Wednesday, May 19, 2010

Screwy and Sociable

“Righty-tighty-lefty-loosey” is a handy mnemonic for pretty much every screw you’ll ever encounter. If you’ve ever met a backwards-threaded screw, it’s usually a signal that there’s something different that you need to pay attention to. The near-universality of a certain “handedness” also applies at the molecular level to all living things, and couple of recent discoveries show how a deviation from standard “handedness” functions as a signal that many Bacteria use to get each others’ attention.

A normal screw is the mirror image of a backwards-threaded screw, the same way your left hand is the mirror image of your right: they have identical composition but mirrored arrangement. Amino acids, the small building-blocks that make up every protein, can also be found in mirror-image forms. A molecule of right-handed alanine (or D-alanine, for Dextro alanine) is the mirror image of left-handed alanine (or L- alanine, for Laevo- alanine).

All the protein that you’re made of, and all the protein in every cell, is made of strings of L-amino acids. Very early in the history of life, L-amino acids became the standard for everything (the explanation for this is one of the nagging mysteries of the origin of life; there’s no reason why L-amino acids would work better than D-amino acids, and non-biological processes that produce amino acids produce a 50-50% mix of the two). One of the only places in the living world to find D-amino acids—the backwards-threaded screws of the living world—is in the cell walls of Bacteria.

The Bacterial cell wall is a meshwork of strings sugars woven together with chains of amino acids to make an astonishingly tough capsule (I think of the cell wearing a very snug sweater woven of Kevlar and wire). The amino acids found in the cell wall are all L-form, except D-alanine and D-glutamate.

The cell has dedicated enzymes to tweak the normal L-alanine and glutamate into their D- forms, and until recently this was what we knew about these odd amino acids.

A group led by Matthew Waldor found a previously unsuspected role for D-amino acids in the life of Bacteria. Their finding also undermines the common view that Bacteria are solitary organisms, but instead suggests that they have a social life that includes their siblings as well as bacteria of completely different species.

As is often the case in microbiology, the first clue came from an odd mutant bacterium. Normal cells are rod shaped, like a can of tennis balls, whether or not they are well fed or starving. The mutant was different: when it was well fed, it was rod shaped, but when they stopped growing because there was no more food, they swelled up into little spheres:

One odd thing about this phenomenon: if some of the “used” medium inhabited by spherical cells was added to well-fed mutant cells, they lost their rod shape and turned spherical within minutes. The mutants responded the same way to “used” medium from normal cells. This suggested that there was something produced by both normal and mutant cells as they ran out of food that acted as a signal; the mutant cells responded inappropriately, by turning into balls. It was fairly simple to purify the signal from the medium, and identify it as the D-form of four amino acids: Methionine, Leucine, Valine, and Isoleucine. So, as these bacteria begin to starve, they start pumping out lots of D-amino acids.

It takes a lot of energy for a cell to fill the growth medium with a high concentration of specially made D-amino acids, and energy is scarce as the cell is running out of food. However, it makes sense if the D-amino acids are a signal from one cell to its neighbors. Waldor’s group needed to find out the meaning of this signal, and the mutant cells provided a big hint. The mutant cells either can’t “hear” this signal or they misinterpret it, causing them to balloon out into spheres. Experience told the researchers that this was a sign that something was wrong with cell wall synthesis: changes in cell wall construction change the shape of the cell, and incidentally, some D-amino acids are found in the cell wall. The expectation was that going into starvation conditions would demand a thicker, tougher cell wall, and that the D-amino acids were a signal from one cell to another to strengthen the wall.

An analysis of the cell wall showed that this conjecture was only partly true. D-amino acids cause cells to significantly decrease cell wall synthesis; however, the composition of the cell wall changes, incorporating D-methionine as well as D-alanine and D-glutamate. The resulting cell wall, despite being thinner, is somewhat tougher than the cell wall of well-fed cells.

This result is cool, because the cells are acting in a seemingly altruistic manner: they are spending their own energy to “warn” other cells of impending hard times so they can change their cell walls. In studying animal behavior, such altruistic behavior is usually ascribed to “kin selection”—as long as an “altruistic” behavior benefits your relatives, the fitness of your genes will be improved. So, it makes a lot of sense to help your siblings, somewhat less sense to help your cousins, and no sense at all to help a total stranger. These bacteria, however, produce a signal that not only warns their own kind, but also just about every type of bacteria Waldor’s group checked.

Bacteria are usually grown in the laboratory, as in this study, as pure cultures suspended in liquid, but in the “real world”—for example, your bathtub, or on your teeth—they grow in diverse communities in a layer of slime called a biofilm. A biofilm is structured like raisin bread: individual bacterial cells of different types are arranged like raisins and nuts in a matrix of fibrous proteins and sugars. Biofilms are great for the Bacteria as long as there’s plenty of food. The cells get to stay in a good place, and are protected from threats in their environment (biofilms are notoriously resistant to antibiotics) and from drying out. However, if the food runs out, cells must escape the biofilm in order to survive.

The same D-amino acids that tell cells that hard times are coming also tell the cell that it’s time to leave the biofilm. Rich Losick and Roberto Kolter’s groups have extended Waldor’s discovery, and hinted at a way that this signal works. They exposed well-fed biofilms to “used” medium, and found that the biofilms rapidly dispersed. As with Waldor’s group, they found that only D-amino acids that were not part of the “well fed” cell wall had this effect, and that the unusual D-amino acids were being built into the cell wall as if the cells were being starved.

A key part of the biofilms that Losick and Kolter study is a long, stringy, sticky protein called Tas A. The Tas A protein can actually be seen using an electron microscope, and Losick and Kolter found that adding D-amino acids caused the cell to release the Tas A filaments.

Each Tas A protein filament is attached to the amino acids in the cell wall by a specific protein, YqxM. This protein is sensitive to D-amino acids—and once again, mutants help to show how. Losick and Kolter were able to find mutant cells that stayed in a biofilm even though D-amino acids were present. The only difference between these mutant cells and normal cells was one DNA base change in the gene that encodes YqxM.

The picture that emerges from these studies is that D-amino acids, the non-standard backwards screws of the biological world, are an important part of the life of Bacterial cells. Bacteria talk to each other in a language of D-amino acids. As starvation approaches, the cells in a biofilm release large amounts of D-amino acids; responding to this signal, the cells remodel and strengthen their cell walls and sever their ties to the biofilm. Losick and Kolter conclude by noting that this seems to be a general phenomenon in the bacterial world, and understanding the language of bacteria may allow us to tell problematic bacteria to go away. Infectious bacteria such as Staph aureus and Pseudomonas form medically troublesome biofilms that are quite resistant to antibiotics. However, they are sensitive to D-amino acids—and once expelled from a biofilm, these bacteria can be more easily killed.

Hubert Lam, Dong-Chan Oh, Felipe Cava, Constantin N. Takacs, Jon Clardy, Miguel A. de Pedro, Matthew K. Waldor (2009). D-Amino Acids Govern Stationary Phase Cell Wall Remodeling in Bacteria. Science 325: 1552-1555.

Ilana Kolodkin-Gal, Diego Romero, Shugeng Cao, Jon Clardy, Roberto Kolter, Richard Losick (2010). D-Amino Acids Trigger Biofilm Disassembly. Science 328: 627-629.

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