The Dangerous Double Life of a Distinctive Diazotroph

For many people around the world, tonight is one of the most anticipated nights of the year. In that spirit, here’s some new science, not under the sun, but under the moonlight and starlight. New research from Wegener, Nagarajan and Pakrasi* describe something new about the Photosystem II D1 protein, an unexpected source that photosynthesis researchers had long written off as conquered or fully explained.

Why would we care about photosynthesis at night anyway? It all comes down to the distinctive lifestyle of the cyanobacterium Cyanothece, which belongs to an interesting class called unicellular diazotrophs (word of the day). Like all cyanobacteria, they perform oxygenic photosynthesis but they have the bonus biochemical ability of nitrogen-fixation.

Nitrogen-fixers are capable of pulling nitrogen gas (N₂) out of the air and turning it into ammonia which the cells can use as a nitrogen source for important molecules like protein and DNA. Other microbes can perform nitrogen-fixation, and you may remember them as the root nodule symbionts of legumes. If you’re not a nitrogen-fixer, you have to rely on ammonia or other nitrate compounds present in the soil, sea or your diet (depending on what kind of organism you are) to make the required nitrogenous biomolecules. Since the Earth’s atmosphere is 78% N₂, you don’t have to be great at math to figure out that acquiring the ability to tap into the atmospheric nitrogen source advances your species in the game of life a considerable number of squares.

What does this have to do with photosynthesis? Why don’t more plants and cyanobacteria get in on this nitrogen-fixation biochemistry? There’s a critical fatal flaw in the photosynthesis/nitrogen-fixation biochemical tango. Nitrogenase, the enzyme responsible for the nitrogen-fixation reaction is hopelessly and irreversibly killed by oxygen. Oxygen destroys the protein enzyme that’s already been made and tells the genome not to even bother wasting energy trying to make it new. If you’ve been paying attention to the blog, oxygenic photosynthesizers like cyanobacteria and plants make oxygen via their PSII enzyme every time light shines on them. So for nonphotosynthetic soil bacteria and other microbes, it’s not a big deal to avoid oxygen and go anaerobic to fix nitrogen. Cyanobacteria, however, must lead dangerous double lives to combine these two opposing biochemical processes.

Anabeana spherica via Wikimedia Commons The more round cell in the center of the string is the nitrogen-fixing heterocyst.

Some diazotrophic cyanobacteria solve this problem by separating these two processes in space. Species like Anabeana can go through a special type of development where the cells divide themselves, but incompletely to form a connected network like beads on a string. Every tenth cell (heterocyst) gets an extra layer of cell wall, destroys its photosynthetic machinery, and commits itself to a life of nitrogen-fixation. These cells share their fixed nitrogen compounds with their neighbors, which in turn share their fixed-carbon from photosynthesis. This is a fascinating process still hasn’t given up all of its secrets and remains an active area of research.

Cyanothece 51142 by Michelle Liberton, Pakrasi lab

Cyanothece, on the other hand, is a more ruggedly individualistic species that manages to perform both oxygenic photosynthesis and nitrogen-fixation within a single cell. It accomplishes this through a carefully controlled circadian rhythm. During the day, oxygenic photosynthesis occurs. During the night, nitrogen-fixation occurs. It sounds so simple, yet this elegance is far from easy. This is a true circadian process, in that, while some cues are definitely derived from light signals, the cells also have a strong internal clock that keeps the time of day and controls what biochemical machinery is active. Thus, near the hour of dawn and dusk, the cells are actively preparing for the metabolism to come. Also, if you were to train a culture of these cells under a certain day/night cycle for several days then switch them to continuous light, they would still maintain their circadian biochemical cycle for several days after the switch.

One issue that has puzzled researchers for some time is that the photosynthetic machinery, like PSII, is actively shut down at night. It’s not just that it’s dark, so there won’t be any photosynthetic oxygen production. Apparently, that’s not good enough. The cells actively shut it down. When researchers take culture samples of these cells during their dark period and shine light on them to check photosynthetic capability, there is no signal. Sure, there’s probably little chance any significant photosynthesis is occurring at night, but Cyanothece isn’t taking any chances. For these cells, no moonlight, floodlights from passing ships, comets, or even the blessed Eastern Star is going to interfere with their nocturnal nitrogen fixation.

Shutting down photosynthesis is no small feat. Again, if you’ve been paying attention to the blog, you will note that I’ve ranted on numerous occasions about how complex PSII is and how difficult it is to assemble  and reassemble from its individual parts. As far as energetics are concerned, it would be a losing battle for Cyanothece to completely disassemble its PSII at dusk only to resynthesize it again the next morning. I don’t care how hard-up it is for a nitrogen source. As it turns out, Cyanothece and other unicellular diazotrophic cyanobacteria solve this problem in a more graceful way, for which Wegener et al have provided new experimental evidence.

Cyanothece, like all other oxygenic photosynthesizers, contains multiple copies of the psbA gene encoding the D1 protein, which is a core subunit of PSII and critical for function. Since the advent of the genomic era, the genomes of numerous photosynthetic organisms have been sequenced revealing these multiple psbA genes, most of which have little or no protein sequence differences. These subtle changes have been linked to cells’ ability to fine-tune photosynthesis under certain conditions. However, Cyanothece and its cousins have a psbA gene copy that is particularly different- psbA4. If one were to take all of the site-directed mutants that photosynthesis researchers made to sort out PSII function over the last few decades since molecular biology techniques were available and threw them altogether into a single mutant gene, you would get psbA4. If PSII contained the D1 protein encoded by psbA4, there would be no C-terminal processing and no active site for water-splitting and oxygen production.

Why would any self-respecting photosynthetic organism retain such an utterly non-functional PSII subunit? The answer is because it does serve a critical function in the daily cycle of Cyanothece, just not during the day. The D1 protein encoded by the psbA4 gene, called a sentinel D1 protein (sD1), is made only for the night, when it does the only thing it still can do- fit right in the center of the PSII complex and prevent PSII function of any kind. There’s no chance of any oxygen production while sentinel D1 is on duty, but the cells can maintain most of the other PSII components in a stable complex (meaning they don’t have to be degraded and made fresh every morning). Then, Cyanothece can turn photosynthesis back on in the morning by replacing a single protein in its PSII complexes. The report by Wegener et al gives the first biochemical characterization of PSII complexes containing a sentinel D1 protein, providing evidence that this strategy works for controlling PSII oxygen production.

Performing oxygenic photosynthesis and nitrogen-fixation within the same cell is biochemically problematic, but the sentinel D1 protein appears to be the secret weapon for this dangerous double life. Through this strategy oxygenic photosynthesis and nitrogen fixation are “Always together, eternally apart.”** Many questions remain regarding the regulation of the transition from functional D1 to sentinel D1 at dusk and the reverse replacement at dawn. There are still outstanding questions regarding the D1 turnover event for any PSII in any photosynthetic organism, so it’s not at all clear what parts of this process Cyanothece uses or whether new factors are involved. Obviously, there must also be some sophisticated regulation to prevent the synthesis of sentinel D1 during the day.

While this work focuses on an organism you’ve never heard of, it has some interesting biotech implications for the future. As I mentioned, being able to both fix CO₂ through photosynthesis and N₂ through nitrogenase puts you at a significant metabolic advantage. And no, I don’t mean you. Remember, we’ve been through this- people are not photosynthetic. It would be great if one day we could engineer certain crop plants to combine these traits. Legumes already have this trick covered through symbiosis, but many other staple crops require tons of nitrogen fertilizer. If researchers could unlock the secrets of Cyanothece’s double life and translate it to plants, then it may be possible to engineer versions of crop plants that don’t require so much nitrogen fertilizer. Thus, one night in the future, engineered crop plants may be fixing nitrogen thanks to the addition of a variant gene they already had.

