Diversity and abundance of phosphonate biosynthetic genes in nature

Authors: Labeda, David; YU, XIAOMIN - University Of Illinois; DOROGHAZI, JAMES - University Of Illinois; JANGA, SARATH CHANDRA - University Of Illinois; ZHANG, JUN KAI - University Of Illinois; CIRCELLO, BENJAMIN - University Of Illinois; GRIFFIN, BENJAMIN - University Of Illinois; METCALF, WILLIAM - University Of Illinois

Submitted to: Proceedings of the National Academy of Sciences (PNAS)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/8/2013
Publication Date: 12/17/2013
Publication URL: http://handle.nal.usda.gov/10113/60342
Citation: Yu, X., Doroghazi, J.R., Janga, S., Zhang, J., Circello, B.T., Griffin, B.M., Labeda, D.P., Metcalf, W.W. 2013. Diversity and abundance of phosphonate biosynthetic genes in nature. Proceedings of the National Academy of Sciences. 110(51):20759-20764.

Interpretive Summary: Phosphonates, molecules that contain directly linked carbon and phosphorus atoms, comprise an extremely diverse group of biologically produced compounds. The potential for production of these compounds in nature and the estimated range of all possible types of phosphonate-containing molecules that could be produced had not been done to date. Because it was known that one key enzyme, phosphoenolpyruvate mutase (pepM), is necessary for the biosynthesis of molecules containing the phosphonate structure we postulated that this gene could be used as a molecular marker to estimate ability to produce phosphonates. Genome sequences in public databases, including sequences determined from metagenomes, bulk DNA samples prepared from environment samples, as well as DNA prepared from a diverse set of microbial isolates were screened for the presence of the pepM gene resulting in the discovery that pepM is relatively common (5 to 7% positive), particularly in the actinomycetes, that are typical soil microorganisms. An estimate of the diversity of potentially novel compounds produced was obtained by sequencing the pepM gene and the 3 genes before and after it on chromosomal DNA from 25 different actinomycete strains and then comparing these sequences to the genome data in the public databases which confirmed that sequence of pepM could reliably predict the capability to produce different compounds. Evaluation of the number of different pepM sequences found in DNA isolated from local soils predicted that there were microorganisms present in the soil with the ability to produce dozens of potentially novel phosphonate-containing compounds, and the potential to produce hundreds of new phosphonate structures were observed in the public metagenome sequence databases from various environmental sources. These microbially-produced phosphonate-containing molecules could play an important role in the global cycling of phosphorus in soil and water and, more importantly, provide an untapped source of novel biologically active natural products with the potential for significant impact in agriculture or human and veterinary medicine.

Technical Abstract: Phosphonates, molecules containing direct C-P bonds, comprise a structurally diverse class of natural products with interesting and useful biological properties. Although their synthesis in protozoa was discovered more than fifty years ago, the extent and diversity of phosphonate production in nature remains poorly characterized. The rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate, catalyzed by the enzyme PEP mutase (PepM), is shared by the vast majority of known phosphonate biosynthetic pathways. Thus, the pepM gene can be used as a molecular marker to examine the occurrence and abundance of phosphonate-producing organisms. Based on the presence of this gene, phosphonate biosynthesis is common in microbes, with ca. 5% of sequenced genomes and 7% of genome equivalents in metagenomic datasets carrying pepM. Similarly, we detected the pepM gene in ca. 5% of random actinomycete isolates. The pepM-containing gene neighborhoods from twenty-five of these isolates were cloned, sequenced and compared with those found in sequenced genomes. PEP mutase sequence conservation is strongly correlated with conservation of other nearby genes, suggesting that the diversity of phosphonate biosynthetic pathways can be predicted by examining PEP mutase diversity. We used this approach to estimate the range of phosphonate biosynthetic pathways in nature, revealing dozens of discrete groups in pepM amplicons from local soils, while hundreds were observed in metagenomic datasets. Collectively, our analyses show that phosphonate biosynthesis is both common and diverse in nature, suggesting that the role of these molecules in a phosphorus-limited biosphere may be more important than commonly recognized.