Study Quantifies the Size of Holes Antibacterials Create in Cell Walls to Kill Bacteria

The rise of antibiotic-resistant bacteria has initiated a quest for alternatives to conventional antibiotics. One potential alternative is PlyC, a potent enzyme that kills the bacteria that causes strep throat and streptococcal toxic shock syndrome. PlyC operates by locking onto the surface of a bacteria cell and chewing a hole in the cell wall large enough for the bacteria’s inner membrane to protrude from the cell, ultimately causing the cell to burst and die.

Research has shown that alternative antimicrobials such as PlyC can effectively kill bacteria. However, fundamental questions remain about how bacteria respond to the holes that these therapeutics make in their cell wall and what size holes bacteria can withstand before breaking apart. Answering those questions could improve the effectiveness of current antibacterial drugs and initiate the development of new ones.

Researchers at the Georgia Institute of Technology and the University of Maryland recently conducted a study to try to answer those questions. The researchers created a biophysical model of the response of a Gram-positive bacterium to the formation of a hole in its cell wall. Then they used experimental measurements to validate the theory, which predicted that a hole in the bacteria cell wall larger than 15 to 24 nanometers in diameter would cause the cell to lyse, or burst. These small holes are approximately one-hundredth the diameter of a typical bacterial cell.

“Our model correctly predicted that the membrane and cell contents of Gram-positive bacteria cells explode out of holes in cell walls that exceed a few dozen nanometers. This critical hole size, validated by experiments, is much larger than the holes Gram-positive bacteria use to transport molecules necessary for their survival, which have been estimated to be less than 7 nanometers in diameter,” said Joshua Weitz, an associate professor in the School of Biology at Georgia Tech. Weitz also holds an adjunct appointment in the School of Physics at Georgia Tech.

The study was published online on Jan. 9, 2013 in the Journal of the Royal Society Interface. The work was supported by the James S. McDonnell Foundation and the Burroughs Wellcome Fund.
Common Gram-positive bacteria that infect humans include Streptococcus, which causes strep throat; Staphylococcus, which causes impetigo; and Clostridium, which causes botulism and tetanus. Gram-negative bacteria include Escherichia, which causes urinary tract infections; Vibrio, which causes cholera; and Neisseria, which causes gonorrhea.

Gram-positive bacteria differ from Gram-negative bacteria in the structure of their cell walls. The cell wall constitutes the outer layer of Gram-positive bacteria, whereas the cell wall lies between the inner and outer membrane of Gram-negative bacteria and is therefore protected from direct exposure to the environment.

Georgia Tech biology graduate student Gabriel Mitchell, Georgia Tech physics professor Kurt Wiesenfeld and Weitz developed a biophysical theory of the response of a Gram-positive bacterium to the formation of a hole in its cell wall. The model detailed the effect of pressure, bending and stretching forces on the changing configuration of the cell membrane due to a hole. The force associated with bending and stretching pulls the membrane inward, while the pressure from the inside of the cell pushes the membrane outward through the hole.

A transmission electron microscope image of a Streptococcus  pyogenes cell experiencing lysis after exposure to the highly active enzyme PlyC. (Credit: Daniel Nelson, UMD)

“We found that bending forces act to keep the membrane together and push it back inside, but a sufficiently large hole enables the bending forces to be overpowered by the internal pressure forces and the membrane begins to escape out and the cell contents follow,” said Weitz.
The balance between the bending and pressure forces led to the model prediction that holes 15 to 24 nanometers in diameter or larger would cause a bacteria cell to burst. To test the theory, Daniel Nelson, an assistant professor at the University of Maryland, used transmission electron microscopy images to measure the size of holes created in lysed Streptococcus pyogenes bacteria cells following PlyC exposure.

Nelson found holes in the lysed bacteria cells that ranged in diameter from 22 to 180 nanometers, with a mean diameter of 68 nanometers. These experimental measurements agreed with the researchers’ theoretical prediction of critical hole sizes that cause bacterial cell death.
According to the researchers, their theoretical model is the first to consider the effects of cell wall thickness on lysis.

“Because lysis events occur most often at thinner points in the cell wall, cell wall thickness may play a role in suppressing lysis by serving as a buffer against the formation of large holes,” said Mitchell.
The combination of theory and experiments used in this study provided insights into the effect of defects on a cell’s viability and the mechanisms used by enzymes to disrupt homeostasis and cause bacteria cell death. To further understand the mechanisms behind enzyme-induced lysis, the researchers plan to measure membrane dynamics as a function of hole geometry in the future.
source:http://www.gatech.edu/newsroom/release.html?nid=182231

Cheating — and getting away with it

We would all like to believe that there is a kind of karma in life that guarantees those who cheat eventually pay for their bad behavior, if not immediately, then somewhere down the line. But a study of a new gene in the amoeba Dictyostelium discoideum suggests that, at least for amoebae, it is possible to cheat and get away with it.