Johnna

*I know I’ve posted frequently about Pakrasi lab (my thesis lab) research, but this topic was just developing as I was leaving the lab and it’s too cool to pass up.

**Bonus points to you if you get the movie reference.

References and Links:

http://www.jbc.org/content/early/2014/12/18/jbc.M114.604124.full.pdf+html?sid=8f38f7c6-96c1-4085-bbb0-efc6ca425624

Dangerous Photosynthesizers

Because photosynthetic organisms are the energetic foundation of our biosphere, we always tend to think of them as allies, organisms with a positive connotation. Their trademark color green is universally linked with goodness, growth, and life. However, there are some bad apples in the bunch that just seem to have it out for us heterotrophs. Well, maybe not apples (though I’m sure there are some poisonous apples out there somewhere*), but nature is filled with examples of poisonous plants. Many toxins and pharmaceuticals have botanical origins.

The focus of today’s blog post sinks even lower- algae, pond scum, cyanobacteria. Most of these aquatic photosynthesizers quietly convert sunlight to biochemical energy without any ill effects to anyone. I’ve written previously, that under the right conditions (warm and nutrient-rich waters) these otherwise inconspicuous organisms bloom in great numbers and overwhelm their environments. Like all life on earth, algae are programmed to capitalize on favorable conditions for reproduction. The ultimate crashes of these blooms can result in aquatic dead zones, areas with dissolved oxygen levels too low to support life.**

Algal bloom in Lake Erie 2011 from the NASA Earth Observatory Credit: Jesse Allen and Robert Simmon via Wikimedia

However, in some cases, the effects of these algal ‘blooms’ go beyond sheer numbers. Some algae produce toxins which cause serious health problems for those of us heterotrophs sharing their environment. The individual constituents of these ‘harmful algal blooms’ (HABs) measure in at ~1-2 µm, but they can wreak all kinds of havoc on the scale of large cities. HABs can stop your summer fun by forcing beaches to close or eliminating certain shellfish from your diet, but this summer the problems went beyond recreation to something much more fundamental- potable water.

Clean, safe drinking water is a fundamental service of human civilization. In our modern society, just turn on the tap, cook, clean, bathe, drink. It has been such a staple of American cities that it is taken for granted. That is, until it’s no longer available. That was the exact situation in Toledo earlier this month. A large American city, in the year 2014, was without safe drinking water for a whole weekend. Approximately, 500,000 citizens were affected. All because of toxic algae.

Microcystin-LR chemical structure Credit: cacycle via Wikimedia

In this case, the culprit was a bloom of cyanobacteria which produce the toxin microcystin. This molecule is harmful to the health of humans, pets and wildlife by acting as a liver toxin. It also has neurotoxic effects. The toxicity of microcystins has been extensively characterized and long-known to be associated with certain cyanobacterial species. Because of the potential adverse effects of microcystin-producing cyanobacteria on modern water supplies, treatment facilities routinely check the levels of this toxin. Only one part per billion of this molecule is considered acceptable. When a microcystin-producing algal bloom occurs near the intake of a municipal water supply (as it did for Toledo this month) the facilities can quickly be overwhelmed causing the water supply to exceed acceptable microcystin levels. The situation is compounded by the fact that microcystins are resistant to boiling. While boiling water may destroy other toxins or contaminating bacteria, it only concentrates microcystins. In order to bring the toxin levels down, the problem must be addressed at the water treatment facility (using methods like activated carbon, ozone treatment and membrane filtration). By adjusting the normal procedures to account for increased microcystins, the water supply can be treated to once again safe levels. All of this is accompanied by exhaustive analysis of microcystin levels and vigilant monitoring after the incident.

Scientists and government agencies are always working to monitor our water systems for HABs. Check out some of the links below for descriptions of continuous efforts to monitor our environment for HABs. How do we get to the point of 500,000 people without water for a weekend with everyone watching out for it? I mean, we’re watching it from space! Even with all of these sophisticated tools and models, nature can be surprisingly swift. Check out these images and reports from the NOAA Great Lakes Environmental Research Lab on Aug 1, 2014 and Aug 4, 2014 showing false-colored images tracking the algal growth.

Simultaneously in the news cycle with the Toledo water crisis, two Ebola patients were being treated in premier isolation facilities on American soil. The nation’s attention was rapt with the details of their treatment and speculation was rampant as to the possibility of an outbreak in America (an infinitesimally small probability not worth talking seriously about among scientists and epidemiological experts, but a great ratings-driver). Worldwide, the Ebola death-toll numbers in the thousands, not just from this year, but ever. Based on pure body count, there are many deadlier infectious diseases, which we as the public dismiss more easily. Beyond those numbers, the lack of clean water for drinking, food preparation and sanitation results in the deaths of ~3 million people every year across the globe. A safe and reliable water supply, as a basic right, continues to elude human civilization.

Water, water everywhere, but not a drop to drink… Credit: de:Benutzer:Alex Anlicker

HABs are only a part of the world’s water problem. However, the disruption of Toledo’s water supply should have been an event that caught our attention and held it for a while longer. It may be easy to turn on the tap, but getting the clean water to that point takes a significant amount of effort with infrastructure maintenance, monitoring and treatment. All of these things are largely invisible to us in modern society. Unfortunately, all of these things are affected by other societal choices like economics, aesthetics, environmental regulations, and the practices of our agricultural systems and other industries. As a society, we should start having the longer, difficult conversations necessary to attack this complicated problem rather than the transient chats that occur when we are in crisis mode. Find out about your community’s water situation and the issues related to your supply. Talk to your community leaders today to ensure that safe water is part of your future.

 

 

Johnna

*Hey, it’s hard to transition away from the summer’s Disney theme completely in a single post. However, I’m not just talking about the evil queen’s poisoned apple from the fairy tale. Apples have a huge amount of genetic diversity and I’m sure there are some varieties out there that are poisonous or so foul-tasting that you would think they are. After all, apples concentrate toxic substances in their seeds.

http://www.thepoisongarden.co.uk/atoz/malus_john_downie.htm

**In case you’re wondering, this year’s Gulf of Mexico hypoxic zone measured ~5500 square miles. That’s not breaking any records for size, but still about as large as the state of Connecticut. Read more about it here.

 

References and Links:

http://oceanservice.noaa.gov/hazards/hab/

http://www.cop.noaa.gov/stressors/extremeevents/hab/current/noaaHab.aspx

http://www.cop.noaa.gov/stressors/extremeevents/hab/current/HAB_Econ.aspx

http://www.cdc.gov/nceh/hsb/hab/default.htm

http://www.whoi.edu/redtide/page.do?pid=14898

http://www2.epa.gov/nutrientpollution/harmful-algal-blooms

http://www2.epa.gov/nutrientpollution/effects-human-health

http://www.dispatch.com/content/stories/local/2014/08/04/this-bloom-is-in-bad-location.html

http://www.lakeeriewaterkeeper.org/

http://www.gulfhypoxia.net/news/default.asp?XMLFilename=201408111452.xml

http://en.wikipedia.org/wiki/Microcystin

http://iaspub.epa.gov/tdb/pages/contaminant/contaminantOverview.do?contaminantId=-1336577584

http://www.weather.com/health/what-you-need-know-about-microcystin-toledos-water-toxin-20140804

http://water.org/water-crisis/water-facts/water/

http://water.org/water-crisis/one-billion-affected/

http://www.cdc.gov/healthywater/global/wash_statistics.html

April Berry Go Round: Plant Colors

When your life depends on light, as it does for plants and other photosynthetic organisms, color is important. Even the most flamboyant displays are functional not frivolous. Beyond being a consequence of the biophysics of photosynthesis, these exhibitions are used to attract pollinators, to send warnings to would-be herbivores, and to adapt to their surroundings. Of course, humans find these colors fascinating for reasons unrelated to their purposes for the plants. As a result, these beautiful botanicals have become entwined with human culture as well- in our gardens, in our kitchens, and in our artistic expressions. This edition (#69) of the Berry Go Round blog carnival explores the diverse topic of plants’ use of color.

