Enzymatic manganese(II) oxidation by metabolically dormant spores of diverse Bacillus species

Abstract

Bacterial spores are renowned for their longevity, ubiquity, and resistance to environmental insults, but virtually nothing is known regarding whether these metabolically dormant structures impact their surrounding chemical environments. In the present study, a number of spore-forming bacteria that produce dormant spores which enzymatically oxidize soluble Mn(II) to insoluble Mn(IV) oxides were isolated from coastal marine sediments. The highly charged and reactive surfaces of biogenic metal oxides dramatically influence the oxidation and sorption of both trace metals and organics in the environment. Prior to this study, the only known Mn(II)-oxidizing sporeformer was the marine Bacillus sp. strain SG-1, an extensively studied bacterium in which Mn(II) oxidation is believed to be catalyzed by a multicopper oxidase, MnxG. Phylogenetic analysis based on 16S rRNA and mnxG sequences obtained from 15 different Mn(II)-oxidizing sporeformers (including SG-1) revealed extensive diversity within the genus Bacillus, with organisms falling into several distinct clusters and lineages. In addition, active Mn(II)-oxidizing proteins of various sizes, as observed in sodium dodecyl sulfate-polyacrylamide electrophoresis gels, were recovered from the outer layers of purified dormant spores of the isolates. These are the first active Mn(II)-oxidizing enzymes identified in spores or gram-positive bacteria. Although extremely resistant to denaturation, the activities of these enzymes were inhibited by azide and o-phenanthroline, consistent with the involvement of multicopper oxidases. Overall, these studies suggest that the commonly held view that bacterial spores are merely inactive structures in the environment should be revised.

FIG. 1.

Unrooted neighbor-joining trees based on 16S rRNA sequences (A) and MnxG amino acid sequences (B) obtained from 15 Mn(II)-oxidizing sporeformers (boldface). Additional sequences in panel A include diverse representatives within the genus Bacillus as well as the most closely related database sequences. The Bacillus species that have been tested for Mn(II) oxidation are given in Table 1. Percentages of bootstrap support (>60%) from 1,000 replicates are indicated at the branch points. The boldface branches highlight the topological similarities between the two trees. The strain designations of the Mn(II) oxidizers are based on the location of isolation: MB, Mission Bay; PL, off Point Loma; and SD, San Diego Bay.

FIG. 2.

SDS-PAGE gels of outermost spore layer extracts from phylogenetically diverse Mn(II)-oxidizing sporeformers. Following electrophoresis, replicate gels were incubated in either (i) Coomassie blue as a general protein stain (left), (ii) HEPES buffer containing 200 μM Mn(II) (after washing to remove SDS [see Materials and Methods]) to stain for Mn(II) oxidation activity (right), or (iii) HEPES buffer as for incubation ii but with pretreatment of the gel in HEPES buffer containing the copper chelator o-phenanthroline (50 μM) for 15 min prior to addition of Mn(II) to inhibit copper oxidases (resulting in a completely blank gel [data not shown]). A sufficient quantity of protein was loaded in each lane to yield visible Mn(II)-oxidizing proteins in gels incubated in Mn(II), as evidenced by the formation of brown Mn oxide bands.