Genetic analysis of manganese(II) oxidation by spores of the marine Bacillus sp. strain SG-1

The marine Bacillus sp. strain SG-1 forms spores in which a component of the spore coat catalyzes the oxidation of manganese(II). Methods for genetic manipulation of SG-1 including protoplast transformation and transposon Tn917 mutagenesis were developed. Transposon mutants were isolated that still form spores but no longer oxidize manganese. Restriction mapping showed that the majority of insertions generating this phenotype clustered in two loci of the chromosome (Mnx regions). Complementation of one of the mutants confirmed that the area plays a role in manganese oxidation.;Spores from nonoxidizing mutants showed no changes in their resistance or germination properties, but transmission electron microscopy of the mutant spores suggested that the outermost ridged spore coat layers, where manganese oxide normally precipitates, were more loosely fitting when compared to wild-type. The latter suggests that oxidation activity may play a role in the assembly of this structure. The transposon had inserted in the correct orientation in one of the mutants from the largest Mnx region to create a transcriptional lacZ fusion. Measurement of ;Sequence analysis of the two Mnx regions revealed a total of eight genes, three with predicted products of extremely hydrophobic nature and, in general, showed other spore coat protein-like properties. Seven of the genes clustered within the largest region and showed an operon-like structure. Two of the genes showed similarity to the family of multi-copper oxidases, specifically in the areas involved in copper binding. These results suggest (1) that manganese oxidation is carried out by one or more spore coat proteins, probably residing in the outermost spore coat, and (2) that at least two of the genes identified in the Mnx regions are good candidate proteins to be the actual oxidizing factors and that copper centers may be involved in the oxidation of manganese(II) by the spores. Study of SG-1 spores will allow further understanding of both bacterially mediated manganese oxidation and bacterial spore coat structure and assembly.