Before we delve into the details of how plants use their colors or the extreme colors plants employ, start with this post from the As many exceptions as rules blog, which describes the extensive biochemical repertoire of plant pigments. Now let’s take a journey across the wavelengths of the spectrum of visible light. As you will see, plants don’t let any wavelengths go to waste. There is a purpose for every color and then some.

Spectrum of visible light via Wikipedia Original source: hi:Image:Srgbspectrum.png

When it comes to the color red, this vibrant color serves as an attractant that is perfectly adapted to the visual systems of their bird pollinators. The color red is also a strong attractant for humans because plants like Rubia tinctorium became so popular at one point as to be synonymous with empires.

Colors can be simultaneously beautiful and delicious as described in this post by Sarah Shailes. If you’ve ever wondered why saffron is so expensive, you should definitely click the link. A common flash of yellow in Louisiana these days is the yellow iris, which is the subject of this post by Dave Spier at the Northeast Naturalist blog.

Green is so ubiquitous among plants that it is often taken for granted. Yet plants do not take it so lightly, as explored in this post Beyond Green at the Postdoc Street blog. This post on the Plants And Rocks blog also describes how green bark can help give aspen trees a head start on photosynthesis before their leaves develop.

In the literary world, violets may be blue, but other botanicals come much closer to true blue in real life. One striking example that you may not be familiar with are the seeds of the Malagasy traveller’s tree (Ravenala madagascariensis) described in this post from Kew Millennium Seed Bank blog. When it comes to indigo, the plant and the color are one in the same. Find out more about the plant behind this pigment in this post by Sushmitha on a Blog of Scientific Nature.

There’s a new kid on the block when it comes to purple plants- the tomato. There are some heirloom varieties of purple tomatoes, but recently genetic engineering has been used to increase the amount of anthocyanins (antioxidant pigments) in tomatoes. Read more about them (and other genetically engineered plants) in this post by Izzy Webb on the John Innes SVC blog. Of course, other naturally-occurring pigments are found in our favorite edible plants. Check out the chemistry in these posts on Beetroot and Grapefruit from the Compound Interest blog.

Plants also display other colors beyond the typical rainbow of the visible spectrum. In fact, one species of tree, the rainbow eucalyptus, lives up to its name in a display of color more akin to the neon colors of an ‘80s music video than nature.

A grove of Rainbow Eucalyptus Eucalyptus deglupta trees, planted along the hana highway, Hawaii. Credit: Amelia via Wikipedia http://www.flickr.com/photos/amelia525/303049143/

Other plants are studies in monochrome. Ornamentals like the black pearl peppers described in this post by Mark Dwyer at the blog of Rotary Botanical Gardens in Wisconsin are entirely ink black– leaves and fruit. Emma Cooper offers you a steamy list of fifty shades of grey in the garden. Don’t worry, these are suitable for public display. Rebecca Deatsman describes a plant on the other end of the spectrum on her blog Rebecca in the Woods- the completely white Indian Pipe. It may look like a fungus, but it’s really a plant despite the fact that is eschews a photosynthetic lifestyle. I’ve mentioned before on this blog that it lacks all pigments required for photosynthesis and therefore lives a shameful (for a plant) heterotrophic existence.

Black Pearl Pepper by Mark Dwyer, Rotary Botanical Garden reused with permission

Montropa Uniflora stem detail. Matthew S. Staben via Wikipedia

Over at The Botanist in the Kitchen blog, Jeanne L. D. Osnas serves up a colorful nasturtium salad with a helping of explanations on the patterns plants use as ‘nectar guides’ to direct their pollinators to their sweet spots. There are also some great examples of how plants use colors that human eyes can’t see. When the pollinators are insects with the ability to see ultraviolet colors, some plants color their flowers with pigments that reflect UV rays. Take some time to chew on the fact that the flowers that bees see have patterns on them that you cannot see.

Mimulus flower photographed in visible light (left) and ultraviolet light (right) showing a nectar guide visible to bees but not to humans. By Plant Surfer via Wikipedia

Simon Norton Museum via Wikipedia

Think it’s silly that plant patterns would create such a frenzy in a species from another kingdom? Before you start to feel too superior, consider the tulip. Fortunes were traded over the newest colors and patterns of tulips in the late 17^th century in the Netherlands. Shown at the right is a picture of the Semper Augustus. This tulip is famous for being the most expensive tulip sold during the tulipomania in the Netherlands in the 17^th century. The highest sums were traded in speculation over bulbs producing the striped or variegated varieties, but the underlying cause wasn’t superior genetics. Read this post by Suzi Claflin on the Direct Transmission blog that describes the virus that caused the hullabaloo.

It isn’t all elaborate chemistry and genetics behind the colors plants use. The most interesting expressions of color are the polish of shine and the shimmer of iridescence, where the illusions are a trick of physics. For more on what this is, check out Anne Osterrieder’s post on structural color on the AoB blog. It may seem like something from science fantasy, but this earthly phenomenon is real and the research subject of Dr. Heather Whitney as she writes in this blog post. The shiniest living things on Earth are the fruit of Pollia condensate. As Ed Yong writes on his Not Exactly Rocket Science blog, they look more like Christmas decorations than edible fruit. Even certain seaweeds and algae are iridescent as illustrated in this post on the Coastal Pathogens blog by Michiel Vos.

Pollia condensata Credit: Juliano Costa via Wikipedia

For some plants, a single color is not good enough, and they change color when environmental conditions change. The most familiar of these is the spectacular display deciduous forests put on each autumn. I’ve written about the biochemistry behind that event on this blog previously. Gardeners may also be familiar with the fickle hues of hydrangeas. Read this post at the Reaction of the Day blog for a refresher on pH and this plant’s pigments. Poinsettias are also good at telling the pH as described in this post at the Compound Interest blog.

Hydrangeas in France Credit: Ookwormbay7 via Wikipedia

Plants are only one class of photosynthetic organism, but they are far from the only ones prone to pageantry when it comes to pigments. Remember the horse of a different color from the Emerald City in Oz? The cyanobacterium Fremyella diplosiphon is a real-life version that changes its color based on its environment. If you’re curious how and why they do it, check out my post from earlier this week. Even on a macroscopic scale, there is plenty of algal color diversity to be found under the sea as shown on this post by Michiel Vos on the An Bollenessor blog.

I think this linkfest has literally spanned the spectrum on the use of color by photosynthetic organisms. If you enjoyed this month’s Berry Go Round, check back next month for the next edition at the Roaming Naturalist’s blog exploring important backyard plants.

UPDATE: Be sure to check out this link from Jessica Budke at Moss Plants and More. It’s not just about the color plants use, but the colors plants ‘see.’ And this latest post from The Botanist in the Kitchen blog about botanical dyes.

Johnna

Prokaryotic Picassos

Photosynthetic organisms are brilliant artists when it comes to their use of colors. Most people are familiar with the wide range of colors plants display. Roses are red, violets are blue, anyone? Well, cyanobacteria package prodigious amounts of pigments in prokaryotic form. Their name is derived from their blue-green color. It’s kinda their trademark. As photosynthetic organisms, they have plenty of chlorophyll in their photosynthetic machinery. Their blue color comes from their light-harvesting antenna, the phycobilisomes (PBS), which are comprised of proteins with blue pigments.

