Surviving in the Pacific Ocean, bacterial style

September 4, 2014 § Leave a comment

Scientists collected samples during a research cruise in October 2011 along a 2,500-mile stretch in the Pacific Ocean, from Hawaii to Samoa. The transect cut across regions with widely different concentrations of nutrients, from areas rich in iron to the north to areas near the equator that are rich in phosphorus and nitrogen but devoid of iron. [Credit: Brian Dimento, University of Connecticut]

Scientists collected samples during a research cruise in October 2011 along a 2,500-mile stretch in the Pacific Ocean, from Hawaii to Samoa.
[Credit: Brian Dimento, University of Connecticut]

Oceans cover 70 percent of the earth’s surface. Given this vast area, how do you thoroughly study how a particular organism survives in it? In a paper just out in Science, researchers analyzed how a type of cyanobacteria ekes out an existence in a 2,500-mile stretch of the Pacific Ocean. The researchers were able to measure for the first time changes in absolute protein concentrations expressed by the community of this cyanobacterium as it weathered scarcities of different nutrients. In another paper in the same issue of Science, a different group of researchers focused on a particular enzyme that helps marine cyanobacteria and other microorganisms survive under conditions of nutrient deficiency and discovered what makes the enzyme tick.

The oceans harbor “much of Earth’s biological diversity,” points out Mak Saito at the Woods Hole Oceanographic Institution who was the first author on the first Science paper. Researchers want to know how the microorganisms, which are the foundation of the marine food web and are essential to the cycling of biologically important elements, survive in oceans. The researchers want to understand how changes in carbon, phosphorus, nitrogen and other elements, caused by natural means or human activity, affect the survival of these critical microorganisms.

But Saito says experiments to analyze the effects of nutrients on marine microorganisms are difficult to do and tend to only give a glimpse of what’s going on. So Saito’s group turned to proteomic technologies because they could use them to quantitatively study the details of the biochemical changes happening in the microorganisms across the Pacific Ocean. The investigators spent a month on a ship, traveling across the Central Pacific Ocean, from Hawaii to Samoa, and collecting microbial protein samples from as deep as 1 kilometer from the ocean. The path they traveled cut through the northern regions that were rich in iron to areas near the equator that were plentiful in phosphorus and nitrogen but lacked iron. For each sample, the investigators filtered  300-800 liters of seawater over 4-6 hours through 0.2-micron filters and froze the samples.

When they got back to Woods Hole, they used two different proteomic methods to study how the protein content changed in their samples that they took from the 2,500-mile stretch of the Pacific Ocean. Saito says that previous studies identified many proteins in the oceans and their relative abundances. In contrast, the measurements he and his colleagues carried out are the first quantitative marine protein concentration measurements “in units of femtomoles of protein per liter of seawater,” he says. “By measuring the concentrations of proteins, we can map changes in the microbial biochemistry across the ocean basin.”

From their data, Saito and colleagues showed that multiple nutrient scarcities affected the cyanobacterial community they chose to track. Their conclusion refutes the notion on which previous work in the field was based, which is microbial growth and protein production was at the mercy of a single nutrient that was scarcest.

Indeed, “biogeochemists have realised that the availability of more than one inorganic nutrient may simultaneously restrict growth of microorganisms, particularly if the concentrations of the nutrients are linked by biological processes,” says Ben Berks at the University of Oxford in the U.K. who led the team in the second Science paper that identified a critical cofactor for an alkaline phosphatase found in cyanobacteria and other microorganisms. The team on the second Science paper is unaffiliated with Saito’s team on the first Science paper.

The phosphatase, PhoX, was reported to be a calcium-dependent enzyme. “However, we noticed that the purified protein had a purple color and we knew this could not arise from calcium ions,” says Berks. “This observation prompted us to investigate the nature of the PhoX cofactor.”

Although PhoX activity is critical in many microorganisms, the enzyme has not been characterized in detail.  “Possibly it reflects the fact that, although the enzyme is widespread in environmental organisms, it is not present in commonly studied model organisms,” suggests Berks.

The investigators crystallized the enzyme and then used an X-ray spectroscopic technique called micro-PIXE as well as electron paramagnetic resonance spectroscopy to identify the metals that were a part of the enzyme. They identified an iron-calcium cofactor.

Previously PhoX was thought to be a simple calcium-dependent enzyme. Calcium is abundant in seawater. If calcium was readily available, Saito says, “people wondered how microbes were maintaining the PhoA zinc alkaline phosphatase.”

Zinc is a rare commodity in marine environments. Why would a microorganism go through the trouble of relying on zinc when there was plenty of calcium to spare for enzyme activity? Now that Berks and colleagues have shown that PhoX depends on iron and calcium to function, says Saito, it now becomes clear the microorganisms are forced to make do with two different scarce elements.

The discovery of a new enzyme cofactor also means that the work of marine biochemists has a long way to go. As Berks notes, “The work demonstrates that there are still novel biological cofactors to discover within the pool of currently unstudied microbial proteins.”

A biomaterial with neuron and muscle properties

April 4, 2013 § Leave a comment

Printing of extended droplet network: Time-lapse movie of an extended droplet network being printed in bulk lipid-in-oil solution. The interval between frames is 20 s. Each frame of the video was cropped around the constructed-network for ease of viewing, as the original recording focused on the stationary capillary-tip, whilst the oil-well was moved in relation to the tip during printing. [Video courtesy of Alexander Graham]

In a paper just out in Science, researchers at the University of Oxford in the U.K. describe making materials in three dimensions that can transmit electrical signals and move in ways similar to how neurons and muscles do. The goal is to make biomaterials  compatible with humans for controlled drug release and repair of damaged organs.

Hagan Bayley says the work began as a basic science project. The investigators were using lipid-coated aqueous droplets in a miniaturized platform  to carry out single-channel recordings of membrane channels and pores. “We quickly realized that we could make interesting devices from collections of droplets,” says Bayley. “Our initial efforts were with just a few droplets, less than 10, and in two dimensions.”

To set up droplets in a network in three dimensions, Gabriel Villar, one of the investigators, made a special 3D printer from scratch. Then taking advantage of the well-established fact that oil and water don’t mix, Villar, Bayley and Alex Graham injected aqueous picoliter droplets into an oil bath. The investigators were able to set up tens of thousands of these droplets and remove most of the oil to form a 3D network of droplets connected by single lipid bilayers.

Each droplet can carry its own specific set of chemicals or biochemicals. When the investigators added a membrane protein called staphylococcal alpha-hemolysin, a pore that incorporates into lipid bilayers, a droplet could use the membrane protein to communicate with its neighbors. It did so by allowing an electric current to flow through the membrane proteins. In this manner, the investigators were able to send a rapid electrical signal along a specific path through the droplet network, much like a neuron.

A rectangular printed droplet network spontaneously folding into a circle.[Image courtesy of Gabriel Villar, Alexander D. Graham and Hagan Bayley (University of Oxford)]

A rectangular printed droplet network spontaneously folding into a circle.
[Image courtesy of Gabriel Villar, Alexander D. Graham and Hagan Bayley (University of Oxford)]

The investigators also demonstrated that by tweaking the osmolality of solutions within the network, they could get the droplet network to fold in a way reminiscent of muscle movement.

Bayley says that the investigators would like to move onto making larger networks. (The ones described in the paper were on the order of a few hundred micrometers.) They aim to make more intricate patterns of droplets and fill the droplets with more complex solutions.”The basic principles of how this might be achieved are now clear and further progress will just require time and patience,” says Bayley. “Our ultimate goal is to make materials that can replace or enhance living tissues, but which lack the problems associated with the use of living, replicating cells.”

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