Cells of Leptothrix discophora SS1 released Mn2+-oxidizing factors into the medium during growth in batch culture. Manganese was optimally oxidized when the medium was buffered with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) at pH 7.5. Manganese-oxidizing activity in the culture medium in which this strain had been grown previously was sensitive to heat, phosphate, Tris, NaN3, HgCl2 NaCl, sodium dodecyl sulfate, and pronase; 0.5 mol of 02 was consumed per mol of MnO2 formed. During Mn2+ oxidation, protons were liberated. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two protein-containing bands were detected in the spent culture medium. One band had an apparent molecular weight of 110,000 and was predominant in Mn2+-oxidizing activity. The second product (Mr 85,000) was only detected in some cases and probably represents a proteolytic breakdown moiety of the 110,000-Mr protein. The Mn2+-oxidizing factors were associated with the MnO2 aggregates that had been formed in spent culture medium. After solubilization of this MnO2 with ascorbate, Mn2+-oxidizing activity could be recovered.

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The Mn 2؉ -oxidizing bacterium Pseudomonas fluorescens GB-1 deposits Mn oxide around the cell. During growth of a culture, the Mn 2؉ -oxidizing activity of the cells first appeared in the early stationary growth phase. It depended on the O 2 concentration in the culture during the late logarithmic growth phase. Maximal activity was observed at an oxygen concentration of 26% saturation. The activity could be recovered in cell extracts and was proportional to the protein concentration in the cell extracts. The specific activity was increased 125-fold by ammonium sulfate precipitation followed by reversed-phase and gel filtration column chromatographies. The activity of the partly purified Mn 2؉ -oxidizing preparation had a pH optimum of circa 7 and a temperature optimum of 35°C and was lost by heating. The Mn 2؉ -oxidizing activity was sensitive to NaN 3 and HgCl 2 . It was inhibited by KCN, EDTA, Tris, and o-phenanthroline. Although most data indicated the involvement of protein in Mn 2؉ oxidation, the activity was slightly stimulated by sodium dodecyl sulfate at a low concentration and by treatment with pronase and V8 protease. By polyacrylamide gel electrophoresis, two Mn 2؉ -oxidizing factors with estimated molecular weights of 180,000 and 250,000 were detected.

Pseudomonas putida GB-1-002 catalyzes the oxidation of Mn2+. Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designatedcumA) encoding a protein homologous to multicopper oxidases. Addition of Cu2+ increased the Mn2+-oxidizing activity of the P. putidawild type by a factor of approximately 5. The growth rates of the wild type and the mutant were not affected by added Cu2+. A second open reading frame (designatedcumB) is located downstream from cumA. BothcumA and cumB probably are part of a single operon. The translation product of cumB was homologous (level of identity, 45%) to that of orf74 ofBradyrhizobium japonicum. A mutation in orf74resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the cumAmutant, suggesting that the cumA mutation may have a polar effect on cumB. This was confirmed by site-specific gene replacement in cumB. The cumB mutation did not affect the Mn2+-oxidizing ability of the organism but resulted in decreased growth. In summary, our data indicate that the multicopper oxidase CumA is involved in the oxidation of Mn2+ and that CumB is required for optimal growth of P. putida GB-1-002.

Coccoliths are delicate calcified structures produced by marine unicellular algae. In the species Emiliania huxleyi the calcium carbonate (mostly calcite) is closely associated with a complex, acidic polysaccharide which binds calcium ions specifically, interferes with the in vitro crystallization of calcium carbonate, and appears to be bound to a positively charged protein before the crystallization process is finished. Ultra-high resolution electron microscopy of the coccoliths reveals that the crystallographic structure differs in different parts of the constituent calcite elements. The synthesis of the coccoliths takes place intracellularly, and when this process is ended the coccoliths are extruded and incorporated into the so-called coccosphere surrounding the cell. Transmission electron microscope studies reveal the localization of polysaccharides in the calcifying organelle by means of cytochemical staining technique. The results are combined in a putative scheme describing coccolithogenesis.