Phycocyanoobilin from Wikipedia

Phycocyanin from Wikipedia

Phycobiliproteins bind blue bilin pigments and assemble into discs; these discs assemble into the large phycobilisome complexes that sit on top of the photosystems and funnel light energy to them. The PBS looks like a giant blue octopus with a central core and a number of ‘arms’ of phycobiliprotein discs that extend out in all directions. Because these complexes are particularly good at absorbing orange light, which chlorophyll does not, the PBSs extend the wavelengths of light (and therefore the energy) available to cyanobacterial photosystems.

 

Some cyanobacteria like Fremyella diplosiphon can take color to another level. They can change the composition of their PBSs according to the color of light available to them in a process called complementary chromatic adaptation (CCA). This is possible because some species of cyanobacteria have two varieties of phycobiliproteins, each of which coordinates a distinct pigment. Phycocyanin binds the blue phycocyanobilin chromophore, which efficiently absorbs orange and red light and appears blue in color. Phycoerythrin binds the pigment phycoerythrobilin, which efficiently absorbs green light and appears pink to red in color. Thus, these cyanobacteria have blue PBSs in red light and have a typical blue-green appearance. However, in green light, they produce PBSs containing phycoerythrin and have a rose or red-colored appearance.

Phycobiliproteins, bilin variation, and group III CA regulation. (A) Phycocyanin and phycoerythrin (blue and red lines, and in vials) absorb in regions of the visible spectrum not well absorbed by chlorophyll or carotenoids. Attached bilins: PEB, phycoerythrobilin; PCB, phycocyanobilin. (B) Natural diversity in coloration of many different cyanobacterial species due to variation in their bilin content [photograph by Christophe Six. Reproduced with permission from Six et al. (2007) (Copyright 2010, Biomed Central Ltd.)]. (C) Group III CA regulation model for F. diplosiphon in red light, showing the asymmetric regulation of red-light active genes (orange) and green-light active genes (yellow) by the Rca and Cgi systems. Dashed line represents proposed repression by the Cgi system; yellow balls, phosphoryl groups; blue boxes, RcaC binding sites.

To achieve this dramatic color change, the cells have elaborate systems for sensing the incident light wavelengths in their environment and eliciting the appropriate response. Changing out the PBS antenna requires changes in gene expression of the protein components as well as the pathways that synthesize the necessary pigment (phycocyanin vs. phycoerythrin). It also means that the existing phycobiliproteins and their pigments must be degraded and their components recycled. The PBS antenna complexes comprise a significant portion of soluble protein within cyanobacterial cells, so it is important that the pieces be reused, no matter the light condition. All of these requirements add up to a sophisticated system of multiple players working to make this simple-looking color change happen on cue. This phenomenon is the research area of a number of groups working on cyanobacteria and photosynthesis.

In this way, cyanobacteria are similar to Picasso, going through a blue period and later a rose period. In the case of Fremyella, the change is not due to swings in mood from melancholy to cheer, nor is it purely aesthetic. In biology, even beauty is functional. In the case of complementary chromatic adaptation, it allows cyanobacteria to most efficiently harvest the wavelengths of light available to them. You may be wondering when cyanobacterial cells would be exposed to differently colored light sources outside of an elementary school science fair project. Well, this scenario is more commonplace than you would think. For example, cyanobacteria cells close to the surface of a pond or ocean would be exposed to higher amount of red light compared to those deeper in the water column.* Those cyanobacterial cells that find themselves deeper in the water column would have much less red light because it does not penetrate as well as green light and other photosynthetic organisms living closer to the surface have already absorbed all of the red wavelengths of light allowing only green to pass through.**

Light microscopy image of a normal ‘Fremyella diplosiphon’ colony (green, left side) adjacent to a ‘F. diplosiphon’ mutant capable of only producing the light-harvesting pigment phycoerythrin (red, right side), growing on an agar plate in red light. Credit: David Kehoe, used with permission

The image on the left may appear to be some kind of impressionism masterpiece or abstract work by a yet-to-be-discovered artist, but really it’s the combination of two cultures of Fremyella. The green side is the normal strain, while the red side is a mutant that contains only the red phycoerythrin pigment.

This post was written for the April Berry Go Round blog Carnival. Plants aren’t the only photosynthetic organisms that use color beautifully and efficiently.

 

 

 

Johnna

*For those of you paying attention, this means they would use the blue phycocyanin-pigmented PBSs and appear blue-green.

**This means that cyanobacterial cells deeper in the water would adapt to use phycoerythrin in their PBSs and appear red.

References and Links:

http://www.prl.msu.edu/faculty/montgomery_beronda

http://en.wikipedia.org/wiki/Cyanobacteria

http://www.pnas.org/content/107/20/9029.full

http://www.psi.cz/ftp/publications/others/david/Grossman2003.pdf

http://www.bio.indiana.edu/faculty/directory/profile.php?person=dkehoe

http://sites.bio.indiana.edu/~kehoelab/research.html

http://www.pnas.org/content/107/20/9029.full

http://en.wikipedia.org/wiki/Phycocyanobilin

http://en.wikipedia.org/wiki/Phycocyanin

http://en.wikipedia.org/wiki/Phycoerythrin

 

Some Answers for Q

Today’s post features new work from Liu et al published in PNAS (paywall). Since the subject is near and dear to my scientific heart, it’s also a mash-up of some of my work too. My favorite enzyme Photosystem II (PSII) uses sunlight to split water to make oxygen and shuttle electrons down the photosynthetic electron transfer chain. So, yeah, it’s kinda important and lots of scientists (really, not just me) would like to know everything about how it functions.

Knowing the structure of this large complex helps our understanding of how it works. PSII has an extensive parts list (more than 20 proteins and even more cofactors like pigments and metals). The proteins are named PsbA-PsbZ, Psb27 and the numbers increase numerically as we continue to identify subunits. It’s quite the alphabet soup. This post is brought to you by the letter Q- PsbQ, that is. I’ll bring you up to speed on what we know about this protein and what new answers we have from Liu et al’s new publication.

PsbQ is a small protein associated with the lumenal side of PSII. There are quite a few differences in extrinsic protein composition in plants vs. cyanobacteria. Researchers have long known about the PsbQ protein in plants, but a similar protein has only more recently been identified in cyanobacteria.

Differences in the lumenal extrinsic proteins, plants vs. cyanobacteria

Some of my thesis work involved characterizing the specific defects of cyanobacterial PSII in the absence of the PsbQ protein. When PsbQ isn’t around, the effects are subtle but significant. The PSII complex isn’t quite as stable and has lower activity under stress conditions like nutrient limitations. To truly peg the PsbQ protein as a legitimate PSII subunit and not just some co-purifying protein with modest effects on PSII function, I resorted to biochemistry (of course!). The common way of purifying PSII with a his-tagged CP47 (PsbB) protein results in PsbQ as a co-purifying protein. I created a his-tagged version of PsbQ and purified PSII. This reciprocal purification experiment demonstrated that PsbQ was indeed a PSII subunit. Not only that, but the PSII complexes I purified were much more stable and had higher activity than the CP47-tagged complexes. However, I recovered much less PsbQ-tagged PSII relative to my purifications of CP47-tagged PSII. These results were interpreted to mean that the PsbQ-containing PSII complexes were the subset of PSII in a cell that were fully-assembled and functional. On the other hand, CP47-containing PSII complexes represent a broader spectrum of PSII complexes in varying states of assembly; as such, their overall activity and stability is lower than the PsbQ-containing PSII. My work also showed some effects of the loss of PsbQ on the PsbV protein, so I made a cartoon model for PSII assembly that looked like this:

The PSII subunits are colored as follows, D1 (light green); D2, Cyt b559, CP43 (dark green); CP47 (cyan); Psb27 (pink); PsbO (teal); PsbV (dark blue); PsbU (light blue); PsbQ (purple); and damaged D1 (yellow). The top half of the cycle represents steps in the synthesis half of the pathway resulting in fully assembled dimers on the far right, and the bottom half of the cycle shows the disassembly of the complex and removal of the damaged D1 protein.

