Genomic insights into Mn(II) oxidation by the marine alphaproteobacterium Aurantimonas sp. strain SI85-9A1

Abstract

Microbial Mn(II) oxidation has important biogeochemical consequences in marine, freshwater, and terrestrial environments, but many aspects of the physiology and biochemistry of this process remain obscure. Here, we report genomic insights into Mn(II) oxidation by the marine alphaproteobacterium Aurantimonas sp. strain SI85-9A1, isolated from the oxic/anoxic interface of a stratified fjord. The SI85-9A1 genome harbors the genetic potential for metabolic versatility, with genes for organoheterotrophy, methylotrophy, oxidation of sulfur and carbon monoxide, the ability to grow over a wide range of O(2) concentrations (including microaerobic conditions), and the complete Calvin cycle for carbon fixation. Although no growth could be detected under autotrophic conditions with Mn(II) as the sole electron donor, cultures of SI85-9A1 grown on glycerol are dramatically stimulated by addition of Mn(II), suggesting an energetic benefit from Mn(II) oxidation. A putative Mn(II) oxidase is encoded by duplicated multicopper oxidase genes that have a complex evolutionary history including multiple gene duplication, loss, and ancient horizontal transfer events. The Mn(II) oxidase was most abundant in the extracellular fraction, where it cooccurs with a putative hemolysin-type Ca(2+)-binding peroxidase. Regulatory elements governing the cellular response to Fe and Mn concentration were identified, and 39 targets of these regulators were detected. The putative Mn(II) oxidase genes were not among the predicted targets, indicating that regulation of Mn(II) oxidation is controlled by other factors yet to be identified. Overall, our results provide novel insights into the physiology and biochemistry of Mn(II) oxidation and reveal a genome specialized for life at the oxic/anoxic interface.

FIG. 1.

Phylogeny of Mn(II)-oxidizing Alphaproteobacteria based on the16S rRNA gene. Mn(II) oxidizers are denoted with “+,” and isolates that have been tested and do not oxidize Mn(II) are denoted with “−.” 16S rRNA gene sequences and Mn(II) oxidation data were compiled from references , , , , and .

FIG. 2.

(A) Growth of SI85-9A1 on glycerol with 100 μM MnCl₂ (closed circles), without added Mn (open circles), and with 100 μM MnCl₂ added to replicates of the no-Mn-added culture in the middle of the growth curve (closed triangles). Growth on glucose is much faster and is not stimulated by Mn(II) (data not shown; cultures reach optical densities of ∼0.1 by 200 h and ∼0.8 by 600 h). (B) Mn oxide formation by the glycerol plus MnCl₂ culture shown in panel A. Error bars represent standard deviations for triplicate cultures.

FIG. 3.

SDS-PAGE analysis of the SI85-9A1 extracellular fraction during growth on glycerol. The gel was cut and one-half stained with Coomassie (left) and one-half screened with an in-gel Mn(II) oxidation activity assay (right). The latter was then immersed in LBB, which reacts specifically with Mn oxide to produce a cobalt blue color (H and I).

FIG. 4.

Duplicated regions surrounding the mox genes; shown are the Mox-1 region (top) and the Mox-2 region (bottom). Predicted open reading frames are represented by arrows. Percentages indicate identity of predicted protein sequence between duplicated genes.

FIG. 5.

Neighbor-joining phylogenetic trees based on 16S rRNA gene (A) and predicted MoxA protein sequences (B). Bootstrap values are the result of 1,000 replicates. Similar tree topologies resulted from all phylogenetic methods tested, including parsimony, minimum evolution, and the unweighted-pair group method using average linkages. In panel A, Burkholderia pseudomallei includes strains 1106b, 1710b, and K96243. In panel B, MoxA sequences with phylogenies apparently different from those of the 16S rRNA gene from that organism are indicated with symbols representing their 16S rRNA lineages: α, Alphaproteobacteria; γ, Gammaproteobacteria.

FIG. 5.

Neighbor-joining phylogenetic trees based on 16S rRNA gene (A) and predicted MoxA protein sequences (B). Bootstrap values are the result of 1,000 replicates. Similar tree topologies resulted from all phylogenetic methods tested, including parsimony, minimum evolution, and the unweighted-pair group method using average linkages. In panel A, Burkholderia pseudomallei includes strains 1106b, 1710b, and K96243. In panel B, MoxA sequences with phylogenies apparently different from those of the 16S rRNA gene from that organism are indicated with symbols representing their 16S rRNA lineages: α, Alphaproteobacteria; γ, Gammaproteobacteria.