The experimental work was conducted by then graduate student Lorenzo Santorelli as part of a collaboration between evolutionary biologists David C. Queller and Joan E. Strassmann of Rice University and Gadi Shaulsky and Adam Kuspa of Baylor College of Medicine. Santorelli has since moved to Oxford University and his advisors to Washington University in St. Louis, where Queller is the Spencer T. Olin Professor of Biology and Strassmann is a professor of biology, both in Arts & Sciences.
The cheat in question is putting more than your clone’s fair share of cells into a communal spore body, so that your genome dominates the next generation of amoebae. The idea has always been that cheating clones pay a price in the form of reduced evolutionary fitness in some other chapter of their lives.

A slice through a culture plates shows slugs (clumps) of the social amoebae D. discoideum (at left) on their way to becoming fruiting bodies (right). The photograph was shot in the lab of Joan Strassmann and David Queller by entomologist and photographer Alex Wild. For more of Wild’s photos, visit http://www.alexanderwild.com/.

In work described in the Jan. 9 issue of BMC Evolutionary Biology, the scientists tested the fitness of a knockout mutant (an amoeba with one disabled gene) called CheaterB. When mixed with equal parts of a wild-type clone, the cheater clone contributed almost 60 percent of the cells in the spore body, 10 percent more than its fair share.

The scientists ran CheaterB cells through exhaustive tests of their ability to grow, develop, form spores and germinate. CheaterB did just as well in these tests as its ancestor wild strain. Under laboratory conditions, at any rate, CheaterB didn’t seem to be paying a fitness cost for cheating.
The study raises important questions about the tension between cooperation and cheating. Why would breaking something that is presumably functional (by knocking out a gene) confer an advantage in the first place? And if cheating benefits the cheater and has no hidden cost, what holds cheating in check?

Cheating is surprisingly easy
D. discoideum spend most of their lives as predatory single cells hunting bacteria through the leaf litter and upper soil layers of forests in eastern North America. But when they can’t find bacteria and begin to starve, they gather to form fruiting bodies, a thin stalk of cells with a ball of spores at the top, like a miniature Space Needle. The amoebae that end up in the stalk die, giving up their lives to benefit the amoebae that become spores.
Importantly the cells that stream together to form the fruiting body can be clonal (genetically identical) or have two (or more) genetic makeups. If each clone in a two-clone fruiting body contributes half the cells to the spore body, both clones gain from cooperating because each must sacrifice fewer cells to the stalk.
But game theory suggests the clones should sometimes evolve strategies that allow them to gain the benefits of cooperation without paying the costs.
In 2008 Queller and Strassmann published a genome-wide screen of D. discoideum that found roughly 180 cooperation genes, genes that might produce cheaters if they mutated. The number of genes, and the number of different biological pathways they affected, suggested it might be easy to evolve cheating and difficult to control it fully.
At the time cheaters were believed to be held in check by mechanisms that made non-cooperation costly. The first D. discoideum cheater to be scrutinized, CheaterA, described in 2000, is not able to form fruiting bodies on its own. This is a crippling disability that would prevent it from surviving in the wild.
But the screen from 2008 selected only clones able to produce clonal fruiting bodies, thus passing a basic test of evolutionary fitness. These clones were what is called facultative cheaters, cheating only under favorable conditions, and not obligate cheaters, forced to cheat no matter what.
The overall robustness of knockout mutant CheaterB deepens the mystery. “No measurable laboratory trait revealed an Achilles heel,” Strassmann says, “but that doesn’t mean there isn’t one in natural environments. Otherwise, why would a naturally occurring mutation that duplicated the knockout not take over amobae populations?”
source:https://news.wustl.edu/news/Pages/24754.aspx

Virus caught in the act of infecting a cell

The detailed changes in the structure of a virus as it infects an E. coli bacterium have been observed for the first time, report researchers from The University of Texas at Austin and The University of Texas Health Science Center at Houston (UT Health) Medical School this week in Science Express.
To infect a cell, a virus must be able to first find a suitable cell and then eject its genetic material into its host. This robot-like process has been observed in a virus called T7 and visualized by Ian Molineux, professor of biology at The University of Texas at Austin, and his colleagues.