So this is what I thought the PsbQ-containing complexes looked like:

Despite the experimental evidence that PsbQ is a legitimate PSII subunit, it is not present in our current structural models. For a number of technical reasons, the PsbQ protein just doesn’t exist in the PSII material that forms crystals that are used to make our structural models. This is a problem for researchers like me because I’d like to know where PsbQ is relative to the rest of PSII. Liu and co-authors* addressed this question using a different experimental technique. They treated PsbQ-containing PSII complexes with chemicals that would covalently crosslink protein components with one another. Then they used mass-spectrometry to identify which protein fragments got linked to one another and where.** Once a handful of crosslinks were identified (PsbQ+PsbO, PsbQ+CP47, even PsbQ+PsbQ***), these data create constraints on the position and orientation of the PsbQ protein.

Synechocystis PsbQ structure http://www.rcsb.org/pdb/explore.do?structureId=3LS1

How is this possible? Other researchers have experimentally determined the structure of the PsbQ protein by itself (not part of the PSII complex). I’ve mentioned before that we know the structure of cyanobacterial PSII without PsbQ.

These structures are combined with one another and modeled to take into account the constraints of the cross linking data to show where PsbQ resides within PSII. So, where is it? Sitting right on top of the PsbO protein near the interface of the PSII dimer. (yes, there’s a much better figure in the paper.) How does that compare to my cartoon model from 2007? Way off. (See above)

So what new answers do we have for PsbQ?

Evidence suggests that the PsbQ protein interacts closely with the PsbO and CP47 proteins (and even with another copy of itself) at the PSII dimer interface. Liu et al did some additional biochemical work to sort out the relationship between PsbO and PsbQ. As it turns out, the PsbO protein is required for PsbQ association.

This isn’t the end of the story for PsbQ. In science, the quest continues. Here are my new Q’s for PsbQ:

What about the relationship between PsbQ and PsbV? There is no evidence from cross-linking that these proteins physically interact, but other biochemical analyses suggest that the absence of PsbQ affects the stability of PsbV. Is there a physical association that could not be detected using this crosslinking method (i.e. no reactive residues on both proteins within the right distance or other technical issues with detection)? Alternatively, are there widespread conformational changes within PSII when PsbQ binds that also effect PsbV?

What’s really going on with the N-terminus of PsbQ? In the structure and the latest model, it just floats off from the helices into oblivion. It’s unlikely that this represents the physiological state of the N-terminus of PsbQ in vivo. The structure probably tightens up once it makes contact with other components of PSII. Also, the N-terminus of the mature protein has a lipid modification that anchors it within the membrane. The model of PSII with PsbQ probably still has some wiggle room in it to stretch the N-terminus so that the end reaches the thylakoid lipid bilayer.

What does this mean for higher plants? Is PsbQ in an analogous position close to PsbO in plants? These proteins are common between plants and cyanos, but their sequences are quite different. Could the overall structure be conserved even though the specifics are not?

We may have some new answers, but there are always more questions to pursue.

Johnna

*This is work Haijun did while in the Pakrasi Lab (my thesis lab) at WashU. It’s a good thing my lab notebooks so clearly described everything I had done and where my strains were in the freezer!

**If this is starting to sound like déjà vu, you probably read my post Photosynthesis Goes Voltron. It was also work by Haijun Liu using chemical crosslinking and mass spec techniques to identify interactions among other protein complexes. It truly is a powerful technique with lots of applications especially in the realm of difficult-to-work-with protein complexes. Also, this makes him the first author to be highlighted twice on my blog. I’m sure he’s updating that under the ‘awards’ section of his CV.

***Is that legal? Yes, proteins can crosslink to themselves if two reactive residues are close enough to one another. In this case, they are nowhere near one another and can only be explained by an interaction between two separate PsbQ molecules, which must be in close contact with one another.

References and Links:

http://www.pnas.org/content/early/2014/02/13/1323063111.abstract

Sidekick (Cont’d Adventures of Superhero PhD)

Science doesn’t stop for holidays, so Superhero PhD must always be vigilant- even if the Ivory Tower is officially closed. For biology research labs, something is always growing and can’t be left unattended for two weeks or more. For these working holidays in the lab, Superhero PhD has a special sidekick- PhD Junior. No really, he is a literal sidekick. One of his superpowers is stealthily climbing into bed with Superhero PhD and SuperChef whereupon he flops and kicks the both of us until it is approximately 30 minutes before the alarm clock buzzes (Yes, just enough time to slip back into such a deep sleep state so as to achieve maximum aggravation by the alarm). Below is his character fact sheet:

Alter ego: Deuce* (4 yr old male)

Tag line: If I have to come to the lab today, we’re going to see Mike the Tiger. It’s really not as much like Monster’s University as Superhero PhD promised.

Special powers: Pushing elevator buttons, adorable pronunciations of Chlamydomonas and Thermosynechococcus

Transportation: A booster seat with cup holder in the backseat of the PhD-mobile

Sidekicks and Associates: Legos, Zoobooks and a Kindlefire loaded with StarWars Angry Birds and Netflix

Nemeses: stairwells, vegetables

Backstory (read with the gravitas of a movie-trailer voice-over): PhD Junior is the spark of chaos in Superhero PhD’s life that pushes her to be increasingly efficient. His sense of wonder and non-stop questions about the world around are an inspiration of Superhero PhD.

Skills Breakdown:

Speed 20

Endurance 20

Stealth 30

Agility 30

During one short visit to check on cultures, Superhero PhD and PhD Junior approach the locked Ivory Tower. It is practically abandoned on the cold holiday, but bacterial cultures must be checked. But first, a potty break. As the mission resumes, Superhero PhD makes for the stairwell. The growth chambers are in the basement and it is only one floor down. PhD Junior balks at the dim and somewhat sinister-looking stairwell. Apparently, they are like kryptonite to him. Anyway, PhD Junior’s special skills include pushing elevator buttons. Superhero PhD says, “Of course we can take the elevator.” But silently calculates how long it might be before someone could come extricate them from the elevator on a holiday (Superhero was stuck for a while on said elevator once when she was still pregnant with PhD Junior). Fortunately, today the elevator functions without incident.

Since PhD Junior only makes rare appearances in the lab, his skill set it not often helpful for the experiments at hand. However, he is honing his own superpowers of logic, reasoning and knowledge by incessantly inquiring, “What’s that? What’s this? Why did you do that?” Superhero PhD is able to answer all of these questions with ease as she carries out her tasks. She peers at the plates of cyanobacteria evaluating their growth. Deep in thought for just a moment, then she snaps back quickly because the questions have stopped from PhD Junior. Silence is always suspicious. Out of the corner of her eye, there is a sudden dimming, and she focuses her attention like a laser on PhD Junior who is now very close to the fluorescent light banks and shaking cultures of Chlamydomonas that belong to the Moroney lab. “Did you just pull the chain on those lights?” asks Superhero PhD. A shy smile broadens across PhD Junior’s face as he nods. Superhero PhD turns the bank of lights back on. “Don’t touch anything” becomes the mantra of the mission.

