Precarious balance of genome stability

Living organisms adapt to altered environments by the stepwise selection of genomic changes that lead to the optimization of fitness under the altered conditions. Conversely, what would happen if one tinkers with the genome while outside conditions remain more or less unaltered? In the case of critical genes (whose functionality is maintained by strong purifying selection), loss of function rapidly results in the accumulation of compensatory secondary mutation/s, a phenomenon known as suppression. What about deletion of non-essential genes?

Under laboratory settings, we often create targeted deletions of “non essential” genes, in order to understand their function. What are the consequences of such genomic perturbations under normal lab conditions (normal in this context means the absence of deliberate selection)? Deleting an apparently, “non-essential” gene might not threaten survival under laboratory conditions but is likely to lead to a reduction in fitness under specific natural environments, a consequence of disrupting millions of years of natural selection at work.

Xinchen Teng et al from Johns Hopkins University School of Medicine, Baltimore, (http://dx.doi.org/10.1016/j.molcel.2013.09.026) have systematically explored the consequence of genome-wide single gene knockouts available in yeast. They have come up with the startling conclusion that “mutation of any single gene may cause a genomic imbalance with consequences sufficient to drive adaptive genetic changes”. They consider this to be a “logical consequence of losing a functional unit originally acquired under pressure during evolution”.

They have screened for hidden heterogeneities in the survivability of the knockout strains by observing heat stress response as well as nutrient sensing under low amino acid conditions using replicates of the deletion strains obtained from different colonies. The presence of secondary mutations was confirmed by following their segregation in tetrads, confirming by whole genomes sequencing in specific cases. Strains carrying deletions in the same gene, obtained from different sources, were evolved under non-stress conditions to determine whether they accumulate the same secondary mutation.

Crux of the study:

Genomic analysis reveals that these heterogeneities are due to secondary genomic changes and not due to stochastic changes in gene-expression or other epigenetic changes, as both are often used to explain the heterogeneity in presumably isogenic populations. Moreover, the driver for these secondary changes is the original gene that is knocked out as evident from the observation that independently constructed deletions of the same gene most often accumulated the same secondary mutations or mutations in the same complementary group. In many cases, the secondary mutations arose while growing the replicates of the deletion strains from individual colonies without selection whereas in some cases they preexisted in the original deletion strain.

 These results reinforce the fact that one must be cautious while interpreting the gene interaction studies involving deletions. Though the rest of the background is supposedly isogenic, the deletions may have unexpected consequence on the fitness of the strain resulting in the accumulation of second site suppressor mutations that are not documented. Next time you are struggling to reproduce your previous result with a knockout, testing multiple biological replicates might help, well…to some extent. I know it is more work but it is better than discarding everything. In fact, you might get a hint about the pathway in which you original gene (that is knocked out) works without the bias of strong selection. For details, check out the original article!

Advertisements

The promoter-search mechanism of Escherichia coli RNA polymerase is dominated by three-dimensional diffusion

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.

Sirturo : A novel anti-Mycobacterium antibiotic after 40 years.,

Sirturo (bedaquiline) – a diarylquinalone – acts against Mycobacterium  and it is approved by FDA  by December 2012. Report claims that the approval is based on phase 2 clinical trial with 394 patients. Its acts by inhibiting the Mycobacterial F1F0-adenosine triphosphate (ATP) synthase – this MoA is novel among other anti tuberculosis drug.

Sirturo is expected to generate  revenue between $400 and $500 million and it was developed by (tibotec) Johnson & Johnson (J&J) and the TB Alliance.

For more information please visit the report by Randy Osborne .

 

Promiscuous restriction is a cellular defense strategy that confers fitness advantage to bacteria

The primary function of Restriction Modification  systems is to restrict the foreign DNA and protect the host bacterium from potent invading life forms, such as bacteriophages.  Type II R-M system is often considered to be highly specific for the foreign DNA. However, bacteria harboring a promiscuous REN (Restriction endonuclease) compared to the one which carries high fidelity REN confers more fitness advantage when challenged with bacteriophages, says a recent study carried by a group of scientists at IISC and JNCASR, Bangalore.

The authors prove that the  ability of the R.KpnI to recognize and cleave noncanonical sequences in vivo confers additional protection to the host against the modified (methylated) infectious genome elements. At the same time they also prove that the self Vs nonself is taken care by the topological state of the naive DNA.

Even though the RM system in bacteria is stringent, the phages evade the defense statergy employed by the bacterium, by various means. In order to cope up with the phages defense mechanism, the bacterium has to counteract  these antirestriction strategies by acquiring additional R-M systems with distinct recognition specificities or by acquiring restriction activity with broader specificity through mutation.

The study says that

The retention of the promiscuous cleavage characteristicsof a type II REase that is normally expected to possess exquisite sequence specificity provides a selective advantage for the bacterial genome in the coevolutionary arms race between phages and bacteria.

Antibiotic Transport in Resistant Bacteria: Synchrotron UV Fluorescence Microscopy to Determine Antibiotic Accumulation with Single Cell Resolution

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.

Experimental evolution of bet hedging

Surviving under fluctuating environments poses a challenge. One solution is to sense the environment and modulate the phenotype accordingly. An alternative solution is to diversify or randomize the phenotype, which can increase the likelihood of survival.

Bet hedging is an often loosely used term to describe a risk spreading strategy that increases a population’s fitness in unpredictably fluctuating environments.Consider microbes competing in not so harsh environment most of the time but encounter harsh environment unpredictably. In the not so harsh environment a heterogeneous population will be less fit than a single phenotype tuned to that environment. However, when there is a transition to a harsh environment the heterogeneous population will contain some individuals that are able to cope better with harsh environment and outcompete the single phenotype population.

Bet hedging is found in organisms ranging from bacteria to humans but evidence for how it originates in an adapting population is lacking. Beaumont et al. demonstrate de novo evolution of bet hedging in Nature Nov.2009 paper.

Bacterial populations were subjected to two contrasting environments (static and shaking). In static conditions genotype diversifies into specialist genotypes by mutation and selection. Diversification is constrained in shaken conditions.Twelve replicates were subjected to 16 alternating selection in static and shaken conditions. Populations were propagated by serial dilution until the existing cell culture formed colonies of distinct morphology and is heritable. Once emerged cells were transferred to opposing environment for next round of selection. This mode of experimentation imposed selection for high growth rate in static and shaken conditions and simultaneously fluctuating selection for colony innovation.

The paper reports that initially the regime generated novel phenotypes but in two out of twelve replicates the trend was broken by evolution of a genotype that could randomly switch between two phenotypes. Genome sequencing of this switching types revealed nine mutations that distinguished it from ancestor. The authors report that only a single mutation (capsule synthesis) is required for switching behavior but other earlier acquired mutations are necessary for conferring fitness. Statistical analysis corroborates epistatic interactions between these mutations.

So the paper provides a mechanistic understanding of adaptive evolution of bet hedging. The findings suggest that

    bet hedging may have been among the earliest evolutionary solutions to life in variable environments ,perhaps even preceding the evolution of environmentally responsive mechanisms of gene regulation.