Friday, December 28, 2007

The complexities of gene regulation in E. coli

We are currently identifying genes that belong to the Sxy regulon in E. coli. The only other well-characterized Sxy regulon was identified by our work in H. influenzae (link). E. coli’s genome is over 2-times larger than H. influenzae’s and, not surprisingly, the E. coli Sxy regulon contains more genes. The E. coli regulon has an additional level of complexity because many of the Sxy-regulated genes are likely to have additional protein regulators (ie. Sxy-regulated genes also belong to other regulons).

This additional complexity is a consequence of lifestyle. E. coli is a more versatile organism than H. influenzae: it can make most of its organic molecules from scratch (aka. from simple sugars plus a few inorganic nutrients) and it can survive in various different environments.

Because a bacterium must at all times satisfy multiple metabolic requirements, it needs to continuously balance its internal functions while exploiting a potentially ever-changing external environment. Bacteria that inhabit very stable niches (such as H. influenzae, which lives in the cavities of a human host) have a small number of transcription factors, whereas bacteria in more complex environments employ a much larger number of regulators. This relationship has been shown to scale as a power-law in which the number of transcription factors doubles twice as fast as does the total number of genes in a genome (van Nimwegen,E., 2003, Trends Genet., 19, 479), indicating that large bacterial genomes employ disproportionately more complex regulatory networks.

Thus, E. coli’s lifestyle necessitates more sensory and response systems than does that of H. influenzae. Consequently, the genes in E. coli’s Sxy regulon are much more likely to belong to multiple (possibly non-overlapping) regulatory networks in order to fine-tune their expression. This unfortunately makes studying the E. coli Sxy regulon more complicated; we can’t be confident that overexpression of the Sxy protein results in induction of all Sxy-regulated genes.

For H. influenzae, gene expression data coupled with bioinformatic analysis revealed that most genes in the Sxy regulon require only CRP and Sxy for transcription activation in standard culture conditions. In E. coli, some Sxy regulon genes will likely be repressed during growth in standard culture conditions, regardless of whether CRP and Sxy are trying to turn them on. Perhaps conducting E. coli gene induction studies in minimal medium will reduce the activities of repressor proteins and so improve our ability to detect some members of the Sxy regulon.

Fortunately, a substantial body of knowledge surrounds the regulation of some of the genes in E. coli’s Sxy regulon. Thus, although we will have trouble identifying genes that can’t be induced by Sxy in standard lab conditions, we will at least be able to integrate our Sxy regulon data with other regulatory networks.

Monday, December 10, 2007

Latest ICAP results

I have now quantified EcCRP and HiCRP affinity for ICAP and four designer variants, and am currently replicating the experiments. The data looks good and supports my hypotheses, but some additional interesting features of CRP-DNA interactions have revealed themselves.

For example, CRP binding causes DNA to assume a very sharp bend of around 90º, which is achieved through two major kinks near the centre of the DNA site (each ~40º) and lesser kinks at each edge of the binding site (each ~10º). Two of the ICAP variants were designed (in part) to address the importance of the small secondary kinks for HiCRP affinity. As I predicted, HiCRP appears to need favourable interactions with a longer stretch DNA than does EcCRP, possibly to stabilize kinking, but what I didn’t expect is that I can readily detect variations in the degree to which DNA is bent by CRP. The second surprise is that when multiple CRP molecules bind to bait DNA at high protein concentrations in bandshift reactions, HiCRP appears to bind in a stepwise fashion: first one protein and then two (possibly in a cooperative manner). EcCRP, on the other hand, goes very quickly from having only one protein bound to a stage were more than two proteins bind to the same piece of DNA, seemingly in a more haphazard fashion. I suspect this is consistent with my model in which HiCRP is highly selective for DNA sites, whereas EcCRP is much less choosy and will bind all sorts of less favourable sites when the good ones are saturated.

These are interesting results, but they require much more thinking before I make good sense of them. Also, they beg for more experiments and my plate is looking pretty full considering the number of different DNA species (ie. different natural promoters) I still want to test in "simple" affinity experiments.