Metagenomic Analyses of the Autotrophic Fe(II)-Oxidizing, Nitrate-Reducing Enrichment Culture KS

Abstract

f Nitrate-dependent ferrous iron [Fe(II)] oxidation (NDFO) is a well-recognized chemolithotrophic pathway in anoxic sediments. The neutrophilic chemolithoautotrophic enrichment culture KS originally obtained from a freshwater sediment (K. L. Straub, M. Benz, B. Schink, and F. Widdel, Appl Environ Microbiol 62:1458 -1460, 1996) has been used as a model system to study NDFO. However, the primary Fe(II) oxidizer in this culture has not been isolated, despite extensive efforts to do so. Here, we present a metagenomic analysis of this enrichment culture in order to gain insight into electron transfer pathways and the roles of different bacteria in the culture. We obtained a near-complete genome of the primary Fe(II) oxidizer, a species in the family Gallionellaceae, and draft genomes from its flanking community members. A search of the putative extracellular electron transfer pathways in these genomes led to the identification of a homolog of the MtoAB complex [a porin-multiheme cytochrome c system identified in neutrophilic microaerobic Fe(II)-oxidizing Sideroxydans lithotrophicus ES-1] in a Gallionellaceae sp., and findings of other putative genes involving cytochrome c and multicopper oxidases, such as Cyc2 and OmpB. Genome-enabled metabolic reconstruction revealed that this Gallionellaceae sp. lacks nitric oxide and nitrous oxide reductase genes and may partner with flanking populations capable of complete denitrification to avoid toxic metabolite accumulation, which may explain its resistance to growth in pure culture. This and other revealed interspecies interactions and metabolic interdependencies in nitrogen and carbon metabolisms may allow these organisms to cooperate effectively to achieve robust chemolithoautotrophic NDFO. Overall, the results significantly expand our knowledge of NDFO and suggest a range of genetic targets for further exploration.

Insights into Nitrate-Reducing Fe(II) Oxidation Mechanisms through Analysis of Cell-Mineral Associations, Cell Encrustation, and Mineralogy in the Chemolithoautotrophic Enrichment Culture KS

“…accounts for only 42% of the total community under autotrophic conditions (31). It has to be taken into account, however, that this culture has been transferred with a 10% inoculum for years while ours was transferred with a 1% inoculum, also for years (31). Previous studies found evidence that the remainder of the community in culture KS consists of strains that are not capable of autotrophic NRFeO but are heterotrophic nitrate reducers (10,30).…”

“…This strain, here referred to as Gallionellaceae sp., was designated the autotrophic Fe(II) oxidizer in the culture (30). In the culture used in this study, it accounts for 96% of the total community under autotrophic growth conditions with Fe(II) and nitrate (31). In another version of culture KS from a different laboratory, which also has different community members, Gallionellaceae sp.…”

“…In another version of culture KS from a different laboratory, which also has different community members, Gallionellaceae sp. accounts for only 42% of the total community under autotrophic conditions (31). It has to be taken into account, however, that this culture has been transferred with a 10% inoculum for years while ours was transferred with a 1% inoculum, also for years (31).…”

“…Previous studies found evidence that the remainder of the community in culture KS consists of strains that are not capable of autotrophic NRFeO but are heterotrophic nitrate reducers (10,30). However, a recent metagenomic study also found genes for CO 2 fixation in the genome of these strains (31 (31). In addition, it can oxidize poorly soluble Fe(II)-bearing minerals (10,32,33).…”

“…Although potential mutual dependencies between the different bacterial strains in culture KS are not fully understood, recent metagenomics analyses suggest a dependence of Gallionellaceae sp. on the other bacteria for complete denitrification to N 2 (31).…”