Researchers found that the T7 virus has six tail fibers that are folded back against its capsid.  The fibers extend as the virus locates  a suitable host and as it “walks”  across its host cell surface to find  a site to infect.

The researchers show that when searching for its prey, the virus briefly extends — like feelers — one or two of six ultra-thin fibers it normally keeps folded at the base of its head.
Once a suitable host has been located, the virus behaves a bit like a planetary rover, extending these fibers to walk randomly across the surface of the cell and find an optimal site for infection.
At the preferred infection site, the virus goes through a major change in structure in which it ejects some of its proteins through the bacterium’s cell membrane, creating a path for the virus’s genetic material to enter the host.
After the viral DNA has been ejected, the protein path collapses and the infected cell membrane reseals.

“Although many of these details are specific to T7,” said Molineux, “the overall process completely changes our understanding of how a virus infects a cell.”
For example, the researchers now know that most of the fibers are usually bound to the virus head rather than extended, as was previously thought. That those fibers are in a dynamic equilibrium between bound and extended states is also new.
Molineux said that the idea that phages “walk” over the cell surface was previously proposed, but their paper provides the first experimental evidence that this is the case.

The top images are tomograms of the virus in action. The illustrations show T7 using its fibers to “walk” across the cell surface and infect the cell.

This is also the first time that scientists have made actual images showing how the virus’s tail extends into the host — the very action that allows it to infect a cell with its DNA.
“I first hypothesized that T7 made an extended tail more than 10 years ago,” said Molineux, “but this is the first irrefutable experimental evidence for the idea and provides the first images of what it looks like.”
The researchers used a combination of genetics and cryo-electron tomography to image the infection process. Cryo-electron tomography is a process similar to a CT scan, but it is scaled to study objects with a diameter a thousandth the thickness of a human hair.

source :http://www.eurekalert.org/pub_releases/2013-01/uota-vci011013.php

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Research opportunities at TIFR

Research Opportunities for exceptionally talented and strongly motivated students.

The Tata Institute of Fundamental Research is India’s premier institution for advanced research in fundamental sciences. The Institute runs a graduate programme leading to the award of Ph.D. degree, as well as M.Sc. and Integrated Ph.D. in certain subjects. With its distinguished faculty, world class facilities and stimulating research environment, it is an ideal place for aspiring scientists to initiate their career.

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In Epigenomics, Location is Everything

In a novel use of gene knockout technology, researchers at the University of California, San Diego School of Medicine tested the same gene inserted into 90 different locations in a yeast chromosome – and discovered that while the inserted gene never altered its surrounding chromatin landscape, differences in that immediate landscape measurably affected gene activity.
The findings, published online in the Jan. 3 issue of Cell Reports, demonstrate that regulation of chromatin – the combination of DNA and proteins that comprise a cell’s nucleus – is not governed by a uniform “histone code” but by specific interactions between chromatin and genetic factors.

An x-ray micrograph of a yeast cell, Saccharomyces cerevisiae, as it buds before dividing. Courtesy of  Carolyn Larabell, UC San Francisco, Lawrence Berkeley National Laboratory and the National Institute of General Medical Sciences.

“One of the main challenges of epigenetics has been to get a handle on how the position of a gene in chromatin affects its expression,” said senior author Trey Ideker, PhD, chief of the Division of Genetics in the School of Medicine and professor of bioengineering in UC San Diego’s Jacobs School of Engineering. “And one of the major elements of that research has been to look for a histone code, a general set of rules by which histones (proteins that fold and structure DNA inside the nucleus) bind to and affect genes.”
The Cell Reports findings indicate that there is no singular universal code, according to Ideker. Rather, the effect of epigenetics on gene expression or activity depends not only on the particular mix of histones and other epigenetic material, but also on the identity of the gene being expressed.
To show this, the researchers exploited an overlooked feature of an existing resource. The widely-used gene knockout library for yeast, originally created to see what happens when a particular gene is missing, was built by systematically inserting the same reporter gene into different locations. Ideker and colleagues focused on this reporter gene and observed what happens to gene expression at different locations along yeast chromosome 1.
“If epigenetics didn’t matter – the state of histones and DNA surrounding the gene – the expression of a gene would be the same regardless of where on the chromosome that gene is positioned,” said Ideker. But in every case, gene expression was measurably influenced by interaction with nearby epigenetic players.
Ideker said the work provides a new tool for more deeply exploring how and why genes function, particularly in relation to their location.
source http://ucsdnews.ucsd.edu/pressrelease/in_epigenomics_location_is_everything

Rare Form of Active ‘Jumping Genes’ Found In Mammals

Little brown bat (Myotis lucifugus) J.N. Stuart

Much of the DNA that makes up our genomes can be traced back to strange rogue sequences known as transposable elements, or jumping genes, which are largely idle in mammals. But Johns Hopkins researchers report they have identified a new DNA sequence moving around in bats — the first member of its class found to be active in mammals. The discovery, described in a report published in December on the website of the Proceedings of the National Academy of Sciences, offers a new means of studying evolution, and may help in developing tools for gene therapy, the research team says.

