a system with more degrees of freedom. Sure enough, it is well documented that intrinsic noise in protein expression (that would be roughly variation in the protein expression between the two independent genes in one bacterial cell) is much less than extrinsic one (roughly - variation between different cells), but still - two genes is more than one!

And what about bugs having more RSHs? Streptococcus mutans has three! From classic microbiological studies it seems that similar regulation could be present there as well, linking stress tolerance and genetic competence.

However fruitful, systems biology investigations of the stringent response are suffering from one big limitation: using current approaches, only a few readouts can be followed in the same cell at the same time. Stringent response, however, involves many different systems being rewired, and microarrays in the combination with metabolomics can follow many, many things happening in the cell (though lacking in the single cell resolution!).


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Mendeley group on stringent response

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