The class Zetaproteobacteria is the sixth and most recently described class of the Pseudomonadota. Zetaproteobacteria can also refer to the group of organisms assigned to this class. The Zetaproteobacteria were originally represented by a single described species, Mariprofundus ferrooxydans, which is an iron-oxidizing neutrophilic chemolithoautotroph originally isolated from Kamaʻehuakanaloa Seamount (formerly Loihi) in 1996 (post-eruption). Molecular cloning techniques focusing on the small subunit ribosomal RNA gene have also been used to identify a more diverse majority of the Zetaproteobacteria that have as yet been unculturable. Regardless of culturing status, the Zetaproteobacteria show up worldwide in estuarine and marine habitats associated with opposing steep redox gradients of reduced (ferrous) iron and oxygen, either as a minor detectable component or as the dominant member of the microbial community. Zetaproteobacteria have been most commonly found at deep-sea hydrothermal vents, though recent discovery of members of this class in near-shore environments has led to the reevaluation of Zetaproteobacteria distribution and significance.
Significance The Zetaproteobacteria are distributed worldwide in deep sea and near shore environments at oxic/anoxic interfaces. With this wide distribution, the Zetaproteobacteria have the potential to play a substantial role in biogeochemical cycling, both past and present. Ecologically, the Zetaproteobacteria play a major role in the engineering of their own environment through the use of the controlled deposition of mineralized iron oxides, also directly affecting the environment of other members of the microbial community. Prevalence of the Zetaproteobacteria in near-shore metal (e.g. steel) coupon biocorrosion experiments highlights the impact of these marine iron oxidizers on expensive problems such as the rusting of ship hulls, metal pilings and pipelines.
Discovery
The Zetaproteobacteria were first discovered in 1991 by Craig Moyer, Fred Dobbs and David Karl as a single rare clone in a mesophilic, or moderate temperature, hydrothermal vent field known as Pele's Vents at Kamaʻehuakanaloa Seamount (formerly Loihi), Hawaii. This particular vent was dominated by sulfur-oxidizing Campylobacterota. With no close relatives known at the time, the clone was initially labeled as Gammaproteobacteria. Subsequent isolation of two strains of M. ferrooxydans, PV-1 and JV-1, along with the increasing realization that a phylogenetically distinct group of Pseudomonadota (the Zetaproteobacteria) could be found globally as dominant members of bacterial communities led to the suggestion for the creation of this new class of the Pseudomonadota.
Cultivation Neutrophilic microaerophilic Fe-oxidizing bacteria are typically cultivated using an agarose-stabilized or liquid culture with an FeS or FeCO3 plug. The headspace of the culture tube is then purged with air or a low concentration of oxygen (often 1% or less O2). Fe-oxidizers have also successfully been cultivated in liquid culture with FeCl2 as the Fe source. These cultivation techniques follow those found in Emerson and Floyd (2005). Recently, researchers have been able to culture the Zetaproteobacteria using graphite electrodes at a fixed voltage. Researchers have also aimed to improve cultivation techniques using a high-biomass batch culturing technique.
Morphology One of the most distinctive ways of identifying circumneutral iron oxidizing bacteria visually is by identifying the structure of the mineralized iron oxyhydroxide product created during iron oxidation. Oxidized, or ferric, iron is insoluble at circumneutral pH, thus the microbe must have a way of dealing with the mineralized "waste" product. It is thought that one method to accomplish this is to control the deposition of oxidized iron. Some of the most common morphotypes include: amorphous particulate oxides, twisted or helical stalks (figure), sheaths, and y-shaped irregular filaments.
These morphologies exist both in freshwater and marine iron habitats, though common freshwater iron-oxidizing bacteria such as Gallionella sp. (twisted stalk) and Leptothrix ochracea (sheath) have only extremely rarely been found in the deep sea (not significant abundance). One currently published morphotype that has been partially resolved is the twisted stalk, which is commonly formed by M. ferrooxydans. This bacteria is a gram-negative kidney-bean-shaped cell that deposits iron oxides on the concave side of the cell, forming twisted stalks as it moves through its environment. Another common Zetaproteobacteria morphotype is the sheath structure, which has yet to be isolated, but has been identified with fluorescence in situ hybridization (FISH). Iron oxidation morphotypes can be preserved and have been detected in ancient hydrothermal deposits preserved in the rock record. Some current work is focused on how the Zetaproteobacteria form their individual biominerals in the modern environment so that scientists can better interpret Fe biominerals found in the rock record.
Ecology
Biodiversity An operational taxonomic unit, or an OTU, allows a microbiologist to define a bacterial taxa using defined similarity bins based on a gene of interest. In microbial ecology, the small subunit ribosomal RNA gene is generally used at a cut off of 97% similarity to define an OTU. In the most basic sense, the OTU represents a bacterial species. For the Zetaproteobacteria, 28 OTUs have been defined. Of interest were the two globally distributed OTUs that dominated the phylogenetic tree, two OTUs that seemed to originate in the deep subsurface, and several endemic OTUs, along with the relatively limited detection of the isolated Zetaproteobacteria representative.
Classification Zetaproteobacteria OTUs can now be classified according to the naming scheme used in McAllister et al. (2011). The program ZetaHunter uses closed reference binning to identify sequences closely related to the established OTUs in addition to identifying novel Zetaproteobacteria OTUs. ZetaHunter's feature list continues to grow, but includes: 1) stable OTU binning, 2) sample comparison, 3) database and mask management options, 4) multi-threaded processing, 5) chimera checking, 6) checks for non-database-related sequences, and 7) OTU network maps. The ZetaHunter software can be downloaded at: https://github.com/mooreryan/ZetaHunter
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