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.

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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.

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

Making a RNA polymerase out of a DNA polymerase

DNA polymerases build DNA from dNTPs and enable replication, whereas RNA polymerases make RNA from NTPs, permitting transcription. Though the subunit structures of DNA and RNA polymerases appear different, there are several single-subunit RNA polymerases in mitochondria and T-odd bacteriophages, which are thought to have evolved from an ancestral DNA polymerase. Now the question that arises is what are the changes that can make a DNA polymerase into an RNA polymerase; should be a difficult change to make given that in the presence of a vast excess of NTPs over dNTPs, DNA polymerases have to powerfully discriminate between the two substrates.

Though several mutations have been described so far, which allow a DNA polymerase to bind to NTPs and produce short stretches of RNA (typically <10nt) at which point the mutant polymerase simply stalls, the answer to the question above has remained elusive.

In a recent paper published in PNAS, Cozens and colleagues from the MRC-LMB and the NIH demonstrate that two targeted mutations in the replicative polymerase from Thermococcus gorgonarius can make an RNA polymerase out of it. The determining mutation is located at about 25 Angstroms from the active site in what is called a ‘thumb’ subdomain. The combination of the two mutations makes the DNA polymerase synthesise RNA upto 1.7kb long, and use various unnatural nucleotide substrates as well. The mutation hardly affects any of the enzyme kinetic parameters associated with its natural dNTP substrate. However, for NTPs (the RNA substrate), the mutation pair increases the Vmax 4-fold and decreases the Km (thus increasing substrate affinity) by three orders of magnitude. These effects might be explained by steric effects and charge differences in the thumb subdomain between the two forms of the polymerase.

The residue at the determining position in the thumb subdomain, though conserved in related organisms, varies in distant organisms. On the basis of the high-temperature adaptation of Thermococcus, the authors state that this residue (referred to as a ‘steric gate’)

may therefore be a specific adaptation to prevent the deleterious consequences of NTP mis-incorporation or replication of genomic lesions at high temperature. Conversely,…, in analogy  to the diverse nature of steric-gate residues, the second gate may therefore be elaborated in different ways in the context of different thumb domain structures.

Bacterial charity work leads to population-wide resistance

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.