When PhD Junior is in the lab, Superhero PhD must kick her superpowers up a notch to channel his excess energy and dangerous curiosity. For longer missions, “Don’t touch anything” and “Do you need to potty” become regular utterances for Superhero PhD. Monday was a longer mission fraught with danger. OK, not as dangerous as if he would have gone to work with SuperChef where everything in the kitchen is hot and sharp, but pouring gradient acrylamide gels and silver staining another gel are not small tasks in the presence of a 4 year old. Supplies are brought in to keep PhD Junior occupied- books, legos, snacks in a T-rex lunchbox, cash for the vending machine on the first floor, a Kindle fire loaded with apps for Netflix and Star Wars Angry Birds (Pro tip: Turn off one-click ordering for Amazon.com and the appstore if a preschooler will be using your Kindle fire unattended for long periods of time.)

PhD Junior is first taken aback when Superhero PhD dons her uniform (lab coat). “Don’t be a Dr.!” PhD Junior pleads. In his defense, the only people wearing lab coats that he interacts with are medical professionals of some kind trying to give him a shot or look down his throat or ears with an uncomfortable instrument. Superhero PhD quickly diverts his attention with the Kindle and bag of distractions. He plays Angry Birds for an hour. Superhero PhD dives into her experiments with lightning speed, arriving at a critical moment just as PhD Junior wanders over to her bench. Let’s just say that loading an acrylamide gel while fending off a 4 year old playing ninja fight is now one of Superhero PhD’s superpowers.** The critical gel was loaded. No one was exposed to any toxins.

Superhero PhD successfully manages to again divert PhD Junior’s attention with games and Scooby Doo on Netflix as other experiments are done. However, just as a critical stage is reached in silver-staining an acrylamide gel, PhD Junior announces, “I’m hungry!” Superhero PhD  is at first taken aback by this because one of her superpowers is working without eating (or going to the bathroom) for hours on end as experiments are executed. Nevertheless, she materializes food from the lunchbox and sets up lunch time just outside the open door of the lab (no food or drink allowed inside of course). Superhero PhD’s experiment continues.

By mid-afternoon the mission is almost complete, which is perfect timing because PhD Junior has eaten through all of the provisions packed by Superhero PhD. The last thing that must be done is scan the stained gel. This must be done in a separate lab room, but PhD Junior is happily assembling Legos in the main lab room. Superhero PhD explains the dilemma to PhD Junior and offer options- stay with Legos or go with Superhero PhD. “I’m building a Lego car!” is the terse reply. After 10 minutes of scanning and saving the image, Superhero PhD’s work is done for the day. She returns to the lab, but PhD Junior is nowhere to be seen. It is quiet. Superhero PhD nervously calls for PhD Junior. No answer, she detects the slightest movement out of the corner of her eye. Then she sees small grey tennis shoes under the bench where the Legos are. First Middle Last Name (redacted)! PhD Junior sheepishly emerges from under the lab bench and says, “I was hiding for you.” Superhero PhD is relieved that PhD Junior is found and her anxiety is eased only slightly by PhD Junior’s misuse of prepositions. They have a serious discussion about Not. Hiding. Evar.

Mission accomplished, it is time to leave the lab. It takes 20 minutes to pack all of PhD Junior’s gear. This includes finding a way to hand-carry the Lego Car and House constructed by PhD Junior. He adamantly refused to disassemble them (ala Johnny Five) and place the pieces back in their case. Everything makes its way back to the PhD mobile and PhD Junior is sleeping before they reach their secret lair.

Johnna

*For those of you paying attention his nickname is Deuce Roose.

**Remind me to update my CV and LinkedIN profile.

Cyanobacteria Shed

Here’s something new under the sun… cyanobacteria shed in the ocean.

Most people are well aware of the fact that dogs and cats shed, but did you know that oceanic cyanobacteria also shed?* OK, so the cyanobacteria don’t shed hair, and you won’t need a lint brush. In a report in this week’s Science, researchers from the Chisholm lab show that cyanobacteria living in the world’s open oceans are shedding zillions of membrane vesicles. Before you accuse them of pollution, allow me to explain why they are the most important bacteria you’ve never heard of– the Prochlorococci. Don’t worry about being too late to this fountain of knowledge, scientists only discovered them about 25 years ago.

Prochlorococcus species are cyanobacteria that live in the oligotrophic ocean, areas of the world’s open oceans that are nutrient poor when it comes to sources of nitrogen, phosphorous and bioavailable forms of essential metals like iron. These bacteria dominate these otherwise ocean deserts and are possibly the Earth’s most populous species by sheer numbers of individuals. Remember about half of the world’s primary productivity comes from aquatic sources. Prochlorococci are a major contributor to global nutrient cycles and a significant number of the oxygen molecules you are constantly breathing in and out were produced by a Prochlorococcus.

You may be curious as to why scientists have only recently discovered these numerous and critically important photosynthetic organisms. The answer is because of their other trademark- their size.  Measuring in with widths and lengths well under a micrometer, they are the world’s smallest photosynthesizers. Prochlorococci pass through many of the standard filters used for trapping microbes from aquatic samples. They were detected using a cell-sorting technique that could just detect their faint chlorophyll fluorescence signal. These fascinating microbes have been studied in the ocean as well as the laboratory for the last couple of decades, but it is a labor of love. Since Prochlorococci live in such a nutrient-limiting environment, they don’t have much competition. Consequently, these cyanobacteria don’t have to grow very fast, and their small size is another way of conserving their resources. In the laboratory, they don’t grow as fast as the other model cyanobacteria and their extremely small size means scientists have to wait quite a while before significant biomass is achieved for experiments.

So, it is quite a remarkable discovery that something so tiny that has toiled so diligently to fix carbon dioxide into biological molecules would just shed them as tiny vesicles into the ocean. The vesicles released by the Prochlorococci and Synechococci** are small membrane-closed vesicles that contain proteins and even small fragments of DNA and RNA. Surely, the bacteria are not just throwing away precious resources. It’s unlikely that science has stumbled upon the first eating disorder among primary producers, so the obvious question arising from this observation is why the bacteria shed these vesicles.  The authors offer some hypotheses to justify this seemingly wasteful phenomenon.

It could be an example of the cyanobacteria ‘casting their bread upon the waters.’ Preliminary results suggest that- as the saying goes- they find it again. The open ocean environment is a hard place to live, but cyanobacteria are not the only organisms living there. Heterotrophic microbes are also part of the biological community and previous reports have demonstrated that they can stimulate the growth of Prochlorococcus strains. Perhaps the vesicles are a way for the cyanobacteria to feed their beneficial heterotrophic neighbors without putting their cells on the menu.

The vesicles could also serve as bacterial countermeasures against cyanophages, viruses that infect cyanobacteria. For all intents and purposes, the vesicles look like miniature bacteria complete with receptor proteins the phages use to recognize their bacterial victims. The authors were able to observe ‘fooled phages’ that had bound and injected their genetic material into vesicles. In this way, the shed vesicles would be a good defense against these oceanic pathogens. From the phage perspective, an ocean filled with zillions of vesicles, each appearing like a potential host would seem like a lottery they can’t lose. However, the cyanobacteria have ensured the odds are in their favor by shedding many, many more vesicles into the environment relative to the susceptible cells.

Finally, the vesicles could just be the clichéd ‘message in bottles’ of seafaring lore. Even an ocean teeming with trillions and trillions of other Prochlorococci is isolating when reproduction is asexual. For microbes, setting your genetic material adrift on the ocean swells is a hopelessly romantic gesture. That DNA may eventually make its way to another cyanobacterial cell that will find it useful and incorporate the DNA into its genome. It is also possible that the messages are not necessarily genetic. The vesicles could also be used to store and transport specialized metabolites as a defensive or offensive strategy. For now, only the cyanobacteria seem to be able to decipher the messages.

