The authors of this recently published paper in nature structural & molecular biology (http://www.nature.com/nsmb/journal/v20/n2/full/nsmb.2472.html) provide many arguments against contribution of facilitated diffusion (1D hopping/sliding along the DNA) as a promoter search mechanism for Escherichia coli RNA polymerase. According to them the contribution of 3D diffusion, especially at physiological protein concentrations outweighs the contribution of any form of facilitated diffusion.
Their experimental system involves a curtain of λ dna molecules tethered at both ends in the same orientation. Using quantum dot tagged RNAP they were able to visualize the RNAP molecules at the DNA curtain using TIRFM. Based on the lifetimes of the quantum dot labeled single molecules of RNAP they discriminate various intermediates: (in order of increasing lifetimes) random diffusion in absence of DNA interaction, random interactions with DNA, closed complexes and open complexes. They find that most events where RNAP engages the promoter were preceded by 3D diffusion and 1D diffusion was virtually not seen.
They also come up with a theoretical model to determine the significance of contribution of the various forms of diffusion to promoter search. They find that with greater concentrations of the protein, 3D diffusion overcomes any possible accelerating effects of 1D diffusion and thus come up with the concept of ‘facilitation threshold’, the concentration of (any) DNA-interacting protein below which facilitated diffusion would be faster in target search than 3D diffusion. They surmise that for the levels of RNAP in the cell 3D diffusion would be a faster mechanism for promoter search.
To demonstrate the significance of facilitation threshold experimentally they use the lac repressor and insert tandem lac operator sequences in the λ DNA curtain. Under conditions where non-specific DNA binding and hence facilitated diffusion is favoured they see that the lac repressor at low concentrations engages its operator mainly by 1D diffusion, however when the concentration of the repressor was increased there was an increase in the number of events in which operator binding was preceded with 3D diffusion of repressor rather than 1D diffusion clearly adding weight to the concept.
Finally the authors also discuss how under various in-vivo conditions seen by the RNAP like presence of nucleoid associated proteins and higher chromatin architecture as well as molecular crowding why 3D diffusion would be a more prevalent mechanism for promoter search rather than 1D diffusion.
The synchrotron at CERN might be close to revealing the Higgs boson but the one at SOLEIL seems to have revealed the concentration of antibiotics that accumulate within drug treated bacterial cells. Perhaps not as exciting but nevertheless important.
One way in which cells acquire resistance to antibiotics is by lowering its intracellular concentration either by active efflux or by preventing its accumulation by altering membrane permeability. However, what was lacking was a way to directly demonstrate this due to the inability to measure levels of antibiotic within single cells. Most methods that attempted to measure intracellular antibiotic concentrations, could achieve this only for a population of cells. Also the methods were invasive- employing cell lysis to release antibiotic and then measure their levels using their natural fluorescence; or they would involve modification of the antibiotic which could affect its efficacy. A recent study published in PLoS one, employed an improvement on an existing fluorimetric method by using synchrotron radiation D(deep)UV imaging and DUV microspectroscopy to measure concentration of antibiotics within single cells.
The authors manage to measure fluorescence levels as well as spectra of certain antibiotics within single cells, taking into account the large amounts of fluorescence given by other cellular components like NADH, proteins rich in tyrosine and tryptophan, etc. Using their technique, they manage to demonstrate that a multi-drug resistant(MDR) strain of Enterobacter aerogenes does not accumulate fluoroquinolone antibiotics within cells, while an antibiotic sensitive derivative of this strain does. Since a non specific drug efflux pump is responsible for the MDR in the particular strain, they thus demonstrate directly that the efflux pump was responsible for decreasing the levels of antibiotic within the cell, a fact hitherto taken for granted (the sensitive strain lacks the gene encoding this pump).
The authors argue, that by being able to reduce the level of antibiotic within cells, such populations of cells (which would otherwise have been killed) can now achieve increased resistance by mutating. Hence it is essential to dissect the molecular mechanisms required to reduce antibiotic levels in bacterial cells. In turn, to study these mechanisms it would be useful to have a direct assay that measures intracellular antibiotic concentration in a non invasive manner.
The possibility of detecting antibiotics within cells, opens avenues for addressing more difficult questions (like what is the subcellular localization of the antibiotic in the cell?) and is bound to make significant impact on the research on clinical antimicrobials.
Can bacteria employ a population based resistance mechanism to counter drugs?
A study published in Nature, Sept. 2010 suggests this is likely.
Lee et al. show that when a population of E. coli cells was evolved in the presence of norfloxacin, it developed resistance, the level of which was not explained by the resistance of its individual components. That is, this evolved resistant population was composed of a larger proportion of cells that were less resistant to the antibiotic and a smaller proportion that had greater resistance. Their speculation that the high resistant isolates were generating benefits for the low resistant ones led them to find that indole, secreted by the resistant cells was the mediator.
The high resistant isolates had mutations that conferred drug resistance, but were unrelated to indole production. It seems that because these cells are resistant they can produce indole in the presence of antibiotic while the others are inhibited. Furthermore if the gene producing indole is deleted from the high resistant isolate, it can grow better in the presence of antibiotic suggesting that indole production has costs associated with it. But a mixed population with less highly resistant cells and a higher proportion of less resistant cells, could grow in the presence of norfloxacin to substantial levels, only if the highly resistant cells were capable of producing indole. Indole seems to enable antibiotic detoxification in the less resistant isolates by up-regulating export pathways and oxidative stress protective mechanisms.
Hence, the altruistic production of indole by the high resistant isolate enables the population as a whole to grow in the presence of otherwise inhibitory concentration of norfloxacin.
The authors suggest that:
This altruism allows weaker constituents to survive and concurrently explore the space of beneficial mutations, a phenomenon similar in character to kin selection. These few drug-resistant mutants, by enhancing the survival capacity of the overall population in stressful environments, may also help to preserve the potential for the population to return to its genetic origins should the stress prove transient.
What’s more, probing into ways in which a population reacts to the presence of antibacterial substances to gain resistance will help develop means to intervene this phenomenon.