The marine, neutrophilic, and chemolithoautotrophic iron-oxidizing bacteria: Insights into the physiology of zetaproteobacteria and the discovery of novel iron-oxidizing gammaproteobacteria

Iron-oxidation performed by bacteria at or near neutral pH is a biological reaction that has been known since the early 1800's. Despite the number of years since its documentation and its global contribution to the biogeochemical cycle of iron, the biological mechanism of bacterial, neutrophilic, iron-oxidation has remained an enigma. Mariprofundus ferrooxydans is the first marine, neutrophilic, chemolithoautotrophic, iron-oxidizing bacteria (FeOB) that has been isolated. Its genome has been sequenced and insights about its physiology were inferred by identifying potential genes in the electron transport chain but no definite mechanism of iron-oxidation was proposed. Here, different approaches involving large-scale culturing and proteomics were combined in order to provide more definite answers about the iron-oxidation mechanism of M. ferrooxydans and FeOB in general. In-situ incubations and proteomics were also combined and applied in the marine environment to study FeOB communities colonizing iron-sulfide minerals.;FeOB are historically difficult organisms to work with that usually produce low-biomass. In order to produce enough biomass for proteomic analysis, a large-scale culturing technique was developed for M. ferrooxydans. Proteins released from these cultures were found to interact strongly with the iron mats of M. ferrooxydans and thereby affect protein extractions. Therefore, a method to circumvent protein binding to the mats is described herein. These methods were used to produce a proteomic profile of actively-growing M. ferrooxydans. The resulting proteomic profile identified numerous components of the electron-transport chain, including an abundant periplasmic cytochrome c as well as cbb3-type cytochrome oxidases. As a result, a more specific pathway for electron transport in M. ferrooxydans is described.;In order to test the developed methods in the marine environment, FeOB communities colonizing iron-sulfide minerals in shallow waters of Catalina Islands, CA were analyzed. In general the results indicate that in-situ enriched iron-sulfides host species-rich communities that are different from the background seawater and similar to inactive hydrothermal vent chimney sulfides. Many of the clones recovered from the iron-sulfide mineral were closely related to deep-sea clones, indicating that this in-situ incubation method is appropriate for the study of microorganisms that are usually seen in deep-sea habitats such as hydrothermal vents and exposed ocean crust. From these in-situ incubations a microorganism previously known only for sulfur oxidation, Thiomicrospira spp., was isolated and shown to be capable of performing Fe oxidation. It is also shown for the first time that the sheath-Zetaproteobacteria can be grown in the laboratory. The data herein presented reveals that environmental proteomics of hard substrates such as iron-sulfide mineral and mild steel can be successfully achieved, opening the door to similar analyses in the ocean floor.