Johnna

* Hopefully, you weren’t unwittingly gifted any pet cyanobacteria for Christmas.

**larger cyanobacteria than the Prochlorococci, but still not in a position to freely give away hard won nutrients

Fun fact: Dr. Chisholm has also co-authored a couple of children’s books on photosynthesis. Check them out here.

References:

http://www.sciencemag.org/content/343/6167/143.full

http://www.sciencemag.org/content/343/6167/183.full

www.ncbi.nlm.nih.gov/pubmed/10066832

http://www.nature.com/ismej/journal/v5/n7/full/ismej20111a.html

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0016805

http://web.mit.edu/newsoffice/2008/microbe-fest-tt0521.html

http://chisholmlab.mit.edu/index.html

The Dark Side Comes to Life in Fluorescent Colors

Something new under the sun… carboxysome assembly in real time.

Today’s post features research out of the Kerfeld Lab from a close colleague of mine, Jeff Cameron.* After doing his thesis work on redox homeostasis in cyanobacteria, he moved on to a postdoc on the dark side of photosynthesis.** His new paper just out in Cell today sheds new (fluorescent) lights on a longstanding question in cyanobacterial carbon fixation.

Cameron et al. Cell Volume 155, Issue 5 2013 1131 – 1140

Schematic for cyanobacterial cell structure showing membrane systems and carboxysomes.

I’ve mentioned before that of the oxygenic photosynthetic organisms, cyanobacteria have an elaborate system for concentrating CO₂ at the site of Rubsico. This prevents Rubisco from getting confused and using O₂ instead, a costly mistake for the cells. Sure, these cells have an array of transporters as well as carbonic anhydrases to interconvert CO₂ and bicarbonate. But the real heart of the cyanobacterial Carbon concentrating mechanism (CCM) is the carboxysome- an intracellular microcompartment where the cells corral all of their Rubsico. This structure allows the cells to handily funnel all of their acquired CO₂ so that it can be fixed by Rubisco. No, I didn’t say it’s an organelle. Cyanobacteria are prokaryotes; while they may break the rules by having an internal thylakoid membrane system, they do not have true organelles in the sense that eukaryotes do.

So what are they exactly? They are protein-based structures, in which an array of Rubsico is encapsulated by hexameric and pentameric shell proteins. Carboxysomes have been well-studied from a structural standpoint and the major protein components have been identified.

Schematic model of the α-carboxysome assembly containing RuBisCO small (dark green) and large (green) subunits and carbonic anhydrase (red). The shell is composed of hexamers (blue), pseudohexamers (light blue, magenta, and light green), and pentamers (yellow) From Kinney et al 2011 Photosynthesis Research

However, all of this beautiful structure work doesn’t provide any information on the dynamics of these components as the cells must construct them.

Cameron and co-authors describe an experimental system that allowed them to follow the assembly of carboxysomes from scratch. This system also allowed them to eliminate carboxysome subunits one at a time and monitor the effects on assembly. From all of these results, they were able to piece together a model for carboxysome biogenesis, something that wasn’t possible from looking at static pictures from single mutants.

Graphical Abstract
Cameron et al 2013 Cell
Biogenesis of a Bacterial Organelle: The Carboxysome Assembly Pathway

Here’s how the method breaks down:

Observations: Mutants of Synechococcus elongatus PCC 7942 that lack the carboxysome structural proteins cannot confine their Rubisco to small areas and require higher than air-levels of CO₂ to grow photosynthetically. Individual mutants of the various carboxysome subunits have variable effects on carboxysome structure, but these are not useful for understanding how the carboxysome subunits come together in a temporal sequence during assembly.

Hypothesis: If a system could be generated to synchronize carboxysome assembly (going from no carboxysomes present to fully assembled carboxysomes), then researchers could probe the effects of the presence/absence of structural components on assembly. This information will allow researchers to deduce the order of assembly events.

Experiment: A synchronized carboxysome assembly system was generated by starting from a mutant of Synechococcus elongatus PCC 7942 lacking all carboxysome structural subunits (no carboxysomes). These structural subunits were added back to this mutant background (as a complete set and as with individual missing subunits) under the control of an inducible promoter. These various cell lines were also engineered to express a fluorescent version of Rubisco were produced to serve as a marker for carboxysome assembly (diffuse fluorescence = no assembly; tight fluorescent spots = assembly).

Results: The control cell line (fluorescent Rubisco + complete set of structural carboxysome subunits under inducible control) showed a diffuse localization of Rubisco throughout the cells prior to the induction of carboxysome genes. After the addition of the inducer chemical, the fluorescence from Rubisco focuses into small green spots indicative of carboxysome assembly. The cases where less-than-the-complete-set of carboxysome subunits were added back to the original mutant cell line, the induction yielded variable results for carboxysome assembly. Some did not assemble any carboxysomes (diffuse Rubisco fluorescence) while others showed intermediate patterns of Rubisco fluorescence (abnormal or partially assembled carboxysomes).

Conclusions: The authors deduced a new model for carboxysome assembly. First Rubisco proteins and the subunit CcmM aggregate together to for pro-carboxysomes (PCs). CcmN is then added to the PCs and begins to associate with the outer shell proteins CcmK2 and CcmO. Finally, when the CcmL protein is incorporated, the carboxysome completely enclosed by its protein shell and released from the PC.

Think Ahead: Carboxysomes are a prototype for other bacterial microcompartments. Other bacteria use similar protein-based microcompartments for specialized metabolism. This system and the insights on the timing sequence of its assembly can applied to these other systems. In any case, these experiments lay the groundwork for engineering similar microcompartments for medical or biotech purposes. For instance, plants might be coerced to make carboxysomes for more efficient carbon fixation. Alternatively, it may be possible to create novel microcompartments in other bacteria for the purposes of efficiently making useful metabolites.

Check out the link below for the fluorescence microscopy video of carboxysome biogenesis. Sorry, there’s a paywall for 12 months.

http://www.sciencedirect.com/science/MiamiMultiMediaURL/1-s2.0-S0092867413013627/1-s2.0-S0092867413013627-mmc3.mp4/272196/FULL/S0092867413013627/fe4fe2db6252ffa45cbd984455447380/mmc3.mp4

UPDATE: There is a free version of Supplemental Video #2. Thanks JGI!

This is after all carboxysome genes have been turned on and you can watch them move and even see some bud off after awhile. (I still prefer the other video where you can watch them form from nothing.) Also, in the current edition of the video caption, they credit James Cameron instead of Jeff Cameron. Sure, James does great cinematic work- just not this one. (It should be fixed on Monday.)

Jeff Cameron, the James Cameron of carboxysome cinema.

 

Johnna

*Jeff and I were graduate students together in Himadri Pakrasi’s lab at WashU. I think he still hears my voice in his head when he does biochemical purifications. “Fast and Cold! Jeff. Fast. And. Cold.”

**Yes, yes, I know. Light-independent. Whatever. ‘Dark’ makes for catchier writing and storytelling.

References:

http://www.sciencedirect.com/science/article/pii/S0092867413013627

http://www.microbemagazine.org/index.php?option=com_content&view=article&id=1863:the-carboxysome-and-other-bacterial-microcompartments&catid=464&Itemid=826

http://link.springer.com/article/10.1007%2Fs11120-011-9624-6/fulltext.html

Room for Improvement on the Dark Side of Photosynthesis

Today’s post highlights another plenary talk from the Midwest Photosynthesis Meeting last weekend. Dr. James Moroney from our very own Louisiana State University presented work from his lab on investigating and improving the dark side of photosynthesis. Click here for a general review on the dark reactions (yeah, yeah they’re really just light-independent).

