Phosphonates Utilization in Marine and Freshwater Picocyanobacteria

A PCR-based assay is described, designed to detect expression of the phosphonate assimilation gene phnD from picocyanobacteria. The phnD gene encodes the phosphonate binding protein of the ABC-type phosphonate transporter, present in many of the picocyanobacterial genome sequences. Detection of phnD expression can indicate a capacity of picoplankton to utilize phosphonates, a refractory form of phosphorus that can represent 25% of the high molecular weight dissolved organic phosphorus pool in marine systems. Primer sets were designed to specifically amplify phnD sequences from marine and freshwater Synechococcus spp., Prochlorococcus spp. and environmental samples from the ocean and Laurentian Great Lakes. Marine strain Synechococcus WH8102 and some freshwater Synechococcus strains are able to grow on phosphonates as a sole P source; particularly, freshwater Synechococcus ARC-21 is able to utilize synthetic phosphonate herbicide (glyphosate). Quantitative RT-PCR from cultured marine Synechococcus sp. strain WH8102 and freshwater Synechococcus sp. ARC-21 demonstrated induction of phnD expression in P-deficient media, suggesting that phn genes are regulated coordinately with genes under phoRB control. However, pho box was not found in the putative promoters of phosphonate utilization genes in picocyanobacteria. Last, RT-PCR of environmental RNA samples from the Sargasso Sea, Pacific Ocean, and the Baltic Sea detected phnD expression from the endemic picocyanobacterial population. Synechococcus spp. phnD expression yielded a depth-dependent pattern following gradients of P bioavailability, and addition of phosphate to natural sample resulted in deactivation of Synechococcus phnD expression. By contrast, the Prochlorococcus spp. primers revealed that in all samples tested, phnD expression was constitutive. In overall, this study demonstrated the significance of phosphonates as a phosphorus source in the DOP pool for picocyanobacteria in P-depleted environments. The method described herein will allow future studies aimed at understanding the utilization of naturally-occurring phosphonates in the ocean as well as monitoring the acquisition of synthetic phosphonate herbicides (e.g. glyphosate) by picocyanobacteria in fresh waters