September 4, 2014 § Leave a comment
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.”