The carbon fixation reactions seem unnecessarily convoluted, but this is the only way biological systems fix carbon dioxide into organic compounds for energy storage. Beyond this, the process suffers from other inefficiencies. The enzymatic entry point for carbon dioxide into the cycle is Rubisco, which takes carbon dioxide and 1,5-RuBP and forms two molecules of 3-phosphoglycerate. The problem is that Rubisco isn’t a very good enzyme. It’s slow and has a very low turnover, and it often gets confused about its substrates. If it uses oxygen instead of carbon dioxide, it yields only one molecule of 3-phosphoglycerate and one molecule of phosphoglycolate. Cells must then dedicate additional energy recycling the phosphoglycolate into 3-phosphoglycerate and carbon dioxide so these regenerated substrates can re-enter the carbon fixation cycle.

Preventing the oxygenase pathway is particularly important for photosynthetic organisms that are constantly making oxygen as a product of the light reactions. To do this and simultaneously combat the inherent enzymatic slowness of Rubsico, photosynthetic organisms have elaborate systems to raise the local concentration of carbon dioxide in the vicinity of Rubisco. These systems are collectively called the ‘Carbon Concentrating Mechanism’ or CCM. Some mechanisms include trapping carbon dioxide as bicarbonate inside cells. Whereas CO₂ can traverse biological membranes easily, the charged bicarbonate molecule cannot. Among the photosynthesizers, cyanobacteria and algae have much better CCMs than plants. They have elaborate machinery for uptake of both CO₂ and bicarbonate as well as a series of carbonic anhydrases strategically placed to interconvert CO₂ and bicarbonate as necessary. These organisms also physically isolate large quantities of the Rubsico enzyme into specialized locations within the cell.

CHLAMYDOMONAS a green alga             ACTIVA (Photo credit: PROYECTO AGUA** /** WATER PROJECT)

The Moroney lab is interested in figuring out how CO₂/bicarbonate is taken up, partitioned among cellular compartments and eventually fed to Rubisco in both plant and algal systems. This kind of basic understanding of the systems is necessary to eventually improve upon them. For instance, can CCM components from cyanobacteria and algae work in plants to make plants more efficient at carbon fixation. However, before researchers can begin to put these systems together, they must first understand how the independent systems work. A critical limitation in understanding the algal CCM is a lack of available mutants in known or putative CCM components. Moroney described a genetic screen that was performed by his group to isolate the necessary mutants in the algal Chalmydomonas reinhardtii. By sampling tens of thousands of mutants, they were able to identify some known genes they needed mutants in as well as some additional interesting mutants based on phenotype. They have also developed a system for efficiently mapping the positions of the insertion mutations so that novel genes can be identified.

The carbon fixation reactions are the rate-limiting step for photosynthesis and represent the area with the largest margin for improvement. A number of different presentations at the Turkey Run meeting were focused on figuring out the CCM secrets of cyanobacteria and algae.

Johnna

Capturing the Soul of Photosystem II

Something new under the sun… techniques for ‘photographing’ the PSII water splitting reaction.

Some cultures believe that taking your photograph captures part of your soul, and believers shun having their images captured to avoid any potential metaphysical rifts. If this is the case, I suppose it’s safe to say that the world is quite soulless based on the number of selfies floating around the internet these days. As it turns out scientists in my field are having a similar problem with a longstanding question regarding my favorite enzyme, Photosystem II (PSII). We need a technique for photographing PSII that doesn’t destroy its soul (catalytic center- that is).

Note, this is not the actual set up devised by Kern and colleagues

PSII is a multi-subunit membrane protein complex that uses light energy to split water into protons (H⁺) and molecular oxygen (O₂). This important reaction makes it possible for aerobic life on earth.* In order to drive this reaction, a huge redox potential is required and P680, the reaction center of PSII, is the most powerful biological oxidant (1.3 V) identified to date. Mechanistically, this is an extremely difficult reaction to do. Using four successive photons, PSII must sequentially pull four electrons from two water molecules and form an O-O bond. All of this is accomplished by the Oxygen-Evolving Complex within PSII, an inorganic cluster of four manganese atoms and one calcium atom (Mn₄CaO₅). This works because manganese is a transitional metal that can exist in a number of different oxidation states Mn²⁺, Mn³⁺ and Mn⁴⁺. This kind of stable redox space makes it the perfect element for holding onto the four electrons required to split water and form O₂. Unraveling the mechanism by which PSII does this is one of the holy grails in our field. If we only knew how it worked, we could make artificial systems that take advantage of the mechanism for coupling light energy to electron extraction from water (translation = ‘free’ electrical energy from water).

Because of the potential applications, many scientists have been working for many years to figure out how this works. We know some things about the mechanism, but the details we need to know rely on having high resolution structural data on PSII as it is working. We’re not there yet. The currently available experimental tools fall into one of two broad categories. The first is X-ray diffraction structure data or protein crystal structures that represent a static picture of the entire complex (proteins and cofactors). The second is X-ray spectroscopy (absorption or emission). These experiments provide detailed information on the Mn₄CaO₅ cluster, but not really a picture- just distance constraints between atoms.

Over the past decade, photosynthesis researchers have made great strides in improving the pictures we have using these techniques, but there are some significant limitations in the data. The problem is twofold. The X-rays used to generate our beautiful high resolution (1.9 Å) crystal structure model destroys the Mn₄CaO₅ cluster during the process of collecting data. We know from other techniques that the energy in those X-rays reduces the Mn₄CaO₅ cluster to a combination of oxidation states that are not useful for water splitting. On the other hand, the other X-ray spectroscopy techniques are not damaging to the Mn₄CaO₅ cluster, but it doesn’t give a singular picture and multiple models can fit into the distance constraints provided by those experiments. Thus, the pictures that we get from these experiments are inaccurate or inadequate to answer the question of how PSII splits water. Plus, we would like to visually capture the process as the enzyme turns over at physiological conditions (as in not cryogenic temperatures).

Today’s post highlight’s a presentation from the Midwest Photosynthesis Meeting presented by plenary speaker Jan Kern from Lawrence Berkley National Laboratory (LBNL). He along with numerous other teams of researchers have been working to develop the techniques necessary to take snapshots of PSII as it is splitting water in a way that doesn’t destroy the enzyme. Their system will simultaneously collect data using the two different techniques (X-ray diffraction and X-ray Emission) on the same sample in such a way that the PSII Mn₄CaO₅ cluster isn’t destroyed. The secret to this is the use of ultra-short high-intensity X-ray pulses. It’s like having a camera that captures the image so fast you don’t even have time to blink before the flash. Also, the system is designed such that the sample can be illuminated with light for varying numbers of flashes to capture images of PSII during different steps of its catalytic cycle. This has been an enormous effort to design the proper experimental set-up and get it working. The preliminary data and proof of concept were published earlier this year in Science. Don’t get ready to start making electricity from water just yet. The system has not achieved the necessary resolution to see the fine structural changes of the Mn₄CaO₅ cluster as it splits water. However, Jan Kern and others at LBNL are working hard to tweak the components of their system so it does have the necessary resolution for the snapshots we need. Check out the links below for more details on how they did it.

Johnna

*That’s you and me!

References:

Simultaneous Femtosecond X-ray Spectroscopy and Diffraction of Photosystem II at Room Temperature

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732582/

Room temperature femtosecond X-ray diffraction of photosystem II microcrystals

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3382516/

Energy-dispersive X-ray emission spectroscopy using an X-ray free-electron laser in a shot-by-shot mode

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511075/