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.

Skin bacteria may protect you from disease

The human body is home to a vast number of bacterial species; in fact, it is thought that the number of bacterial cells in a human body far outnumbers the human cells themselves. Many of these bacteria live in the gut, while others live in saliva, the inside of the eyelids, or the skin.

While most of them have no known effect on us, some are useful to their human hosts. For example, several studies have suggested  that gut bacteria help our immune systems protect us against pathogens. A recent paper by Naik et al. in Science suggests that at least some of the bacteria living in our skin do the same.

These authors found that ‘germ-free’ mice without resident bacteria have reduced levels of interleukins (chemicals produced by white blood cells that help fight infections) in their skin tissues. When bacteria were introduced into the gut of these mice, interleukin levels in the gut increased, but levels in the skin did not. On the other hand, when a species of bacteria that normally resides in skin was introduced into the skin of germ-free mice, the interleukin levels in the skin increased, as did the immune response to the parasite Leishmania major.

It has long been known that gut bacteria produce vitamins that are important for human health. In recent years, a variety of studies have linked resident bacteria to obesity, disease, prevention of allergies, and even behavior. The finding that bacteria in our skin may be important for the human immune system is another step in understanding the importance of this vast and diverse population of cells.

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.

The Black Queen Hypothesis!

Bacteria play Hearts! Yes, the same card game that we play as a pastime. While, depending on our skills we win or lose the game; in bacterial world there are some who always Win while some others who always lose by ending up with the black queen!

A recent paper published in mbio describes how organisms in the ocean might be playing Hearts. In the bacterial version of the game as the paper describes, the black queen is the gene responsible for breaking down HOOH, the gene is named katG. HOOH is dangerous for the survival of all bacteria, so it needs to be degraded and katG gene is responsible for the breakdown of HOOH.So, it came as a surprise to many scientists that the most dominant bacterioplankton in the ocean the  “Prochlorococcus” and the “Candidatus Pelagibacter” lacked the katG gene entirely. These two bacteria lack the katG gene and yet manage to survive in the oceans where HOOH is constantly produced via photo oxidation of organic carbon by sunlight. Clearly, the lack of katG gene is costly for any bacteria, then how does the Prochlorococcus manage to not only survive but also dominate in the oceans?

You know how one hates doing boring jobs but its important that they are done, you just have to do it! Unless…someone else does that job for you!

Prochlorococcus is a winner; other bacteria do the dirty job of degrading HOOH for themselves and for Prochlorococcus. It is important to note that katG gene function is leaky i.e other bacteria keep the concentration of HOOH low (sink effect) making the katG gene function dispensable for Prochlorococcus. Thus, other bacteria ‘help’ Prochlorococcus to survive in a HOOH environment. Prochlorococcus does not bear the cost of maintaining the katG gene but earns the benefit of HOOH being degraded.

Morris and co describe the above-mentioned interaction in an evolutionary context and propose the Black Queen hypothesis to describe this interaction,

the black queen refers to a playing card, in this case the queen of spades in the game Hearts. In Hearts the goal is to score as few points as possible. The queen of spades, however, is worth as many points as all other cards combined, and therefore a central goal of the game is to not be the player that ends up with that card. In the context of evolution, the BQH posits that certain genes, or more broadly, biological functions, are analogous to the queen of spades. Such functions are costly and therefore undesirable, leading to a selective advantage for organisms that stop performing them. At the same time, the function must provide an indispensable public good, necessitating its retention by at least a subset of the individuals in the community—after all, one cannot play Hearts without a queen of spades. The detoxification of HOOH fulfills both of these criteria, and therefore the BQH predicts that this function will be performed by helpers that comprise only a fraction of the community.

Prochlorococcus is successful in its survival and dominance in oceans since the benefits of losing the katG gene outweighs the cost of losing that gene, according to the BQH.

BQH thus provides a unique perspective of looking at interactions between different organisms in a given natural environment. Not only does it successfully explain genome reduction in free-living organisms but also the occurrence of essential yet rare functions!

P.S

The paper has created quite a stir and has grabbed a lot of media attention, links to few  …

http://beacon-center.org/blog/2012/05/21/beacon-researchers-at-work-the-evolution-of-simplicity-and-the-black-queen-hypothesis/

http://io9.com/5897134/researchers-describe-a-new-evolutionary-theory-the-black-queen-hypothesis

http://pleiotropy.fieldofscience.com/2012/05/black-queen-hypothesis.html