“Transposable elements are virtually everywhere in nature, from bacteria to humans,” says Nancy Craig, Ph.D., a Howard Hughes investigator and professor in the Johns Hopkins University School of Medicine’s Department of Molecular Biology and Genetics. “They’re often seen as parasites, replicating themselves and passing from generation to generation without doing anything for their hosts. But in fact they play an important role in fueling adaptation and evolution by adding variability to the genome.”

As their name suggests, jumping genes can move from place to place in the genome, sometimes even inserting themselves into the middle of another gene. Some work by replicating themselves and inserting the copies into new places in the genome — retroviruses such as HIV are comprised of this type of jumping gene, which enables the host cell to be hijacked to make more virus particles. Another class of jumping genes, known as “DNA cut-and-paste,” doesn’t make copies, but instead cuts itself out of one site in the genome before hopping into another. Craig explains that in mammal genomes, most jumping genes are of the copy-and-paste variety, and most of these are fossils, mutated to the point where they can no longer move about. Although some remnants of cut-and-paste jumping genes have been unearthed in mammals, until now, all of them have been inactive.

Craig’s team made its discovery while studying piggyBac, an active cut-and-paste jumping gene from insects. PiggyBac got its name because it hitched a ride from one host to another on a virus. While studying how the jumping gene works, the researchers also used computational methods to search for piggyBac-like DNA sequences in the genomes of some species, including that of the little brown bat. There they found a sequence similar to piggyBac, one that didn’t appear to have collected mutations that would make it inactive. Sure enough, near-identical copies were sprinkled throughout the genome, indicating that the sequence had jumped relatively recently. Craig named the find piggyBat. Her team also found that piggyBat can move within bat cells, other mammalian cells and yeast, showing that it is indeed a still-active DNA element.

Many organisms have developed systems to decrease the frequency at which jumping genes move, Craig says. Such systems are a component of immunity, protecting mammals from retroviruses, as well as from the risk that jumping genes will wreak havoc by interrupting an important gene.

Over time, the protective systems have made most mammalian jumping genes inactive. The finding that a bat species is host to an exception, combined with the fact that bats are particularly susceptible to viruses, may indicate that the systems that protect us from dangerous genetic material are not as well-developed in bats, Craig says. But whatever the reason for its presence, piggyBat “opens up a window for studying jumping gene regulation in a mammal where the element is still active,” she says.

This future research should yield insights on the workings of jumping genes themselves, as well as on the protective systems that keep them in check, Craig says. Ultimately, her group hopes to custom-design jumping genes that can be used for targeted, safe and effective gene therapy, delivering genes needed to treat disease.
source: http://www.hopkinsmedicine.org/news/media/releases/rare_form_of_active_jumping_genes_found_in_mammals

Strange behavior: new study exposes living cells to synthetic protein

One approach to understanding components in living organisms is to attempt to create them artificially, using principles of chemistry, engineering and genetics. A suite of powerful techniques—collectively referred to as synthetic biology—have been used to produce self-replicating molecules, artificial pathways in living systems and organisms bearing synthetic genomes.
In a new twist, John Chaput, a researcher at Arizona State University’s Biodesign Institute and colleagues at the Department of Pharmacology, Midwestern University, Glendale, AZ have fabricated an artificial protein in the laboratory and examined the surprising ways living cells respond to it.
“If you take a protein that was created in a test tube and put it inside a cell, does it still function,” Chaput asks. “Does the cell recognize it? Does the cell just chew it up and spit it out?”  This unexplored area represents a new domain for synthetic biology and may ultimately lead to the development of novel therapeutic agents.
The research results, reported in the advanced online edition of the journal ACS Chemical Biology, describe a peculiar set of adaptations exhibited by Escherichia coli bacterial cells exposed to a synthetic protein, dubbed DX. Inside the cell, DX proteins bind with molecules of ATP, the energy source required by all biological entities.
“ATP is the energy currency of life,” Chaput says. The phosphodiester bonds of ATP contain the energy necessary to drive reactions in living systems, giving up their stored energy when these bonds are chemically cleaved. The depletion of available intracellular ATP by DX binding disrupts normal metabolic activity in the cells, preventing them from dividing, (though they continue to grow).
After exposure to DX, the normally spherical E. coli bacteria develop into elongated filaments. Within the filamentous bacteria, dense intracellular lipid structures act to partition the cell at regular intervals along its length (see figure 1). These unusual structures, which the authors call endoliposomes, are an unprecedented phenomenon in such cells.
“Somewhere along the line of this filamentation, other processes begin to happen that we haven’t fully understood at the genetic level, but we can see the results phenotypically,” Chaput says. “These dense lipid structures are forming at very regular regions along the filamented cell and it looks like it could be a defense mechanism, allowing the cell to compartmentalize itself.”  This peculiar adaptation has never been observed in bacterial cells and appears unique for a single-celled organism.
Producing a synthetic protein like DX, which can mimic the elaborate folding characteristics of naturally occurring proteins and bind with a key metabolite like ATP is no easy task.  As Chaput explains, a clever strategy known as mRNA display was used to produce, fine-tune and amplify synthetic proteins capable of binding ATP with high affinity and specificity, much as a naturally occurring ATP-binding protein would.

