Microaerophilic, neutrophilic, iron-oxidizing bacteria (FeOB) grow via the oxidation of reduced Fe(II) at or near neutral pH, in the presence of oxygen, making them relevant in numerous environments with elevated Fe(II) concentrations. However, the biochemical mechanisms for Fe(II) oxidation by these neutrophilic FeOB are unknown, and genetic markers for this process are unavailable. In the ocean, microaerophilic microorganisms in the genus Mariprofundus of the class Zetaproteobacteria are the only organisms known to chemolithoautotrophically oxidize Fe and concurrently biomineralize it in the form of twisted stalks of iron oxyhydroxides. The aim of this study was to identify highly expressed proteins associated with the electron transport chain of microaerophilic, neutrophilic FeOB. To this end, Mariprofundus ferrooxydans PV-1 was cultivated, and its proteins were extracted, assayed for redox activity, and analyzed via liquid chromatography-tandem mass spectrometry for identification of peptides. The results indicate that a cytochrome c 4 , cbb 3 -type cytochrome oxidase subunits, and an outer membrane cytochrome c were among the most highly expressed proteins and suggest an involvement in the process of aerobic, neutrophilic bacterial Fe oxidation. Proteins associated with alternative complex III, phosphate transport, carbon fixation, and biofilm formation were abundant, consistent with the lifestyle of Mariprofundus. Iron (Fe) is one of the most abundant elements on Earth and a major component of the oceanic crust (1). The biologically catalyzed oxidation of Fe at circumneutral pH with oxygen (O 2 ) as the terminal electron acceptor has remained largely enigmatic, even though neutrophilic Fe oxidation is among the first chemoautotrophic microbial metabolisms described in the literature (2). This lack of data is due, in part, to obstacles such as culturing of fastidious microaerophilic, neutrophilic, Fe-oxidizing bacteria (FeOB); the relatively low cell densities in cultures; and the interference of Fe oxides with sample preparation. In addition to this, aerobic, neutrophilic FeOB have so far been elusive to genetic manipulation. Consequently, these challenges have impeded the ability to understand the mechanisms of neutrophilic Fe oxidation in the presence of O 2 and inhibited the development of molecular diagnostics targeting genetic markers for such a biological function (i.e., molecular probes targeting genes, transcripts, or proteins indicative of activity). Recent genomic analyses of microaerophilic, neutrophilic FeOB (3-6) have suggested genes that might be involved in Fe oxidation; however, evidence of expression of these genes in FeOB has not been shown. This is in contrast with the recent advancements in the elucidation of the mechanisms of Fe oxidation in aerobic, acidophilic bacteria (especially Acidithiobacillus ferrooxidans and Leptospirillum spp.) and neutrophilic, anoxygenic, phototrophic bacteria (Rhodopseudomonas palustris and Rhodobacter spp.) (see reference 7 for a review).Mariprofundus fer...

Seafloor basalts are widely distributed and host diverse prokaryotic communities, but no data exist concerning the metabolic rates of the resident microbial communities. We present here potential extracellular enzyme activities of leucine aminopeptidase (LAP) and alkaline phosphatase (AP) measured on basalt samples from different locations on Loihi Seamount, HI, coupled with analysis of prokaryotic biomass and pyrosequencing of the bacterial 16S rRNA gene. The community maximum potential enzyme activity (V max ) of LAP ranged from 0.47 to 0.90 nmol (g rock) ؊1 h ؊1 ; the V max for AP was 28 to 60 nmol (g rock) ؊1 h ؊1 . The K m of LAP ranged from 26 to 33 M, while the K m for AP was 2 to 7 M. Bacterial communities on Loihi basalts were comprised primarily of Alpha-, Delta-, andGammaproteobacteria, Bacteroidetes, and Planctomycetes. The putative ability to produce LAP is evenly distributed across the most commonly detected bacterial orders, but the ability to produce AP is likely dominated by bacteria in the orders Xanthomonadales, Flavobacteriales, and Planctomycetales. The enzyme activities on Loihi basalts were compared to those of other marine environments that have been studied and were found to be similar in magnitude to those from continental shelf sediments and orders of magnitude higher than any measured in the water column, demonstrating that the potential for exposed basalts to transform organic matter is substantial. We propose that microbial communities on basaltic rock play a significant, quantifiable role in benthic biogeochemical processes. Exposed seafloor basalts comprise a 600,000-km 2 continuous undersea habitat (1). Endolithic basalt microbes are diverse and abundant (2), and several clades of bacteria appear more likely to be encountered on basalts than elsewhere (3). Bacterial phyla display trends in abundance that reflect rock geochemistry, indicating a strong selection of microbial communities by rock composition (4). Genes diagnostic for methanogenesis, nitrogen fixation, anaerobic ammonium oxidation, denitrification, Fe reduction, and dissimilatory sulfate reduction are present in basalt microbial communities (3), indicating the potential for diverse biogeochemical transformations on basalts. However, no data are currently available for metabolic activity rates of basaltic microbes.The relationship between the presence of prokaryotes and the activity and function of enzymes in extreme environments in general and basalts in particular is underexplored. Hydrolytic extracellular enzymes have been shown to be indicators of metabolically active bacteria, and existing data sets from various marine environments can be used for comparison with information from newly explored areas (5, 6). Additionally, in deep sea environments, organic matter is more refractory in nature, and therefore, extracellular enzyme hydrolases should play an important role in the initiation of organic matter recycling. Indeed, recent work showed that the most abundant group of Archaea in marine sediments produces unique...