Collaborative Research: Expanding the diversity of iron oxidation mechanisms via genetics, microscopy and 'omics in Leptothrix

Iron is practically ubiquitous across Earth, making it an important component in the chemistry of life and in the chemistry of soils, sediments, and groundwater. Iron in contact with an oxidant such as oxygen can result in iron oxidation. When iron oxidation occurs in soils, it can lead to the sequestration of nutrients and metals; thus, understanding the mechanisms and controls on iron oxidation is critical.

Microorganisms can catalyze iron oxidation, yet the extent of microorganism contribution to oxidation remains relatively unknown. This project evaluates microbial iron oxidation mechanisms (genes/proteins) to help identify when, and how much, microbial iron oxidation is occurring in the environment. This project specifically uses the iron-oxidizing bacterium Leptothrix cholodnii SP-6 to explore new iron-oxidizing mechanisms.

The advanced knowledge can be applied to determine controls on iron-oxidizing bacterial activity and how to harness it for bioremediation, water treatment, resource recovery, and other applications. This project trains two graduate students and numerous undergraduates. In addition to public lectures, hands-on activities, and lab tours, a major outreach activity includes "Microbes in the Wild" workshops for middle school girls and first-generation college-bound students, who learn about the power of microbes to address environmental challenges.

Culturing, genetics, and 'omics research tasks are being incorporated into college classes as multi-week experiential learning units.

A new genetic system in the heterotrophic iron-oxidizing bacterium Leptothrix cholodnii SP-6 provides an unprecedented opportunity to reveal new iron oxidation mechanisms. L. cholodnii SP-6 is easily culturable and its genome has numerous predicted metal oxidation genes including genes for multiheme cytochromes and multicopper oxidases with homologs in other iron oxidizers.

Yet, the genome is missing well-known iron oxidase genes, making L. cholodnii SP-6 a good model organism to investigate novel iron oxidation mechanisms. This project is using a combination of culturing, comparative genomics, transcriptomics, proteomics, genetics, and microscopy to determine the iron oxidation mechanisms in Leptothrix, notably SP-6. The research aims include (1) discover candidate iron oxidation genes in L. cholodnii SP-6 via transposon sequencing (TnSeq), transcriptomics, and proteomics.

A high throughput pipeline for culturing and assaying iron oxidation is being developed so that as many genes as possible can be screened. (2) Explore the environmental diversity and frequency of Leptothrix iron oxidation genes via isolation and genomic characterization of new Leptothrix spp. followed by comparative genomics study. (3) Validate iron oxidation genes in L. cholodnii SP-6 via genetic methods of knockout and complementation. (4) Determine the role of iron oxidation proteins in biomineralization by labeling and localizing key iron oxidation proteins and imaging cells and biominerals by confocal and electron microscopy. Together, these approaches will establish a larger set of iron oxidation genes/proteins while understanding how these catalyze formation of environmentally important iron oxyhydroxide biominerals.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.