The depletion of ATP in cells of the bacterium Escherichia coli causes them to transition to a filamentous state and form dense lipid structures known as endoliposomes. The structures can be clearly observed in these transmission electron micrographs of increasing magnification.

First, large libraries of random sequence peptides are formed from the four nucleic acids making up DNA, with each strand measuring around 80 nucleotides in length. These sequences are then transcribed into RNA with the help of an enzyme—RNA polymerase.  If a natural ribosome is then introduced, it attaches to the strand and reads the random sequence RNA as though it was a naturally-occurring RNA, generating a synthetic protein as it migrates along the strand. In this way, synthetic proteins based on random RNA sequences can be generated. 

Exposing the batch of synthetic proteins to the target molecule and extracting those that bind can then select for ATP-binding proteins.  But as Chaput explains, there’s a problem: “The big question is how do you recover that genetic information? You can’t reverse transcribe a protein back into DNA. You can’t PCR amplify a protein. So we have to do all these molecular biology tricks.”
The main trick involves an earlier step in the process. A molecular linker is chemically attached to the RNA templates, such that each RNA strand forms a bond with its newly translated protein. The mRNA-protein hybrids are exposed to selection targets (like ATP) over consecutive rounds of increasing stringency. After each round of selection, those library members that remain bound to the target are reverse-transcribed into cDNA (using their conveniently attached RNA messages), and then PCR amplified.
In the current study, E. coli cells exposed to DX transitioned into a filamentous form, which can occur naturally when such cells are subject to conditions of stress. The cells display low metabolic activity and limited cell division, presumably owing to their ATP-starved condition.
The study also examined the ability of E. coli to recover following DX exposure. The cells were found to enter a quiescent state known as viable but non-culturable (VBNC), meaning that they survived ATP sequestration and returned to their non-filamentous state after 48 hours, but lost their reproductive capacity. Further, this condition was difficult to reverse and seems to involve a fundamental reprogramming of the cell.
In an additional response to DX, the filamentous cells form previously undocumented structures, which the authors refer to as endoliposomes. These dense lipid concentrations, spanning the full width of the filamented E. coli, segment the cells into distinct compartments, giving the cells a stringbean-like appearance under the microscope.
The authors speculate that this adaptation may be an effort to maintain homeostasis in regions of the filamentous cell, which have essentially been walled off from the intrusion of ATP-depleting DX. They liken endoliposomes to the series of water-tight compartments found in submarines which are used to isolate damaged sections of the ship and speculate that DX-exposed cells are partitioning their genetic information into regions where it can be safely quarantined. Such self-compartmentalization is known to occur in some eukaryotic cells, but has not been previously observed in prokaryotes like E. coli.
The research indicates that there is still a great deal to learn about bacterial behavior and the repertoire of responses available when such cells encounter novel situations, such as an unfamiliar, synthetic protein. The study also notes that many infectious agents rely on a dormant state, (similar to the VBNC condition observed in the DX-exposed E. coli),  to elude detection by antibiotics. A better understanding of the mechanisms driving this behavior could provide a new approach to targeting such pathogens.
The relative safety of E. coli as a model organism for study may provide a fruitful tool for more in-depth investigation of VBNC states in pathogenic organisms. Further, given ATP’s central importance for living organisms, its suppression may provide another avenue for combating disease. One example would be an engineered bacteriophage capable of delivering DX genes to pathogenic organisms.
 source:http://www.biodesign.asu.edu/news/strange-behavior-new-study-exposes-living-cells-to-synthetic-protein-