Elimination of Manganese(II,III) Oxidation in Pseudomonas putida GB-1 by a Double Knockout of Two Putative Multicopper Oxidase Genes

Abstract

bBacterial manganese(II) oxidation impacts the redox cycling of Mn, other elements, and compounds in the environment; therefore, it is important to understand the mechanisms of and enzymes responsible for Mn(II) oxidation. In several Mn(II)-oxidizing organisms, the identified Mn(II) oxidase belongs to either the multicopper oxidase (MCO) or the heme peroxidase family of proteins. However, the identity of the oxidase in Pseudomonas putida GB-1 has long remained unknown. To identify the P. putida GB-1 oxidase, we searched its genome and found several homologues of known or suspected Mn(II) oxidase-encoding genes (mnxG, mofA, moxA, and mopA). To narrow this list, we assumed that the Mn(II) oxidase gene would be conserved among Mn(II)-oxidizing pseudomonads but not in nonoxidizers and performed a genome comparison to 11 Pseudomonas species. We further assumed that the oxidase gene would be regulated by MnxR, a transcription factor required for Mn(II) oxidation. Two loci met all these criteria: PputGB1_2447, which encodes an MCO homologous to MnxG, and PputGB1_2665, which encodes an MCO with very low homology to MofA. In-frame deletions of each locus resulted in strains that retained some ability to oxidize Mn(II) or Mn(III); loss of oxidation was attained only upon deletion of both genes. These results suggest that PputGB1_2447 and PputGB1_2665 encode two MCOs that are independently capable of oxidizing both Mn(II) and Mn(III). The purpose of this redundancy is unclear; however, differences in oxidation phenotype for the single mutants suggest specialization in function for the two enzymes.

Identification of a Third Mn(II) Oxidase Enzyme in Pseudomonas putida GB-1

“…To test these possibilities, we generated a triple mutant in which the two known Mn(II) oxidase genes and fleQ had each been deleted (⌬mnxG ⌬mcoA ⌬fleQ) ( Table 1). In previous work, the ⌬mnxG ⌬mcoA strain failed to oxidize Mn(II) (21). However, the ⌬mnxG ⌬mcoA ⌬fleQ strain retained a level of Mn(II) oxidase activity similar to that of the ⌬fleQ ⌬mnxR double mutant (Fig.…”

“…Recent work revealed the presence of two Mn(II) oxidase genes in this strain: mnxG, encoding an MCO with a low level of homology to the Bacillus sp. PL-12 Mn(II) oxidase subunit MnxG, and mcoA, encoding a second MCO distinct from MnxG (21). A strain from which both genes had been deleted failed to oxidize Mn(II) under all conditions tested.…”

“…A strain from which both genes had been deleted failed to oxidize Mn(II) under all conditions tested. Quantitative PCR (qPCR) revealed that both mnxG and mcoA are upregulated in the presence of the 54 -dependent response regulator MnxR (21). MnxR is part of a two-component regulatory pathway (the Mnx TCR) that is required for Mn(II) oxidation (22).…”

Mineralogical Diversity in Lake Pavin: Connections with Water Column Chemistry and Biomineralization Processes

“…Most of the Mn oxidation in aquatic environments is attributed to the activity of Mn-oxidizing bacteria, because abiotic Mn oxidation is kinetically limited at circumneutral pH, and Mn-oxidizing bacteria are widespread in aquatic environments, especially at oxic-anoxic interfaces, and contribute to the formation of Mn-oxides [53]. They are widely distributed across the bacterial phylogenetic tree, and various processes of Mn biomineralization have been described, involving either enzymatic processes [54,55] or the export of reactive oxygen species [56], in any case leading to the extracellular formation of Mn-oxides. Specific morphologies of Mn-oxides have been described that are similar to the present observations [57].…”

Sustained Anthropogenic Impact in Carter Saltpeter Cave, Carter County, Tennessee and the Potential Effects on Manganese Cycling

“…Several mechanisms of heterotrophic Mn(II) oxidation have been proposed. Bacteria such as Pseudomonas putida GB-1 (Geszvain et al 2013), various species of Bacilli (Francis and Tebo, 2002), and Leptothrix discophora SS-1 (Corstjens et al 1997) are all thought to use multicopper oxidases for Mn(II) oxidation. Whereas the bacteria Aurantimonas maganoxydans SI85-9A1 and Erythrobacter sp.…”