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!

Viruses turn the tables on their bacterial hosts

Many species of  bacteria – including those that infect humans – are themselves threatened by parasites and predators. A large group of viruses called bacteriophages are known to infect bacteria. On infection, the phage transfers its DNA to the bacterial cell, hijacks the bacterial DNA-replicating machinery to make multiple copies of itself, and then escapes by killing the bacterial cell.

Bacteria have in turn evolved a variety of immune mechanisms to protect themselves against invading phages. About 40% of sequenced bacterial genomes contain a set of CRISPR/Cas genes. These include a set of genes encoding Cas proteins as well as CRISPR loci, which are arrays of short repeats separated by highly variable ‘spacer’ sequences.

These spacer sequences are identical to sequences present in phage DNA. They are transcribed into small RNA molecules called crRNAs, which, with the help of the Cas proteins, bind to and cause degradation of the invading phage DNA. However, a recent study published in Nature has discovered a novel CRISPR/Cas system – not in a bacterium, but in a bacteriophage.

This bacteriophage, called ICP1, attacks a strain of the cholera pathogen Vibrio cholerae. The authors demonstrated that the CRISPR/Cas system in the bacteriophage is fully functional and targets a region of bacterial DNA that is responsible for defense against the phage. Not only is this an example of a supposedly bacterial immune mechanism being used by a phage, but it appears that the phage uses it to counter an entirely different bacterial immune mechanism. This host vs. pathogen arms race suddenly looks a lot more interesting.

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.

Bacteria Cooperate to Survive Overcrowding

For any bacterium, whether living deep in the ground or infecting a human body, overcrowding is one of the greatest threats it faces. A tightly packed environment means a shortage of food, competition for the best spaces, and the accumulation of toxic wastes excreted by its neighbors. And since many species of bacteria multiply very fast – a well-fed Escherichia coli cell can split in two every twenty minutes – overcrowding is almost inevitable.

Nevertheless, bacteria exist all around us, often packed to densities of billions in a single gram of soil. It’s hardly surprising that many scientists have wondered how these creatures manage to survive in such crowded conditions.

Now a joint study by Korean and American biologists has shown that not only do bacteria sense when their surroundings become severely congested, but that the bacterial community works together to help every bacterial cell survive the resultant stress. This involves a process named quorum sensing, by which bacteria can detect and respond to others of their kind.

Imagine every bacterial cell slowly releasing a chemical signal into its surroundings – a cell’s way of declaring ‘I am here’. As the environment grows steadily more crowded, more and more cells pump out their signal into a small space. When the amount of chemical signal present in the surroundings exceeds a certain level, indicating that a large number of cells are present, the quorum sensing system detects this change and directs all the cells to switch certain genes on or off, leading to a coordinated change in their behavior.

Glow-in-the-dark bacteria provide a classic example of the value of quorum sensing. Since a lone bacterium would not glow brightly enough to be noticeable, it keeps its light-producing apparatus switched off most of the time. But as soon as many such bacteria get together, all of them are simultaneously activated, producing a glow that is visible from afar.

In this case and others, bacteria use quorum sensing to avoid wasting energy. Though scientists guessed that quorum sensing might also help bacteria survive the stress of living in a crowded environment, this had not been demonstrated until now.

The study, published in the October issue of the Proceedings of the National Academy of Sciences, tested this idea and found it to be true. Researchers grew three species of bacteria under conditions that forced them to excrete ammonia as a waste product. Ammonia is toxic, and as the bacteria multiplied, the ammonia around them accumulated to potentially lethal levels.

However, as soon as the environment grew dangerously crowded, the quorum sensing system triggered all the bacteria to produce an acid called oxalate. This oxalate was released into the environment where it reacted with ammonia to form a harmless product, allowing the bacterial population to survive far longer than it would otherwise have.

This finding is particularly intriguing to scientists interested in social behavior, for it raises the possibility of interesting community dynamics among these bacteria. Would some bacteria cheat the rest by not producing any oxalate, instead surviving on the efforts of others? Over time, would the community evolve ways to punish these cheaters?

Even more interestingly, some of the bacteria used in this experiment are agents of disease in plants and humans, and the authors suspect that producing oxalate may help them survive the defenses of their hosts. If this is true, it might open the doors to a new strategy for treatment: one based not on killing bacteria, but on allowing them to crowd themselves to death.

“Regulation of cell size in response to nutrient availability by fatty acid biosynthesis in Escherichia coli”

One of the most intriguing questions in biology is how cells control their size. This question has been answered partly by many groups using various cellular models such bacteria, yeast and mammalian cell lines. However, the question is still open for detailed investigation. A recent study by Yao et.al., revealed that fatty acid biosynthesis plays a vital role in regulating the cell size of E.coli in response to nutrient availability.

As the cell size regulation must be connected to the membrane biogenesis, the authors start the investigation by looking for suppressor mutations that overcome the lethality, caused by the defect in Lpt pathway. As a first step,  they isolated such mutants, in which the cell size and growth rate is greatly reduced. These mutations they could map to the gene FabH, a gene that encode enzyme involved in fatty acid biosynthesis.

Secondly they have proved, that FabH is required to adjust the cell size. Furthermore they have proposed a model on cell size regulation by E.coli. “The nutrient availability determines the rate of fatty acid biosynthesis, which in turn controls the cell size, which determines the overall biosynthetic capacity of the cell and therefore the growth rate.”

This is in contrast to the previous thought that the growth rate controls the cell size. They conclude the paper by proposing the above model and by revisiting the Pathway for Type II fatty acid biosynthesis with many open questions.