Gallionellaceae pangenomic analysis reveals insight into phylogeny, metabolic flexibility, and iron oxidation mechanisms

Abstract

The iron-oxidizing Gallionellaceae drive a wide variety of biogeochemical cycles through their metabolisms and biominerals. To better understand the environmental impacts of Gallionellaceae, we need to improve our knowledge of their diversity and metabolisms, especially any novel iron oxidation mechanisms. Here, we used a pangenomic analysis of 103 genomes to resolve Gallionellaceae phylogeny and explore their genomic potential. Using a concatenated ribosomal protein tree and key gene patterns, we determined Gallionellaceae has four genera, divided into two groups: iron-oxidizing bacteria (FeOB) Gallionella , Sideroxydans , and Ferriphaselus with iron oxidation genes ( cyc2 , mtoA ) and nitrite-oxidizing bacteria (NOB) Candidatus Nitrotoga with the nitrite oxidase gene nxr . The FeOB and NOB have similar electron transport chains, including genes for reverse electron transport and carbon fixation. Auxiliary energy metabolisms, including S oxidation, denitrification, and organotrophy, were scattered throughout the FeOB. Within FeOB, we found genes that may represent adaptations for iron oxidation, including a variety of extracellular electron uptake mechanisms. FeOB genomes encoded more predicted c -type cytochromes than NOB genomes, notably more multiheme c -type cytochromes (MHCs) with >10 CXXCH motifs. These include homologs of several predicted outer membrane porin-MHC complexes, including MtoAB and Uet. MHCs efficiently conduct electrons across longer distances and function across a wide range of redox potentials that overlap with mineral redox potentials, which can expand the range of usable iron substrates. Overall, the results of pangenome analyses suggest that the Gallionellaceae genera Gallionella , Sideroxydans , and Ferriphaselus have acquired a range of adaptations to succeed in various environments but are primarily iron oxidizers. IMPORTANCE Neutrophilic iron-oxidizing bacteria (FeOB) produce copious iron (oxyhydr)oxides that can profoundly influence biogeochemical cycles, notably the fate of carbon and many metals. To fully understand environmental microbial iron oxidation, we need a thorough accounting of iron oxidation mechanisms. In this study, we show the Gallionellaceae FeOB genomes encode both characterized iron oxidases as well as uncharacterized multiheme cytochromes (MHCs). MHCs are predicted to transfer electrons from extracellular substrates and likely confer metabolic capabilities that help Gallionellaceae occupy a range of different iron- and mineral-rich niches. Gallionellaceae appear to specialize in iron oxidation, so it would be advantageous for them to have multiple mechanisms to oxidize various forms of iron, given the many iron minerals on Earth, as well as the physiological and kinetic challenges faced by FeOB. The multiple iron/mineral oxidation mechanisms may help drive the widespread ecological success of Gallionellaceae.

Gallionellaceae in rice root plaque: metabolic roles in iron oxidation, nutrient cycling, and plant interactions

“…The rhizosphere genome bin 9BH_112 is closely related to the rice paddy soil isolate Ferrigenium kumadai An22 (90.1% ANI/91.5% AAI); however, GTDB classifies AN22 and bin 9BH_112 as Gallionella, which is also confirmed by our phylogenetic analysis based on 13 concatenated ribosomal protein sequences [ Fig. 6 ; details in methods and ( 35 )]. Thus, going forward, we refer to bin 9BH_112 as Gallionella 9BH_112 with the understanding that it is closely related to F. kumadai An22.…”

“…Support values based on 1,000 bootstraps. Further information on classification and tree construction, including all genomes in the tree, are available in Hoover et al ( 35 ).…”

“…All four 9BB/9BH Gallionellaceae genomes have genes for microaerophilic iron oxidation. The genomes contain the iron oxidase gene cyc2 , which is characteristic of most Gallionellaceae ( 35 ). They lack mtoAB , an outer membrane multiheme cytochrome-porin complex associated with iron oxidation in S. lithotrophicus ES-1 ( 70 – 72 ).…”

“…S. lithotrophicus ES-1 similarly has sox and dsr genes and is able to grow by thiosulfate oxidation. Based on previous phylogenetic analysis, the DsrAB from ES-1 and the four 9BB/9BH Gallionellaceae genomes cluster with reverse DSR (rDSR) ( 35 ).…”

“…( 24 ). While the isolate An22 was named Ferrigenium based on 16S rRNA gene dissimilarity from Gallionella , subsequent full-genome analyses showed that it is a Gallionella ( 35 ). Gallionellaceae have also been identified using 16S rRNA gene analyses including universal and taxa-specific primers in paddy soil and the rice rhizosphere ( 31 , 36 – 39 ).…”